ISOLATION AND QUANTIFICATION OF THE CORTICAL GRANULE LECTIN
LIGAND OLIGOSACCHARIDES AND ELUCIDATION OF THEIR ROLE IN THE
BLOCK TO POLYSPERMIC FERTILIZATION IN XENOPUS LAEVIS
Noah Peter Kiedrowski
B.S., California State University, Sacramento, 2007
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
2010
© 2010
Noah Peter Kiedrowski
ALL RIGHTS RESERVED
ii
ISOLATION AND QUANTIFICATION OF THE CORTICAL GRANULE LECTIN
LIGAND OLIGOSACCHARIDES AND ELUCIDATION OF THEIR ROLE IN THE
BLOCK TO POLYSPERMIC FERTILIZATION IN XENOPUS LAEVIS
A Thesis
by
Noah Peter Kiedrowski
Approved by:
__________________________________, Committee Chair
Dr. Tom Peavy
__________________________________, Second Reader
Dr. Roy Dixon
__________________________________, Third Reader
Dr. Tom Landerholm
iii
Student: Noah Peter Kiedrowski
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
Dr. James W. Baxter
Department of Biological Sciences
iv
_________________
Date
Abstract
of
ISOLATION AND QUANTIFICATION OF THE CORTICAL GRANULE LECTIN
LIGAND OLIGOSACCHARIDES AND ELUCIDATION OF THEIR ROLE IN THE
BLOCK TO POLYSPERMIC FERTILIZATION IN XENOPUS LAEVIS
by
Noah Peter Kiedrowski
The block to polyspermic fertilization in Xenopus laevis is mediated by a
calcium-dependent, galactose specific binding reaction between a lectin derived from
the cortical granules and its ligand partners located in the immediate surroundings
within the egg extracellular matrix. The cortical granule lectin (CGL) ligands have been
shown to possess O-linked oligosaccharides as the functional moieties when binding to
the CGL.
However, it is unknown as to which particular ligand oligosaccharides are
the functional moieties in the binding interaction. Elucidation of the functional
oligosaccharides will be valuable to our understanding of fertilization and cell-cell
binding interactions involving this lectin family of CGL-like proteins.
In the current work, an HPLC based method was developed to profile the
oligosaccharides found on the ligands and to isolate and quantify them for subsequent
functional binding assays. A novel method was developed without derivatization agents
or exact standards utilizing the HPLC-CAD (Charged Aerosol Detection) system
v
interfaced with an amino Prevail Carbohydrate ES HPLC column. The hypothesis for
this study was that the HPLC-CAD methodology will allow the isolation and
quantification of O-linked oligosaccharides released from the CGL ligand which can
then be utilized in functional binding assays to assess which oligosaccharides function
in the lectin-ligand binding interaction during the X. laevis block to polyspermy.
In testing the methodology, it was demonstrated to be sensitive in the picomolar
range or mass detection limits 0.3 to 0.9 ng with regard to oligosaccharides separated
under optimized conditions. Commercially available standard oligosaccharides were
accurately quantified to within less than a 19% average error with excellent
reproducibility. The X. laevis CGL ligand oligosaccharides were chemically released
and profiled using the HPLC-CAD demonstrating the presence of four predominate
oligosaccharide peaks. Oligosaccharides were subsequently separated, isolated and
quantified utilizing the developed quantification methodology employing the Prevail
Carbohydrate ES HPLC column.
A plate binding assay was developed (enzyme-linked lectin assay) to test
whether particular compounds could competitively inhibit the binding interaction of
purified CGL and ligands thereby implicating a functional role in binding. The data
indicated that binding could be inhibited by galactose, fucose and the galactose
containing disaccharides lactose and melibiose. In addition, it was found that whole
vi
CGL ligand oligosaccharide fractions possessed strong inhibition properties. The
neutral CGL ligand oligosaccharide fraction demonstrated strong lectin-ligand
inhibition characteristics eliciting a 95% reduction in binding, whereas the acidic CGL
ligand fraction elicited only a 35% reduction. However, when the isolated and
quantified CGL ligand oligosaccharide fractions were tested at concentrations of 10 and
45 µM, they were unable to elicit an inhibitory response of the lectin-ligand binding
interaction. Based on these observations, the evidence suggests that there are
oligosaccharides that do inhibit lectin-ligand binding that are derived from the ligands,
but that the particular purified oligosaccharides tested were not effective at inhibiting
binding at the concentrations used. Future studies will need to test these compounds at
higher concentrations and to test other oligosaccharide fractions to elucidate which
oligosaccharides are the functional binding moieties on the ligand.
___________________________, Committee Chair
Dr. Tom Peavy
___________________________
Date
vii
ACKNOWLEDGEMENTS
Dr. Tom Peavy
I would like to give a special thanks to Dr. Tom Peavy for recruiting me into the
graduate program and accepting to serve as my thesis advisor and mentor. I would like
to thank him for introducing me into the research laboratory along with other graduate
students from the onset of the graduate program and for proposing this specific research
project for my thesis research. Additionally, I would like to thank Dr. Peavy for writing
the grant proposal in collaboration with Dr. Roy Dixon that provided funding and
support for my thesis research. I would like to thank him for graciously providing the
purified cortical granule lectin (CGL) and Xenopus laevis egg jelly for the biological
aspects of my thesis research. Furthermore, I would like to thank him for his
supervisory role and support in my research efforts and lending his expertise in
biological techniques and data interpretation. Lastly, I would to thank him for his time
and assistance in proof reading, meeting and preparing for my advancement to
candidacy, thesis and thesis defense seminar.
Dr. Roy Dixon
I would like to thank Dr. Roy Dixon for his supervisory role and support
throughout my thesis research. Dr. Dixon was undoubtedly instrumental in bringing my
thesis project to fruition, specifically with regard to the initial stages of the research
viii
project. His expertise in analytical chemistry and HPLC based chromatographic
separations was indispensable to the project as a whole. Additionally, his assistance in
trouble-shooting instrumentation, data interpretation and data analysis was essential in
developing the HPLC-CAD oligosaccharide isolation and quantification methodology. I
would like to thank him for his support in attending both CSU Biotechnology
Symposiums in which our HPLC-CAD preliminary data was presented. Lastly, I would
like to thank him for his assistance and time in preparing for both my advancement to
candidacy and thesis defense seminar.
Dr. Tom Landerholm
I would like to thank Dr. Landerholm for accepting to serve as the final member
of my graduate committee and facilitating my advancement to candidacy. Additionally,
I would like to thank him for his advice throughout graduate school and proof reading
my thesis. I would also like to thank Dr. Landerholm for the invitation and pleasure to
be a guest speaker in his undergraduate biology seminar course.
Simon Helminski
I would like to thank my graduate colleague and friend Simon for providing
laboratory support and advice throughout the graduate program and collaboration on
experiments. I would like to thank him for assisting me with SDS-PAGE gel
techniques, initial binding assay parameters and additional biological techniques and
ix
aspects of my thesis research. Additionally, I would like to thank him for lending his
assistance and time on occasion as needed for some of my experimental questions and
interpretation of results.
Monique Bastidas
I would like to thank Monique for assisting me in some of my quantitative
HPLC-CAD work and for collaborating on both CSU Biotechnology Symposium
presentations.
Tom Boyce
I would like to thank Tom Boyce for his contributions with regard to the initial
HPLC-CAD work that was completed.
My Family
I would like to give a special thanks to my family for their unconditional
support, patience and appreciation throughout graduate school and 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 and
throughout my thesis research. They have been a large part of my overall success in
both the laboratory and in the academic setting throughout the graduate program. I
appreciate their tolerance and patience during periods of frustration and setbacks.
x
Lastly, I would like to thank them for assisting me through times of doubt about
graduate school on a whole and for helping me maintain my perseverance and
optimism.
xi
TABLE OF CONTENTS
Page
Acknowledgements.........................................................................................................viii
List of Tables...................................................................................................................xiii
List of Figures................................................................................................................. xiv
INTRODUCTION .............................................................................................................1
MATERIALS AND METHODS .....................................................................................19
Purification of X. laevis CGL Ligands .................................................................19
Release and Purification of X. laevis CGL Ligand Oligosaccharides .................19
HPLC-CAD Methodology Development ............................................................21
Separation, Fractionation and Isolation of CGL Ligand Oligosaccharides .........29
Quantification and Secondary Purification ..........................................................30
Procurement and Preparation of X. laevis Egg Jelly and CGL ............................31
SDS-PAGE Gel Characterization of X. laevis Egg Jelly and CGL .....................32
Competitive Enzyme-Linked Lectin Assays .......................................................34
RESULTS ........................................................................................................................37
DISCUSSION ................................................................................................................104
Literature Cited ..............................................................................................................121
xii
LIST OF TABLES
Page
Table 1. Initial characterization and profiling of CGL ligand oligosaccharides .............58
Table 2. HPLC-CAD profile of X. laevis CGL ligand oligosaccharides. .......................62
Table 3. CGL ligand oligosaccharide fractions with concentrations. .............................72
xiii
LIST OF FIGURES
Page
Figure 1A. Distribution of X. laevis egg jelly layers ........................................................6
Figure 1B. Diagrammatic representation of the frog egg .................................................6
Figure 2. Diagrammatic representation of the HPLC-CAD system ...............................23
Figure 3. Diagrammatic representation of the electrical aerosol size-analyzer. .............26
Figure 4. A Gradient method chromatogram (oligosaccharide standards) .....................38
Figure 5. Gradient calibration plot (oligosaccharide standards). ....................................41
Figure 6. ‘A’ Term calibration plot.................................................................................43
Figure 7. ‘b’ Term calibration plot. ................................................................................44
Figure 8. Quantification of surrogate oligosaccharide standards....................................47
Figure 9. Short chain oligosaccharide structures. ...........................................................49
Figure 10. Quantification of short chain oligosaccharides .............................................52
Figure 11. Biological test oligosaccharide structures. ....................................................54
Figure 12. Quantification of biological test oligosaccharides ........................................56
Figure 13. Prevail chromatogram of X. laevis ligand oligosaccharides. .........................60
Figure 14. Hypercarb chromatogram of X. laevis ligand oligosaccharides ....................61
Figure 15. Prevail chromatogram overlay with oligosaccharide standards. ...................66
Figure 16. Prevail purity and quantification fraction analysis (CAD signal) .................68
Figure 17. Prevail purity and quantification fraction analysis (UV signal). ..................70
Figure 18. X. laevis egg jelly SDS-PAGE gel ................................................................75
xiv
Figure 19. X. laevis native CGL and biotinylated CGL SDS-PAGE gel. .......................78
Figure 20. Reactivity of the enzyme-linked lectin assay ................................................81
Figure 21. Monosaccharide inhibition of the enzyme-linked lectin assay......................84
Figure 22. Monosaccharide inhibition of the enzyme-linked lectin assay......................86
Figure 23. Monosaccharide inhibition of the enzyme-linked lectin assay......................87
Figure 24. Disaccharide inhibition of the enzyme-linked lectin assay ...........................89
Figure 25. Disaccharide inhibition of the enzyme-linked lectin assay. ..........................91
Figure 26. Monosaccharide structures and binding specificity of the CGL ...................94
Figure 27. Disaccharide structures and binding specificity of the CGL. ........................95
Figure 28. Enzyme-linked lectin assay (whole ligand oligosaccharide fractions)..........97
Figure 29. Enzyme-linked lectin assay (isolated ligand oligosaccharide fractions). ....101
Figure 30. Enzyme-linked lectin assay (isolated ligand oligosaccharide fractions). ....103
xv
1
INTRODUCTION
Fertilization is a highly choreographed event that is essential for the
reproductive success of a species so as to produce viable offspring. Fertilization
typically involves the union between a nucleus of a male haploid (N) gamete and a
nucleus of a female haploid (N) gamete to reconstitute the normal diploid (2N)
chromosome number. Species have evolved a highly stringent regulatory process with
regard to sperm selection and entry into the egg to ensure successful fertilization.
