UNDERSTANDING THE FUNCTIONAL ROLE OF THE XENOPUS LAEVIS
CORTICAL GRANULE LECTIN OLIGOSACCHARIDES IN THE BLOCK TO
POLYSPERMIC FERTILIZATION
Peter Simon Helminski
B.S., California State University, Sacramento, 2005
THESIS
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
in
BIOLOGICAL SCIENCES
(Molecular and Cellular Biology)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2012
© 2012
Peter Simon Helminski
ALL RIGHTS RESERVED
ii
UNDERSTANDING THE FUNCTIONAL ROLE OF THE XENOPUS LAEVIS
CORTICAL GRANULE LECTIN OLIGOSACCHARIDES IN THE BLOCK TO
POLYSPERMIC FERTILIZATION
A Thesis
by
Peter Simon Helminski
Approved by:
__________________________________, Committee Chair
Thomas R. Peavy, Ph.D.
__________________________________, Second Reader
Tom Landerholm, Ph.D.
__________________________________, Third Reader
Hao Nguyen, Ph.D.
___________________________
Date
iii
Student: Peter Simon Helminski
I certify that this student has met the requirements for format contained in the
University format manual, and that this thesis is suitable for shelving in the Library and
credit is to be awarded for the thesis.
__________________________, Graduate Coordinator
Ronald M. Coleman, Ph.D.
Department of Biological Sciences
iv
_________________
Date
Abstract
of
UNDERSTANDING THE FUNCTIONAL ROLE OF THE XENOPUS LAEVIS
CORTICAL GRANULE LECTIN OLIGOSACCHARIDES IN THE BLOCK TO
POLYSPERMIC FERTILIZATION
by
Peter Simon Helminski
The fertilization layer of Xenopus laevis eggs is formed by the cortical granule
lectin (CGL) binding to its ligand. This lectin is released from the egg cortical granules
at fertilization and biologically functions in providing a block to polyspermy, an
essential step in the fertilization process. Xenopus laevis CGL has a calcium requiring
binding specificity for galactose, and exists as a large oligomeric complex composed of
10-12 CGL subunits. The carbohydrate moiety consists of three types of N-linked
glycans: high mannose, hybrid, and complex types. In addition, sialic acid residues are
known to be present on the hybrid and complex glycans. Because sialic acids often play
an important role in binding interactions, this study was undertaken to evaluate the
hypothesis that the binding of the Xenopus laevis CGL glycoprotein to its ligand
counterpart is dependent on its N-linked hybrid/complex oligosaccharides. Elucidation
of the role of the xlCGL glycans on binding will be not only informative for
understanding the block to polyspermy, but for a variety of other functions performed
by homologues of xlCGL such as pathogen surveillance and allergic responses in
humans.
v
In the current study, two different strategies were employed to modify the Nlinked oligosaccharides of the xlCGL and evaluate their role in binding. The first
strategy used enzymatic methods to remove different types of N-linked oligosaccharides
from xlCGL, whereas the second modified the N-linked oligosaccharides found on
xlCGL by expressing a recombinant form of CGL in a yeast (Pichia pastoris)
expression system which could only make simple mannose structures and not
hybrid/complex oligosaccharides. Modified CGL was then evaluated for binding to its
jelly ligand using a plate binding assay.
Yeast CGL (yCGL) clones generated previously were evaluated for secretion
and purification to enable the binding studies. Although the yCGL clone was found to
have an extra 21 amino acids added to its C-terminus from the vector, the clone was
found to secrete yCGL into the culture medium at a level of ~1mg/ml, and was partially
purified by ammonium sulfate precipitation. Immunoblotting analysis revealed that
deglycosylated yCGL (PNGaseF treated) was the appropriate size for the polypeptide
(~35kD) whereas glycosylated yCGL had an apparent size of ~65kD, significantly
larger than xlCGL (~45kD). Ligand binding activity of yCGL was found to be
significantly reduced, but since the preparation was only partially purified due to
difficulties, a quantitative analysis was not feasible.
Complete removal of xlCGL N-linked glycans by PNGaseF resulted in a 91%
decrease of binding indicating that the N-glycans were indeed important. Treatment
with two sialidases that remove sialic acid residues reduced binding by 86%
vi
with the α2-3 linkage of sialic acid being the most important. Competitive inhibition of
xlCGL binding to its ligand using a sialic acid analog, N-acetylneuraminic acid,
decreased binding to ~80% at a 2mM concentration, indicating that sialic acid plays a
direct role in binding. It is likely that sialic acid is involved in coordinating calcium
and facilitating oligomer formation, since calcium promotes oligomer formation and
yCGL was unable to form oligomers. Treatment with two mannosidases to remove
mannose residues in a variety of linkages (α1-2,3,6) also reduced binding by 86%,
which indicated that mannose residues were also important for binding. It is unclear as
to the role mannose residues play in binding, but it is hypothesized that they play a
structurally supportive role for proper presentation of the binding pocket.
Taken together, enzymatic, genetic, and inhibition experiments demonstrate that
the N-glycans of the Xenopus laevis cortical granule lectin are required for binding to its
ligand. Specifically, terminal sialic acid residues on CGL play an important role in the
binding mechanism to its ligand. These experiments have enhanced our knowledge of
the CGL-ligand binding mechanism, and are likely to be applicable to the binding
interactions of other CGL homologues.
___________________________, Committee Chair
Thomas R. Peavy, Ph.D.
___________________________
Date
vii
ACKNOWLEDGEMENTS
I would like to give a special thanks to Dr. Tom Peavy for accepting to serve as
my thesis advisor and mentor. I would like to thank him for introducing me into the
research laboratory and for proposing this specific research project for my thesis
research. I would like to thank him for kindly providing the purified cortical granule
lectin (CGL) and Xenopus laevis egg jelly. Furthermore, I would like to thank him for
his supervisory role and support in my research efforts and lending his expertise in
bench techniques, data interpretation, proof reading, and preparing me for my thesis
advancement and defense. His guidance showed me many of the qualities a good
scientist should have, including inquisitiveness, resourcefulness, skepticism, and most
importantly, perseverance. I look forward to continuing our friendship for many years to
come.
I would like to thank Dr. Hao Nguyen and Dr. Tom Landerholm for accepting to
serve as members of my graduate committee, and facilitating my advancement to
candidacy. I have enjoyed the many conversations in the basement regarding life as a
grad student, and the resiliency necessary to succeed in research. Their advice was
much appreciated. I am thankful for Dr. Nguyen’s undergraduate molecular biology
course, which was a catalyst for me to pursue a career in molecular biology.
viii
I would like to thank my graduate colleague and friend Noah Kiedrowski for
providing laboratory support and collaborations on experiments. I would like to thank
him for assisting me with the binding assay parameters, and advice on writing the
thesis. Working with Noah represented one of the best aspects of graduate school,
which is making lifelong friendships.
I would like to thank Breanna Wallace for her work in screening the clone
library, and teaching me how to express yCGL.
I would like to thank Ravi Singh and Matt Sugimoto for their hard work in
expressing the recombinant glycoprotein, optimizing glycoprotein concentration and
desalting by ultrafiltration under pressure, and the subsequent analysis by SDS-PAGE
and Bradford assay. Their dedication to doing what was necessary to get results was
much appreciated.
I would like to give a special thanks to my brother and mother for their
unconditional love, support, and patience throughout my thesis research. They have
been extremely supportive and helpful during times when I have encountered difficult
academic, research and/or personal obstacles during graduate school. They have been a
large part of my overall success as both a researcher and as a person. I appreciate their
tolerance and patience during periods of frustration and setbacks. Lastly, I would like to
thank them for assisting me through times of doubt about graduate school, and for
helping me maintain my perseverance and optimism.
ix
TABLE OF CONTENTS
Page
Acknowledgements.........................................................................................................viii
List of Figures................................................................................................................. xiii
INTRODUCTION .............................................................................................................1
MATERIALS AND METHODS .....................................................................................23
yCGL Transformants ...........................................................................................23
yCGL DNA Purification ......................................................................................23
PCR Analysis of yCGL ........................................................................................24
Sequencing of PCR Amplified yCGL cDNA ......................................................25
Secreted Expression of Recombinant CGL in Pichia pastoris ............................25
Quantification of Protein Concentration by Bradford Assay...............................26
Protein Precipitation with Trichloroacetic Acid ..................................................27
Procurement and Preparation of Xenopus laevis Egg Jelly and CGL ..................27
Purification of IgG Antibody Specific to Deglycosylated xlCGL .......................28
Titering of Antigen and Antibodies .....................................................................29
CGL Analysis by SDS-PAGE and Immunoblotting ............................................29
Membrane Dialysis and Lyophilization ...............................................................30
Centrifugal Filter Device Concentration and Desalting.......................................31
Ultrafiltration Through Membrane Disks Under Nitrogen Gas Pressure ............31
x
Ammonium Sulfate Precipitation ........................................................................32
Sephadex G-25 Gel Filtration ..............................................................................32
Purification of yCGL by Anion Exchange Column Chromatography.................33
Purification of yCGL by Con A Affinity Column Chromatography ...................33
Purification of yCGL by HPLC Size Exclusion Column Chromatography ........34
Deglycosylation of CGL ......................................................................................34
Enzyme-Linked Plate Binding Assays.................................................................35
Effect of Varying Buffer pH on xlCGL ...............................................................37
Inhibition Experiments.........................................................................................37
Statistics ...............................................................................................................38
RESULTS ........................................................................................................................39
PCR and DNA Sequence Analysis of yCGL .......................................................39
Optimization of Secreted Expression of CGL in Pichia pastoris ........................48
Optimization of Immunoblotting .........................................................................50
Concentration of yCGL by Filtration ...................................................................52
Ammonium Sulfate Precipitation of yCGL .........................................................55
Purification of yCGL ...........................................................................................61
Optimization of Enzyme-Linked Lectin Assays ..................................................67
Competitive Inhibition Studies of CGL-ligand Binding ......................................74
pH Effects on CGL-ligand Binding .....................................................................75
xi
Glycosidase Removal of CGL Oligosaccharides and Binding Effects ................75
Competitive Inhibition using Sialic Acid Analogs ..............................................78
Binding of yCGL to the Jelly Ligand ..................................................................78
DISCUSSION ..................................................................................................................87
Literature Cited ..............................................................................................................101
xii
LIST OF FIGURES
Figures
Page
1. Structure of the sea urchin egg during fertilization ......................................................4
2. Cortical granule exocytosis ...........................................................................................6
3. Simple high mannose and complex oligosaccharides. ................................................14
4. Vector used for the insertion of recombinant Xenopus laevis cortical
granule lectin into the genome of the yeast Pichia pastoris for
secreted expression ....................................................................................................41
5. Agarose gel electrophoresis of PCR products from Pichia CGL integrants. .............42
6. yCGL DNA and deduced amino acid sequence .........................................................43
7. DNA sequence alignment comparing xlCGL to yCGL. .............................................44
8. Comparison of amino acid sequences of xlCGL and yCGL.......................................46
9. Amino acid sequence alignment of four members of the eglectin family. .................47
10. SDS-PAGE of protease inhibitor effects on yCGL expression ................................57
11. Immunoblotting analysis of yCGL time course expression. .....................................58
12. Immunoblotting analysis of ammonium sulfate precipitation of yCGL. ..................59
13. Immunoblotting analysis of sephadex G-25 desalted yCGL after
ammonium sulfate precipitation. .............................................................................60
14. Size exclusion chromatography profile of xlCGL ....................................................64
15. Size exclusion chromatography comparing yCGL to xlCGL ...................................65
16. Size exclusion chromatography of yCGL with or without calcium .........................66
xiii
17. Schematic of enzyme-linked lectin assay and two potential CGL-ligand
binding mechanisms..................................................................................................70
18. Tittering of rabbit antibodies to de-glycosylated xlCGL. .........................................71
19. Evaluation of blocking reagents for ELLA ...............................................................72
20. Reactivity of the enzyme-linked lectin assay............................................................73
21. Monosaccharide inhibition of the xlCGL ELLA ......................................................80
22. pH and calcium effect on xlCGL binding activity. ...................................................81
23. N-glycan variation and glycosidase cleavage sites ...................................................82
24. Reduction in binding activity of glycosidase treated xlCGL. ...................................83
25. Immunoblotting analysis of glycosidase treated xlCGL...........................................84
26. Competitive inhibition of xlCGL with sialic acid analog. ........................................85
27. Comparison of yCGL and xlCGL binding activity...................................................86
xiv
1
INTRODUCTION
Fertilization is the critically important process in which two gamete cells from
different parents fuse together to create a new organism. The goals of fertilization are
to pass on genes from parent to offspring, and to initiate the molecular reactions that
allow development of the egg to proceed (Gilbert, 2000). One of the major
evolutionary conserved events in fertilization is the regulation of sperm entry into the
egg. In most species, for successful fertilization to occur, only one sperm nucleus can
fuse with the egg. In normal monospermy, a haploid (1N) sperm nucleus and a haploid
egg combine to form the diploid nucleus of the fertilized egg (zygote), thus restoring the
chromosome number to a full complement of genes (2N). One important aspect in the
regulation of the sperm-egg interaction is that as soon as one sperm has bound to the
egg, the characteristics of the egg membrane which made it so able to fuse with the
sperm becomes a harmful vulnerability to multiple sperm entry.
In most organisms, the entrance of multiple sperm known as polyspermic
fertilization (polyspermy), leads to catastrophic consequences, such as cell death or
abnormal development. If multiple sperm are able to fuse with an egg, the
overabundance of genetic material usually results in faulty segregation of chromosomes
and the termination of the polyploid embryo. If the embryo is able to survive to term,
the polyploid condition usually leads to serious consequences, such as mental
retardation and physical defects. For instance, polyspermy appears to be the cause of
the majority of triploid human embryos. Triploidy occurs in about 1-2% of all human
2
conceptuses, is detected in approximately 10-20% of spontaneously aborted embryos,
and results in the termination of embryo development (no survivors reported beyond
10.5 months of age) (Forrester and Merz, 2003; Gardner and Evans, 2006; Sherard et
al., 1986).
To avoid this outcome, organisms have evolved mechanisms to prevent the
union of more than two haploid nuclei. Most species have evolved two molecular and
structural mechanisms to ensure monospermy: 1) a “fast reaction” which lasts about a
minute and is accomplished by an electric change in the egg cell membrane, and 2) a
second “slow reaction” which is due to modification of the extracellular matrix of the
egg (Gilbert, 2000). The fast block to polyspermy is achieved by changing the ionic
concentration within the egg so that it differs greatly from that of its surroundings which
causes an electrical potential change of the egg plasma membrane thereby preventing
sperm from being able to fuse with the egg’s membrane (Gilbert, 2000). However, the
egg’s membrane potential shift is brief, lasting only about a minute, and is not sufficient
to prevent polyspermy permanently. Thus, polyspermy can still occur if the sperm that
are penetrating through the egg extracellular matrix are not prevented from reaching the
egg plasma membrane (Gilbert, 2000). This prevention is accomplished by a second,
slower block to polyspermy that becomes active shortly after the fast block is
established and ensures that multiple sperm do not reach the surface of the egg (Gilbert,
2000).
To prevent polyploid associated conditions, most vertebrates use the
extracellular matrices surrounding the egg as a vital defense mechanism in preventing
3
more than one sperm from penetrating the egg. These extracellular matrices are
composed of glycoprotein-rich layers overlying the egg plasma membrane (Figure 1).
The egg plasma membrane encloses the cytoplasm of the egg and must regulate the
flow of certain ions during fertilization (Gilbert, 2000). The egg plasma membrane is
surrounded by the extracellular matrix (a fibrous layer termed the vitelline envelope in
non-mammals and the zona pellucida in mammals). In many non-mammalian species, a
layer of jelly surrounds the vitelline envelope which is comprised of highly glycosylated
proteins which are used to attract sperm as well as to contribute to other protective
functions (Gilbert, 2000).
Sperm-egg binding occurs between molecules located on the outer surfaces of
both the sperm plasma membrane and the vitelline envelope. Just after the fusion of a
single sperm, the sperm-binding glycoproteins found on the vitelline envelope are
modified by the contents of secretory vesicles called cortical granules that reside just
beneath the egg plasma membrane. The sperm induced exocytosis of these cortical
granules sets up the slow block to polyspermy and is termed the cortical granule
reaction.
4
Figure 1. Structure of the sea urchin egg during fertilization. Structurally speaking,
sea urchin eggs are very similar to frog eggs. Cortical granules are shown just
underneath the plasma membrane, surrounded by the vitelline envelope (extracellular
matrix). The drawing also shows the relative sizes of egg and sperm. From Gilbert:
Developmental Biology, Ninth Edition, Sinauer Associates, Inc, MA 2010. Used with
permission.
5
In the African clawed frog, Xenopus laevis, the cortical granule reaction occurs
about a minute after the first sperm penetrates the egg and is the beginning of the slow
block to polyspermy (Gilbert, 2000). Sperm penetration triggers the release of
intracellular free calcium throughout the egg, which induces cortical granule exocytosis.
Release of these cortical granule contents leads to a series of reactions which causes the
envelope of the egg to expand and become a hardened layer called the fertilization
envelope (Figure 2), which is impenetrable to sperm (Nishihara et al., 1986). The
fertilization envelope modifications include envelope lift-off from the egg surface and a
decrease in solubility compared to the vitelline envelope (Grey et al., 1974). In addition
to preventing sperm entry, the cortical granule reaction creates an extremely stable
protective layer that encapsulates the developing embryo without dissociating (ArranzPlaza et al., 2002).
A variety of proteins are released during cortical granule exocytosis, including
lectins (class of carbohydrate binding proteins), proteases, and glycosidases, all of
which contribute simultaneously to establish this slow block and prevent polyspermy
(Gilbert, 2000). These cortical granule proteins modify different components in the
vitelline envelope all leading to the inability of sperm to bind and/or penetrate through
the extracellular matrix. It is thought that multiple mechanisms to prevent polyspermy
exist so as to increase the chances of successful fertilization. In mammals, the cortical
granule reaction serves the same purpose. Released cortical granule enzymes modify
the zona pellucida (structurally homologous to the frog vitelline envelope) and render
sperm receptors non-functional (Gilbert, 2000).