Regulation of sperm entry into the egg is essential in disallowing more than one sperm
from penetrating the egg in the majority of species. The penetration of multiple sperm
into the egg results in polyspermic fertilization and subsequent deleterious effects such
as spontaneous abortion, premature death or developmental abnormalities [1]. Polyploid
conditions (i.e. greater than 2N) in human embryos are usually fatal. If more than one
sperm is successful in fertilizing an egg, the abundance of genetic material results in
faulty segregation of chromosomes and the resulting polyploid embryo is aborted. If
survival beyond parturition occurs, then many detrimental polyploid conditions
manifest themselves in which mental retardation along with a host of physical defects
are observed.
To prevent polyploid associated conditions, most species have evolved
molecular and structural mechanisms to ensure the fusion of only one sperm with the
female pronucleus. In nearly all vertebrates, the extracellular matrices surrounding the
2
egg are utilized as an essential defense mechanism in disallowing more than one sperm
from penetrating the egg. These extracellular matrices are composed of glycoproteinrich layers overlying the egg plasma membrane. The innermost layer, termed the zona
pellucida in mammals and the vitelline envelope in most non-mammals (in general, this
innermost layer will be referred to as the egg envelope), participates in a number of
essential functions during fertilization. These functions include sperm-egg binding,
induction of the acrosome reaction and formation of the block to polyspermy [2,3].
In particular, sperm-egg binding occurs between molecules located on the outer
surface of the sperm plasma membrane and outer surface of the egg envelope.
Subsequent to a single sperm entry into the egg, these egg envelope sperm-binding
glycoproteins become structurally modified by a series of events triggered by the
release of secretory vesicles that reside just beneath the egg plasma membrane. These
secretory vesicles, termed cortical granules, release their contents by exocytosis (fusion
with the overlying plasma membrane) and the subsequent interaction of their contents
with the egg envelope constitutes the egg cortical reaction. These cortical granules are
triggered to fuse with the egg plasma membrane. Cortical granule fusion is initiated
near the site of sperm contact, facilitating a wave of calcium release which sweeps
around the egg, followed by a wave of cortical granule fusion [2,3].
Contents of the egg cortical granules vary with species however they typically
include enzymes and structural proteins. The enzymatic contents consist of glycosidases
3
and proteases which remove carbohydrate and cleave proteins respectively and are
targeted to destroy sperm binding receptors and thus the ability of sperm to bind and
penetrate through the egg envelope. Additionally, non-enzymatic components are
released which include a glycoprotein, termed the cortical granule lectin (CGL). Lectins
are broadly classified as sugar-binding proteins, which are highly specific for their
sugar moieties and typically play a role in binding mechanisms involving cells and
proteins. In fertilization, a binding reaction occurs between the cortical granule derived
lectin, CGL, and glycoproteins found on the outer surface of the egg envelope, termed
CGL ligands. When CGL binds to these ligands, they form a large complex which
induces a conformational change in the extracellular matrix, thereby providing a
physical barrier to additional sperm penetration. Collectively, these released cortical
granule contents (enzymes and CGL) result in a “hardening” by means of chemically
modifying the structure of the egg envelope, thereby establishing a permanent block to
polyspermy [2,3,4].
Nearly all of what is currently known about the block to polyspermy in
vertebrate organisms originally were derived from experiments utilizing the model frog
Xenopus laevis and then subsequently were discovered to be conserved in mammals as
well [13]. The South African frog X. laevis has served as an excellent model for
fertilization studies due to its large egg size (1 mm in diameter), ease of obtaining large
quantities of gametes (inducing ovulation via hormone injection and yielding ~3000
eggs) and visibility of the process since fertilization is external (typically in pond
4
water). Much of what has been found in X. laevis is translatable to mammals. With
respect to the mammalian zona pellucida and the X. laevis, vitelline envelope, they both
consist of homologous proteins which perform identical functions during fertilization
[5,6,7]. Additionally, in both mammals and X. laevis these egg envelope protein
homologues originate from the egg itself during oogenesis and follicle maturation [7,8].
Subsequent to ovulation in X. laevis, the egg transits through the oviduct where
mucous-like glycoproteins secreted from the oviduccal tissue are sequentially deposited
around the egg, forming a thick outer jelly coat [7,8]. Mammals are dissimilar in this
respect as they do not possess a thick outer jelly coat layer per se, however the
mammalian egg does consist of oviduct-derived glycoproteins that associate with the
egg and mucous-like secretions derived from follicle cells termed cumulus cells [2,3].
Many functional parallels have been established between X. laevis and mammals with
regard to fertilization, thus much of what we learn about the molecular interactions
involved during fertilization using X. laevis may be informative with regard to
mammalian fertilization.
In X. laevis, three morphologically distinct jelly coat layers, J1, J2 and J3 from
innermost to outermost, are deposited around the egg sequentially as the egg transverses
through the oviduct (Figure 1). These egg jelly coat layers have been demonstrated to
play an essential role in fertilization, whereby removal of the jelly layers resulted in the
inability of sperm to fertilize the egg. Interestingly, this observation was reversible in
5
the presence of solubilized egg jelly [6,7]. This finding has been attributed to
chemotactic molecules, calcium-binding reservoirs and other structurally and/or
functionally important glycoproteins residing in the egg jelly [5,6,14].
6
Figure 1 – A. Distribution of X. laevis egg jelly layers containing glycoproteins
involved in fertilization [18]. B. Diagrammatic representation of the frog egg and the
cortical granules lying beneath the plasma membrane.
7
It has been well documented that the contents of the egg cortical granules, alter
the properties of the jelly coat layers when released by sperm-induced exocytosis,
thereby establishing the functional block to polyspermy [5,6,7,9]. In particular, the CGL
binds to jelly coat ligands at the innermost jelly coat layer, J1, and thereby forms an
electron dense fertilization layer which results in a physical compositional and
conformational change of the extracellular matrix that becomes impenetrable by sperm
[7,10,11]. The CGL ligands are large highly glycosylated extracellular matrix proteins
that are derived from the superior portion of the oviduct and possess an affinity for the
CGL. This fertilization layer leads to “hardening” of the egg as measured by an
increased resistance to physical deformation, proteolysis, thermal dissolution and
solubility of the VE [5,6,7,9].
CGL is the major constituent of the cortical granule exudate, accounting for
77% of the total glycoproteins released [9]. In addition to the CGL, the cortical granule
exudate consists of several other proteins such as proteases and glycosidases which
collectively act to simultaneously produce a permanent block to polyspermy, as
mentioned previously. CGL is a carbohydrate binding glycoprotein that serves in
binding recognition involving cells and proteins. The binding interaction with its jelly
coat ligands has been shown to be specific for terminal galactose residues and also
dependent on the presence of calcium [9,10,11].
8
Further analysis of this specific lectin-ligand glycoprotein binding interaction
has valuable implications since the existence of the X. laevis CGL gene was found to be
present in a variety of vertebrate genomes, including humans. The presence of the CGL
homologue was found to be expressed in the eggs of mice, pigs and rhesus macaque
[13,14]. The human and mouse homologue, termed intelectin, displayed high amino
acid identity (> 60%) relative to the X. laevis CGL polypeptide [15]. Interestingly, invitro fertilization experiments with mice demonstrated further functional homology
when exogenous X. laevis CGL was incubated with mouse eggs prior to the addition of
sperm and it was observed that the CGL prevented sperm entry into the mouse eggs
[13,14]. Additionally, the CGL effect on the unfertilized mouse eggs was found to be
concentration dependent and could be inhibited when galactose and/or galactosecontaining compounds were competitively introduced [13,14].
Furthermore, human and mouse intelectin was found to be expressed not only in
the egg, but in many different tissues. Human intelectin gene expression has been
shown to occur in at least 24 different tissues including; heart, liver, kidney, lung, brain,
prostate, bone marrow, small intestines and notably the ovary [16]. Expression of the
CGL homologue in many human adult tissues suggests that CGL participates in a wide
variety of functions in the fully developed organism in addition to the block to
polyspermy. Expression of the CGL gene in X. laevis has been shown to be present at
other developmental stages in non-ovarian tissues, suggesting a possible role of the
CGL in somatic tissues as well [15].
9
Several other CGL-like proteins have since been identified in organisms as
diverse as ascidians [15]. Since CGL and CGL-like proteins are not significantly
related to any of the other known lectin families, they have been classified as a new
family of lectins termed the eglectins [15]. All of the eglectins found to date appear to
function in cell-cell recognition mechanisms and can be found as both soluble lectins or
as cell-surface receptors. The eglectins possess four significant characteristics; 1)
binding is calcium-dependent; 2) binding is specific for terminal galactose residues; 3)
exist as large oligomers; and 4) are glycosylated proteins [15]. Collectively, this
evidence suggests that the structural and functional properties of eglectins and in
particular CGL are likely to be conserved including their binding specificity.
Although CGL has been purified, cloned and well characterized, little is know
about the molecular identity of its ligand partners [6,9,15,17]. Yet, previous work has
demonstrated that the X. laevis CGL ligands, isolated from the fertilization layer, are
high molecular weight glycoproteins of 450 and 630 kDa [6,11,12]. The amino acid
sequence of these proteins is unknown and thus it is unclear as to how these two ligands
are related. The CGL ligands have been shown to possess O-linked oligosaccharides
which are characterized by the addition of sugars to the hydroxyl (-OH) groups of
selected serine or threonine side chains of the polypeptide backbone. The CGL ligands
have been demonstrated to possess O-linked oligosaccharides with terminal galactose
residues as the functional moieties in binding activity [9,11,12]. Previous research has
demonstrated that under hydrolysis conditions in which β-elimination was employed to
10
cleave O-linked oligosaccharides, ligand function was rendered non-functional [11].
Furthermore, exposure to α-galactosidase (cleavage of terminal α-galactosyl moieties
from glycoproteins) conditions, ligand binding capabilities and its affinity for its lectin
partner were rendered ineffective, resulting in a 75% decrease in binding association
[11].
In addition, monosaccharide competitive inhibition analysis of the lectin-ligand
binding reaction utilizing an enzyme-linked lectin assay (ELLA) demonstrated that
galactose was able to inhibit the binding interaction, while no significant inhibition was
observed with mannose [11,12]. Competitive inhibition studies utilizing galactosecontaining disaccharides such as lactose and melibiose exhibited complete inhibition of
the lectin-ligand binding reaction as well [9,13]. These data provide strong evidence
that terminal galactose residues may be the essential terminal carbohydrate component
that is recognized by the CGL and that the binding interaction is dependent on ligand Olinked oligosaccharide moieties.
O-linked oligosaccharides are distributed throughout the X. laevis egg
extracellular matrix as well as the three distinct jelly coat layers; J1, J2 and J3 from
innermost to outermost, respectively (Figure 1) [18,19]. Each jelly coat layer is
composed of 50-60% O-linked oligosaccharides by weight, therefore these
oligosaccharide rich layers in combination with the egg envelope are likely to play a
critical role in many events leading to fertilization such as sperm binding, sperm
11
selection, the acrosome reaction and the block to polyspermy [5,6,7,8,19,20]. Structural
elucidation of the vastly distributed O-linked oligosaccharides throughout the egg jelly
coats have been performed by utilizing nuclear magnetic resonance (NMR) and mass
spectrometry (MS) [19,21,22]. The primary structural motifs of neutral oligosaccharides
released by β-elimination from the egg jelly coats were determined by NMR analysis,
and it was found that a subset of these oligosaccharide structures contained terminal
galactose and fucose residues [19,21].
In addition, structural determination of both neutral and anionic O-linked
oligosaccharides derived from the whole egg jelly coat extract was elucidated by MS
[22]. The MS analysis demonstrated the presence of O-linked oligosaccharide
components with conjugated anionic sulfate groups throughout the egg jelly coat [22].
In addition to the sulfated O-linked oligosaccharides, the structure of many neutral
oligosaccharides were elucidated and demonstrated that terminal galactose and fucose
residues are prevalent throughout these neutral species. It was also found that neutral
oligosaccharides from whole jelly glycoproteins ranged in size from trisaccharides to
octasaccharides [18,22]. A complementary study on X. laevis employed MS to
characterize the distribution and structure of O-linked oligosaccharides with respect to
the three distinct egg coat jelly layers [18]. The sulfated O-linked oligosaccharides were
found to be present in both the combined J1 and J2 layers of the egg jelly; however,
these sulfated oligosaccharides were entirely absent in the J3 layer [18]. The J3 layer
was composed primarily of neutral oligosaccharides and the combined J1 and J2 layers
12
contained approximately half the number of neutral species but contained all the
sulfated species.
Substantial progress has been obtained in elucidating the role of the ligand Olinked oligosaccharides as the functional moieties in binding to its CGL partner.
Interestingly, the 450 and 630 kDa CGL ligands have been shown to stain with alcian
blue, a dye that stains sulfated (acidic) glycoconjugates [14,18,23,24,25]. In addition,
MS studies have shown that these ligands contain sulfated groups attached to their
oligosaccharide structures. Collectively, O-linked oligosaccharide components present
within the egg jelly coat layers contain terminal galactose and fucose residues along
with associated sulfate groups, all of which may be biologically relevant structures
recognized by the CGL during the block to polyspermy [11,12,18,19,21,22].
Lectins in general are known to have more residues involved in their binding
interactions than merely a single terminal monosaccharide (oligosaccharide specificity).
This is demonstrated by the mannose-binding lectin, a free blood plasma protein
instrumental in the pathogen-recognition system of innate immunity. The mannosebinding lectin recognizes bacterial surfaces that display a specific spatial arrangement
of both mannose and fucose residues [26]. Regarding the CGL specifically, it has been
demonstrated to possess specificity for galactose-containing saccharides and the relative
affinities are modulated by secondary structural features such as terminal sugar,
anomeric configuration and the linkage pattern of branching sugars [9,11,13,27]. As
13
mentioned previously, CGL exists naturally in an oligomeric conformation (~10-12
subunits) and thus possesses multivalency for ligand oligosaccharides. It has been
postulated that the biological role of multivalency is utilized to compensate for weaker
binding affinities for the monovalent binding interactions of single subunits and thus
increases the overall binding strength of the interaction. The dissociation constants
measured for lectins generally are in the range of Kd = 10-6 whereas antigen-antibody
and other receptor-ligand interactions are in Kd = 10-9 or one thousand-fold stronger. A
useful analogy for the accumulated strength attained by multivalent binding interactions
is velcro. A single velcro hook and loop interaction is not very strong and can be easily
pried apart, whereas the overall bond strength is increased by possessing more
individual hook and loop fasteners involved in the interaction thereby increasing the
amount of surface area available for contact. Thus, the spatial orientation and surface
area of the lectin binding sites provided by the natural ligand may also be important
with respect to producing the block to polyspermy [26,27].
Although quite a bit has been learned about the CGL-ligand interaction, it is
unknown which of the ligand O-linked oligosaccharides are functionally relevant.
Therefore, further analyses such as isolation and quantification of the oligosaccharide
functional moieties and determining which structures bind with the highest relative
affinity to the CGL will contribute to a more definitive molecular mechanism and role
in the block to polyspermy. Elucidation of the CGL-ligand molecular specificity of
oligosaccharide binding will likely advance our understanding of fertilization and cell-
14
cell binding interactions involving eglectins, since the binding properties of the CGL
and its biological role appear to be conserved.
Challenges arise when studying the biological properties of oligosaccharides
since isolation and quantification methods often involve multiple analytical instruments
and derivatization procedures, respectively [28]. Quantification of oligosaccharides
typically requires large quantities for assays and/or derivatization procedures that
chemically modify their structures. Previous methods employed to separate
oligosaccharides liberated from their polypeptide backbone by either chemical or
enzymatic
means
have
included
different
HPLC
techniques
and
capillary
electrophoresis along with subsequent analysis from MS and NMR to elucidate
structural characteristics [18,28,29]. Since oligosaccharides lack strong chromophores,
or lack chromophores all together, sensitive quantification analysis methodologies have
utilized derivatization by incorporating a UV-absorbing or fluorescing group into the
oligosaccharide for subsequent detection [28]. Derivatization procedures require the
removal of such chemical modifications to reestablish the native compound for
subsequent biological assays. Thus, derivatization possesses limitations and can be a
laborious process.
Methods have been employed that do not require derivatization, such as highperformance/high-pH anion exchange chromatography (HPAEC) coupled to pulsed
amperometric detection (PAD) and HPLC equipped with UV detection, however these
15
methodologies have limitations. The HPLC-PAD requires a high salt content for this
methodology to be effective which can interfere with subsequent analysis, while the
HPLC-UV is limited to oligosaccharides that contain an N-acetyl group in the
hexosamine residues of chromophores in order to absorb at a specified wavelength and
the sensitivity is poor [18,28,30].
Previously, quantification of underivatized O-linked oligosaccharides derived
from the X. laevis egg jelly coat layers was attempted using measured UV absorbance
of chromophores at 206nm [18]. However, researchers were unable to profile all
possible oligosaccharides since the absence of N-acetylhexosamine residues would
result in no detectable signal. Interestingly, no linear relationship was found to exist
between UV absorbance and the number of N-acetylhexosamine residues present,
which would limit the ability to accurately quantify the oligosaccharide chains [18]. It is
noteworthy to point out that a relative quantification of the O-linked oligosaccharides
was determined and compared to the most abundant component within the egg jelly
coats, and it was found that the most abundant oligosaccharides within the combined J1
and J2 layers contained terminal galactose and fucose residues, further corroborating
previous NMR and MS findings [18].
To circumvent these limitations, a superior method to isolate and quantify
underivatizated oligosaccharides is needed. Recently, a charged aerosol based detection
(CAD) method coupled with HPLC has been developed that has the potential to
16
quantify underivatized oligosaccharides independently of their structural properties
[31,32]. The direct detection without fluorescent derivatization of biologically relevant
oligosaccharides has been achieved by employing this novel HPLC-CAD device
utilizing a hydrophilic interaction liquid chromatography column [30]. This detection
method has successfully demonstrated sensitivity in the picomolar range or mass
detection limits of 0.3 to 0.7 ng with regard to monosaccharides and oligosaccharides
[33,34]. Both UV and derivatization procedures have demonstrated sensitivities in the
picomolar range as well, however the HPLC-CAD method was demonstrated to be
approximately five times more sensitive than conventional UV absorbance detection.
Conversely, the HPLC-CAD was demonstrated to be approximately ten times less
sensitive than derivatization with fluorescent detection with regard to oligosaccharides
[30].
Thus, this HPLC-CAD system is likely to provide a universal detection method
along with greater sensitivity than most conventional HPLC-detection based methods
while circumventing complications associated with derivatization procedures. Since the
detection method is based on detecting non-volatile charged particles, which can be
applied more universally to compounds, quantification is predicted to be possible
without exact oligosaccharide standards [30,31,33]. Surrogate oligosaccharide standards
possessing similar properties as the compounds to be analyzed are utilized and thus a
relationship between calibration standards and the output signal of the instrument is
expected to be quantifiable and translatable to biologically-derived oligosaccharides.
17
The aim of this research is to accurately determine the relative quantities of Olinked oligosaccharides present on the CGL ligand and to utilize isolated
oligosaccharides in functional binding assays to determine which oligosaccharides
function in the CGL-ligand binding interaction. HPLC-CAD profiles and purification
thereof of the CGL ligand oligosaccharides will assist in elucidating the preferential
oligosaccharides that participate in CGL binding and their relative abundance.
Separation and quantification of the ligand oligosaccharide constituents can be used to
generate an oligosaccharide profile of the ligand and reveal the predominate
components. Predominate peaks will be isolated, quantified and then used in an
enzyme-linked lectin assay (ELLA) to assess whether these particular oligosaccharides
can bind to CGL. This in-vitro functional binding assay may be able to estimate the
relative binding affinity of particular isolated oligosaccharides to purified CGL. In
addition, this research will re-evaluate the inhibition effects of commercially available
saccharides (i.e. galactose, lactose, melibiose, etc.) using our refined ELLA assay.
This research has two major implications. Firstly, the contribution of this
developed oligosaccharide isolation and quantification methodology may have many
other applications throughout the scientific community. This novel HPLC-CAD
technique should allow sensitive and universal detection of any sugar and allow
quantification of biologically relevant oligosaccharide concentrations in their native
state thus circumventing any derivatization procedures that may interfere with
18
subsequent biological assays. Secondly, this research will assist in identification of the
ligand oligosaccharides that functionally bind to CGL and thus participate in the block
to polyspermy. Further implications with regard to the molecular binding mechanism of
CGL homologues may be deduced since compelling evidence suggests its calciumdependent, galactose-specific binding properties are conserved in this lectin family.
Provided all this information, the following hypothesis and objectives have been
set forth.
Hypothesis:
HPLC-CAD will allow the isolation and quantification of O-linked oligosaccharides
released from the CGL ligand which can then be utilized in functional binding assays to
assess which oligosaccharides function in the lectin-ligand binding interaction during
the Xenopus laevis block to polyspermy.
Objectives:
1) Develop an HPLC-CAD methodology for quantification and isolation of biologically
relevant oligosaccharides.
2) Apply the developed methodology to profile, quantify and permit the purification of
X. laevis CGL ligand O-linked oligosaccharides.
3) Develop and perform enzyme-linked lectin assays (ELLA) to assess whether isolated
CGL ligand oligosaccharide fractions are involved in the CGL binding interaction.
19
MATERIALS AND METHODS
Purification of X. laevis CGL Ligands
Recently, an Alcian blue dye binding methodology has proven advantageous for
the purification of the X. laevis CGL ligands [6,25]. Cationic dyes, such as Alcian blue
have been demonstrated to selectively stain the highly glycosylated acidic ligand
glycoproteins within X. laevis egg jelly which can be visualized after separation by
SDS-PAGE [25]. This method provides a rapid and effective way to distinguish acidic
glycoproteins from other proteinaceous components which can be utilized for
subsequent purification of these components. The Alcian blue dye preferentially binds
to the sulfated ligands under acidic conditions within the crude egg jelly extract. An
Alcian blue precipitation procedure was developed by Dr. Peavy so as to generate a
resin of purified ligands bound to the Alcian blue dye which can then be separated by
either electrophoresis (SDS-PAGE and electroelution) or used directly in the alkalineborohydride reduction methodology described below since this chemical procedure
disrupts dye binding to the oligosaccharides.
Release and Purification of X. laevis CGL Ligand O-linked Oligosaccharides
Alcian blue pellets complexed with the CGL ligands were subjected to a
protocol for the release and purification of O-linked oligosaccharides, thus dissociating
the protein and carbohydrate moieties [18,22]. This protocol utilizes alkalineborohydride reduction to achieve the desired dissociation. The Alcian blue pellets were
20
dissolved in a 1.0 M NaBH4/0.1 M NaOH solution which was prepared by dissolving
the appropriate amount of NaBH4 solid into 0.1 M NaOH solution and incubated for 24
hours in a water bath at 45°C. Following the reduction protocol, the resulting solution
was neutralized with a 1.0 M HCl solution to stop the reaction and destroy the excess
NaBH4 in an ice bath. The resulting neutralized solution consisting of reduced
oligosaccharides was stored at 4°C.
Removal of impurities along with isolation of the O-linked oligosaccharides was
performed utilizing centrifugation and column separation using porous graphitized
carbon solid-phase extraction (PGC-SPE) 4.0 mL cartridges (Grace Davison Discovery
Science, Deerfield, IL) [18,35]. Neutralized solution was centrifuged at 10,000 rpm for
12 minutes and the resulting supernatant was aspirated and applied to a PGC-SPE
cartridge.