6
Figure 2. Cortical granule exocytosis. Schematic diagram showing the events leading
to the formation of the fertilization envelope. As cortical granules undergo exocytosis,
they release cortical granule serine protease (CGSP), which cleaves the proteins linking
the vitelline envelope to the cell membrane. Mucopolysaccharides released by the
cortical granules form an osmotic gradient, thereby causing water to enter and swell the
space between the vitelline envelope and the cell membrane. Peroxidases (OVOP and
Udx1) and transglutaminases (TG) then harden the vitelline envelope, now called the
fertilization envelope. From Gilbert: Developmental Biology, Ninth Edition, Sinauer
Associates, Inc, MA 2010. Used with permission.
7
The focus of this study is on one particular lectin glycoprotein that contributes to
this slow block to polyspermy which is found in the Xenopus laevis egg cortical granule
exudate, termed the cortical granule lectin (CGL). Lectins are a type of protein (or
glycoprotein) that are categorized by their specificity of binding to particular
oligosaccharide structures often found on cell surfaces, extracellular matrices, and
secreted glycoproteins (Matejuk and Dus, 1998). CGL is a major constituent of the egg
cortical granules, being 77% of the total glycoproteins in the cortical granule exudate
(Nishihara et al., 1986). The Xenopus laevis CGL (xlCGL) is a metalloglycoprotein
with approximately 20% total carbohydrate by weight, and has a molecular weight of
approximately 45 kD under reducing conditions with size heterogeneity due to variation
of conjugated N-linked oligosaccharides. The glycosylation of CGL solely consists of
N-linked oligosaccharides with no O-linked oligosaccharides having been detected.
CGL forms a tertiary structure that is comprised of 10-12 non-covalently bound
monomers with identical polypeptide backbones, meaning it forms a large oligomeric
complex, with a molecular weight of approximately 450 kD under non-denaturing and
non-reducing conditions (Chamow and Hedrick, 1986). CGL has a binding specificity
for terminal galactoside residues, and requires calcium for its binding activity.
The
translated cDNA for CGL has a signal peptide, a structural sequence of 298 amino
acids, a polypeptide size of 32.7 kD, a fibrinogen domain, and contains two to three
consensus sequence sites for N-glycosylation depending on the sequence variant.
8
After xlCGL is released into the perivitelline space following egg activation by
sperm fusion, CGL diffuses through the vitelline envelope and binds to its ligand found
in the innermost aspect of the jelly coat layer which surrounds the vitelline envelope
and is considered part of the egg’s extracellular matrix. Binding of CGL to its jelly
ligand is non-covalent and produces a heteropolymer called the fertilization layer at the
outer edge of the vitelline envelope (Hedrick, 2007). The fertilization layer is
impenetrable by sperm and is thought to provide a mechanical block to polyspermy.
Experiments have shown that the fertilization layer can be dissociated by treatment with
galactose and EDTA (chelates or removes divalent cations such as calcium) which is
consistent with the properties of CGL-ligand binding (Hedrick and Nishihara, 1991). In
addition, the ligand for CGL was isolated from solubilized egg jelly, and determined to
be an oviductal secretory product containing a large amount of O-linked
oligosaccharides (Quill and Hedrick, 1996). Using mass spectrometry, it was
determined that the oligosaccharides of the ligand glycoprotein contain terminal
galactosyl residues and that some also have fucosyl and sulfate residues attached in
various positions (Hedrick et al., 1993; Tseng et al., 2001). Currently, it is unclear as to
which of these O-linked ligand oligosaccharides are the functional moieties that bind to
CGL.
However, it is clear that CGL homologues do exist in mammalian species and
can be found immunologically in mammalian egg cortical granules (Chang et al, 2004;
Hedrick, 2007; Peavy and Hedrick, unpublished). Using mouse and human ovarian
cDNA libraries, full length cDNAs were cloned, and it was found that the human and
9
mouse translated cDNA sequences exhibited 63% identity to the xlCGL sequence.
Using the CGL sequence, 21 human and 23 mouse cDNA sequences in EST databases
were identified. This shows that genes homologous to that of xlCGL exist in mouse and
human genomes, and that the CGL gene is expressed in the ovary (Chang et al, 2004).
Further investigation using antibodies to the xlCGL polypeptide revealed that CGL was
indeed found in the cortical granules of eggs of from mice (Mus muscularis), pigs (Sus
scrofa) and rhesus macaque monkeys (Macaca mulatta) using confocal laser
fluorescence microscopy and transmission electron microscopy (Peavy, unpublished).
Subsequently, the mouse and pig CGL homologues were isolated from egg extracts
using the same affinity chromatography and immunoprecipitation methods that were
used to purify xlCGL and shown to have about the same subunit molecular weight
(Hedrick, 2007).
In addition, the functional role of the mammalian CGL homologues appears to
be conserved. CGL was observed to be released from mouse and pig egg cortical
granules after the cortical reaction and localized immunologically to the perivitelline
space and the zona pellucida (Hedrick, 2007).
Interestingly, CGL was found within
the zona pellucida itself and not on the outermost aspect of the vitelline envelope like in
Xenopus laevis. The calcium-dependence and galactose specificity of the mammalian
CGL homologues was also observed by treatments of activated eggs or embryos with
EDTA and melibiose (galactose containing disaccharide) which eliminated the
immunolocalization of CGL (Hedrick, 2007). Using mouse in vitro fertilization assays,
the addition of xlCGL to mouse eggs prior to the addition of sperm inhibited
10
fertilization and could be reversed when treated with galactose or melibiose. These
results provide strong evidence for the conservation of a CGL-ligand block to
polyspermy mechanism in mammals, and thus studies of the mechanism in the frog
Xenopus laevis are likely pertinent to mammals.
Interestingly, the expression of the mammalian CGL homologue is not limited to
the egg (Chang et al, 2004). For instance, an identical mammalian CGL cDNA was
found to be expressed in the intestines of mice and humans and thus was termed
intelectin, which is now the official name for the gene (Komiya et al., 1998; Tsuji et al.,
2001). Analysis of cDNA expression data has revealed the presence of intelectin in at
least 24 different human tissues including heart, testes, stomach, small intestine,
thyroid, liver, kidney, lung, brain, mammary gland, and ovary (Chang et al., 2004).
Expression of CGL homologues in these other tissues suggests that this lectin likely
participates in a wide variety of functions in addition to the block to polyspermy.
CGL homologues have also been found in species as diverse as ascidians and
lamprey (Chang et al, 2004). Homologues can also be found both as soluble lectins
and as glycophosphatidyl inositol-anchored surface receptors (post-translationally
attached glycolipid to a protein’s C-terminus) (Suzuki et al., 2001). Additionally, the
peptide sequences from the Xenopus laevis blood group B-active glycophosphatidyl
inositol-anchored membrane glycoprotein nearly match X. laevis CGL, which suggests
that the glycoprotein is either a product of the same gene or a closely related paralog
(Chang et al., 2004).
11
All of these CGL homologues share four major characteristics: 1) specific
binding to galactose-containing oligosaccharides, 2) oligomeric conformations, 3)
calcium-dependence for binding, and 4) is glycosylated (Chang et al., 2004). The
requirement for calcium in order to bind to oligosaccharides is indicative of the C-type
family of lectins, whereas galactose specific binding is indicative of the Galectin family
of lectins. However, the sequence of CGL homologues does not contain the sequence
motifs required for inclusion in these respective lectin families. Therefore, the
conserved binding and structural properties that xlCGL and its homologues share have
resulted in the creation of a new family of related glycoproteins called the eglectin
family of lectins (Chang et al., 2004), or also known as X-lectins (Lee et al., 2004). In
addition, it has been found that eglectins have a region that appears related to a
fibrinogen-like motif that could be involved in carbohydrate binding (Lee et al., 2004).
In humans, two CGL homologues have been identified and have become
increasingly important areas of research (Tsuji et al., 2001). Human intelectin-1 and 2, have been characterized as secretory glycoproteins that bind to a specific form
(furanose) of galactose residues. Human intelectin-1 transcripts were found in the heart,
small intestine, colon, thymus, ovary, and testis; whereas human intelectin-2 was found
solely in the small intestine (Lee et al., 2004). The carbohydrate-binding properties of
human intelectin-1 was investigated, and it was found to bind to galactose and be
calcium dependent similar to xlCGL (Tsuji et al., 2001). Interestingly, the galactose
containing disaccharides lactose and melibiose could not competitively inhibit the
binding activity of intelectin-1 (Tsuji et al., 2001), which is a distinct difference from
12
xlCGL. These results imply that while the carbohydrate binding specificities of human
intelectin-1 and xlCGL are closely related, they do have some notable differences.
Human intelectin-1 is the homologue identified in the ovary and likely participates in
the formation of the fertilization envelope (Tsuji et al., 2001).
In addition, intelectins are thought to be involved in the other functions such as
the innate defense against microorganisms and as important mediators of asthma
(allergic airway inflammation). Intelectins were found to be housed in specialized
vesicles that are thought to be released after infection by pathogens and bind to
oligosaccharides from bacterial pathogens (Lee et al., 2004). Therefore, the human
eglectins likely participate in pathogen surveillance as part of the innate immune system
in addition to the block to polyspermy. As for airway allergic responses, it has been
proposed that blocking the binding of intelectin to allergens would be a viable
therapeutic strategy for preventing asthma (Gu et al., 2010).
However, while the biological functions of these lectins has become an
increasingly important area of research, it is unclear as to what ligands the lectins are
binding to and the molecular mechanism of the binding activity. Studies using the
Xenopus laevis CGL and jelly ligand are likely to be informative towards the binding
mechanism of eglectins as a whole. Furthermore, a more complete understanding of
eglectin-ligand binding interactions could lead to the development of novel
antimicrobial and asthmatic therapeutics, immunocontraception, improved in vitro
fertilization success, and many other unforeseen advances. Towards this end, the
structural properties and binding interactions of Xenopus laevis CGL and its ligand have
13
been the most studied of all the eglectins due to the ease of purification and amounts of
material that can be gathered for biochemical studies.
With respect to glycosylation of xlCGL, it has been shown that the
oligosaccharides found on the polypeptide are separated into simple mannose, complex,
and hybrid (combination of simple mannose and complex) N-linked oligosaccharides
with no O-linked oligosaccharides found (An et al., 2003) (Figure 3). The Nglycosylation sites of xlCGL have been determined and were found to be
heterogeneous, meaning that every molecule of xlCGL does not always have the same
oligosaccharides conjugated to it (An et al., 2003). Also, in addition to the original
report of CGL with two N-linked glycosylation sites, a second form of CGL was found
that has a third N-linked site due to a sequence polymorphism generating an amino acid
substitution (Lys  Asp) for a consensus N-linked site (An et al., 2003).
14
Figure 3. Simple high mannose and complex oligosaccharides. A pentasaccharide
core (shaded) is common to all N-linked oligosaccharides and serves as the foundation
for a wide variety of N-linked oligosaccharides. Simple high mannose (left) and
complex (right) type oligosaccharides are shown attached to an asparagine (Asn)
residue. Man = mannose. GlcNAc = N-Acetylglucosamine. Gal = galactose.
15
In addition, a published preliminary report (Hedrick et al., 1993) indicated that
the terminal residue on many of the xlCGL complex oligosaccharides was sialic acid, a
negatively charged sugar. Sialic acid residues on oligosaccharides have been shown to
play important roles such as in the stabilization of molecules and membranes, as well as
in modulating interactions with the environment (Varki, 1999). For example, the
negative charge provided by sialic acids on human erythrocytes and other cell types
provides charge repulsion, which prevents unwanted interactions of cells in the blood
circulation (Varki, 2008). It has also been shown that sialic acid residues are critical
factors for assessing when to remove certain glycoproteins from circulation by receptormediated clearance in the liver and other organs (Varki, 2008; Weigel and Yik, 2002).
In addition, sialic acids have been found to be directly involved in the binding
interaction of various pathogens and toxins including human and avian influenza A
(Varki, 2008). There is also evidence that several such viruses recognize the linkage,
and the type of sialic acids, and that these differences can determine the species
preference of various viruses (Nicholls et al., 2008; Varki and Chen, 2011).
Additionally, many pathogens (e.g. Influenza virus) express sialidases, which are
enzymes that remove terminal sialic acids from glycoproteins (and other
glycoconjugates), as receptor-destroying enzymes (Corfield, 1992; Varki and Chen,
2011).
16
Extending current knowledge of sialic acid biology to the CGL-ligand block to
polyspermy has led to the hypothesis that sialic acid plays a direct role in the molecular
mechanism of xlCGL binding to its ligand. Sialic acid residues are typically found at
the terminal position of N-linked glycans and confer a negative charge, whereas most
carbohydrate residues are neutral. It is widely appreciated that when negatively charged
sialic acids are exposed as terminal residues on glycoproteins, as they appear to be in
xlCGL, they are ready to interact with other molecules, and therefore often play an
important role in biological functions (Zhuo and Bellis, 2010). In particular, sialic acid
is known to bind calcium (Jaques et al., 1977). It is possible that sialic acid residues
may help CGL to sequester calcium which is essential for its ligand binding activity.
Understanding the CGL-ligand binding interaction does present challenges.
Preliminary studies from the Hedrick lab at UC Davis have provided three relevant
pieces of information as to CGL-ligand binding activity. Firstly, when the
oligosaccharides of xlCGL are enzymatically removed using PNGaseF (glycosidase that
removes all N-linked sugars), a 10 fold reduced binding of CGL to its ligand was
observed (data not published). This is significant because it suggests that the
oligosaccharides are important for binding. Secondly, enzymatic removal of
oligosaccharides appears to cause the xlCGL to become less soluble and cause
considerable precipitation and insoluble complexes. Lastly, when CGL was treated in
acidic conditions, binding activity was irreversibly lost. This is important because
many glycosidases (enzymes that remove oligosaccharides), including those that
remove sialic acid, have pH optimums that are in the acidic range and therefore it is
17
difficult to determine whether a decrease in binding activity is due to the removal of
oligosaccharides, or simply the acidic conditions involved in the enzyme incubation.
This study is designed to follow up on these observations and study the relative
importance that the CGL oligosaccharides have on CGL binding to its jelly ligand.
To this end, this study will employ two different strategies to modify the Nlinked oligosaccharides of the xlCGL and evaluate their role in binding. The first
strategy will use enzymatic methods to remove different types of N-linked
oligosaccharides from xlCGL. PNGaseF will be used to release the entire N-glycan
chain attached to the protein backbone at a neutral pH. In addition, this study will also
take advantage of other glycosidases that can function at neutral pH that cleave at only
specific positions in the oligosaccharide chain and thus display high specificity. These
specific glycosidases will allow for a finer analysis of which types of N-link structures
and specific residues such as whether sialic acid residues are important for binding.
The four additional glycosidases to be used in this study are the following: α1-2,3
mannosidase, α1-6 mannosidase, α2-3,2-6,2-8 sialidase, and α2-3 sialidase. These
glycosidases will be used separately, and in combination with each other, to provide a
set of tools to achieve the desired oligosaccharide removal without protein degradation.
While the pH optimum for mannosidases and sialidases are reportedly acidic (pH 4.56.5), they can function reasonably well (~10-20% decrease in activity) at pH 7.0
(personal communication, New England Biolabs Technical Support).
The second strategy to modify the N-linked oligosaccharides of the xlCGL will
be to express a recombinant form of CGL in a yeast protein expression system which
18
can only make simple mannose structures and not complex oligosaccharides (Figure 3).
Yeast appears to be the most “primitive” eukaryote making the simple high-mannose Nglycans, and this pathway has been well preserved in mammals (Varki, 1999). Both
yeast and higher eukaryotic glycoproteins normally have these simple high-mannose Nglycan structures, however, eukaryotes also add hybrid and/or complex N-glycans to
glycoproteins with the majority being the complex type (Figure 3). Since the yeast
expression system can only add simple mannose structures to the CGL polypeptide,
secreted glycosylated yeast CGL (yCGL) will be used in binding studies to determine
whether the loss of the complex oligosaccharides will affect binding. Fortunately, the
yeast expression media has a neutral pH (~6.9) since it was noted previously that xlCGL
binding was negatively affected by acidic pH.
This expression system strategy circumvents the issue that most glycosidases
function in acidic pH which would impair CGL binding when trying to remove sugars.
An added benefit is that if the yeast CGL can bind to its ligand, then the recombinant
yCGL could be used to generate enough glycoprotein for structural studies such as xray crystallography to determine its structure. Many proteins have been expressed in
the Pichia expression system while maintaining function including enzymes, proteases,
protease inhibitors, receptors, single-chain antibodies, and regulatory proteins
(Invitrogen, 2001). In addition, expression of the glycoprotein in the media facilitates
purification since these yeast do not secrete appreciable amounts of host proteins.
Pichia pastoris offers several other advantages for the expression of CGL when
compared to other expression systems. Firstly, E. coli is not really an option for
19
expression of CGL since it does not have the ability to produce glycosylated proteins
and it was mentioned previously that CGL binding and solubility properties are
dramatically affected without oligosaccharides. Other more advanced expression
systems such as Chinese Hamster Ovary cells (and other mammalian cell lines) would
produce a glycosylation profile very similar to the native CGL, so this would not
provide any meaningful difference. Saccharomyces cerevisia (bakers yeast) would be
an option as it is also used as an expression system, however, Pichia pastoris has
several major advantages over its relative. The first is that Pichia is a methylotroph,
meaning it can grow with only methanol as its energy source. This property has been
exploited for the Pichia expression system by engineering the promoter for methanol
metabolism into the expression vector, and methanol is a much cheaper reagent than
other induction reagents used for expression (e.g. IPTG). Secondly, Pichia can grow to
much higher densities and provide a greater protein yield than Saccharomyces
(Brondyk, 2009). Lastly, Saccharomyces recombinant glycoproteins are more
susceptible to hyperglycosylation than Pichia, which could result in excessive
oligosaccharide modifications that might complicate the results of binding assays.
Another option would be to use S2-cells from Drosophila melanogaster, however, this
expression system provides lower yields and requires complex rich media which can
significantly increase the cost of expression (Brondyk, 2009). In summary, Pichia
pastoris is likely to provide a high yield of recombinant CGL with the desired
oligosaccharide modification in a system that is cost effective to set up and maintain.
20
Previous research (Peavy, unpublished; Breanna Wallace senior honors thesis
project, 2006) used a X. laevis ovary cDNA library to obtain the CGL cDNA by PCR
amplification. The cDNA was cloned into the Pichia pastoris genome using an
expression vector with a signal peptide for secretion. Clones were screened by PCR to
detect which clones were expressing CGL, with 14 clones testing positive. CGL
positive clones are to be selected and expressed in this study, and a screening process
will be done to select the clone that produces the highest quality and quantity of yCGL.
After successful expression of yCGL, it will be necessary to purify yCGL in
preparation for plate binding assays, which have already been established for a variety
of parameters (Quill and Hedrick, 1996). Plate binding assays will be used to compare
the binding activity of CGL to the ligand counterpart using the following CGL
preparations: xlCGL, yCGL, glycosidase treated xlCGL, and sialic acid analog treated
xlCGL. This will allow us to clarify if the complex sugars present in the xlCGL, but
missing in yCGL and glycosidase treated xlCGL are important in binding. If yCGL
with only simple mannose oligosaccharides has a weaker binding association when
compared to xlCGL, then complex sugars will be implicated as important for the
binding of CGL to the ligand. In addition, binding assays will be used to confirm the
preliminary reports that treatment of xlCGL with PNGaseF and acidic buffers
significantly reduce the binding interaction. As for the sialic acid analog, competitive
inhibition binding experiments will be used to test whether the analog can compete with
sialic acid and reduce binding of CGL to its ligand therefore implicating that sialic acid
has a direct role in binding.
21
Provided all this information, the following hypothesis and objectives have been
set forth.
Hypothesis: The binding of the Xenopus laevis CGL glycoprotein to its ligand
counterpart is dependent on its N-linked hybrid and complex oligosaccharides.
Objectives:
1) Grow recombinant yeast clones with Xenopus laevis CGL cDNA and optimize
expression of yCGL.
2) Purify recombinant yCGL
3) Enzymatically remove specific oligosaccharides from xlCGL by glycosidase
digestion.
4) Develop plate binding assays
5) Use the plate binding assays to compare ligand binding activity of xlCGL, yCGL,
glycosidase treated xlCGL, and sialic acid analog treated xlCGL to assess relative
importance of oligosaccharides on binding.
The results of these CGL binding studies will have a significant impact towards
the fields of fertilization biology and more broadly, to research on other eglectin family
members and sialylated glycoproteins. With respect to fertilization, this study would
contribute to further potential application towards clinical diagnoses of infertile couples,
assisted reproductive technologies, and contraceptive strategies because approximately
1% of natural human conceptions and about 10% of IVF trials result in polyspermy
22
(Wessel et al., 2001). One contraceptive strategy could be to produce an antibody to the
oligosaccharides of the ligand which would function in an analogous manner to CGL
binding to the jelly ligand. This would likely prevent sperm from penetrating through
the egg extracellular matrix and constitute a viable contraceptive strategy. Therefore,
the glycoproteins involved in the lectin-ligand polyspermy block can potentially be used
as targets for contraception. Thus, this study will contribute to a better understanding of
the molecular mechanisms of fertilization and the block to polyspermy which should be
translatable to humans and other mammals.
In addition, these results will also impact the field of lectins and more
specifically, other eglectin family members since there have been no reports that other
types of lectins need particular oligosaccharides for their binding activity to ligands
(Varki, 1999). Therefore, this would be a significant finding if complex N-linked
oligosaccharides were important in CGL binding. It is possible that these CGL
oligosaccharides might be directly involved in binding or they may simply assist other
functions such as coordinating calcium or facilitating folding, solubility, or other
structurally related aspects. Thus, studies of CGL binding are likely to impact many
other fields since eglectins all share similar binding characteristics and appear to be
have many other biological roles such as pathogen surveillance and the innate immune
response. Finally, the biological significance of sialic acids found on oligosaccharide
chains is only beginning to be appreciated and these studies are likely to contribute to
this field.
23
MATERIALS AND METHODS
yCGL Transformants
Previous work by Dr. Peavy and students cloned the xlCGL into the pPICZα
expression vector, introduced the xlCGL containing vector into GS115 Pichia pastoris
cells (Invitrogen) by electroporation, and transformants were grown on YPD plates
which where supplemented with 50 g/ml Zeocin (Invitrogen) as a selection marker, and
clones were stored in 20% glycerol at -80°C.
yCGL DNA Purification
Purification of total DNA was done using the Qiagen DNeasy Blood and Tissue
Kit (#69504) modified for yeast. Buffers ATL, AL, AW2, AE, proteinase K, and spin
columns are supplied with the kit. A single Mut+ colony carrying the xlCGL cDNA
was cultured at 30ºC overnight in 10mls of YPD (Yeast extract peptone dextrose
medium). Culture (500µl) was collected and cells were harvested by centrifuging for 10
minutes at 5000 x g. Supernatant was discarded and the pellet was resuspended in
600µl sorbitol buffer (1M sorbitol, 100mM EDTA, 14mM β-mercaptoethanol).
Lyticase (200 units, Sigma #L2524) was added to the pellet to remove their cell wall
and was incubated at 30ºC for 60 minutes, with shaking every 15 minutes. Spheroblasts
(yeast without cell walls) were pelleted by centrifugation for 10 minutes at 300 x g, and
resuspended in 180µl Buffer ATL. Proteinase K (20µl) was added and the sample was
incubated at 56°C for 1 hour with shaking at 500rpm. After vortexing for 15 seconds,
24
200µl Buffer AL was added, mixed, and then 200µl ethanol was added, and mixed.
Mixture was loaded into the DNeasy Mini spin column and centrifuged at 6000 x g for
1 minute. Flow-through was discarded, and the spin column was placed in a new
collection tube, and 500µl Buffer AW2 was added, followed by centrifugation for 3
minutes at 20,000 x g. Flow-through was discarded, column was placed in a new
collection tube, and 200µl Buffer AE was loaded onto the membrane, followed by
incubation for 1 minute at room temperature, and then centrifuged for 1 minute at 6000
x g to elute, and DNA concentration was measured using a NanoDrop
spectrophotometer.
PCR Analysis of yCGL
Analysis by PCR was conducted to confirm whether the Xenopus laevis cDNA
was actually integrated into the Pichia pastoris genome. Three clones from the Pichia
pastoris GS115 (mut+) host strain were tested. DNA was extracted from cells
transformed with the vector carrying the Xenopus laevis cDNA using the DNeasy Blood
and Tissue Kit as described above. Pichia recombinants were confirmed by PCR for
the integration of the xlCGL gene into the Pichia genome. PCR amplification of the
xlCGL gene was carried out with 5'- and 3'-AOX vector primers (5'-AOX1, 5'GACTGGTTCCAATTGACAAGC-3'; 3'-AOX1, 5'GCAAATGGCATTCTGACATCC- 3'). PCR was performed using the Promega
GoTaq PCR kit and reaction mixes consisted of the following (working concentration
given in parenthesis): 500 ng of genomic template DNA, 10µl 5X Green GoTaq
25
Reaction Buffer, 3µl MgCl2 (1.5mM), 1µl dNTP Mix (0.2mM), 0.78µl upstream primer
(1.0µM), 0.78µl downstream primer (1.0µM), 0.25µl GoTaq DNA Polymerase
(1.25units), nuclease-free water to a total volume of 50µl. A thermal cycler (BioRad
MyCycler) was programmed to run for 1 cycle of heat soak at 94°C for 2 min, 25 cycles
of denaturation at 94ºC for 1 min, annealing at 55ºC for 1 min, and extension at 72ºC
for 1 min, and 1 cycle of final extension at 72ºC for 7 minutes. Afterwards, 10µl of the
50 µl PCR reaction was analyzed by gel electrophoresis using an 0.8% TAE gel.
Sequencing of PCR amplified yCGL cDNA
An aliquot of the yCGL PCR product amplified from the purified yeast genomic
DNA was loaded onto an 0.8% low melt TAE agarose gel for electrophoretic separation
(100 volts for 35 minutes). After electrophoresis, gels were stained in ethidium
bromide (1µg/ml) and separated bands were visualized using ultraviolet light.
Appropriately sized PCR products were then excised from gel and then purified for
sequencing using the Montage DNA Gel Extraction Kit ( Millipore Billerica, MA).
Purified PCR products were then commercially sequenced (Sequetech, Mountain View,
CA).
Secreted Expression of Recombinant CGL in Pichia pastoris
Yeast expression was based on protocols in the Invitrogen EasySelect Pichia
expression kit. Chosen recombinant yCGL Pichia clones were grown on YPD agar
containing zeocin for 2-3 days in the dark at room temp. Individual colonies were
26
selected for inoculation into 25 ml of BMGY medium (Yeast extract 1%, peptone 2%,
potassium phosphate, pH 6.0, 100 mM, YNB 1.34%, 0.00004% biotin, glycerol 1%) in
a 250ml baffled flask. Growth was performed at 29ºC in a shaking incubator (275 rpm)
until the cultures reached an OD600 of 2-6 (log phase growth). Cells were harvested by
centrifuging at 2,420 g for 5 min. Supernatant was decanted and the cell pellet was
resuspended to an OD600 of 1.0 in BMMY (yeast extract 1%, peptone 2%, potassium
phosphate, pH 6.0, 100 mM, YNB 1.34%, 0.00004% biotin , methanol 0.5%) and
protease inhibitors (1% casamino acids, 1mM EDTA, SIGMAFAST Protease Inhibitor
Cocktail Tablets, EDTA-free) to induce expression. Cells were grown in a shaking
incubator, and 100% methanol was added every 24 hr to a final concentration of 0.5%.
After 0hr, 24 hr, 48 hr, 72 hr, and 96 hr of incubation, cells were pelleted, and the
culture supernatant (presumably containing the secreted protein) was harvested and
stored at -80ºC. To determine cell culture density, a standard curve was generated
plotting OD600 values (Promega Glo-Max-Multi+ Detection system) against cell counts
(hemacytometer). Media was collected at desired time points: Time = 0hr (T0), 24hr
(T24), 48hr (T48), 48hr (T72), 96hr (T96), and analyzed by a Bradford assay, SDSPAGE, and immunoblotting (methods described below).
Quantification of Protein Concentration by Bradford Assay
The protein concentration of culture supernatants, column purified samples, and
other samples possibly containing protein were determined by the Bradford (Sigma)
protein assay method using bovine serum albumin (BSA, Fract V; Fisher Scientific) as a
27
standard with concentrations ranging from 100µg/ml to 1400µg/ml. The absorbance
was measured with a microplate reader either at 630nm (Opsys MR, DYNEX
Technologies), or 600nm (Promega Glo-Max-Multi+ Detection system)
Protein Precipitation with Trichloroacetic Acid
In order to properly assess yCGL expression levels, the secreted proteins in the
expression media had to be concentrated prior to SDS-PAGE to ensure enough protein
was loaded in the gel for visualization. TCA (250µl of a 100% solution) was added to
1.0ml of expression supernatant in a 1.5ml tube. Sample was incubated for 10min at
4ºC, and then centrifuged at 13.3K rpm for 5min. Supernatant was removed, leaving
protein pellet intact. Pellet was washed with 200µl ice-cold acetone, and then
centrifuged at 13.3K rpm for 5min. Supernatant was removed and the acetone wash
was repeated. Pellet was dried by placing the tube in a 100ºC heat block for 5min to
remove residual acetone, and solubilized in loading sample buffer.
Procurement and Preparation of Xenopus laevis Egg Jelly and CGL
Purified and concentrated egg jelly preparations containing the CGL ligand, and
purified and lyophilized xlCGL were obtained prior to this research by Dr. Peavy. The
egg jelly preparation were diluted 1:800 in 100mM sodium carbonate, pH 9.5. xlCGL
was solubilized in either 1X TBS (10mM Tris, 150mM NaCl, pH 7.4) or ELLA wash
(1X TBS with 10mM Ca2+).
28
Purification of IgG Antibody Specific to Deglycosylated xlCGL
Previous work collected 50mls of antisera from rabbits that were injected with
deglycosylated xlCGL (chemical deglycosylation by trifluoromethanesulfonic acid)
which generated antibodies directed to the polypeptide of xlCGL. Preliminary results by
a former student in Dr. Peavy’s lab (Breanna Wallace, honor project) indicated that the
crude antisera generated background signal during immunoblotting analyses. Thus, it
was deemed necessary to purify the IgG fraction from the antisera for future immunoblots and plate binding assays. IgG purification was performed using Protein A agarose
(Santa Cruz Biotech). The antiserum was passaged through the Protein-A (2mls)
column 3 times at a flow rate of 2ml/min. The column was then washed with 30ml
TBS. A 50ml tube with 600µl neutralization buffer (1M Tris-HCl, 1.5M NaCl, 1mM
EDTA, pH 8.3) was prepared to prevent denaturation of IgG. The next step was to add
15mls of elution buffer (50mM Glycine-HCl, pH 2.7) at room temp to the column and
then collect the eluant. Another 50ml tube with 700µl neutralization buffer was
prepared, and 10ml of elution buffer (50mM Glycine-HCl, pH 1.9) was added to the
column, followed by collection. The eluted fractions were then dialyzed in 50mM
ammonium bicarbonate overnight with two buffer changes, lyophilized overnight, and
then solubilized in TBS.
29
Titering of Antigen and Antibodies
The tittering and optimization of the enzyme-linked lectin assay components
were performed on 96 Immunolon HHBX plates (Dynex Technologies) using a
standard checkerboard dilution series strategy of the jelly ligand (solid phase), xlCGL
(antigen), primary antibody to de-glycosylated xlCGL, biotin-SP-conjugated AffiniPure
goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories), and peroxidaseconjugated streptavidin (Jackson ImmunoResearch Laboratories). The secondary
antibodies were solubilized in water following manufacturers recommendations, then
100% glycerol was added for a 1:2 dilution, for a final concentration of 50% glycerol.
Antibodies were then aliquoted and stored at -20ºC.
CGL Analysis by SDS-PAGE and Immunoblotting
Protein samples were reduced and denatured (10min at 70ºC in 4X NuPage
Lithium dodecyl sulfate sample buffer) and separated by SDS-PAGE in NuPage 10%
Bis-Tris, 1.5mm x 10 gels (Invitrogen) at 200V for 50 min in MOPS SDS running
buffer using the Invitrogen XCell SureLock electrophoresis system. Proteins were then
transferred to a 0.45µm nitrocellulose membrane in Invitrogen NuPage transfer buffer
(25mM Bicine, 25mM Bis-Tris, 1mM EDTA, pH 7.2) at 30mA for 75 minutes at 4ºC in
the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell. For detection with the
primary antibody to deglycosylated xlCGL, the membrane was washed 2 x 10min in
TBS, and was then blocked with western blocking buffer TBST (3% BSA, 0.1% tween,
TBS pH 7.5) for 1 hour at room temp with rocking. Membrane was then washed (3 x
30
5min with TBST) and then incubated with primary antibody diluted 1:4000 in western
blocking buffer for 1 hour at room temp with rocking. Membrane was washed and then
incubated in biotin-SP-conjugated AffiniPure goat anti-rabbit IgG (H+L) diluted
1:100,000 in western blocking buffer for 1 hour at room temp with rocking. Membrane
was washed and then incubated in peroxidase-conjugated streptavidin diluted 1:1000 in
western blocking buffer and incubated for 15min at room temp with rocking.
Membrane was washed and then bands were visualized using the Vector NovaRED
substrate kit for peroxidase.
Prior to using NuPage gels, samples were reduced (10min at 100ºC in LSB) and
separated using freshly made gels. The Biorad Multi-Casting chamber was used to cast
10% resolving gels (41% water, 33% bis-acrylamide, 25% 0.5M Tris/HCl, 1% SDS)
followed by 4% stacking gel (61% water, 13% bis-acrylamide, 25% 1.5M Tris/HCl, 1%
SDS ). The gels were electrophoresed using the Mini-Protean 3 Electrophoresis
Module Assembly at 50mA for 90min. The gels were then stained with Coomassie
Brilliant Blue Staining dye (0.5g R250, 46% MeOH, 7% HAc) for 1 hour, and then
destained overnight (46%MeOH, 7.5% HAc).
Membrane Dialysis and Lyophilization
Subsequent purification methods required that the protein be concentrated and
that the salts in the expression buffer be removed. There are a variety of ways to
accomplish these objectives, and the first method attempted was to use Spectra/Por
Regenerated Cellulose Membranes (Spectrum Labs, 12-14kD MW cutoff) for dialysis,
31
followed by lyophilization. Eight inches of dialysis tubing was cut and hydrated in
water for 10min to remove residual glycerol. A dialysis closure clip was applied, and
7mls of expression of supernatant were pipetted into the tubing. A second dialysis clip
was added, leaving ~1inch of air in the tubing. The sample was then put into a 2L flask
with dialysis buffer (50mM ammonium bicarbonate) on a stirring plate and left
overnight at 4ºC. Dialysis buffer was changed two times. After dialysis, sample was
removed from the tubing, pipetted into a freeze-drying flask, frozen at -80ºC,
lyophilized until fully dehydrated, resolubilized in 1X TBS pH 7.4, and stored at -20ºC.
Centrifugal Filter Device Concentration and Desalting
Expression supernatant (10mls) was concentrated and desalted by ultrafiltration
through low-adsorptive, hydrophilic membranes with a 50kD molecular weight cutoff
(Amicon Centriplus). Expression supernatant was added to the sample reservoir, and
was centrifuged (Sorvall RC 5B plus) at 3000g for 45min at 4ºC. To desalt the sample,
the volume was reconstituted to 10ml with TBS pH 7.4 and centrifuged. To recover the
retentate, the vial was separated from the reservoir, inverted into collection tube, and
centrifuged at 2,000g for 3min. Concentrate was then stored at -20ºC.