Most of the Alcian blue is not retained in this neutralized supernatant
solution. Prior to use, the PGC-SPE cartridge was conditioned with three column
volumes (5 mL) of 80% acetonitrile in 0.1% trifluoroacetic acid followed by three
column volumes of water. When applied, monosaccharides and salts pass through the
cartridge (flow through fraction), whereas peptides, oligosaccharides and the remaining
Alcian blue dye bind to the solid-phase matrix [25,35]. The cartridge was washed with
three column volumes of water to completely remove salts while the O-linked
oligosaccharides remain bound to the stationary carbon phase. The neutral and acidic
CGL ligand O-linked oligosaccharides were successfully eluted from the PGC-SPE
cartridge with three column volumes of 25% acetonitrile/75% water solvent and 25%
21
acetonitrile/0.05% trifluoroacetic acid/water solvent, respectively, while the peptides
and Alcian blue dye remained entrapped [18,35]. After preliminary HPLC analysis of
the CGL ligand oligosaccharides (described below), it was observed that an early
eluting, large peak suspected to be comprised of salts, monosaccharides and other
impurities was consistently present. Therefore, in an effort to rid subsequent GCL
ligand oligosaccharide samples of this initial contaminant, an additional “clean-up” step
consisting of a 5% acteonitrile/95% water wash was added to the PGC-SPE procedure
prior to the 25% acetonitrile/75% water solvent in order to reduce the amount of this
initial peak.
Furthermore, the resulting PGC-SPE neutral oligosaccharide fractions were
subjected to a Millex-LG 0.20µm hydrophilic 4mm syringe driven filtration unit
(Millipore, Billerica, MA) for fine particle removal. Purified neutral oligosaccharide
samples were centrifuged under heated conditions to evaporate the acteonitrile in the
sample followed by a subsequent round of centrifugation under a vacuum (speed-vac).
The resulting dried oligosaccharide samples were stored at –20°C for subsequent HPLC
analysis.
HPLC-CAD Methodology Development for the Quantification of Oligosaccharides
An Agilent (Santa Clara, CA) 1100 series high performance liquid
chromatograph outfitted with a custom-built charged aerosol detection system (HPLCCAD) in tandem was employed to develop a sensitive method for quantification of
22
neutral oligosaccharides (Figure 2). Chem Station software was used to operate the
instrument. A Prevail Carbohydrate ES HPLC column (250 x 4.6 mm – 5µm particle
size) along with a Prevail guard column (Grace Davison Discovery Science, Deerfield,
IL) was used for HPLC separation of neutral oligosaccharides.
23
Figure 2 – A diagrammatic representation of the HPLC-CAD system and associated
components.
24
The custom-built CAD system utilizes similar equipment as described in Dixon
and Peterson [31] but with some modifications. Following separation, analytes from the
HPLC column enter the nebulizer (Meinhard, Santa Ana, CA) which in turn produces
spray droplets along with a flow of nitrogen gas. Solvent is evaporated away from the
droplets in the heated drift zone and converted into particles, upon which, detection of
aerosol particles occur (Figure 2). The HPLC-CAD system detects particles that have
been charged during the nebulization stage, in a process termed spray electrification.
Spray electrification produces approximately equal amounts of both positively and
negatively charged particles.
Aerosol detection is carried out with an electrical aerosol size-analyzer (EAA)
(TSI, Shoreview, MN) (Figure 3). The ion filter includes a negatively charged rod and
grounded surrounding wall that is necessary to preferentially remove negatively charged
particles. The ion filter permanently removes small positivity charged and most
negatively charged particles that travel to the rod or walls. The greater efficiency in
removal of negatively charged particles occurs as a result of the particles entering the
ion filter close to the wall. The remaining charged aerosol particles that reach the
aerosol filter are trapped and subsequently detected with an electrometer. The flux of
charged aerosol particles of net positive charge produces a current that is measured by
the electrometer. Greater voltages applied to the EAA’s ion filter result in a decreased
signal due to removal of more positively charged particles prior to reaching the detector,
resulting in a decreased overall positive charge flux reaching the electrometer. Thus, the
25
sensitivity of chromatographic analyses as well as the dynamic range (minimum
detectable concentration to maximum detectable concentration) can be modulated by
adjusting voltage.
26
Sample Aerosol In
(stays in shaded
region)
EAA
Sheath Air Flow 1
Corona Discharge
Ion Filter
Sheath Air Number 2
Aerosol Filter
To Electrometer
Figure 3 – A diagrammatic representation of the electrical aerosol size-analyzer (EAA)
with associated components.
27
Surrogate standards consisting of cyclodextrins (α-cyclodextin, β-cyclodextrin
and 6-O-α-maltosyl-β-cyclodextrin) and linear chain oligosaccharides ranging from
glucose to maltooctaose (all α1→6 linked glucose-containing oligosaccharides with a
degree of polymerization from one to eight) were prepared at 1, 4, 10, and 25 µg/mL
concentrations in 50% water/50% acetonitrile mixtures for calibration purposes.
Prepared samples were transferred to 2 mL amber autosampler vials using 9 mm closure
vial caps (Fisher Scientific, Pittsburgh, PA) for HPLC analysis. Initial testing of the
developed quantification methodology was explored with four test standards consisting
of three trisaccharides and one tetrasaccharide, which were prepared at 10 µg/mL
concentrations (isomaltotriose, raffinose, melezitose and stachyose). The linear chain
oligosaccharide standards ranging from a degree of polymerization from three to seven
and the initial four test standards were prepared from a Supelco oligosaccharide kit
(Bellefonte, PA), whereas maltooctaose was prepared from Carbosynth (Beedon
Newbury, Berkshire, UK). The cyclodextrins, glucose and maltose were prepared from
Sigma-Aldrich (St. Louis, MO).
All sample runs with regard to quantification work were performed at -300 volts
(ion filter voltage reading on the EAA) utilizing 20 µL injection volumes during each
chromatographic analysis on the HPLC-CAD system. Two methods were developed for
quantification analysis: an isocratic method in which solvent conditions remained
constant and a gradient method in which solvent conditions changed gradually over
time throughout the chromatographic runs. The isocratic method consisted of 62%
28
acetonitrile/38% water solvent conditions over 30 minutes, while the gradient method
was performed with a decreasing percentage of acetonitrile from 65% to 50% over 25
minutes throughout the sample runs. The flow rates were 1.0 mL/min for both
developed methodologies.
Additional oligosaccharide standards more representative of typical structures
found on glycoproteins including branched structures of comparable molecular structure
relative to the CGL ligand oligosaccharides were prepared to test the developed
quantification methods. These commercially available branched oligosaccharides
contained specific residues (monosaccharides) expected to be present in our selected
fraction isolates, such as terminal fucose and galactose residues. Additionally, core
glycoprotein structures such as branched N-acetylglucosamines and mannose hybrids
were utilized to test the quantification method. All biological test structures were
prepared at 10 µg/mL concentrations and consisted of Lacto-N-hexose (LNH), Lacto-NFucopentaose
(LNFP
II),
oligomannose-3
(MAN-3),
Asialo-
galactosylated-
biantennary (NA2), Asialo- galactosylated- tetraantennary (NA4), mannopentaose and
lactodifucotetraose (LDFT). LNH, LNFP II and NA4 were prepared from
Prozyme/Glyco (San Leandro, CA). NA2 and MAN-3 were prepared from V-labs
(Covington, LA). Mannopentaose was prepared from Sigma-Aldrich (St Louis, MO)
and LDFT was prepared from Glycotech (Gathersburg, MD).
29
Multiple sugar standards, including the various branched structures were
prepared at different concentrations to serve in calibrating and optimizing the HPLC
technique for quantification of these oligosaccharides. The optimization consisted of
adjusting the eluent program and the CAD’s ion filter voltage, where ion voltage has an
inverse relationship to response sensitivity. Once all the necessary standards were
completed and methodology optimized, the selected neutral O-linked oligosaccharides
derived from the CGL ligand were analyzed on the HPLC-CAD system to quantify the
specific oligosaccharide constituents of interest.
Separation, Fractionation and Isolation of X. laevis CGL Ligand Oligosaccharides
Separation of CGL ligand oligosaccharide constituents was performed on a
Hypercarb PGC (100 x 4.6 mm – 3µm particle size) HPLC column (Thermo Scientific,
Waltham, MA) utilizing a gradient method consisting of an increasing percentage of
acetonitrile from 5% to 35% over 45 minutes. The purified CGL ligand neutral
oligosaccharide samples were reconstituted in 100% water for the Hypercarb PGC
chromatographic analysis. Prepared oligosaccharide samples were transferred to 100 µL
polyspring inserts and placed into 1.5 mL autosampler vials (National Scientific,
Rockwood, TN). The flow rate was 0.800 mL/min and 10 µL injection volumes were
used for each chromatographic analysis when separating and isolating the neutral
oligosaccharides derived from the CGL ligands.
30
Fraction collections from the HPLC-CAD system were permitted by an
analytical flow splitter (Grace Davison Discovery Science, Deerfield, IL) with a split
ratio of 3:1. This split ratio allowed fraction collection of 75% of the sample while the
other 25% was used for detection on the CAD instrument. Installation of the analytical
flow splitter and its effects on operating efficiency were determined and compensated
for, such as the flow rates and initial pressure in the HPLC-CAD system. Collection of
oligosaccharide fractions was accomplished by an Agilent 1200 series fraction collector
on a time-based trigger mode by accounting for lag times between the CAD and the
fraction collection. The Agilent 1200 series fraction collector was interfaced with the
Agilent 1100 series instrument. Selected peaks were collected and individual fractions
were pooled together and centrifugally evaporated for subsequent HPLC-CAD
quantitative analysis and competitive plate binding assays.
Quantification and Secondary Purification of Selected CGL Ligand Oligosaccharides
Selected X. laevis oligosaccharide fractions were isolated, corresponding to
specific peaks and respectively pooled together throughout multiple chromatographic
analyses on the Hypercarb PGC HPLC column. A secondary purification (as necessary)
along with quantification of specific fractions was performed on the Prevail
Carbohydrate ES column. Specific fractions that displayed more than one peak with
significant peak area were selected to undergo a secondary purification on the Prevail
Carbohydrate ES column. The different modes of separation and selectivity between the
two HPLC columns provided further fraction purity as indicated by the presence of a
31
single oligosaccharide constituent. Pooled fractions from the Hypercarb PGC
chromatographic runs were dried under a vacuum using centrifugation and subsequently
reconstituted in a 50% acetonitrile/50% water solvent. Aliquots were removed from
each selected fraction for HPLC-CAD quantification analysis and the remaining
sample, if needed underwent a secondary round of purification performed in an
identical manner as described in the previous section with regard to fractionation and
isolation of the CGL ligand oligosaccharides. The quantification analysis was
performed under identical conditions as described for the developed Prevail
Carbohydrate ES HPLC gradient methodology. The purity and amount of
oligosaccharide mass remaining within each respective fraction vial was determined and
used in subsequent competitive plate binding assays.
Procurement and Preparation of X. laevis Egg Jelly and CGL
Egg jelly samples, enriched for CGL ligands, were obtained along with purified
CGL prior to this research by Dr. Tom Peavy. CGL was biotinylated utilizing a Biotin
Tag Micro Biotinylation Kit (Sigma-Aldrich, St Louis, MO). CGL in the amount of
0.37 mg (quantified as described below) was solubilized in 0.500 mL of 0.1 M
NaHCO3, pH 8.4. To this, 13 µL of biotinamidohexanoic acid 3-sulfo-Nhydroxysuccinide ester (5 mg/mL in 30 µL DMSO and 970 µL of 0.1 M sodium
phosphate buffer pH, 7.2 prepared fresh) was added at a molar ratio of 13:1. The
resulting solution was incubated at 4°C for 2 hours with gentle agitation and
subsequently dialyzed in 4 liters of TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5). The
32
buffer was exchanged three times within a 24 hour period. Afterwards, the sample was
removed from the dialysis bag and stored at -20°C.