Ultrafiltration Through Membrane Disks Under Nitrogen Gas Pressure
An ultrafiltration membrane disc (YM10, Amicon, MWCO 10,000 kD) was
hydrated in nanopure water for 1hour, changing the water three times, and then loaded
into the stirred cell of the ultrafiltration unit (Amicon 8200, Millipore). The stirred cell
32
was filled with 150ml of expression supernatant and 50ml of TBS pH 7.4 to the
maximum operating volume. The unit was placed on a magnetic stirrer and stirring rate
was adjusted until vortex created was approximately one-third of the depth of liquid
volume. The stirred cell was connected to a nitrogen gas cylinder and pressurized to
75psi (5.3kg/cm2). TBS (pH 7.4) was periodically added to remove salts.
Concentration was continued until volume reached 1/10th of the original volume (15ml).
The sample was collected and stored at -20ºC.
Ammonium Sulfate Precipitation
Expression supernatant (22.5mls) was chilled on ice, and 2.5ml of 1M Tris-HCl
pH 8.0 was added to adjust pH. Ammonium sulfate was slowly added to 10%
saturation, and stirred on ice for 30min. Sample was then centrifuged (Sorvall RC 5B
plus) for 20min at 23,426g at 4ºC. Supernatant was returned to beaker for continued
stirring, and the process was repeated for the desired ammonium sulfate saturation (20,
30, 40, 50, 60, 70, and 100%) to determine the optimal precipitation and concentration
conditions. The pellet containing precipitated proteins was resuspended in either 1X
TBS pH 7.4, ELLA wash buffer (defined below), or 100mM sodium carbonate, pH 7.5.
Sephadex G-25 Gel Filtration
PD-10 desalting columns (GE Healthcare) containing 8.3mls of sephadex G-25
medium (particle size range 85-260µm, MW cutoff 5000kD) were used to rapidly
separate high molecular proteins from low molecular weight substances and impurities.
33
A PD-10 column was prepared by removing storage solution and equilibrating column
with equilibration buffer (TBS pH 7.4). The resolubilized ammonium sulfate fraction
(2.5mls) was slowly pipetted into the middle of the packed bed. The sample was eluted
by centrifugation at 1000 x g for 2 minutes at RT. Eluent was collected and stored at 20ºC.
Purification of yCGL by Anion Exchange Column Chromatography
A fractionated ammonium sulfate pellet containing yCGL was solubilized in
20mM Tris-HCl, 20mM NaCl, pH 7.5 and desalted using a PD-10 column. Ten mls of
Q Sepharose Fast Flow (Sigma, Q1126) was loaded into a 1.6cm (inner-diameter) x
10cm econo-column and pre-equilibrated with 50ml of wash solution (20mM Tris-HCl,
20mM NaCl, pH 7.5). One ml of the sample was loaded and pre-equilibrated with wash
solution at a flow rate of 1.0ml/min. A single step-wise elution was done with 20mM
Tris-HCl, 500mM NaCl, pH 7.5), and twenty fractions (500µl/fraction) were collected
in 1.5ml tubes, and analyzed by Bradford assay. All solutions were kept on ice.
Column regeneration was performed following manufacturer’s indications.
Purification of yCGL by Con A Affinity Column Chromatography
A fractionated ammonium sulfate pellet containing yCGL was solubilized in
20mM Tris-HCl, 500mM NaCl, pH 7.5 and desalted in a PD-10 column. One ml of
Concanavalin A-Sepharose 4B (Sigma) was loaded into a 0.7cm (inner-diameter) x
15cm econo-column and pre-washed with 20ml of wash solution (1m NaCl, 5mM
34
MgCl2, 5mM MnCl2, 5mM CaCl2) at a flow rate of 0.5ml/min. The column was then
equilibrated with 20mls of equilibration buffer (500mM NaCl, 50mM Tris-HCl, pH
7.8). After equilibration, 1ml of the sample was loaded, washed with 20ml
equilibration buffer, and eluted using 500mM methyl α-D-mannopyranoside (Sigma).
Twenty fractions (500µl/fraction) were collected in 1.5ml tubes, and analyzed for
protein using the Bradford assay. All solutions were kept on ice. Column regeneration
was performed following manufacturer’s specifications.
Purification of yCGL by HPLC Size Exclusion Column Chromatography
A silica-based gel filtration column (Phenomenex BioSep-SEC-S 4000, 300 x
7.80mm) was connected to a model 6000A solvent delivery HPLC system, with a
mobile phase of ELLA wash buffer (10mM Tris, 150mM NaCl, 10mM CaCl2, pH 7.5).
Two samples were prepared: purified xlCGL solubilized in ELLA wash buffer, and a
fractionated ammonium sulfate pellet containing yCGL solubilized in 1ml ELLA wash
buffer. The sample (20µl) was injected into the column with a flow rate of 0.5ml/min.
UV detection was monitored at 280nm. Protocol was conducted at RT.
Deglycosylation of CGL
The digestion of N-linked oligosaccharides attached to xlCGL was performed
under non-denaturing conditions at 37ºC for 15hr in a 10µl reaction. General reactions
consisted of 4µl water, 1µl 10X G7 (1X = 50mM sodium phosphate, pH 7.5), 1µl 10X
BSA, 1µl xlCGL (0.794µg), and 3µl of specific enzyme. Exoglycosidases were
35
purchased from New England Biolabs: α1-2,3 mannosidase, α1-6 mannosidase, α2-3
neuraminidase, and α2-3,6,8 neuraminidase. PNGaseF digestion of xlCGL was done in
a 25µl reaction volume with 12µl water, 2µl 10X G7, 1µl xlCGL, and 10µl enzyme
(5000 units). For a control, xlCGL was heat inactivated by heating a 10µl reaction (9µl
water, 1µl xlCGL) for 10min at 100ºC. The digestion of yCGL with PNGaseF was
done under denaturing conditions. Six mls of yCGL T24 expression supernatant was
concentrated and pooled together by TCA precipitation. The pellet was solubilized in
40µl of 100mM Tris-HCl, pH 8.0. Eleven µl of this solution was combined with 1.5µl
of 10X denaturing buffer and incubated at 100ºC for 10min. Then 2µl 10X G7, 2µl
10% NP40, and 5µl PNGaseF were added, and the sample was incubated for 15hr at
37ºC. Preparation of the glycosidase reactions for the enzyme-linked lectin assay was
done by the following: for the 10µl reactions, 5µl was added to 45µl of ELLA wash for
a 1:10 dilution. For the PNGaseF reaction, 12.5µl of reaction was added to 140.6µl
ELLA wash for a 1:10 dilution. Of this 1:10 dilution, 3.74µl was added to 316µl ELLA
wash, and 100µl of this final dilution (0.0928ng/µl) was added to three wells of the 96
well plate.
Enzyme-Linked Plate Binding Assays
An enzyme-linked lectin assay (ELLA) developed initially by Quill and Hedrick
(1996) was used for binding studies of CGL and jelly ligand. ELLA is a sandwich
ELISA-based technique used to detect and quantify specific lectin binding using a 96well format. Xenopus laevis egg jelly (previously solubilized and filter concentrated
36
from Xenopus laevis eggs by Dr. Peavy) was diluted 1:800 in 100mM sodium
carbonate, pH 9.5 and 100µl was coated to the bottom of Immunolon 96 well microtiter
plates at 4ºC overnight. Wells were then washed four times with 300µl
washing/binding buffer (10mM Tris, 150mM NaCl, 10mM CaCl2, pH 7.5). For studies
coating wells with Con A, 5mM MnCl2 and MgCl2 were also included in the
washing/binding buffer. The wells were then blocked for 3 hours at room temp with
rocking using 0.5% polyvinyl alcohol (PVA; MP Biomedicals; MW 22,000 g/mol) in
1X TBS, 0.1% tween 20, pH 7.4. Then 100µl of the test sample (xlCGL, yCGL,
glycosidase treated xlCGL) diluted in washing/binding buffer (final concentration of
92.8ng/ml, 9.28ng/well) was added to each well and then incubated with rocking for 1
hour at room temp. After incubation, wells were washed, blocked again, and washed
once more. Then purified 1ºab (specific for the dg-xlCGL, 1.48 mg/ml) was diluted
1:4000 in western blocking buffer and added to each well and incubated with rocking at
room temp for one hour and then washed. Then 100µl Biotin-SP-conjugated AffiniPure
Goat anti-Rabbit IgG (diluted 1:100,000 in blocking buffer) was added and incubated
with rocking at room temp for one hour and then washed. Then 100µl peroxidase
conjugated streptavidin (diluted to 1:1000 in blocking buffer) was added and then
incubated with rocking at room temp for 10 minutes followed by washing. Then 100ul
TMB blue substrate was added and then incubated with rocking at room temp for about
20 minutes. The OD at 630nm was read using an Opsys MR microplate reader, which
measured the product (conjugated secondary antibody) that is directly proportional to
the binding activity of the CGL to the ligand.
37
Effect of Varying Buffer pH on xlCGL
Citric acid – sodium citrate buffer solutions were used for pH 3.0, 4.0, 5.0, and
6.0. Buffers were composed of 50mM citric acid (Fisher Scientific), 50mM sodium
citrate (Sigma), and 150mM NaCl. Tri-HCl buffers were used for pH 7.5, 8.0, and 9.0.
Buffers were composed of 50mM Tris. pH adjustments were made with 12N HCl or
12N KOH. A 100µl reaction volume was composed of 5µl of xlCGL (0.0397 µg/µl),
1µl 100X BSA, and 94µl buffer. For reactions containing calcium, 1µl 500mM CaCl2
was added. Samples were incubated at 37ºC for 15hours. The reaction (23.37µl) was
added to 476.6µl ELLA wash for a final concentration of 0.0928ng/µl. One-hundred µl
of this reaction was added to 3 wells of the 96 well plate.
Inhibition Experiments
N-acetylneuraminic (Acros Organics) acid was diluted in washing/binding
buffer to the desired concentration in 1.5ml or 0.5ml microcentrifuge tubes. This test
inhibitor was added to xlCGL and incubated for 20 minutes at room temp prior to
addition to the jelly coated wells for ELLA analysis as described previously. Relative
binding was compared to control solutions of xlCGL without inhibitor which was
considered 100% binding (0% inhibition). The percentages of inhibition were
calculated with the equation: % inhibition = (A(no inhibitor) - A(with inhibitor) ) / A(no inhibitor) x
100, where A = absorbance at 630nm.
38
Statistics
Glycosidase treatment effects on xlCGL were assessed using a two-way
ANOVA (without replication). The fixed factor was each glycosidase and each reaction
was treated as a block effect. This allowed an assessment of the differences among
glycosidases while controlling for differences among reactions. Each reaction was
measured in triplicate, and the average was used in this test The statistical analysis was
performed using IBM SPSS, version 19.
39
RESULTS
PCR and DNA Sequence Analysis of yCGL
To investigate the functional role of Xenopus laevis CGL oligosaccharides in the
block to polyspermic fertilization, previous work cloned the xlCGL cDNA into the
Pichia pastoris expression vector pPICZα (Figure 4) to allow recombinant CGL protein
secretion into the medium. The primers were designed to use the XhoI restriction site to
express the recombinant CGL protein using the α-factor signal peptide for secretion in
one set of integrants (GS115 strain), and without for cytoplasmic expression in another
set (KM71H strain). Previous work by Dr. Peavy and students screened 14 zeocin
resistant colonies by PCR, resulting in 4 recombinant clones, as determined by
preliminary PCR confirmation. Yeast DNA purification prior to PCR was previously
not pure, so the current study developed a yeast DNA purification scheme to confirm
the PCR results with a more purified preparation of DNA (Figure 5a). Purification of
total DNA was done using the Qiagen DNeasy Blood and Tissue kit modified for yeast,
which involved the inclusion of the enzyme lyticase, which removes the yeast cell
walls. In order to confirm the sequence of the CGL cDNA and determine insert
orientation in the vector, GS115 yCGL clones 1 and 9 (sec1 and sec9) were PCR
amplified using two different high fidelity Taq polymerases (Fig 5b). The PCR
products were electrophoretically separated, excised from the gels, purified, quantified,
and then commercially sequenced using the 5’ AOX1 and 3’ AOX1 primers. The PCR
40
amplimers had the expected size of ~1353bp, which included the 1080bp from the
insert, and 273bp of vector sequence.
Sequence analysis of the PCR products revealed that the CGL cDNA insert was
in frame with the α-factor signal peptide as designed (Figure 6). The sequence
alignment of yCGL and xlCGL revealed that they were 99% identical with 11 sequence
differences of which three resulted in amino acid substitutions (Figure 7). Analysis also
showed that three N-linked consensus sequences were observed in the yCGL (Figure 8)
which is consistent with the xlCGL sequence reported by Pierce et al., (XL35, Genbank
accession no. U86699.1) A second variant of xlCGL has also been reported that has
only 2 N-linked sites (Chang et al, 2001; Genbank accession no. X82626.1 ). Thus,
yCGL has 3 potential sites for the attachment of N-linked oligosaccharides.
Unexpectedly, the yCGL sequence data revealed that the stop codon was 63 nucleotides
(21 amino acids) downstream of the location expected for the xlCGL cDNA. Although
this peptide addition was found, expression studies were pursued since it was not likely
to affect binding. Many recombinant proteins are routinely expressed as fusion proteins
at the C-terminus and the presence of these foreign peptides do not affect the functional
role of the inserted gene.
41
Figure 4. Vector used for the insertion of recombinant Xenopus laevis cortical granule
lectin into the genome of the yeast Pichia pastoris for secreted expression. The 1080bp
CGL gene was cloned in frame with the α-factor signal sequence open reading frame of
the 3.6kb pPICZα vector. Since a stop codon was not present at the end of the xlCGL
cDNA, an additional 21 amino acids were added and is termed an insertion. The
alcohol oxidase (AOX1) promoter allows for induction using methanol.
42
A)
B)
Figure 5. Agarose gel electrophoresis of PCR products from Pichia CGL integrants.
A) PCR analysis of two strains of Pichia integrant clones (GS115 & KM71H) for the X.
laevis CGL cDNA. Lanes 1 & 6 were loaded with DNA size standards (ladder 0.07 –
12.2Kb), whereas lanes 2-4 were loaded with the PCR of GS115 secreted yCGL clones
#1 & 9, & KM71H intracellular yCGL #1 using yeast vector specific AOX primers,
respectively. Lane 5 was a “no template” control PCR using AOX primers. Lanes 7-9
were the same clones but PCR amplified using CGL specific primers (3/3R); lane 10
was purified CGL cDNA bacterial plasmid amplified using CGL primers; and Lane 11
was a “no template” control with CGL primers. B) PCR amplification of GS115 clones
# 1 and 9 (Sec1 and Sec9) using the AOX primers and two different Taq polymerases,
Invitrogen Platinum Taq HI fidelity and GoTaq. PCR from each clone was loaded in
duplicate and predominant bands excised for gel extraction and subsequent DNA
sequencing. Final DNA concentrations are listed. The middle lane is a size standard.
43
atgagatttccttcaatttttactgctgttttattcgcagcatcctccgcattagctgct
M R F P S I F T A V L F A A S S A L A A
ccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggt
P V N T T T E D E T A Q I P A E A V I G
tactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaat
Y S D L E G D F D V A V L P F S N S T N
aacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggta
N G L L F I N T T I A S I A A K E E G V
tctctcgagaaaagacagtcttgtgaacctgttgtaatagtcgcctcaaaaaacatggtg
S L E K R Q S C E P V V I V A S K N M V
aagcagctggattgtgataaattcagaagctgcaaggagatcaaggattcaaacgaagaa
K Q L D C D K F R S C K E I K D S N E E
gcacaagatggaatatacacactgacctcttcagatgggatatcctaccagaccttctgt
A Q D G I Y T L T S S D G I S Y Q T F C
gacatgactacaaatggaggaggatggactttggtggccagtgttcatgagaacaacatg
D M T T N G G G W T L V A S V H E N N M
gcagggaagtgcactataggggatcgctggtccagccaacaggggaatcgagctgactat
A G K C T I G D R W S S Q Q G N R A D Y
ccagagggcgatggcaactgggcaaactataatacatttggatcagctggtggcgcaact
P E G D G N W A N Y N T F G S A G G A T
agtgatgactacaagaatcctggctattatgatattgaagcatataaccttggggtgtgg
S D D Y K N P G Y Y D I E A Y N L G V W
cacgtgcccaacaagactcccctgagtgtttggaggaattcatcactacagagataccgt
H V P N K T P L S V W R N S S L Q R Y R
acaacagatggcatccttttcaaacatggaggaaacctcttcagtctgtatcggatctat
T T D G I L F K H G G N L F S L Y R I Y
ccagtgaaatatggtataggaagctgctcaaaggacagtggcccaactgtgccagtagtg
P V K Y G I G S C S K D S G P T V P V V
tacgatcttggaagtgctaatttaacggcttctttctactctccaggtttcagaagtcag
Y D L G S A N L T A S F Y S P G F R S Q
tttacccctggctatatccaatttcggccaattaacactgaaaaagctgctctggcgcta
F T P G Y I Q F R P I N T E K A A L A L
tgtccgggaatgaagatggagccatgcaatgtggaacatgtgtgcataggaggaggtggc
C P G M K M E P C N V E H V C I G G G G
tactttccagaagcagaccctcggcaatgtggagactttgcagcctatgattttaatgga
Y F P E A D P R Q C G D F A A Y D F N G
tatggaacgaaaaagtttaacagtgcgggcatagagataactgaggccgctgtattactt
Y G T K K F N S A G I E I T E A A V L L
ttctatctatgctcgagccgcggcggccgccagctttctagaacaaaaactcatctcaga
F Y L C S S R G G R Q L S R T K T H L R
agaggatctgaatag
R G S E -
Figure 6. yCGL DNA and deduced amino acid sequence. Gray highlighting specifies
XhoI restriction sites where gene was inserted. Solid underlining denotes the signal
peptide sequence. Black highlighting specifies a nucleotide which was meant to encode
a stop codon, however, it resulted in a cysteine residue, abbreviated by a “C”. Bold
lettering denotes additional sequences past expected stop codon.