The CGL was quantified by a Bradford assay prior to and following the
biotinylation procedure for determination of protein concentration using Bovine Serum
Albumin (BSA) as the standard. BSA standards ranged from 0.1 to 1.4 mg/mL for all
calibration curves and absorbance was measured at 595 nm. X. laevis egg jelly was
quantified based on carbohydrate and protein concentration by a phenol-sulfuric acid
assay using galactose as the standard and a Bradford assay, respectively [6,25]. The
galactose standard used in the phenol-sulfuric acid assay was prepared at 1 µg/µL, in
which calibration curves were generated in the range of 10 µg to 60 µg and absorbance
was measured at 490 nm.
SDS-PAGE Gel Characterization of X. laevis Egg Jelly and CGL
Additionally, egg jelly preparation and CGL were further characterized by SDSPAGE analysis. A Bio-Rad Multi-Casting chamber and a Mini-Protean 3
Electrophoresis Module Assembly was used to cast all gels and conduct the
electrophoresis runs, respectively (Hercules, CA). X. laevis egg jelly was analyzed on a
3.25% stacking gel (63.5% water/25% 0.5 M Tris-HCl pH 6.8/11% Acrylamide/Bis/1%
SDS) and 7.5% resolving gel (48.5% water/25% 1.5 M Tris-HCl pH 8.8/25%
Acrylamide/Bis/1% SDS). Egg jelly samples were heated for 10 minutes at 95°C in
loading sample buffer prior to loading wells for a total loading sample volume of 100
µL. Egg jelly SDS-PAGE gels were run at 10 mA at 4°C until the tracking dye
33
approached the bottom of the gel. Gels were stained utilizing a thiosulfate-silver/Alcian
blue staining procedure as described by Bonnell et al. (1999) [25]. Gels were fixated in
40% methanol/10% acetic acid for three 15 minutes incubations. Gels were then washed
in 40% methanol for 10 minutes and soaked in 0.05% sodium thiosulfate for 45
seconds. Gels were rinsed in distilled water for 2 seconds and subsequently soaked in
0.12% silver nitrate for 5 minutes followed by another rinse with distilled water for 2
seconds. Gels were developed with 60 µL of 36.5% formaldehyde in 100 mL 3%
sodium carbonate. The reaction was stopped with 20% acetic acid for 5 minutes.
Finally, the gels were soaked in 0.05% Alcian blue in 40% methanol/10% acetic acid
for 60 minutes and destained in 40% methanol/10% acetic acid overnight or until clear.
CGL was analyzed on a 4% stacking gel (61% water/25% 0.5 M Tris-HCl pH
6.8/13% Acrylamide/Bis/1% SDS) and 10% resolving gel (41% water/25% 1.5 M TrisHCl pH 8.8/33% Acrylamide/Bis/1% SDS). CGL samples were heated for 10 minutes
at 95°C in loading sample buffer prior to loading wells for a total loading sample
volume of 50 µL. CGL gels were stained utilizing Coomassie Brilliant Blue stain (0.1%
R250/46% methanol/7% acetic acid) for 1 hour and destained (40% methanol/10%
acetic acid) until clear. The CGL gels were run at 50 mA for 90 minutes or until the
tracking dye reached the bottom of the gel. All reagents used were electrophoretic grade
and purchased from Fisher Scientific (Pittsburgh, PA). Bio-Rad broad range molecular
weight standards (200 kDa – 6.5 kDa) were utilized for both egg jelly and CGL gel
analyses.
34
Competitive Enzyme-Linked Lectin Assays
Once isolation and quantification of the selected neutral CGL ligand
oligosaccharide fractions were completed, further elucidation of these oligosaccharides
and their role in the block to polyspermic fertilization were investigated by a
competitive enzyme-linked lectin assay (ELLA). These competitive plate binding
assays were based on previous protocols utilized in the detection and disruption of the
CGL-ligand binding, where sensitivity in detection of the CGL ligand was demonstrated
to be at 1-2 ng/mL [11,12].
Initial tittering and optimization of the enzyme-linked lectin assays were
performed utilizing a standard dilution series approach of the biotinylated CGL and
ExtrAvidin Peroxidase conjugate and incorporating selected monosaccharide and
disaccharide inhibitors as positive and negative controls, respectively. The tittering
consisted of a starting concentration of 10 µg/mL and 5 µg/mL of biotinylated CGL and
ExtrAvidin Peroxidase, respectively. Serial dilutions of the biotinylated CGL were
performed from 10 µg/mL to 0.04 µg/mL across columns 1 through 10 which were
incubated with a fixed concentration of ExtrAvidin Peroxidase at 5 µg/mL. Serial
dilutions of ExtrAvidin Peroxidase were performed from 5 µg/mL to 0.08 µg/mL down
rows A through H which were incubated with a fixed concentration of biotinylated CGL
at 10 µg/mL. Monosaccharide and disaccharide controls were selected based on
previous research involving the binding specificity of the CGL. Selected
monosaccharides included galactose, glucosamine, glucose and fucose. Selected
35
disaccharides consisted of maltose, lactose, lactulose, sucrose and melibiose. All
monosaccharides and disaccharides were purchased from Sigma-Aldrich (St. Louis,
MO) with the exception of lactulose, which was purchased from ACROS organics
(New Jersey, USA).
Egg jelly test samples (500 ng carbohydrate/well) were bound to an Immulon
4HBX ultra-high binding 96 well flat bottom polystyrene microtiter plate (Thermo
Scientific, Waltman, MA) in 100 µL of 100 mM Na2CO3, pH 9.5 at 4°C overnight. The
plate was washed three times with CTBST dilution/wash buffer (10 mM Tris-HCl, 150
mM NaCl, 10 mM CaCl2, 0.05% Tween 20, pH 7.5). Blocking buffer (10 mM TrisHCl, 150 mM NaCl, 3% BSA, 0.05% Tween, pH 7.5) was added for 30 minutes to 1
hour at room temperature. Biotinylated CGL, 0.74 mg/mL was diluted to 0.75 µg/mL
with dilution/wash buffer and a 100 µL aliquot was added to each well and incubated
for
1
hour
at
25°C.
Selected
inhibitors
(monosaccharides,
disaccharides,
isolated/quantified CGL ligand oligosaccharide fractions and SPE derived neutral and
acidic whole CGL ligand oligosaccharide fractions) were solubilized in CTBST and
included during the biotinylated CGL incubation to assess inhibitive properties. The
excess biotinylated CGL was removed by washing three times with dilution/wash
buffer, followed by an additional blocking step as described above. A 100 µL aliquot of
0.2% (v/v) ExtrAvidin Peroxidase conjugate was diluted to 500 ng/mL in dilution/wash
buffer and added to each well and incubated for 1 hour at 25°C. The excess ExtrAvidin
Peroxidase conjugate was removed by washing three times with dilution/wash buffer.
36
Following the washing procedure, 100 µL of TMB (3,3’,5,5’-tetramethylbenzidine)
Blue (Lab Vision, Fremont, CA) substrate was added to all wells and incubated for 1020 minutes. The chromophore, which was cleaved by the enzyme conjugate, was
developed and measured at 630 nm using an Opsys MR (DYNEX Technologies,
Chantilly, Virginia) microplate reader. TMB Blue product formation measured at an
optical density (OD) of 630 nm is directly proportional to the binding activity of the
CGL to its ligand partners contained in the egg jelly.
37
RESULTS
Quantification of Oligosaccharides
To investigate the functional
role of
Xenopus
laevis
CGL ligand
oligosaccharides in the block to polyspermic fertilization, a novel method for
quantification of biologically relevant oligosaccharides was developed. The
quantification methodology was developed without derivatization agents or exact
standards utilizing the HPLC-CAD system. The HPLC-CAD system demonstrated
excellent sensitivity, separation and a relatively uniform response in the analysis of
surrogate oligosaccharide standards ranging from a degree of polymerization (DP) of
one to eight (glucose to maltooctaose) and 6-O-α-maltosyl-β-cyclodextrin, respectively
(Figure 4).
38
15.390
500
17.748
14.210
12.953
11.575
600
10.040
8.507
5.930
7.109
ADC1A, ADC1CHANNELA(NOAH\052009000002.D)
mV
400
300
200
100
DP 1 DP 2
2
4
6
DP 3
8
DP 4
10
DP 5 DP 6
12
DP 7 DP 8
14
16
18
min
Figure 4 – A gradient method chromatogram comprising a mixed linear oligosaccharide
standard (glucose to maltooctaose at retention times of 5.930 through 15.390 minutes)
and 6-O-α-maltosyl-β-cyclodextrin (last peak at 17.748 minutes) at 25 µg/mL.
39
Multiple surrogate oligosaccharide analyses were performed and calibration
curves were generated from each linear glucose oligomer and the cyclodextrin standard
throughout their respective concentrations (Figure 5). It is noteworthy to point out that
maltosyl-β-cyclodextrin was utilized as an additional surrogate oligosaccharide standard
despite its structural dissimilarity to the other glucose oligomers. This was deemed
necessary in order to extend the quantification calibration curves to accommodate the
quantification of the larger biological test oligosaccharide structures. Although it would
have been preferred, a linear glucose oligomer possessing a degree of polymerization
(DP) of nine was not commercially available therefore this compound was selected in
an effort to substitute for the projected retention time that a linear DP 9 would possess.
A non-linear calibration scheme was generated and utilized since the response of the
surrogate standards did not display a true linear relationship utilizing the traditional
linear equation (y = mx + b). Therefore, response curves were fitted utilizing a power-fit
model:
y = ACb
Where y is the peak area, C is the concentration in µg/mL, and ‘A’ and ‘b’
values are fixed parameters that are derived from the calibration power-fit equations.
The non-linear relationship is observed utilizing the power-fit model as well,
demonstrated by ‘b’ terms (power variable) possessing values significantly greater than
1.0 (Figures 5 and 7). Although the three standards highlighted in figure 5 display R 2
40
values of 1.0, these values were rounded from 0.999 and the R2 range of all nine
standards was 0.993 to 0.999 (Figure 5).
41
Gradient Calibration Plot
12000
10000
Maltotetraose
8000
y = 288.31x1.1049
R2 = 1
Glucose
y = 325.61x1.0474
R2 = 1
Maltose
Peak Area
Glucose
y = 292.18x
R2 = 1
Maltotriose
Maltotetraose
1.0836
Maltopentaose
6000
Maltohexaose
Maltosyl-β-cyclodextrin
Maltoheptaose
Maltooctaose
4000
Maltosyl-cyclodextrin
2000
0
0
5
10
15
20
25
30
Concentration (ug/mL)
Figure 5 – Representative plot of peak area to concentration relationship. Power-fit
calibration equations derived from throughout the prepared concentration range (1, 4,
10 and 25 µg/mL) for glucose (DP 1), maltotetraose (DP 4) and 6-O-α-maltosyl-βcyclodextrin.
42
Therefore, a correlation between ‘A’ and ‘b’ terms and retention times were
applied as the appropriate quantification calibration terms (Figures 6 and 7). Each
individual surrogate oligosaccharide ‘A’ and ‘b’ values, collectively obtained from 1
through 25 µg/mL concentrations, were plotted from the power-fit calibration equations
(Figure 5) with respect to retention time (Figures 6 and 7). ‘A’ and ‘b’ parameters
showed definitive trends as a function of retention time, allowing the prediction of
calibration parameters for test and unknown oligosaccharides of interest.
43
Figure 6 – ‘A’ terms derived from the surrogate oligosaccharide standards and
corresponding 2nd order polynomial curve relative to retention time. ‘A’ values for
glucose (325), maltotetraose (288) and maltosyl-β-cyclodextrin (292) are highlighted.