44
yCGL
xlCGL
CAGTCTTGTGAACCTGTTGTAATAGTCGCCTCAAAAAACATGGTGAAGCAGCTGGATTGT 60
CAGTCTTGTGAACCTGTTGTAATAGTAGCCTCAAAAAACATGGTGAAGCAGCTGGATTGT 60
**************************.*********************************
yCGL
xlCGL
GATAAATTCAGAAGCTGCAAGGAGATCAAGGATTCAAACGAAGAAGCACAAGATGGAATA 120
GATAAATTCAGAAGCTGCAAGGAAATCAAAGATTCAAACGAAGAAGCACAAGATGGAATA 120
***********************.*****.******************************
yCGL
xlCGL
TACACACTGACCTCTTCAGATGGGATATCCTACCAGACCTTCTGTGACATGACTACAAAT 180
TACACACTGACCTCTTCAGATGGGATATCCTACCAGACCTTCTGTGACATGACTACAAAT 180
************************************************************
yCGL
xlCGL
GGAGGAGGATGGACTTTGGTGGCCAGTGTTCATGAGAACAACATGGCAGGGAAGTGCACT 240
GGAGGAGGATGGACTTTGGTGGCCAGTGTTCATGAGAACAACATGGCAGGGAAGTGCACT 240
************************************************************
yCGL
xlCGL
ATAGGGGATCGCTGGTCCAGCCAACAGGGGAATCGAGCTGACTATCCAGAGGGCGATGGC 300
ATAGGGGATCGCTGGTCCAGCCAACAGGGGAATCGAGCTGACTATCCAGAGGGCGATGGC 300
************************************************************
yCGL
xlCGL
AACTGGGCAAACTATAATACATTTGGATCAGCTGGTGGCGCAACTAGTGATGACTACAAG 360
AACTGGGCAAACTATAATACATTTGGATCAGCTGGTGGCGCAACTAGTGATGACTACAAG 360
************************************************************
yCGL
xlCGL
AATCCTGGCTATTATGATATTGAAGCATATAACCTTGGGGTGTGGCACGTGCCCAACAAG 420
AATCCTGGCTATTATGATATTGAAGCATATAACCTTGGGGTGTGGCACGTGCCCAACAAG 420
************************************************************
yCGL
xlCGL
ACTCCCCTGAGTGTTTGGAGGAATTCATCACTACAGAGATACCGTACAACAGATGGCATC 480
ACTCCCCTGAGTGTTTGGAGGAATTCATCGCTACAGAGATACCGTACAACAGATGGCATC 480
*****************************.******************************
yCGL
xlCGL
CTTTTCAAACATGGAGGAAACCTCTTCAGTCTGTATCGGATCTATCCAGTGAAATATGGT 540
CTTTTCAAACATGGAGGAAACCTCTTCAGTCTGTATCGGATCTATCCAGTGAAATATGGT 540
************************************************************
yCGL
xlCGL
ATAGGAAGCTGCTCAAAGGACAGTGGCCCAACTGTGCCAGTAGTGTACGATCTTGGAAGT 600
ATAGGAAGCTGCTCAAAGGACAGTGGCCCAACTGTGCCAGTAGTGTACGATCTTGGAAGT 600
************************************************************
yCGL
xlCGL
GCTAATTTAACGGCTTCTTTCTACTCTCCAGGTTTCAGAAGTCAGTTTACCCCTGGCTAT 660
GCTAAATTAACAGCTTCTTTCTACTCTCCAGATTTCAGAAGTCAGTTTACCCCCGGCTAT 660
*****:*****.*******************.********************* ******
yCGL
xlCGL
ATCCAATTTCGGCCAATTAACACTGAAAAAGCTGCTCTGGCGCTATGTCCGGGAATGAAG 720
ATCCAATTTCGGCCAATTAACACTGAAAAAGCTGCTCTGGCGCTATGTCCGGGAATGAAG 720
************************************************************
yCGL
xlCGL
ATGGAGCCATGCAATGTGGAACATGTGTGCATAGGAGGAGGTGGCTACTTTCCAGAAGCA 780
ATGGAGTCATGCAATGTGGAACATGTGTGCATAGGAGGAGGTGGCTACTTTCCAGAAGCA 780
****** *****************************************************
yCGL
xlCGL
GACCCTCGGCAATGTGGAGACTTTGCAGCCTATGATTTTAATGGATATGGAACGAAAAAG 840
GACCCTCGGCAATGTGGAGACTTTGCAGCCTATGACTTTAATGGATATGGAACCAAAAAG 840
*********************************** ***************** ******
yCGL
xlCGL
TTTAACAGTGCGGGCATAGAGATAACTGAGGCCGCTGTATTACTTTTCTATCTATGCTCG 900
TTTAACAGTGCGGGCATAGAGATAACTGAGGCCGCTGTATTACTTTTCTATCTATGA--- 897
********************************************************.
yCGL
xlCGL
AGCCGCGGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAG 960
------------------------------------------------------------
Figure 7. DNA sequence alignment comparing xlCGL to yCGL.
45
Figure 7 (continued). The Clustal alignment program was used to compare the yCGL
DNA sequence to xlCGL GenBank accession # X82626.1 which has only two N-linked
glycosylation sites. Black highlighting specifies locations of missense mutations
(amino acid changes). In the Clustal program, asterisks below sequence positions
indicate identical matches, colons indicate conservation of residue properties, periods
indicate weakly similar properties of residues, and no symbol indicates no similarity of
residues.
46
yCGL
xlCGL
QSCEPVVIVASKNMVKQLDCDKFRSCKEIKDSNEEAQDGIYTLTSSDGISYQTFCDMTTN 60
QSCEPVVIVASKNMVKQLDCDKFRSCKEIKDSNEEAQDGIYTLTSSDGISYQTFCDMTTN 60
************************************************************
yCGL
xlCGL
GGGWTLVASVHENNMAGKCTIGDRWSSQQGNRADYPEGDGNWANYNTFGSAGGATSDDYK 120
GGGWTLVASVHENNMAGKCTIGDRWSSQQGNRADYPEGDGNWANYNTFGSAGGATSDDYK 120
************************************************************
yCGL
xlCGL
NPGYYDIEAYNLGVWHVPNKTPLSVWRNSSLQRYRTTDGILFKHGGNLFSLYRIYPVKYG 180
NPGYYDIEAYNLGVWHVPNKTPLSVWRNSSLQRYRTTDGILFKHGGNLFSLYRIYPVKYG 180
************************************************************
yCGL
xlCGL
IGSCSKDSGPTVPVVYDLGSANLTASFYSPGFRSQFTPGYIQFRPINTEKAALALCPGMK 240
IGSCSKDSGPTVPVVYDLGSAKLTASFYSPDFRSQFTPGYIQFRPINTEKAALALCPGMK 240
*********************:********.*****************************
yCGL
xlCGL
MEPCNVEHVCIGGGGYFPEADPRQCGDFAAYDFNGYGTKKFNSAGIEITEAAVLLFYLCS 300
MESCNVEHVCIGGGGYFPEADPRQCGDFAAYDFNGYGTKKFNSAGIEITEAAVLLFYL-- 298
**.*******************************************************
yCGL
xlCGL
SRGGRQLSRTKTHLRRGSE-R 320
---------------------
Figure 8. Comparison of amino acid sequences of xlCGL and yCGL. Clustal was used
to align the protein sequence of yCGL to xlCGL (accession # Q91719). Gray
highlighting specifies N-linked consensus sites. Asterisks below sequence positions
indicate identical matches, colons indicate conservation of residue properties, periods
indicate weakly similar properties of residues, no symbol indicates no similarity of
residues, and dashes indicate an insertion in one of the two sequences.
47
Mouse_Intelectin_1
Human_Intelectin_1
X.laevis_CGL_#1
X.laevis_CGL_#2
MTQLGFLLFIMVATRGCSAAEENLDTNRWGNSFFSSLPRSCKEIKQEHTKAQDGLYFLRT
MNQLSFLLFLIATTRGWSTDEANTYFKEWTCSSSPSLPRSCKEIKDECPSAFDGLYFLRT
MLVHILLLLVTGGLSQSCEPVVIVASKNMVKQLDCDKFRSCKEIKDSNEEAQDGIYTLTS
MLVHILLLLVTGGLSQSCDPVVIVASKNMVKQLDCDKFRNCKEIKDSNEEAQDGIYTLTS
*
:**::
.
:.
.
. *.*****:. .* **:* * :
60
60
60
60
Mouse_Intelectin_1
Human_Intelectin_1
X.laevis_CGL_#1
X.laevis_CGL_#2
KNGVIYQTFCDMTTAGGGWTLVASVHENNMRGKCTVGDRWSSQQGNRADYPEGDGNWANY
ENGVIYQTFCDMTSGGGGWTLVASVHENDMRGKCTVGDRWSSQQGSKAVYPEGDGNWANY
SDGISYQTFCDMTTNGGGWTLVASVHENNMAGKCTIGDRWSSQQGNRADYPEGDGNWANY
PDGISYQTFCDMTTNGGGWTLVASVHENNMAGKCTIGDRWSSQQGNRADYPEGDGNWANY
:*: ********: *************:* ****:*********.:* ***********
120
120
120
120
Mouse_Intelectin_1
Human_Intelectin_1
X.laevis_CGL_#1
X.laevis_CGL_#2
NTFGSAEAATSDDYKNPGYFDIQAENLGIWHVPNKSPLHNWRKSSLLRYRTFTGFLQHLG
NTFGSAEAATSDDYKNPGYYDIQAKDLGIWHVPNKSPMQHWRNSSLLRYRTDTGFLQTLG
NTFGSAGGATSDDYKNPGYYDIEAYNLGVWHVPNKTPLSVWRNSSLQRYRTTDGILFKHG
NTFGSAGGATSDDYKNPGYYDIEAYNLGVWHVPNKTPLSVWRNSSLQRYRTTDGILFKHG
****** .***********:**:* :**:******:*: **:*** **** *:*
*
180
180
180
180
Mouse_Intelectin_1
Human_Intelectin_1
X.laevis_CGL_#1
X.laevis_CGL_#2
HNLFGLYKKYPVKYGEGKCWTDNGPALPVVYDFGDARKTASYYSPSGQREFTAGYVQFRV
HNLFGIYQKYPVKYGEGKCWTDNGPVIPVVYDFGDAQKTASYYSPYGQREFTAGFVQFRV
GNLFSLYRIYPVKYGIGSCSKDSGPTVPVVYDLGSAKLTASFYSPDFRSQFTPGYIQFRP
GNLFSLYRIYPVKYGIGSCSKDSGPTVPVVYDLGSANLTASFYSPGFRSQFTPGYIQFRP
***.:*: ****** *.* .*.**.:*****:*.*. ***:*** : :**.*::***
240
240
240
240
Mouse_Intelectin_1
Human_Intelectin_1
X.laevis_CGL_#1
X.laevis_CGL_#2
FNNERAASALCAGVRVTGCNTEHHCIGGGGFFPEGNPVQCGDFASFDWDGYGTHNGYSSS
FNNERAANALCAGMRVTGCNTEHHCIGGGGYFPEASPQQCGDFSGFDWSGYGTHVGYSSS
INTEKAALALCPGMKMESCNVEHVCIGGGGYFPEADPRQCGDFAAYDFNGYGTKKFNSAG
INTEKAALALCPGMKMESCNVEHVCIGGGGYFPEADPRQCGDFAAYDFNGYGTKKFNSAG
:*.*:** ***.*::: .**.** ******:***..* *****:.:*:.****:
*:.
300
300
300
300
Mouse_Intelectin_1
Human_Intelectin_1
X.laevis_CGL_#1
X.laevis_CGL_#2
RKITEAAVLLFYR
REITEAAVLLFYR
IEITEAAVLLFYL
IEITEAAVLLFYL
:**********
313
313
313
313
Figure 9. Amino acid sequence alignment of four members of the eglectin family.
Xenopus laevis #1 is the cDNA that was cloned into Pichia pastoris. The following
translated cDNA sequences were aligned using ClustalW2: Xenopus laevis #1
(Genbank accession no. CAA57946.1), Xenopus laevis #2 (XL35, Genbank accession
no.AAB47537), Mouse Intelectin 1 (NP_034714.1), Human Intelectin 1
(NP_060095.2). Gray highlighting specifies N-linked consensus sites. Asterisks below
sequence positions indicate identical matches, colons indicate conservation of residue
properties, periods indicate weakly similar properties of residues, no symbol indicates
no similarity of residues, and dashes indicate an insertion in one of the two sequences.
48
Optimization of Secreted Expression of CGL in Pichia pastoris
Once recombinant strains were confirmed by DNA sequencing to contain the
CGL insert, the optimal methods and conditions for expressing yCGL were developed,
which included purification of the primary antibody to recognize CGL, and
optimization of the transfer conditions for detecting yCGL by immunoblotting.
Many
parameters were involved in determining the ideal expression conditions for
recombinant CGL. These included aeration, temperature, shaking, and susceptibility to
proteases.
yCGL clones were used to inoculate a starter culture to generate a large enough
cell mass to achieve log phase growth (OD600 ~6). Cells were then harvested and
resuspended in buffered methanol complex medium (BMMY). This phosphate buffered
media had a pH of 6.1, and included yeast extract and peptone to stabilize secreted
proteins and prevent or decrease proteolysis. Pichia pastoris uses the methanolinduced alcohol oxidase (AOX1) promoter for heterologous protein production, and so
expression was induced using 0.5% methanol at 29ºC. Since the inserted gene was
down stream of the secretory signal sequences, the expressed gene product was
expected to be secreted out into the medium. Methanol was added (0.5%) every 24h to
sustain induction up to 96h.
Initial analysis of the expression supernatant by immunoblotting resulted in
profiles with multiple faint bands with a very low intensity, or the complete absence of
bands. Research into the literature provided many examples that some secreted foreign
proteins are unstable in the Pichia pastoris culture medium and are rapidly degraded by
49
vacuolar proteases released during cell lysis due to the high cell density environment
(Cregg, 2005). To test whether degradation by proteases was indeed responsible for the
absence of detectable yCGL, different combinations of proteases were included in the
expression media and examined by SDS-PAGE. As shown in Figure 10, yCGL proved
to be especially susceptible to proteases, and it was determined that a combination of
1% casamino acids, 1mM EDTA, and protease inhibitor tablets significantly reduced
degradation by proteases. Other variations to expression conditions were explored to
increase yield.
The literature suggested that the most important parameter for efficient
expression was adequate aeration during methanol induction. Therefore, baffled flasks
were incorporated into the protocol, which resulted in expression supernatants yielding
protein concentrations in the range of 900-1100µg/ml, which represented a ~20%
increase in yield. While the recommended optimum temperature for shaking is 2830ºC, a few reports showed that decreasing the expression temperature to as low as
20ºC decreased the susceptibility to protease degradation and increased protein stability.
In an effort to increase overall yield, the expression temperature was decreased to room
temp (~24ºC), however, the results displayed the same characteristics as the expression
at 29ºC, so further expressions were conducted at 29ºC for consistency and expedience.
Once the ability to produce full length yCGL had been achieved, an additional
analysis of expression levels was necessary to determine the optimal time postinduction to harvest yCGL. Therefore, a time course study was conducted where the
expression supernatant was collected between the first moment of methanol induction
50
(Time =0, T0) and 96 hours post induction (T96). Immunoblotting revealed that the 48
hour time point provided the highest quantity of yCGL expression relative to the
amount of a ~25kD degradation product (Figure11). The immunoblot indicated that
yCGL had a molecular weight of ~65kD, with the band appearing to be slightly
smeared. The ~65kD size of yCGL was unexpected, because this is ~20kD larger than
the 45kD of the xlCGL.
Optimization of Immunoblotting
Immunoblotting was chosen as the method to detect recombinant CGL because
the primary antibody to deglycosylated xlCGL offered a sensitive and accurate tool to
compare xlCGL to yCGL. It should be noted that since the xlCGL polypeptide was
injected into rabbits, antibodies were generated solely to peptide antigens thereby
avoiding any oligosaccharide detection differences. Initially, immunoblotting analyses
used freshly made 10% tris-glycine gels, and transfers were done overnight at 4ºC,
which did seemingly provide adequate and fairly consistent results. While these
transfer conditions did provide a signal, further optimization was necessary to obtain
consistent results with sharp, intense bands. To this end, optimization of the
immunoblotting transfer required an extensive amount of troubleshooting, as the
transfer conditions of yCGL differed from that of xlCGL. xlCGL was able to
consistently bind to nitrocellulose membrane under a wide variety of voltage and buffer
conditions. However, yCGL was only able to bind to membranes under specific
51
circumstances, and high-quality results were obtained only when the transfer was done
under extremely gentle conditions.
In addition, two types of commercially available 10% gels were used for
immunoblot transfer: Biorad TGX gels, and Invitrogen NuPAGE Bis-Tris Gels.
Despite many attempts with a variety of transfer conditions, the transfers in the trisglycine system showed high-quality bands only for the xlCGL, but failed to show
consistent bands for yCGL. Interestingly, only the NuPAGE system was able to
consistently provide positive results, but only under the most gentle of conditions (30V
for 1.5hours at 4ºC). It is noteworthy to point out that the NuPAGE system operates
under neutral pH conditions (compared to the basic conditions of tris-glycine systems)
which is advertised to minimize protein modifications and is presumed to result in
sharper bands.
It should be noted that in the summer of 2010, the -80ºC freezer housing the
recombinant clones, secondary antibodies, and expression samples experienced a power
failure, and all of the previously mentioned items were incubated at room temperature
for ~3 days. The collected protein samples were lost, and the signal of the secondaries
(per ELISA) were severely reduced. The recombinant clones did survive, and new
cultures were propagated, and new glycerol stocks were created. Therefore, another
round of immunoblotting optimization had to be undertaken to incorporate new
antibodies, and clones that have been exposed to unfavorable conditions.
52
Concentration of yCGL by Filtration
Once expression of yCGL had been confirmed by immunoblotting and
expression relatively optimized, methods to purify secreted yCGL from the expression
media were explored for use in binding assays. Since yCGL was estimated to be
expressed in the media at less than 1mg/ml, further concentration was necessary to
pursue various purification techniques. In addition, another goal when concentrating
yCGL was to exchange the media that it was suspended in since expression media
contains buffers, salts, methanol, and amino acids that would not be compatible in
certain types of purification methods.
There are many different ways to concentrate glycoproteins, but method success
is always dependent on the properties of the particular glycoprotein being studied.
Initially, three different methods were used to concentrate yCGL based on previous
success with xlCGL, cost effectiveness, and time efficiency: 1) centrifugal filter
devices with a molecular weight cutoff, 2) membrane dialysis and lyophilization, and 3)
ultrafiltration through membrane disks under gas pressure. These three methods are
based on the principle of forcing water and low-molecular weight substances (salts and
other contaminants) through a semipermeable membrane or barrier that has a particular
molecular size cutoff while retaining the molecules larger than the pore size,
specifically yCGL. Additionally, ultrafiltration is considered to be far gentler to solutes
than other concentration processes such as precipitation (Phillips and Signs, 2004).
These techniques also offer the advantage of simultaneously exchanging the expression
53
supernatant with the buffer of choice during the process, thereby facilitating
downstream purification.
Previously, membrane dialysis and lyophilization had successfully concentrated
and desalted xlCGL, therefore, this was the first technique chosen for application to
yCGL. Spectra/Por regenerated cellulose membranes of different molecular weight
cutoffs (10kD, 30kD, 50kD) were used with varying concentrations (5 to 50mM) of
ammonium bicarbonate. The protocol included exchanging the buffer every 3-12 hours,
for a total of three buffer exchanges, which resulted in ~12 to 36 hour protocols.
Despite many attempts, this technique repeatedly failed, as both SDS-PAGE and
Bradford assay showed a complete loss of protein. It was presumed that the yCGL was
irreversibly precipitating. It is important to note that a nearly identical protocol for
dialysis and lyophilization had been attempted twice for the purification of the primary
antibody, and both attempts were successful, establishing that user technical error was
not the issue.
The next technique to concentrate and desalt the expression supernatant used
was centrifugal ultrafiltration using Amicon Centriplus disposable centrifugal
concentrators with a 10kD molecular weight cutoff. This technique proved to be more
successful than lyophilization and dialysis, as the final product did yield full length
yCGL. However, the final recovered product also included a significant amount of
protein degradation as determined by immunoblotting analysis. Additionally, this
technique was inconsistent, as four of the seven total attempts resulted in nearly
complete loss of the protein, as determined by Bradford assay. One of the known
54
limitations of this technique is that target proteins can adsorb to the membrane and
reservoir wall of the concentrator, and become unrecoverable. This likely played a role,
as the protocol experienced clogging of the membrane, preventing the transfer of
solutes to the reservoir. These protocols were generally 4-6 hours in length.
The third filtration based technique attempted was stirred-cell ultrafiltration
through asymmetric membrane disks under pressure, also known as ‘dead-end
filtration’. This method uses pressure from nitrogen gas to force molecules through the
molecular weight cutoff, while simultaneously remixing the solution by a stirring
action. This technique was attempted ten times using different volumes of supernatant
and different molecular weight cutoffs. One experiment diluted 50mls of ~1mg/ml
expression supernatant into 350mls of TBS (1:8 dilution), and collected 1ml samples at
different time points during the pressurized filtration for analysis by Bradford assay.
Analysis revealed that all samples that had been exposed to pressure did not have any
protein. Another experiment diluted 50mls of ~1mg/ml expression supernatant into
150mls of TBS (1:4 dilution). SDS-PAGE analysis revealed a faint band of ~65kD,
showing that concentration and desalting by ultrafiltration was possible. However, two
attempts to repeat this experiment under the same conditions resulted in a complete loss
of protein. This protocol took approximately 4-6 hour for each trial.
In summary, all three of these filtration-based methods failed to consistently
yield full length yCGL compatible with downstream processes. While every effort was
made to ensure batch to batch reproducibility, the ability to repeat successful results
remained elusive. Centrifugal and pressurized ultrafiltration were able to produce at
55
least one partially successful result, either by SDS-PAGE or Bradford assay, which
suggested that the respective method might be optimized to produce large quantities of
concentrated yCGL. However, repeated attempts resulted in either complete loss of
protein or excessive degradation, suggesting that the solubility of the yCGL was far
more sensitive than anticipated, and that a different strategy would be required.
Ammonium Sulfate Precipitation of yCGL
Since the stability of yCGL seemed to be of issue during filtration, salt
precipitation techniques were explored. Ammonium sulfate is a common procedure
used to change the solubility of a protein so that it will precipitate at a particular
concentration of the salts. Different proteins or glycoproteins will “salt out” at specific
concentrations of ammonium sulfate and so a mixture of proteins can be separated into
pellet and supernatant fractions. After centrifugation, the protein-salt pellet can be
resolubilized in the buffer of choice while retaining its function as has been documented
for many different proteins (Wingfield, 1998). In addition, such an experiment can be
completed in less than two hours, which is significantly faster than any of the
previously attempted strategies.
Thus, ammonium sulfate was added to expression supernatant in concentrations
ranging from 5% to 100% saturation to determine the optimal precipitation condition,
and immunoblotting analysis revealed that recombinant yCGL would consistently and
efficiently precipitate at a range of 15-20% salt saturation (Figure 12). On average,
22mls of ~1mg/ml expression supernatant would yield, after being resolubilized, 1ml of
56
~1mg/ml fractionated yCGL. The loss of yCGL indicates that much of the yCGL was
not resolubilizing into solution after precipitation. However, the method successfully
demonstrated that concentration and partial purification had been achieved. It should be
noted that consistency using the salt precipitation method was only observed when the
expression supernatant had a concentration of at least 1mg/ml, as ammonium sulfate
precipitation has been document to not work as efficiently with solutions having a lower
protein concentration. Additionally, figure 13 demonstrates that the ammonium sulfate
fractionation protocol had issues with degradation of yCGL, much like the previous
methods, if the experiment was not done quickly and efficiently.
While salt precipitation did successfully concentrate and fractionate yCGL from
other secreted proteins and unwanted materials, the sample still had residual ammonium
sulfate from the solubilized pellet, so an additional desalting step was needed. To this
end, desalting was accomplished by the technique of gel filtration using sephadex G-25
spin columns, which separate low molecular weight impurities from high molecular
weight proteins on the basis of size. However, this filtration method again suffered
from a significant (50-90%) loss in overall yield of yCGL. Nevertheless, enough
concentrated yCGL was obtained to proceed with the purification scheme.
57
Figure 10. SDS-PAGE of protease inhibitor effects on yCGL expression. Lanes 1 and
2 were loaded with two different size standards. The rest of the lanes were loaded with
1mL 48hr expression supernatant that had been precipitated by TCA and were treated in
the following manner during expression: lane 3 had no protease inhibitors; lane 4 had
casamino acids added; lane 5 had casamino acids and sigma protease inhibitor tablets
added; and lane 6 had casamino acids, sigma protease inhibitor tablets, and EDTA
added.
58
Figure 11. Immunoblotting analysis of yCGL time course expression. Supernatants of
yCGL expression were collected over a 96hr time period and then TCA precipitated
prior to gel electrophoresis (Lanes 5-9). Expression was performed without protease
inhibitors but with 1mM EDTA and 1% casamino acids. Lane 1 was loaded with
PNGaseF treated yCGL from a prior expression (to remove N-linked sugars), whereas
lane 3 was loaded with PNGaseF treated xlCGL for comparison. Lane 2 was loaded
with a size standard and lane 10 was loaded with BSA for a blotting control.
59
Figure 12. Immunoblotting analysis of ammonium sulfate precipitation of yCGL.
Ammonium sulfate was added to yCGL expression supernatant to a final concentration
of 5%, 10%, 15%, 20%, and 100%, and resuspended in buffer for immunoblotting
(lanes 4-8, respectively). Lane 1 was loaded with a size standard and lane 2 was loaded
with xlCGL for reference.
60
Figure 13. Immunoblotting analysis of sephadex G-25 desalted yCGL after ammonium
sulfate precipitation. After yCGL expression, ammonium sulfate was added to a final
concentration of 30%, 50% and 70% and the precipitates were analyzed by
immunoblotting after resuspension (lanes 4-6). The 30% ammonium sulfate yCGL
precipitation was further desalted by passage through a sephadex G-25 column and the
eluent was loaded into lane 3. Lane 1 was loaded with a size standard and lane 2 was
loaded with xlCGL for reference.
61
Purification of yCGL
In developing a method to purify yCGL, the goal was to isolate yCGL to
homogeneity in the most efficient way without disrupting its binding functionality.
Previous research has used the jelly ligand to purify xlCGL, however, this binding
affinity option was not explored since it was likely that yCGL would not bind as well to
the jelly ligand due to the loss of complex oligosaccharides. Thus, other promising
chromatographic approaches were attempted that were based on charge (anion
exchange), affinity to Concanavalin A that binds to mannose residues, and size
exclusion columns.
Previous research used Q-sepharose anion exchange column chromatography to
successfully purify xlCGL from a complex egg extract mixture so it was employed to
purify yCGL from the concentrated preparation. Q-sepharose consists of agarose beads
cross-linked to positively charged groups which binds to anionic groups on molecules
(negative charges) thereby allowing for the separation of proteins based on their ionic
and polar properties. Three attempts were made to purify yCGL by anion exchange,
however, all three attempts resulted in very low yields of protein when fractions were
evaluated for protein amounts using Bradford assays (data not shown).
The next purification method attempted was affinity column chromatography
using Concanavalin A (ConA). ConA is a plant lectin that binds specifically to
mannosyl and glucosyl (carbohydrate) residues of glycoproteins which has been used
routinely for purifying glycoproteins from Pichia expression systems due to their high
mannose N-linked oligosaccharides. The coupling of ConA to Sepharose 4B agarose
62
beads can therefore be used to separate glycoproteins from non-glycosylated proteins
(and other impurities). This method was attempted twice, and both attempts failed to
produce any significant amount of glycoprotein in the eluant as determined by Bradford
assays. However, when the eluent was concentrated by TCA precipitation for SDSPAGE analysis, a small pellet was evident and revealed a single band of ~25kD in
eluent fractions, with the highest amounts found in the first three 500uL fractions
collected. This band was indicative of previously identified degradation products,
however, when a immunoblotting analysis was performed, no immunoreactive band
was found. However, this result is likely a problem with the blotting technique since
the blotting technique had not been optimized at this point. While further optimization
may have yielded a suitable final product, resource constraints led to the conclusion that
different purification methods may be more appropriate.
Therefore, a different strategy was employed based on HPLC size exclusion
column chromatography. Size-exclusion chromatography is a method which separates
molecules on the basis of molecular size, whereby the larger molecules elute first from
the column since they spend less time entering the pores of the chromatography beads.
This method offers the advantage that samples do not have to be desalted prior to
separation so the ammonium sulfated yCGL preparations can be used directly. This
strategy would also take advantage of the oligomeric structure that xlCGL is known to
form (10-12mer approximately 450kD), and the fact that yeast do not have any native
proteins in this size range. However, this strategy would only be successful if yCGL
63
forms oligomeric structures similar to xlCGL which was to be determined after
performing pilot experiments.
In order to test the technique, xlCGL preparations were examined using a size
exclusion column that separates molecules in the 1,500kD to 15kD range (Figure 14). It
was found that xlCGL displays three prominent peaks that appear to correspond to the
10-12mer (~6.3min), dimer (~12.4min), and monomer (~13.5min). The effect of
varying the concentration of Ca2+ in xlCGL preparations was examined, and revealed
that increasing the concentration of Ca2+ did increase the amount of the oligomeric form
(Figure 14).
When yCGL was examined without calcium, one major peak was evident and its
elution time corresponded to the xlCGL monomer (~13.5min) after overlaying
chromatograms (Figure 15). This yCGL peak appeared to be heterogenous since the
peak was not uniformly bell-curve shaped but had a shoulder. Notably, no peaks were
observed where the oligomeric or dimeric forms should be present. After adding
calcium to yCGL, oligomeric or dimeric structures were still not observed suggesting
that yCGL does not form these larger subunit structures (Figure 16). This result is in
contrast to an earlier observation found in a immunoblot of yCGL (Figure 12, lane 6),
which showed a faint band at ~130kD, which is the correct size for a dimer of yCGL.
All in all, these results suggested that size exclusion chromatography using the current
column would not be a suitable purification method since the large peak was
heterogenous.
64
Figure 14. Size exclusion chromatography profile of xlCGL. Preparations of xlCGL
were separated by HPLC using a Phenomenex BioSep-SEC-S 4000 size exclusion
column and detected by UV. The black chromatogram is the elution of xlCGL treated
with 10mM calcium whereas the blue chromatogram is xlCGL treated with only 1.5mM
calcium. Peaks eluting at earlier time points represent larger molecules (i.e. the 6.3min
peak represents the larger oligomer whereas the 13.5min peak represents the monomer).
65
Figure 15. Size exclusion chromatography comparing yCGL to xlCGL. Preparations of
xlCGL and yCGL were separated by HPLC using a Phenomenex BioSep-SEC-S 4000
size exclusion column and detected by UV. The blue chromatogram is the elution of
xlCGL treated with 1.5mM calcium whereas the black chromatogram is the elution of
yCGL without calcium.
66
Figure 16. Size exclusion chromatography of yCGL with or without calcium.
Preparations of yCGL were separated by HPLC using a Phenomenex BioSep-SEC-S
4000 size exclusion column and detected by UV. The blue chromatogram is the elution
of yCGL treated with 10mM calcium whereas the black chromatogram is the elution of
yCGL treated with 20mM EDTA, which eliminates the effect of calcium.
67
Optimization of Enzyme-Linked Lectin Assays
Although many complications were encountered while attempting to purify
yCGL from expression supernatants, it was necessary to move forward with the binding
studies due to time constraints. Partially purified yCGL preparations were deemed
ample to provide an assessment as to its relative binding activity to the Xenopus laevis
jelly ligand when compared to xlCGL given the appropriate controls. In addition, there
were many treatments such as glycosidases that could be applied to the native xlCGL
glycoprotein to study the importance of oligosaccharides for binding to the jelly ligand.
Therefore, an enzyme-linked lectin plate binding assay (ELLA) was developed
based on established protocols from previous studies (Mozingo, 1996; Nishihara et al.,
1986; Quill and Hedrick, 1996). ELLA binding studies are performed much in the
same way enzyme-linked immunoassays (ELISA) are done using 96-well plates to
assess antibody binding to antigens followed by detection using a secondary antibody
conjugated to an enzyme for a colorimetric assay. In the case of the ELLA, the lectin of
interest is tested for binding activity to a particular ligand under a specific set of
conditions and then detected by adding an antibody to the lectin followed by a
secondary antibody enzyme conjugate for a colorimetric assay. Soluble preparations of
Xenopus laevis egg jelly (previously collected by T.R. Peavy) were used for the CGL
binding studies since it had been used in prior studies. In short, the design of the ELLA
was the following: 1) coat the bottom of the wells with egg jelly, 2) block all other
available sites in the well not bound by egg jelly with a blocking agent, 3) add CGL
preparations to the well with or without additions such as competitive inhibitors to
68
allow binding, 4) wash away unbound CGL, 5) add primary antibody to the
deglycosylated CGL polypeptide made in rabbits, 6) wash, 7) add secondary antibody
specific to primary antibody which has an biotin conjugate for amplification of signal
(i.e. goat anti-rabbit biotin), 8) wash, 9) add enzyme-linked streptavidin to bind to biotin
(i.e. horseradish peroxidase), 10) wash, 11) add enzyme substrate (i.e. TMB blue), and
11) quantitatively detect the enzymatic formation of a colored product by
spectrophotometry (i.e. 630nm for TMB blue) (Figure 17).
The deglycosylated xlCGL rabbit antibodies used in this study to detect bound
CGL was an important reagent for the assay and requires further description. To obtain
these antibodies, rabbits were injected with xlCGL that had been chemically
deglycosylated (i.e. trifluoromethanesulfonic acid treatment) such that the antibodies
made by the rabbits would be only directed to polypeptide antigenic sites. This was
critical for the design of these experiments since injected rabbits would have likely
generated antibodies to the oligosaccharides of xlCGL (e.g. complex and mannose-type)
in addition to the polypeptide if they had not been removed which would have
confounded the results of the binding studies. Since the cDNA corresponding to xlCGL
was inserted into the yeast as described previously, the antibodies should react with
yCGL in a similar manner. In addition, care was taken to purify the IgG fraction from
the rabbit antisera to reduce background issues derived from the serum, and determine a
suitable working concentration for the primary antibody (Figure 18).
Considerable time was spent optimizing the conditions for ELLA given the
specific reagents used in this study so as to minimize the detection of background levels
69
of non-specific antibody binding. The positive control for all ELLA experiments was
the binding of native xlCGL to the egg jelly in the binding buffer described in previous
studies which includes calcium (10mM Tris-HCl, 150mM NaCl, 10mM CaCl2, 0.05%
Tween 20, pH 7.5) (Mozingo, 1996; Nishihara et al., 1986; Quill and Hedrick, 1996).
Initial ELLA studies were performed using BSA for the blocking reagent as described
in previous assays, however this produced a relatively high level of background signal
(Figure 19). Therefore, it became evident that an evaluation of alternative blocking
reagents would be necessary to achieve better signal to noise ratios. A recent report
using ELLA methodology indicated that 0.5% PVA was preferable to BSA as a blocker
to reduce background (Thompson et al., 2011). When 0.5% PVA was substituted for
BSA in the current study, the majority of the background was eliminated (Figure 19).