44
Figure 7 – ‘b’ terms derived from the surrogate oligosaccharide standards and
corresponding 2nd order polynomial curve relative to retention time. ‘b’ values for
glucose (1.047), maltotetraose (1.105) and maltosyl-β-cyclodextrin (1.084) are
highlighted.
45
Figures 6 and 7 demonstrate that changes in sensitivity and linearity (‘A’ and ‘b’
terms) changed relativity smoothly with a change in retention time, allowing the use of
equations to fit the variation of ‘A’ and ‘b’ terms with retention time. While the R 2
values were less than 1.0 (Figures 6 and 7), the range in both ‘A’ and ‘b’ values is not
very large, displaying ranges of 230 to 340 and 1.05 to 1.13, respectively. The overall
objective of this developed quantification scheme was to accurately determine
concentrations of unknown oligosaccharides and in particular X. laevis CGL ligand
oligosaccharides. Thus, employing this non-linear calibration scheme, an unknown
sample lying within the surrogate oligosaccharide standard range is based on observed
retention time and peak area along with ‘A’ and ‘b’ fixed parameters (‘A’ and ‘b’ terms
are determined for unknown compounds by using the unknown compounds’ retention
time together with the 2nd order polynomial fit equations given in Figures 6 and 7) as
determined from standards (Figures 6 and 7) using the following equation:
C = (y/A) 1/b
Prior to oligosaccharide HPLC-CAD analyses, all commercially available
oligosaccharide standards (i.e. surrogate and biological test standards) in large enough
quantities were prepared by weighing out the required mass on an analytical balance
(mg or µg) to create a stock solution, from which appropriate dilutions were performed.
Although preferred, it was not possible to weigh out some of the test oligosaccharides
since the commercially available amount was only in the tens of micrograms. Therefore
46
in these circumstances, the amount of mass determined by the manufacturer was
utilized in concentration calculations. Selected dilutions were then transferred to
sampling vials for ensuing HPLC-CAD analysis (Figure 4). Additionally, purity of each
standard, as reported by the manufacturer, was compensated for in all final calculations.
Employing this non-linear calibration scheme, surrogate oligosaccharide
standards displayed very low average percent errors to within 5% (excluding
maltooctaose) and excellent reproducibility (Figure 8). Furthermore, a relatively
uniform distribution with regard to variation in instrument response throughout each
surrogate oligosaccharide standard was observed. With regard to average percent error,
six of the surrogate oligosaccharide standards displayed positive values, while two
displayed negative values. There were no correlative relationships between the degree
of polymerization and the average percent error or standard deviation. Interestingly,
maltooctaose displayed the poorest response in average percent error, however the
reproducibility was excellent (-10.7 +/- 1.51).
47
Surrogate Oligosaccharide Standards
35
15
-35
al
to
te
tr a
os
e
M
al
to
pe
nt
os
e
M
al
to
he
xo
se
M
al
to
he
pt
ao
se
M
al
to
oc
M
ta
al
os
to
e
sy
l-c
yc
lo
de
xt
r in
-25
M
M
M
-15
al
to
se
-5
al
to
tr i
os
e
5
G
lu
co
se
Average Percent Error
25
Figure 8 – The mean and standard deviation for three separate trials of surrogate
standards are shown (one trial excluded the maltotetraose data due to contamination).
Non-linear calibration terms used for concentration calculations were based on 10
µg/mL test samples.
48
Once accurate and precise quantification was achieved by the non-linear
calibration scheme for the surrogate oligosaccharide standards under optimized
conditions, four short chain oligosaccharide structures were tested. These short chain
oligosaccharides consisted of three trisaccharides (isomaltotriose, raffinose and
melezitose) and a tetrasaccharide (stachyose), which were utilized in testing the
developed quantification methodology (Figure 9).
49
Isomaltotriose
Melizitose
Stachyose
Raffinose
Figure 9 – Short chain oligosaccharide structures analyzed by the developed HPLCCAD methodology.
50
Collectively, accurate quantification to within less than 11% average error was
obtained, however the reproducibility was more variable relative to the surrogate
oligosaccharide standards (Figure 10). Isomaltotriose and raffinose produced
comparable responses and variations of -10.8 +/- 7.07 and -10.2 +/- 8.18, respectively.
Conversely, stachyose and melezitose produced similar responses and variations of 1.58
+/- 13.7 and -3.81 +/- 11.9, respectively; however the variation was greater relative to
isomaltotriose and raffinose. Increased variance was observed with regard to the
quantification analysis of the initial short chain test oligosaccharides in relation to the
surrogate oligosaccharide standards (Figure 8); however a definitive range of variation,
in excellent agreement to that in Figure 10, was established for each initial test standard
by previous work performed under both the isocratic and gradient conditions used for
quantification methodologies. In brief, the developed quantification methodologies
consisted of isocratic and gradient conditions where the mobile phase remained constant
or changed gradually over time throughout the chromatographic runs, respectively (data
not shown for the isocratic method but the method is described in the materials and
methods section).
The gradient solvent conditions affected the elution of samples from the HPLC
column as compared to the isocratic method, and thus the retention times and
distribution of peak areas were significantly different. The gradient method
demonstrated superiority to the isocratic method in the chromatographic analysis of
oligosaccharides due to the decrease of retention times for later eluting peaks, thus
51
providing sharper peak shapes while the signal-to-noise ratio in later eluting peaks was
greatly improved. Therefore, all data collected with regard to the quantification analysis
of oligosaccharide standards (with the exception of LNFP II and LNH, which were
quantified by both developed methodologies) and with regard to the neutral
oligosaccharides derived from the CGL ligand glycoprotein were analyzed by the
developed gradient methodology.
52
Initial Test Oligosaccharide Standards
35
Average Percent Error
25
15
5
-5
Isomaltotriose
Stachyose
Raffinose
Melezitose
-15
-25
-35
Figure 10 – The mean and standard deviation for three separate trials of initial test
standards are shown. Non-linear calibration terms used for concentration calculations
were based on 10 µg/mL test samples.
53
Once a defined set of parameters were determined that optimized accuracy and
reproducibility of the test oligosaccharides, oligosaccharides more representative of
biological structures found on glycoproteins were analyzed by the developed HPLCCAD quantification methodology (Figure 11). Seven oligosaccharide structures of
varying complexity were analyzed by the developed HPLC-CAD quantification
methodology (Figures 11 and 12). These core and branched oligosaccharide structures
are characteristic of typical O- and N-linked oligosaccharides found on biological
proteins and thus were selected as biological test standards. O-linked oligosaccharides
are characterized by the addition of sugars to the hydroxyl (OH) groups of selected
serine or threonine side chains, whereas N-linked oligosaccharides are conjugated to
amine (NH2) groups on asparagine amino acids which occur in the tripeptide sequence
Asn-X-Ser or Asn-X-Thr (where X can be any amino acid except proline).
54
Galβ1-4GlcNAcβ1
6
Galβ1-4GlcNAcβ1-2Manα1
Manα1
2
Galβ1-4GlcNAcβ1
6
6
Manβ1-4GlcNAcβ1-4GlcNac
Manβ1-4GlcNAcβ1-4GlcNAc
Galβ1-4GlcNAcβ1
3
3
Galβ1-4GlcNAcβ1-2Manα1
4
Manα1
2
Asialo, galactosylated, biantennary oligosaccharide (NA2)
Galβ1-4GlcNAcβ1
Asialo, galactosylated, tetraantennary oligosaccharide (NA4)
Galβ1-4GlcNAcβ1
Manα1
6
6
Galβ1-4Glc
Manβ1-4GlcNAcβ1-4GlcNac
3
Galβ1-3GlcNAcβ1
3
Manα1
Lacto-N-hexaose (LNH)
Fucα1
I
3
Galβ1-4GlcNAcβ1
I
6
Fucα1
Galβ1-4Glc
I
3
4
I
Galβ1-4GlcNAcβ1
Oligomannose 3 (MAN-3)
Galβ1-3GlcNAcβ1-3Galβ1-4Glc
I
Fucα1
Lacto-N-Fucopentaose (LNFP II)
Lactodifucotetraose (LDFT)
Figure 11 – Subset of branched oligosaccharide structures representative of typical Oand N-linked oligosaccharide moieties found on glycoproteins, which were analyzed by
the developed HPLC-CAD quantification methodology.
55
Collectively, accurate quantification to within less than a 19% average error was
obtained along with comparable variance to the initial test oligosaccharide standards
(Figures 10 and 12). There were no correlative relationships between core
oligosaccharides (MAN-3 and LNH) and complex high-mannose type oligosaccharides
(NA2 and NA4) with regard to absolute average percent errors (7.31 vs. 6.99), however
standard deviations increased as complexity increased (4.1 vs. 11.6). Structures
containing residues found to exist in the CGL ligand oligosaccharides by NMR and MS
[19,21,22] such as core glycoprotein oligosaccharide N-acetylglucosamine structures
with terminal fucose residues (LDFT) and N-acetylglucosamine structures with terminal
fucose and galactose residues (LNFP II), displayed very low average percent errors and
high-quality reproducibility (6.63 +/- 8.51 and 3.46 +/- 3.69, respectively).
Mannopentaose, a five-chain high-mannose structure fragment commonly found among
N-linked oligosaccharides displayed excellent reproducibility (-14.3 +/- 3.78). Notably,
both NA2 and NA4 displayed the greatest range in variance 18.4 +/- 7.37 and -4.42 +/15.9, respectively.
56
Biological Test Oligosaccharide Standards
35
15
II*
*
FP
H*
LN
an
no
pe
n
M
-25
LN
ta
os
e*
*
FT
LD
NA
4*
-15
NA
2*
-5
AN
-3
*
5
M
Average Percent Error
25
-35
Figure 12 – Results of biological test standards representing typical branched
oligosaccharides present on glycoproteins. The mean and standard deviation for three
separate trials of biological test standards are shown (two separate trials are shown for
LNFP II and LNH). Non-linear calibration terms used for concentration calculations
were based on 10 µg/mL test samples.
* Based on assumed weight from manufacture.
** Normalized as a result of manufacturer error in sample quantity verified by freeze
dry gravimetric analysis.
57
An even distribution and impartial response from the HPLC-CAD system with
regard to specific biological test structures was observed (Figure 12). With regard to
average percent error, three of the biological test standards displayed positive values,
while four displayed negative values. Taken together, surrogate, short chain and
biological test oligosaccharide standards were quantified to within less than a 19%
average error with excellent reproducibility by the developed HPLC-CAD methodology
(Figures 8, 10 and 12).
Profiling, Selection and Isolation of CGL Ligand Neutral Oligosaccharides
Initial characterization of the CGL ligand neutral oligosaccharides was
performed employing the Prevail Carbohydrate ES HPLC column (Figure 13 and Table
1). These chromatographic runs were intended to profile CGL ligand oligosaccharides
and to subsequently isolate some of the major oligosaccharide constituents. Due to an
unstable/elevated baseline, injection volumes exceeding column capacity limited the
ability to resolve oligosaccharides. Thus, a Hypercarb PGC HPLC column was
substituted for subsequent profiling and isolation of the CGL ligand neutral
oligosaccharides as described in materials and methods. However, the Prevail analysis
demonstrated the presence of approximately 17 oligosaccharide peaks. Both profiles
display four major oligosaccharide peaks, however a specific major oligosaccharide
peak observed on the Prevail column is not correlative to a specific major
oligosaccharide peak observed on the Hypercarb column as a result in their differences
in separation/selectivity mechanisms (Figures 13 and 14).
58
Table 1 – Initial characterization and profiling of the CGL ligand neutral
oligosaccharides on the Prevail Carbohydrate ES HPLC column. Estimated
concentration of the major oligosaccharide peaks are shown along with associated
calibration terms.