Once the blocking agent had been chosen, other controls were performed to ensure that
the colorimetric detection was specific for CGL. These included omitting specific
constituents from the reaction wells such as the CGL, egg jelly, and the primary and
secondary antibodies. Figure 20 demonstrates that binding between the CGL and its
ligand was nearly eliminated when any of the above constituents were omitted. Notably,
there was a small amount of signal in wells lacking egg jelly (~ 8% signal relative to the
native lectin-ligand binding) which suggests that the well was not completely blocked
when treated with CGL and antibodies. This was deemed non-specific and not relevant
since the standard ELLA protocol will be to coat all wells with egg jelly and then test
various preparations of CGL. Tittering of xlCGL revealed that a final concentration of
0.0928ng/µl (9.28ng/well) provided the optimum signal (absorbance at 630nm = ~1.3).
70
Figure 17. Schematic of enzyme-linked lectin assay and two potential CGL-ligand
binding mechanisms.
Jelly is adhered to the well surface by nonspecific absorption
(96 well plate). Sites without jelly bound are blocked by the addition of blocking agents
(polyvinyl alcohol). Preparations of CGL are added to the well and incubated for
binding to jelly. Bound CGL is then detected by the sequential addition of a rabbit
antibody to CGL polypeptide, a goat anti-rabbit secondary antibody conjugated to biotin
(for amplification of signal), streptavidin conjugate to horseradish peroxidase enzyme,
and TMB blue substrate. The TMB substrate produces a blue color when enzymatically
converted and this colorimetric product is measured spectrophotometrically at 630nm.
Scenario A illustrates the possibility that oligosaccharides of CGL directly participate in
the binding interaction to the ligand. Scenario B depicts the possibility that only the
CGL protein interacts with the ligand such that oligosaccharides are simply providing a
supportive or structural role.
71
Absorbance at 630 nm
3
2.5
2
1.5
1
0.5
50000
30000
10000
5000
2500
800
1000
700
600
500
400
300
200
180
160
140
120
100
80
60
40
20
10
0
Dilution fold
Figure 18. Tittering of rabbit antibodies to de-glycosylated xlCGL. Tittering and
optimization of the enzyme-linked lectin assay were performed utilizing a standard
checkerboard two-fold dilution series approach. Optimum concentration for antibody
was deemed between 1:5000 and 1:10000.
72
Figure 19. Evaluation of blocking reagents for ELLA. Different blocking reagents
commonly used in ELISA and ELLA were assessed for their ability to prevent nonspecific binding and reduce background signal. Jelly is adhered to the well surface by
nonspecific absorption (96 well plate). Sites without jelly bound are blocked by the
addition of respective blocking reagent. Preparations of CGL are added to the well and
incubated for binding to jelly, followed by antibody detection.
The “TMB blue”
columns represents wells that contained only TMB blue, and did not have any jelly
ligand, CGL, or any other ELLA constituents. Each reaction was measured in
triplicate, and the error bars represent the standard deviation.
73
Figure 20. Reactivity of the enzyme-linked lectin assay. Specific elements pivotal in the
lectin-ligand binding interaction were removed to identify non-specific binding and
background signal. Each reaction was measured in triplicate, and the error bars represent
the standard deviation.
74
Competitive Inhibition Studies of CGL-ligand Binding
Once the ELLA protocol had been optimized, competitive binding assays were
performed using two previously established monosaccharide competitive inhibitors,
galactose and glucose. These monosaccharides are thought to compete for binding at
the active site where CGL binds to the ligand and interferes with binding in a dosedependent manner (Mozingo, 1996; Nishihara et al., 1986; Quill and Hedrick, 1996).
These controls were performed to compare the binding selectivity and sensitivity to
prior studies. Galactose and glucose were incubated with xlCGL at varying
concentrations (2.5 – 320mM) during the binding step to the jelly ligand to test their
inhibitory properties (Figure 21). Increasing the concentration of galactose resulted in a
decrease of CGL binding to the egg jelly ligand, with maximal inhibition (nearly 100%)
achieved at approximately 150 mM galactose. Glucose demonstrated intermediate
inhibitory characteristics, as increasing concentrations of glucose resulted in a maximal
inhibition of CGL binding to 70% at ~200 mM (Figure 21). These results were
consistent with previous studies.
In addition, controls were performed to confirm the calcium dependence of
CGL-ligand binding. EGTA was used in the incubation medium to strip calcium from
xlCGL since it is a chelating agent with a high affinity for calcium. EGTA proved to be
a potent inhibitor, resulting in total (100%) inhibition of the CGL-ligand interaction and
thus corroborating this observation from prior studies (Nishihara et al., 1986; Quill and
Hedrick, 1996) (Figure 20).
75
pH Effects on CGL-ligand Binding
Since a previous report indicated that acidic pH can irreversibly affect CGLligand binding, xlCGL was pre-treated in buffers that ranged in pH from 3-9 (overnight
incubation at 37ºC) and then tested for its binding activity using the optimized binding
buffer during the CGL-jelly incubation step which has a pH of 7.5 (Figure 22). In
addition, the pH pre-treatments of CGL were performed with and without calcium since
pilot experiments provided evidence that calcium may prevent the loss of binding
activity. The binding assays showed that xlCGL’s binding activity was the highest for
the pH 7.5 tris buffer as expected, and that pre-treatment in acidic pH buffers did indeed
cause a dramatic loss of binding (binding decrease: pH 6=63%, pH 5=84%, pH 4=97%,
pH 3= 85%). Interestingly, calcium was able to protect the binding activity of CGL
when included in the pH 6 and 5 treatments (binding decrease: pH 6=11%, pH 5=34%).
Alkaline pH treatments did cause some reduction of CGL binding activity but not to the
same degree as acidic pH treatments. Calcium also appeared to protect the binding
activity in alkaline treatments.
Glycosidase Removal of CGL Oligosaccharides and Binding Effects
Glycosidases are enzymes that cleave glycosidic bonds at very specific locations
and can be used as tools to remove selected oligosaccharide components found on
glycoproteins. As mentioned in the introduction, most glycosidases have catalytic
76
activity usually in the acidic range which would be problematic for CGL binding as
evidenced by the experiments above. Thus, glycosidases were chosen for selective
cleavage of CGL N-linked oligosaccharides that could be used at a neutral pH even
though it would be outside their optimum range but added in higher amounts to increase
catalysis (as recommended by the manufacturers). The use of selected glycosidases
would then allow an assessment as to the relative importance of particular N-linked
oligosaccharides on the binding activity of CGL to its ligand. The glycosidase PNGase
F was chosen since it cleaves all N-linked chains between the innermost Nacetylglucosamine and asparagine amino acid residue which in effect deglycosylates
CGL. In addition, two mannosidases and two sialidases were also chosen and these are
specific for cleaving at the following glycosidic bonds: α1-6 mannose, α1-2,3 mannose,
α2-3 sialic acid, and α2-3,6,8 sialic acid (Figure 23). The second sialidase was chosen
since it cleaves a broader range of sialic acid glycosidic bonds whereas the α2-3
sialidase was a more specific enzyme (> 500 fold preference for α2-3 linkages over α26 and α2-8 linkages).
After treatment of xlCGL with the glycosidases, half of the reaction mixture was
used for a western blotting analysis to assess whether a change in mass could be
detected (Figure 25). The blot did show a change in the electrophoretic pattern of CGL
for the various glycosidase treatments. In the case of PNGase F, the heterogenous
banding pattern normally observed for native xlCGL shifted to a predominant band at
around 35kD which is the reported polypeptide size (Chang et al, 2004). Although the
77
specific glycosidases did not show dramatic mass shifts, the electrophoretic pattern did
show changes in CGL band prevalence providing evidence of catalytic activity.
The remainder of the glycosidase treated xlCGL preparations were then tested
for binding activity using ELLA, and were found to reduce binding activity
significantly (Figure 24). Removal of all N-linked oligosaccharides with PNGase F had
the largest effect on binding as expected, 91% reduction as compared to the untreated
control. For specific glycosidase treatments, the sialidase specific for α2-3 sialic acid
had the next largest effect on binding with an 86% reduction. Interestingly, removal of
α2-3,6,8 sialic acid decreased binding by 68%, which is 18% less than just the removal
of α2-3 sialic acid. This suggests that α2-3 sialic acid is likely a critical residue for
binding since this cleavage is at the terminal end of the oligosaccharide chain.
Mannosidase treatment of xlCGL also reduced binding significantly but not as much as
the sialidases when used alone. However, combined treatment with the two
mannosidases had an 86% reduction in binding which was comparable to the loss of
sialic acid. Heat inactivation of xlCGL without any enzyme reduced binding by 81%.
The two-way ANOVA (without replication) found significant block effects
(F(2,14) = 5.404, P value =0.018), but more importantly significant differences among
glycosidases (F(7,14) = 18.29, P value < 0.001).
78
Competitive Inhibition using Sialic Acid Analogs
In addition to removing sialic acids by glycosidase treatment, another
experiment assessed the importance of sialic acid to the CGL-ligand reaction using a
different strategy. The sialic acid analog N-acetylneuraminic acid, purified from bovine
milk, was used to test whether it could competitively inhibit binding of CGL to its jelly
ligand (Figure 26). The sialic acid analog convincingly demonstrated an inhibition of
CGL binding with maximum inhibition at around 70-80% at a concentration near 2mM.
The kinetics of inhibition indicate that 50% inhibition was achieved at approximately
1.5mM and was established within a tight window. Increasing the concentration to
150mM did not increase the binding inhibition.
Binding of yCGL to the Jelly Ligand
Lastly, the binding properties of yCGL (as compared to xlCGL) were evaluated
(Figure 27). In a single 96 well plate, xlCGL and four different samples of yCGL were
tested for their binding properties. In addition to performing the ELLA with jelly
coating the wells, Concanavalin A (Con A; a plant lectin that binds to mannose
residues) was bound to the wells to determine if yCGL and xlCGL could bind followed
by regular antibody detection. This was done to confirm that yCGL does indeed have
the mannose chains added by the yeast when secreted.
As another control, yCGL and
xlCGL were bound directly to the wells for direct antibody detection to confirm the
ability to detect yCGL using the antibodies.
79
As noted earlier, the yCGL preparations were not purified to homogeneity,
which presented some limitations, but did result in data that was very informative as to
the ability of yCGL to bind to the jelly ligand (Figure 27). Firstly, figure 27
demonstrates that yCGL is able to bind to both ConA and the jelly ligand, which
confirms that the yCGL is glycosylated, and that the CGL-ligand binding reaction is not
solely dependent on sialic acid residues. However, the yCGL preparations do
demonstrate a significant inconsistency in that they show different binding affinities for
the solid phase, even though they all contain the same amount of protein, as determined
by Bradford assays. This is likely a consequence of the sample not being purified to
homogeneity. While they all have the same total amount of protein, they probably
differ in the either the quality (degraded) or amount of yCGL. However, they do
display similar patterns of relative binding ratios. For instance, yCGL samples #1 and
#2 both bound to the jelly ligand and ConA with equal affinity. In contrast, xlCGL
showed a higher affinity for the jelly ligand than ConA. The yCGL samples likely
contain other secreted proteins in the expression supernatant, however, it does seem
plausible to assume that the majority of the protein is the actual yCGL, particularly
since analysis by SDS-PAGE (Figure 10, lane 6) shows that yCGL is the most
prominent band.
80
Figure 21. Monosaccharide inhibition of the xlCGL ELLA. xlCGL was incubated with the
galactose (black curve) or glucose (gray curve) over a concentration range of 2.5 – 320mM.
Each point represents the average of triplicate assays, with error bars representing the
standard deviation.
81
Figure 22. pH and calcium effect on xlCGL binding activity. The effect of incubating
xlCGL in different pH buffers on binding to jelly ligand was evaluated in the presence or
absence of calcium using the standard ELLA assay. Assay buffer consisted of 50mM
Citrate (pH 3-6) or 50mM Tris (pH 7.5 – 9). After incubation in buffers (37ºC for
~15hours), xlCGL was assayed in the normal pH 7.5 buffer by ELLA to determine the
relative decrease in binding. Each treatment was performed in triplicate (error bars
represent standard deviation).
82
Figure 23. N-glycan variation and glycosidase cleavage sites. The top image represents
a potential simple mannose N-glycan found in the yeast Pichia pastoris; bottom left
image is an example of a complex N-glycan found in vertebrates; and bottom right
image depicts a hybrid N-glycan found in vertebrates. Hyperglycosylation in Pichia
pastoris is demonstrated by the extension commonly found on the α1-6 mannan
backbone. “Asn” is the asparagine residue in the N-glycosylation consensus sequence
of the protein backbone. Purple diamonds represent sialic acid. Green circles represent
mannose. Blue squares represent N-Acetylglucosamine. Yellow circles represent
glucose. Scissors represent potential locations where glycosidases can cleave.
Exoglycosidases can only cleave terminal residues. For example, the α1-6 mannose
linkage (green circle) in the bottom right diagram can only be cleaved after the α1-2 and
α1-3 linkages have been cleaved.
83
Figure 24. Reduction in binding activity of glycosidase treated xlCGL. Enzymelinked lectin assay demonstrating the % reduction in binding activity of glycosidase
treatment on the ability of xlCGL to bind to its ligand. xlCGL was incubated with
respective enzyme under non-denaturing conditions at 37ºC for ~15hr. Half of reaction
(5µl) was diluted in ELLA washing/binding buffer, and was then assayed by ELLA to
determine the relative decrease in binding. Data points reported are mean values of three
independent reactions, with each reaction measured in triplicate. “Manno” =
mannosidase. There were significant differences among glycosidases (- F(7,14) = 18.29,
P < 0.001).
84
Figure 25. Immunoblotting analysis of glycosidase treated xlCGL. For lanes 2-9, half
of glycosidase reaction (5µl, 0.397µg xlCGL) were loaded into a 10% gel. The other
half of the reaction was used for ELLA analysis (Figure 24). Lane 10 was loaded with
PNGaseF treated yCGL (to remove N-linked sugars), whereas lane 9 was loaded with
PNGaseF treated xlCGL for comparison. Lane 1 was loaded with a molecular weight
size standard. “manno” = mannosidase and “sial” = sialidase.
85
A)
B)
Figure 26. Competitive inhibition of xlCGL with sialic acid analog. xlCGL was
incubated with the sialic acid analog over a concentration range of 0.1 – 40mM (Data points
beyond 21mM not shown). Each point represents the average of triplicate assays, with error
bars representing the standard deviation. A) This chart represents one assay done in
triplicate. B) Assay in A overlapped with three additional independent assays.
~75%
inhibition was maintained until the highest concentration tested at 150mM (1st batch, 1st
run, data points not shown). Two separate batches of sialic analog were tested.
86
Figure 27. Comparison of yCGL and xlCGL binding activity. Enzyme-linked lectin
assay comparing the binding properties of xlCGL and yCGL. xlCGL and four different
samples of yCGL were bound to the jelly ligand and Concanavalin A, which are
different solid phase components. The third binding experiment used the CGL as the
solid phase component, which allowed the CGL to bind directly to the surface of the
Immunolon plates. “AS” means the yeast CGL were in 15% ammonium sulfate
solution. “Super” refers to expression supernatant. yCGL #1, 2, and 4 are from the
same expression. An absorbance of 1.3 – 1.5 is considered the maximum value for
which TMB blue is quantifiable. Above this threshold is considered inaccurate due to
limitations in instrumentation. Each point represents the average of triplicate assays,
with error bars representing the standard deviation.
87
DISCUSSION
CGL is an oligomeric metallo-glycoprotein that has the highest affinity for
terminal galactose residues which requires Ca2+ for binding to its ligand. The goal of
the present study was to examine whether the N-linked glycans found on xlCGL play an
important role in the binding activity. Both complex and high mannose type N-linked
oligosaccharides were found to exist on xlCGL, and the complex type was discovered to
have terminal sialic acid residues, a negatively charged sugar. Given that sialic acids
are often involved in receptor-mediated interactions, it was hypothesized that the
complex N-glycans that contain sialic acid are important for the binding activity of
xlCGL to its jelly ligand.
Several different strategies were utilized to test this hypothesis and were chosen
due to preliminary data that indicated that xlCGL binding activity was irreversibly lost
when incubated in non-neutral buffers. The first strategy was to evaluate the binding
properties of a recombinant form of xlCGL that did not contain complex N-linked
glycans, but rather only the high mannose type. This was accomplished by inserting the
xlCGL cDNA into a yeast expression vector (pPICZα) and integrating it into the
methylotroph Pichia pastoris for methanol-induced secretion. The second strategy was
to employ glycosidases (PNGaseF, sialidases and mannosidases) that could function at
neutral pH to enzymatically modify the native xlCGL N-linked oligosaccharides and
evaluate their effect on binding. A third strategy was to competitively challenge the
xlCGL with a sialic acid analog to test whether it could inhibit binding to its jelly
88
ligand. The combination of these strategies provided a multipronged approach to
determine whether the xlCGL N-glycans are important for ligand binding.
As for the first strategy, Pichia expression of CGL (yCGL) turned out to be very
problematic, to say the least. After evaluating DNA sequence information, it was
discovered that the Pichia clones being utilized for secretion had integrated a vector
containing a mutated stop signal at the end of the xlCGL cDNA which added an
additional 21 amino acids onto the length of the protein. When the peptide was
analyzed, it consisted of a high proportion of basic amino acids causing the isoelectric
point (pI) of the xlCGL polypeptide to increase from 5.3 to 6.6 in yCGL. After
searching the sequence databases and determining that the additional peptide was not
related to any known sequence, the project to express the recombinant yCGL was
continued. It is plausible that this peptide did cause some of the expression problems
that will be discussed, but solubility and denaturing issues have historically been a
problem with xlCGL. According to T.R. Peavy, after routine purifications of xlCGL or
concentration by ultrafiltration, a proportion of xlCGL would often be lost due to
precipitation that could be visually observed.