Retention
Time
(minutes)
7.485
9.061
11.455
12.660
14.377
16.533
17.577
18.663
18.696
20.545
21.693
21.721
23.517
24.409
25.584
28.114
29.685
31.298
32.619
Peak
Area
‘A’
Term
‘b’
Term
339
535
2971
2769
928
374
777
1220
1630
1779
1065
1320
653
398
619
1914
1385
3548
372
316
296
274
268
264
267
272
280
280
298
313
314
342
359
383
445
490
541
587
1.08
1.10
1.12
1.12
1.12
1.11
1.11
1.09
1.10
1.07
1.05
1.05
1.01
0.99
0.95
0.88
0.82
0.75
0.70
Predicted
Concentration
(µg/mL)
1.07
1.72
8.42
8.00
3.06
1.35
2.58
3.84
5.00
5.32
3.22
3.95
1.90
1.11
1.65
5.30
3.56
12.15
0.52
Percentage of
Oligosaccharide
Mass
1.45
2.33
11.42
10.85
4.15
1.83
3.50
5.21
6.78
7.22
4.37
5.36
2.58
1.51
2.24
7.19
4.83
16.48
0.71
59
The Hypercarb analysis demonstrated the presence of approximately 20
oligosaccharide peaks which was comparable to the Prevail chromatogram and taken
together, these HPLC profiles illustrate the abundance and complexity of CGL ligand
oligosaccharides consistent with previous NMR and MS studies [19,21,22]. Employing
the Hypercarb PGC column, CGL ligand oligosaccharide constituents that displayed the
greatest peak area (greatest overall oligosaccharide mass) and good baseline resolution
were targeted for fractionation and isolation (Figure 14 and Table 2). Since
oligosaccharide standards and subsequent calibration curves were not performed with
the PGC Hypercarb column, estimation of their concentrations could not be determined.
Although the separation mechanism between the Prevail Carbohydrate ES
HPLC and the Hypercarb PGC HPLC column differ significantly, the chromatographic
profiles are reasonably comparable with regard to the number of major oligosaccharide
peaks and their abundance. The separation mechanism of the Prevail column functions
under normal phase characteristics, exploiting hydrophilic interactions for the primary
basis of interaction. The Hypercarb PGC column on the other hand possess a unique
separation/selectivity mechanism based on a polar retention effect on graphite
producing a highly efficient separation of polar compounds of structural similarity.
60
mV
2.419
ADC1A, ADC1CHANNELA(NOAH\021909000001.D)
11.455
12.660
225
31.298
29.685
28.114
23.516
24.538
25.585
20.545
21.721
14.377
100
9.059
7.487
125
16.5
.662
3
17.762
18.696
150
32.617
175
34.104
200
75
50
5
10
15
20
25
30
35
min
Figure 13 – Prevail Carbohydrate ES HPLC chromatogram of a 1 µL injection of X.
laevis CGL ligand neutral O-linked oligosaccharides. Gradient method consisted of a
water/acetonitrile mobile phase with a decreasing percentage of actonitrile from 65% to
50% over 40 minutes.
61
Figure 14 – Hypercarb PGC HPLC chromatogram of a 10 µl injection of X. laevis CGL
ligand neutral oligosaccharides. Gradient method consisted of a water/acteonitrile
mobile phase with an increasing percentage of acetonitrile from 5% to 35% over 45
minutes. * Indicates peaks that were fraction collected.
62
Table 2 – HPLC-CAD profile of X. laevis CGL ligand oligosaccharides comprising all
targeted major peaks and their relative abundance.
Fraction
Retention Time
(minutes)
Peak Area
1
*
*
2
3
10.428
11.596
12.983
14.150
26.420
1986
1056
3438
2391
2302
Estimated Percentage of
Oligosaccharide Mass (Based on
Peak Area)
12
6
20
14
13
63
It is notable to point out that the initial large broad peak observed in the X. laevis
Prevail chromatogram (Figure 13) has been attributed to unretained non-oligosaccharide
material derived from the PGC-SPE purification procedure and stationary phase
degradation. This identical HPLC profile constituting this large abrupt peak was
observed employing a PGC-SPE blank and to a lesser extent an HPLC solvent blank
which accounted for all solvents, any stationary phase degradation and salts involved
with the SPE and HPLC procedures. Some of these unretained peaks were attributed to
autosampler vials and associated cap debris. Therefore, in an effort to focus on the
oligosaccharide profiles, all subsequent HPLC figures have been fitted to exclude the
large abrupt peak consisting of this unretained material (Figures 13 and 14).
Figures 15 and 16 were fitted in the same manner for a different reason based on
experimental observations during the lyophilization procedure of the CGL ligand
oligosaccharide fractions. During this procedure, it was observed that the sample vials
selected were incompatible with the lyophilization apparatus, which caused minor
etching of the centrifudge lid, which subsequently caused unretained shards of plastic to
enter the sample vials. This unretained material produced an initial large abrupt peak,
similar to that observed in Figure 13 (however to a lesser extent) in all chromatograms
analyzing the collected fractions. Therefore, employing a lyophilization control for the
isolated fractions in Figures 15 and 16 a blank subtraction method was utilized for
purity and concentration analysis.
64
The PGC Hypercarb CGL ligand oligosaccharide peaks displaying retention
times of 10.428, 14.150 and 26.420 minutes (fractions 1 through 3, respectively) were
selected for isolation, fractionation and subsequent quantification for plate binding
assays since they fit the following criteria: major peaks, good baseline resolution and
minimal interference by overlapping compounds (Figure 14 and Table 2). Initially, two
additional major oligosaccharide peaks were targeted (with a retention times of 11.596
and 12.983) however subsequent analysis via a secondary purification resulted in
multiple oligosaccharide peaks on the Prevail Carbohydrate ES HPLC column.
Resolution of any predominate components within these oligosaccharide aggregates to
homogeneity was difficult given the amount of starting material; thus, adequate mass
for subsequent biological assays was projected to be unattainable. As a consequence,
attempts to isolate these oligosaccharide peaks were abandoned.
Selected CGL ligand oligosaccharides were isolated by collecting fractions at
the specified time intervals relative to their maximum peak. By means of conducting
numerous rounds of chromatographic runs, an adequate amount of each oligosaccharide
fraction was collected for subsequent analysis and secondary purification (if deemed
feasible and necessary). This was accomplished by utilizing an analytical flow splitter
which was interfaced with a fraction collector, whereby 75% of the sample was
collected and the remaining 25% was consumed in the HPLC-CAD detection.
65
Quantification of Selected CGL Ligand Oligosaccharide Constituents
Prior to quantification analysis, purified CGL ligand oligosaccharides were run
in parallel to the surrogate quantification standards on the Prevail Carbohydrate ES
HPLC column to ensure complete coverage of the overall X. laevis CGL ligand
oligosaccharide mass (Figure 15).
66
ADC1 A, ADC1 CHANNEL A (NOAH\021909000002.D)
VWD1 A, Wavelength=205 nm (NOAH\021909000002.D)
ADC1 A, ADC1 CHANNEL A (NOAH\021909000001.D)
VWD1 A, Wavelength=205 nm (NOAH\021909000001.D)
10
34.104
32.617
31.298
32.677
28.761
29.685
28.114
25.585
23.516
24.538
21.721
20.545
15
39.376
39.408
18.501
17.762
16.562
16.663
14.377
12.676
11.250
50
4.843
100
9.120 9.059
7.487
150
18.696
200
12.660
11.455
250
24.420
19.509
300
35.679
14.652
350
10.477
7.453
mV
20
25
30
35
min
Figure 15 – Prevail Carbohydrate ES HPLC chromatogram overlay of surrogate
oligosaccharide quantification standards (glucose to maltooctaose at retention times of
7.453 through 35.679) and X. laevis CGL ligand oligosaccharide profile (displaying
retention times of 7.487 through 34.104). Gradient method consisted of a
water/acetonitrile mobile phase with a decreasing percentage of actonitrile from 65% to
50% over 40 minutes.
67
The developed HPLC-CAD quantification methodology was applied to the
selected neutral CGL ligand O-linked oligosaccharide fractions as described previously.
CGL ligand oligosaccharide fractions (fractions 1 through 3) were reconstituted to
produce approximate fraction concentrations at 10 µg/mL, thus allowing accurate
quantification by the non-linear calibration scheme. Additionally, utilizing a blank
subtraction method, to account for any SPE or intrinstic HPLC contaminates as
discussed previously, purity of each CGL ligand oligosaccharide fraction was
determined (Figures 16 and 17).
68
*ADC1A, ADC1CHANNELA(NOAH\060209000007.D- NOAH\060209000002.D)
6.879
mV
250
200
150
100
50
0
6
8
10
12
min
14
A. Fraction 1 with associated CAD signal.
*ADC1A, ADC1CHANNELA(NOAH\060209000009.D- NOAH\060209000002.D)
7.157
mV
300
250
200
150
100
50
0
6
8
10
12
14
16
min
18
min
B. Fraction 2 with associated CAD signal.
*ADC1A, ADC1CHANNELA(NOAH\060209000011.D- NOAH\060209000002.D)
12.994
mV
200
150
100
6.355
50
0
8
10
12
14
16
C. Fraction 3 with associated CAD signal.
Figure 16 – Prevail Carbohydrate ES HPLC chromatogram purity and quantification
analysis utilizing a blank subtraction method with manual integration for fractions 1
through 3 (from A to C panels, respectively). Fractions 1, 2 and 3 correspond to
retention times of 10.428, 14.150 and 26.420 on the Hypercarb HPLC column.
69
Additionally, fractions 1 through 3 were measured by UV for the presence of Nacetyl groups of the hexosamine residues of the isolated oligosaccharides at 205nm
(Figure 17). The UV signals generated for fractions 1 through 3 indicate the presence of
N-acetyl groups within these oligosaccharide fractions, thus further corroborating the
presence of oligosaccharide components in agreement with that of the CAD signal
(Figure 16). In addition to the CAD signal, the UV data demonstrated a single
predominate oligosaccharide component for fractions 1 through 3. Collectively, the UV
and CAD data demonstrate the absence of any significant additional interfering
oligosaccharide components within the isolated and quantified CGL ligand
oligosaccharide fractions 1 and 2. However, fraction 3 did contain an additional, yet
significant oligosaccharide component, Figure 16C at observed retention time of 12.994
minutes.
70
*VWD1A, Wavelength=205nm(NOAH\060209000007.D- NOAH\060209000002.D)
mAU
6.705
5
4
3
2
1
0
5
6
7
8
9
10
12 min
11
A. Fraction 1 with associated UV signal.
*VWD1A, Wavelength=205nm(NOAH\060209000009.D- NOAH\060209000002.D)
mAU
6.981
6
5
4
3
2
1
0
-1
6
7
8
9
10
11
min
B. Fraction 2 with associated UV signal.
*VWD1A, Wavelength=205nm(NOAH\060209000011.D- NOAH\060209000002.D)
12.805
mAU
3
2.5
2
1.5
1
0.5
0
-0.5
10
11
12
13
14
15
16
min
C. Fraction 3 with associated UV signal.
Figure 17 – Prevail Carbohydrate ES HPLC chromatogram of UV signals for fractions
1 through 3 (from A to C panels, respectively).
71
The isolated CGL ligand fractions were respectively pooled together and
subsequently lyophilized (freeze dried). The lyophilized powder was reconstituted in
volumes that yielded approximately 10 µg/mL concentrations in efforts to provide an
accurate quantification analysis utilizing the middle portion of the calibration curve.
Aliquots were removed and subjected to the developed HPLC-CAD quantitative
analysis scheme. Based on the concentrations produced from the quantification analysis
of the removed aliquots, total mass of the reconstituted vials was calculated. The
remaining volume containing known oligosaccharide mass was then lyophilized again
for forthcoming biological assays.
Overall, the successful isolation of hundreds of nanograms to microgram
quantities along with quantification and purity greater than 85% of the three fractions
was achieved with the initial separation and isolation by the Hypercarb PGC HPLC
column (Table 3). Utilizing a blank subtraction method, fractions 1 and 2 demonstrated
excellent purity (> 90%), while fraction 3 had a significant amount of an additional
oligosaccharide component. Thus, fraction 3 was subsequently enriched for the major
oligosaccharide component via a secondary purification (see materials and methods) to
attain approximately 92% purity.