Although it would have been preferable to re-clone the correctly oriented xlCGL
cDNA into Pichia, this would have been a considerable set-back to completing the
Master’s project. In addition, many expression proteins are engineered with C-terminal
epitopes as fusion proteins that do not interfere with its functional activity. Further
analyses could be performed to test this assumption such as purchasing a synthetic
peptide with this same sequence and testing whether it inhibits xlCGL binding to its
89
ligand (i.e. competitive inhibition studies). However, this type of experiment would not
be able to address the possibility that the peptide caused inherent structural problems for
yCGL and instability issues.
With regards to stability, yCGL was notably susceptible to forming insoluble
precipitates after it was expressed in Pichia when attempting to concentrate and/or
purify yCGL. After optimizing the timing of expression (48 hours), temperature (29ºC),
and aeration (baffled flasks), it was estimated that yCGL was being expressed in
concentrations in the 1 mg/mL range, which is a typical yield for Pichia expression.
Unfortunately, the phosphate expression buffer (BMMY) used for yCGL secretion was
not compatible with further purification strategies and included such additives such as
methanol, yeast nitrogen base, peptides and amino acids used for yeast nutrients which
needed to be removed. Membrane filtration techniques, dialysis, and lyophilization
techniques all resulted in huge losses of yCGL and were time consuming. Ammonium
sulfate salt precipitation of yCGL was the most successful, but yields were still not very
high. After using the 1mg/mL expression media for salt precipitation, it took about 22
mLs to yield a 1mg/mL enriched yCGL fraction (~20 fold loss). However, since
expression could be done in liter quantities, the loss was deemed acceptable enough to
continue with purification strategies.
As to the cause of yCGL instability, expression in a phosphate buffer may have
been at the root of the solubility problem since yCGL is likely to have bound calcium
just as xlCGL does and potentially precipitated during the process of concentration. It
is well known that phosphate-based buffers can precipitate with calcium since they can
90
form salts such as calcium phosphate. Calcium was certainly present in the expression
medium since yeast nitrogen base was added as a nutrient and includes salts such as
calcium chloride and calcium pantothenate, albeit at low levels. Filtration techniques
work by concentrating proteins at an interface of a membrane in minimal media
(phosphate buffer initially), which may have led to precipitation.
In searching the literature and discussing this issue with the manufacturers of the
Pichia expression system, only one other buffer system was presented as an option to
use that is compatible with Pichia methanol expression and this was a Minimal
Methanol Medium. This media did not use the phosphate buffer but consisted solely of
YNB, biotin, methanol and water. Unfortunately, this media has a pH of ~5 which was
likely to cause yCGL to irreversibly lose binding activity. Even so, one attempt was
made to use this expression media but no increase in yield and purity was obtained so it
was abandoned. As an alternative to concentrating the phosphate expression
supernatant by filtration, dialysis into ammonium bicarbonate followed by
lyophilization to a powder was attempted. Interestingly, this strategy seemed to still
cause insoluble precipitates and did not improve the yield of yCGL for unknown
reasons.
It is essential to highlight that solubility issues are a common problem when
expressing foreign proteins/glycoproteins in all model systems. For instance, it is
widely documented that inclusion bodies (insoluble complexes) often form when
attempting to express proteins in the bacteria E. coli. The pharmaceutical industry
experiences this problem on a much larger scale with respect to purification and long
91
term stability for delivery of its final product. It has been stated that, “Protein stability
remains one of the most difficult problems in protein science” and that “predicting the
stability of protein mutants remains one of the great unsolved problems of protein
science, proving itself more difficult than the prediction of protein structure or even the
design of fairly efficient enzymes.” (Magliery et al., 2011). It is well known that the
folding of proteins/glycoproteins are influenced not only by their own chemical
constituents, but also by the solution they are suspended in.
Ironically, glycosylation has been reported to improve the stability of many
expressed proteins by preventing proteolytic degradation, oxidation, chemical cross
linking, pH denaturation, chemical denaturation, heating and freezing denaturation,
precipitation, and aggregation (Sola and Griebenow, 2009). In the case of yCGL, this
does not appear to be the case. It has been shown that solubility is minimal near the
isoelectric point of a protein, but increases as the net charge increases at pH values
acidic or alkaline relative to the isoelectric point (Trevino, 2007). The isoelectric point
is the pH value where a protein’s positive and negative charges balance each other, and
the protein bears a net charge of zero (Trevino, 2007). The terminal ends of glycans
are often further functionalized with chemically charged groups (e.g., phosphates,
sulfates, carboxylic acids) that contribute to the stability and solubility of the molecule
(Sola and Griebenow, 2009). For yCGL, the loss of the complex N-glycans may have
adversely affected its solubility since sialic acid would certainly have contributed a
negative charge to the molecule.
92
Protein degradation of yCGL was also a persistent problem during expression
studies. As mentioned, glycosylation is known to protect the protein backbone from
proteases which is often by not allowing access to cleavage sites through steric
hindrance (Sola and Griebenow, 2009).
It is plausible that the loss of complex N-
glycans increased the susceptibility of yCGL to proteases. However, it is also possible
the xlCGL would have succumbed to proteolytic degradation if incubated in the same
expression environment. Even though protease digestion during expression was
minimized by the addition of protease inhibitors, concentration and purification
techniques were often plagued by protein degradation.
Another anomaly that was observed was the apparent size of yCGL by SDSPAGE. Many researchers commonly use SDS-PAGE to estimate the size of target
proteins/glycoproteins due to the ease of comparison to a set of molecular weight
protein standards. However, this technique completely depends on the ability of test
compounds and size standards to adopt the same shapes after SDS denaturing, ideally as
polypeptide “rods” uniformly coated with SDS (micellar-type structure). SDS-PAGE
size anomalies have been noted for a variety of proteins/glycoproteins and has been
traced to the stoichiometry of SDS which changes the charge to mass ratio (Rath et al.,
2009). In essence, less SDS has been found to coat sterically hindered regions of
glycoproteins (glycosylation sites) and hydrophobic helical regions (i.e. membrane
proteins) and cause them electrophoretically to migrate more slowly.
As for yCGL, western blot analyses revealed that yCGL had an apparent size of
~65kD, which is about 20kD larger than that of xlCGL (45kD). The 21 amino acid tail
93
found on yCGL does not appear to account for this size difference since the mass it
should contribute is 2.4 kD and it is hydrophilic in nature (ExPASy Compute pI/Mw)
Thus, the high mannose type N-glycans that yeast are known to add are likely to have
disrupted the ability of SDS to coat the molecule uniformly. Most informatively,
western blot analyses of PNGaseF treated yCGL and xlCGL revealed electrophoretic
sizes very close to one another (~35kD), which supports the hypothesis that the Nglycans were the issue.
Another anomaly of yCGL was that it did not electrophoretically transfer well to
nitrocellulose membranes unless a more neutral gel system (bis-tris buffers) was used.
Although the exact mechanism by which macromolecules bind to nitrocellulose is not
well understood, both electrostatic and hydrophobic properties are thought to play a
role. In this case, the 21 amino acid tail on yCGL might have significantly altered the
transfer conditions since it imparts a significant positive charge to the C-terminus as
mentioned previously. However, it is also possible that the high mannose N-glycans
also altered its transfer properties, especially if hyperglycosylation was involved.
Hyperglycosylation is common in certain strains of yeast that add variable amounts of
mannose sugars during N-glycan modifications, although this was reported by the
manufacturer to be less of a problem in Pichia. Hyperglycosylation is characterized by
a smeared banding pattern when analyzed by SDS-PAGE, however yCGL does not
appear to have much smearing of the 65kD band. It should be noted that the smeared
banding pattern that xlCGL exhibited by SDS-PAGE is due to differential glycosylation
rather than hyperglycosylation. Regardless, when the western blotting conditions were
94
performed at neutral pH, yCGL and xlCGL both transferred well for subsequent
antibody detection.
As for the CGL-ligand binding studies, the ELLA method developed in this
study confirmed previous studies that xlCGL bound to the jelly ligand in a galactose
specific and calcium-dependent manner. In addition, this particular ELLA using the
deglycosylated xlCGL rabbit antibodies was shown to be a very sensitive assay, using
only 9.28ng CGL per well for detection whereas prior CGL ELLA assays used
biotinylated xlCGL with amounts of 75ng (Kiedrowski, 2010) and 2500ng (Quill and
Hedrick, 1996) per well. Detection using biotinylated CGL needed to be altered for this
study since the yeast CGL and other xlCGL treatments (i.e. glycosidase treated) would
have needed to be biotinylated and would not have been directly comparable. The
increased sensitivity of the ELLA used in this study is likely due to detection
amplification achieved by using a biotinylated secondary antibody that reacted to the
purified IgG fraction of the deglycosylated xlCGL, followed by a streptavidinhorseradish peroxidase conjugate. Interestingly, PVA turned out to be a more effective
blocking agent in this assay which may be due to the specific type of plate material
being used (Immunolon) and/or the detection methodology.
As for the kinetics of competitive inhibition, xlCGL binding to jelly was
decreased by 50% when using 5mM galactose, whereas prior studies have reported
about 10mM galactose for this same level of inhibition (Kiedrowski, 2010; Quill and
Hedrick, 1996). This 2-fold difference may be due to differences in sensitivity of the
techniques as aforementioned (biotinylated CGL vs antibodies), nonetheless it is
95
relatively close. Another kinetic difference noticed between these same studies is that
prior studies were able to completely inhibit CGL binding at 75mM (Kiedrowski, 2010)
and 150mM galactose (Quill and Hedrick, 1996), whereas this study was only able to
achieve 95% and 97% inhibition at 150mM and 320mM galactose, respectively. This
difference may again be due to the increased detection sensitivity noted for the current
assay which could be detecting binding of xlCGL to other secondary sugars such as
glucose that could not be removed by galactose competition. However, this study was
able to achieve 100% inhibition when using EGTA providing evidence this residual
xlCGL binding was not due to non-specific binding.
Reduced binding activity of xlCGL was also observed when incubated in buffers
above or below pH 7.5. This loss of binding activity appeared to be irreversible since
binding assays were performed in the normal binding buffer (10mM Tris, 150mM
NaCl, 10mM CaCl2, pH 7.5) after incubations. Interestingly, even the pH 6 buffer
caused a dramatic loss of binding (63% decrease from control) indicating that xlCGL
was prone to denaturing irreversibly in sub-optimal pH buffers. This data suggests that
xlCGL is particularly susceptible to pH effects on its internal electrostatic forces and
charge–charge interactions. It has been shown that pH values far enough from a
protein’s isoelectric point can cause unfolding due to electrostatic repulsions between
similarly charged atoms (Sola and Griebenow, 2009). Additionally, sub-optimal pH
can cause a reduced capability for salt bridge formation between oppositely charged
atoms and lead to unfolding (Sola and Griebenow, 2009).
In the case of xlCGL,
structural unfolding appears to have been unrecoverable with respect to binding activity
96
in these sub-optimal pH buffers. However, the addition of calcium to the mildly acidic
(pH 5 and 6) and alkaline (pH 8 and 9) buffers appeared to protect xlCGL from
denaturation (e.g. pH 6 with Ca2+ only had 11% loss of binding), but not so in the more
acidic buffers. Thus, it is likely that calcium stabilizes xlCGL into its binding
conformation and is not easily disrupted.
As for treatment with glycosidases, xlCGL binding to jelly was nearly abolished
with the loss of N-linked glycans (i.e. 91% with PNGaseF treatment). The immunoblot
of PNGaseF treated xlCGL indicates a shift of the heterogeneous banding pattern
(45kD) to the deglycosylated 35kD band suggesting that the reaction went to
completion, so it is appears that the xlCGL polypeptide alone can bind to the jelly
ligand but at about a 10-fold reduction. Thus, although N-glycans do not appear to be
absolutely required for binding, the relative binding affinity to its ligand would be
reduced at least 10 fold which could make a huge difference biologically.
Further studies treating xlCGL with exoglycosidases also demonstrated dramatic
loss of binding. In particular, the α2-3 sialidase (also termed neuraminidase) used
resulted in an 86% reduction in binding which was similar to the decrease produced by
PNGaseF. Since this enzyme only removes the α2-3 linked sialic acid from the terminal
end of the oligosaccharide (meaning only one cleavage per chain possible), this
indicates a primary role in binding for this sugar residue. Combined treatment of the
α2-3 sialidase with α2-3,6,8 sialidase (removes additional linkages) did not further
decrease binding which further indicated that the α2-3 sialic acid was the critical
linkage type.
97
Furthermore, the competitive inhibition studies using the sialic acid analog, Nacetylneuraminic acid, established that sialic acid plays a direct role in binding activity.
This type of competitive inhibition experiment differs from glycosidase treatments since
the analog should not be modifying the structural content of xlCGL. Glycosidases such
as sialidases cleave the terminal sialic acid residues which could cause a structural
alteration of the xlCGL binding conformation due to the loss of this negatively charged
sugar. However, on the other hand, the sialic acid analog should simply be interfering
with binding by competing for important sites involved in the lectin-ligand interaction
which likely involves an electrostatic charge. The analog convincing inhibited xlCGL
binding with maximal inhibition achieved around 2mM at 80% inhibition. Thus, this
analog disrupted some xlCGL interaction that lead to a dramatic decrease in binding.
This interaction could be actually in the binding pocket itself or with the divalent cation
calcium. Disruption of either is core to xlCGL binding activity, so it can be concluded
that sialic acid residues are important to achieve a high affinity interaction with its
ligand.
As for the importance of mannose residues, the mannosidases used in the study,
α1-2,3 mannosidase and α1-6 mannosidase, reduced xlCGL binding by 86% when both
enzymes were used in combination. These enzymes would cleave mannose residues in
the α1-2, α1-3 and α1-6 linkages but only when found on the terminal ends of
oligosaccharides. This means that mannose residues could be trimmed off one-by-one
from the terminal end of each glycan chain until a linkage other than those defined
above is encountered. These mannose structures have the potential to be found on the
98
high mannose and hybrid type N-glycans (refer to Figure 23). It is possible that after
digestion, only the trisaccharide core linked to the asparagine linkage would be left on
these types of chains, but complex type chains would be unaffected (which includes
sialic acid). Thus, mannose residues do appear to play an important role in binding.
However, it is unclear from these experiments whether the importance of mannose
residues is related to the ability of xlCGL to adopt a binding conformation, or rather
they participate directly in the binding interaction. Significant losses of sugars in
general from the N-glycans would likely cause conformational changes, which could
affect access to the binding pocket.
The binding experiments using partially purified yCGL also shed some light on
the role of mannose residues. Even though the yCGL clone was imperfect and
purification was not homogeneous, the binding studies using yCGL demonstrated that it
could indeed bind to the jelly ligand. However, since 750ng of this partially purified
yCGL were used in the binding assay as compared to 10ng of xlCGL, it suggests that
there was a dramatic decrease in yCGL’s relative affinity to the ligand. Further
evidence to this effect is provided by the ratio of jelly ligand to ConA binding for both
yCGL and xlCGL. It is known that one of the xlCGL N-linked sites has high mannose
type glycans whereas the other 2-3 sites have complex/hybrid types when it was
analyzed by mass spectrometry (An et al., 2003). Thus, ConA should bind to the
mannose residues found on each xlCGL subunit. It was noted that the xlCGL ratio of
jelly ligand to ConA binding of xlCGL was about 2:1, whereas yCGL was 1:1. This
again indicates a reduced capability of yCGL to bind the jelly ligand.
99
Interestingly, yCGL did not seem capable of forming large oligomeric
complexes as evidenced by the size exclusion data. When calcium was added to the
xlCGL preparation, larger oligomeric complexes were observed whereas yCGL
remained as a lower molecular weight complex assumed to the monomeric subunit.
Thus, since calcium is required for ligand binding activity and for subunit organization
into larger oligomeric forms (10-12mers), the oligomeric conformation is important for
high affinity binding to its ligand. The reduced ligand binding activity of yCGL may be
a result of the inability to form larger oligomeric structures. This suggests that yCGL
may not bind calcium as well as xlCGL which may be due to the loss of sialic acids
from complex/hybrid N-glycans. This would need further testing to confirm this
hypothesis.
However, what is clear from the literature is that carbohydrate–protein
interactions (lectin-ligand) are much weaker than protein–protein interactions, by about
a factor of 102–103 from typical antibody equilibrium dissociation constants (Zeng et
al., 2012). In many instances, this limitation has been overcome through the use of
multivalent interactions, which is defined as the simultaneous contact between clustered
carbohydrates and protein receptors that contain multiple carbohydrate recognition
domains (Zeng et al., 2012). In other words, some lectins have more than one
carbohydrate recognition domain on a subunit whereas other lectins have adopted a
strategy of forming oligomeric structures to cluster binding sites. Since the strength of
one lectin-ligand binding site is relatively weak, clustering many of them together
makes the overall strength of interaction much greater. An analogy for this is Velcro
100
where one single contact is weak but the combined interactions of a Velcro sheet
generates a strong interaction. In the case of lectins with more than one carbohydrate
recognition domain, it has been shown that the occurrence of two simultaneous binding
events can increase the avidity of interaction by more than 100-fold (Zeng et al., 2012).
Since xlCGL is found as oligomer of 10-12 subunits (450kD in size), it can
easily be envisioned that the inability of subunits to adopt the larger oligomer would
dramatically affect its binding affinity. As mentioned, yCGL may have lost this ability
due to not having sialic acid residues, but it could also be due to the introduction of the
21 amino acid C-terminus tail. Regardless, the end result is that yCGL can bind to the
ligand but it does not form these larger oligomers.
In summary, this study has provided novel information as to the biological role
of the N-glycans found on xlCGL. The α2-3 linked sialic acid residues found on the
complex/hybrid N-glycans are key players in ligand binding and may be related to
calcium coordination which stabilizes xlCGL into a binding conformation. The
mannose residues found on high mannose and hybrid N-glycans appear to play an
important supportive role for binding so as to facilitate access to the binding site. These
experiments have enhanced our knowledge of the CGL-ligand binding mechanism, and
are likely to be applicable to the binding interaction of other eglectin family members.
101
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