72
Table 3 – CGL ligand oligosaccharide fractions with corresponding concentrations as
determined
by
the
developed
HPLC-CAD
quantification
methodology
and
oligosaccharide mass isolated, accounting for removed quantification aliquots and
estimated purity.
* Fraction containing significant additional oligosaccharide components and subjected
to a secondary purification.
Fraction
Retention Time
(minutes)
1
2
3
6.879
7.157
12.994
Predicted
Concentration
(µg/mL)
9.11
13.1
10.34
Mass Collected
(µg)
Estimated
Purity (%)
1.53
2.06
0.567
96
90
92*
73
Characterization of X. laevis Egg Jelly and CGL Fertilization Constituents
Prior to biological assays involving the isolated and quantified CGL ligand
oligosaccharides, characterization of the X. laevis fertilization constituents (i.e. egg jelly
and CGL) involved in the block to polyspermic fertilization were analyzed by means of
SDS-PAGE gel analysis. X. laevis egg jelly (extracted and enriched for CGL ligands by
Dr. Peavy) was separated and selectively stained for the sulfated CGL ligands utilizing
a thiosulfate silver/Alcian blue staining protocol as described by Bonnell et al. (Figure
18) [25]. The SDS-PAGE analysis of the egg jelly preparation demonstrates the
presence of two heavily glycosylated, high-molecular weight components greater than
200 kDa which stained intensely for the Alcian blue dye and appeared blue on the gel
(lane 2). As a reference, lane 3 contains dextran sulfate, a high-molecular weight sugar
polymer possessing charged sulfated residues exhibiting a molecular weight range in
excess of 500 kDa. The dextran sulfate contained in lane 3 also stained intensely for the
Alcian blue dye and appeared blue on the gel. This work corroborates previous findings
by Bonnell et al., which meticulously demonstrated the presence of two high-molecular
weight glycoconjugates within the J1 egg jelly component possessing molecular
weights of 450 kDa and 630 kDa by means of Alcian blue staining (Figure 18) [6,25].
Taken together, the current study, along with previous research are in agreement
in demonstrating the two CGL ligand glycoproteins of interest, exhibiting molecular
weights of ~ 450 kDa and ~ 630 kDa are present in the egg jelly (Figure 18 lane 2).
Additionally, previous SDS-PAGE analyses of the egg jelly extract demonstrated the
74
presence of multiple jelly components in addition to the two CGL ligands [25]. Figure
18 demonstrates the absence of these additional jelly components demonstrating the
preparation is highly enriched for the CGL ligands.
75
200 kDa
116 kDa
97 kDa
66 kDa
45 kDa
31 kDa
1
Molecular
Weight
Standards
2*
X. Laevis
Egg Jelly
3*
Dextran
Sulfate
Figure 18 – Thiosulfate-silver/Alcian blue staining of a 7.5% SDS-PAGE gel
comprising X. laevis egg jelly illustrating the presence of the CGL ligands (lane 2) and
as a reference, dextran sulfate (lane 3). Lane 1 contains 10µL of broad range molecular
weight protein standards (myosin, 200,000; β-galactosidase, 116,000; phosphorylase b,
97,000; serum albumin, 66,000; ovalbumin, 45,000; and carbonic anhydrase, 31,000).
Lane 2 contains 44 µg of X. laevis egg jelly extract (enriched for the CGL ligands)
based on carbohydrate content and lane 3 contains 5 µg of dextran sulfate (> 500,000).
* In addition to staining for silver, these lanes stained intensely for Alcian blue and
appeared blue on the gel.
76
X. laevis CGL has been isolated and well characterized from chemical analysis
to be a metalloglycoprotein, composed of 84% protein, 15.8% carbohydrate and 0.19%
calcium [9]. Additionally, the CGL metalloglycoprotein is composed of 10-12 subunits
which form a tertiary structure composed of non-covalently bound monomers with
identical polypeptide backbones [36]. These 10-12 subunits form an oligomeric
complex comprising a molecular weight of approximately 450 kDa whereas the
monomeric subunit size is between 46-58 kDa [9,36]. In this work, purified X. laevis
CGL (provided by Dr. Peavy) was biotinylated (refer to materials and methods) in an
attempt to provide a sensitive detection method for subsequent biological binding
assays involving the isolated and quantified CGL ligand oligosaccharides [11,12].
Figure 19 is an SDS-PAGE gel image consisting of purified CGL (lane 2) and biotinconjugated CGL (lane 4) for comparison purposes. Lane 2 contains the native CGL
possessing an apparent molecular weight of approximately 47-55 kDa in agreement
with previous work [9,15,36]
It has been postulated that microheterogeneity in CGL glycosylation is the cause
for the observed broad diffuse bands in SDS-PAGE separations of the CGL. This
heterogeneity appears to be due to variation in N-linked glycosylation since PNGaseF
(enzyme that cleavages off N-linked oligosaccharides from the asparagine residue)
treatment results in a single distinct CGL band [15,36]. The molecular weight of the
CGL polypeptide chain after deglycoslyation by SDS-PAGE was found to be 39 kDa
[9,15,36]. Comparison between the native CGL and the biotinylated CGL via SDS-
77
PAGE analysis, demonstrates a modest increase in molecular weight of the native CGL
as a result of the incorporated biotinylation reagent (Figure 19 lane 2 vs. 4) as indicated
by an upward shift in band migration.
78
200 kDa
116 kDa
97 kDa
66 kDa
45 kDa
31 kDa
1
Molecular
Weight
Standards
2
Native
CGL
3
4
Biotinylated
CGL
Figure 19 – Coomassie Brilliant Blue staining of a 10% SDS-PAGE gel comprising X.
laevis native CGL (lane 2) and biotinylated CGL (lane 4). Lane 1 contains 10µL of
broad range molecular weight protein standards (myosin, 200,000; β-galactosidase,
116,000; phosphorylase b, 97,000; serum albumin, 66,000; ovalbumin, 45,000; and
carbonic anhydrase, 31,000). Lane 2 contains 7µg of native X. laevis CGL and lane 4
contains 9µg of biotinylated CGL.
79
Competitive Enzyme-Linked Lectin Assays (ELLA) and CGL Binding Specificities
After obtaining the preparations of the X. laevis egg jelly ligands, isolated
oligosaccharides and biotinlyated CGL, a plate binding assay (ELLA) was developed
based on the modification of methodology utilized in previous studies [11,12]. In brief,
the refined ELLA was set-up by coating the wells of a 96-well plate with the
preparation of X. laevis egg jelly ligands, blocking all other available binding sites, and
then subsequently incubating with biotinlyated CGL. During the biotinlyated CGL
incubation period, potential inhibitors of the CGL-ligand binding interaction were
included in the binding buffer to measure the relative inhibition of biotinlyated CGL
bound to ligand. This is in essence a competitive inhibition binding assay such that if a
particular sugar (or oligosaccharide) inhibits CGL binding, then it is assumed to be
competing with the ligand oligosaccharides for the binding pocket. After the incubation
period (with or without test inhibitors), the relative amount of biotinylated CGL bound
to the ligand was detected by secondary incubation with an avidin-peroxidase enzyme
conjugate which forms a biotin-avidin complex. The amount of avidin-peroxidase
enzyme bound is then measured by using a peroxidase substrate that when cleaved will
be colorimetrically detected.
Since similar plate binding assays have been performed on CGL (albeit using
different reagents that are predicted to be less sensitive for detection purposes), the
optimal binding conditions with respect to the specific buffer and salt composition
(10mM Tris-HCl, 150mM NaCl, 10mM CaCl2, 0.05% Tween 20, pH 7.5) were adapted
80
from prior studies [11,12]. The positive control for these ELLA studies is the relative
amount of colorimetric product generated when biotinylated CGL is bound to its ligands
in this optimal binding buffer, and is considered maximal or 100% binding. All other
treatments or additions (e.g. potential inhibitors) to the incubation media are then
compared to the positive control to determine the decrease or percent inhibition of
binding.
Other appropriate controls were performed to define the most optimal binding
assay parameters such as omitting specific constituents from the reaction wells such as
the biotinylated CGL, egg jelly, calcium ions (via chelation with EGTA) or the
detection reagents (i.e. ExtrAvidin Peroxidase). Figure 20 demonstrates that binding
between the CGL and its ligand (coating the well) was nearly abolished when any of the
above constituents were omitted. Notably, there was a modest signal in the wells
lacking egg jelly (~ 10% signal relative to the native lectin-ligand binding). However,
this was anticipated to some extent since the biotinylaed CGL itself may exhibit some
adherence (despite the blocking and washing steps) to the microtiter well surfaces, and
thus produce a false-positive signal via non-specific binding. Nevertheless, this nonspecific binding was deemed insignificant.
81
Reactivty of Enzyme-Linked Lectin Assay
Absorbance (630 nm)
1.2
1
0.8
0.6
0.4
0.2
0
Lectin-Ligand
(100% Binding)
No Egg Jelly
No Peroxidase
No CGL
EGTA Treatment
Figure 20 – Reactivity of the enzyme-linked lectin assay in response to omitting
specific fertilization constituents pivotal in the lectin-ligand binding interaction. The
mean and standard deviation for triplicate trials are shown with regard to each
experimental condition.
82
Furthermore, the calcium dependent binding interaction between the CGL to its
ligand partners was demonstrated by the incorporation of 10 mM EGTA into the
binding buffer with biotinylated CGL. EGTA is a chelating agent (binds to or
complexes with metal ions so they are unavailable for other interactions) that is related
to the chemical EDTA. EDTA chelates divalent metal cations in general (e.g. Mn2+,
Mg2+ and Ca2+) wheras EGTA specifically chelates Ca2+ which has been shown to be
an essential co-factor in the lectin-ligand binding interaction. EGTA proved to be a
potent inhibitor, resulting in total inhibition of the CGL-ligand interaction and thus
corroborating this observation from prior studies (Figure 20) [9,11,12].
Plate binding assays were also performed with selected monosaccharide and
disaccharide inhibitors as appropriate controls to establish selectivity and sensitivity as
a comparison to previous studies. Selected inhibitors at varying concentrations were
incorporated during the biotinylated CGL incubation [9,11,12]. Galactose, lactose and
melibiose were utilized since they have all been shown to be potent inhibitors of the
lectin-ligand binding interaction.
In particular, galactose has been well established and widely utilized as a potent
and specific inhibitor of the lectin-ligand binding interaction. Figure 21 illustrates the
percent inhibition of CGL binding (as compared to the positive control) in a dosedependent manner when the binding media includes either galactose or glucosamine.
Increasing concentrations of galactose resulted in increased inhibition of CGL binding
83
to the egg jelly ligands with maximal inhibition (nearly 100%) achieved at
approximately 75 mM galactose. At 10 mM galactose, approximately 50% inhibition
was obtained. When glucosamine (a suspected non-inhibitor) was substituted for
galactose, a < 5% inhibition was obtained at 12 mM, however increasing the
concentration of this monosaccharides up to 100 mM did not result in a significant
increased inhibitory effect (< 20%) as observed with galactose (Figure 21).
84
CGL-Ligand Monosaccharide Inhibition
Percent Inhibition
100
80
60
Galactose
40
Glucosamine
20
0
0
20
40
60
80
100
Concentration (mM)
Figure 21 – Monosaccharide inhibition of the enzyme-linked lectin assay for CGL
ligand. Xenopus laevis egg jelly, 500 ng carbohydrate, was adsorbed to a microtiter
plate and incubated with 75 ng biotinylated CGL in the presence of the indicated
concentrations of monosaccharide. Each point represents the average of triplicate
assays.
85
Glucose was demonstrated to possess intermediate inhibitory characteristics,
increasing concentrations of glucose resulted in increased inhibition of CGL binding to
the adsorbed egg jelly with maximal inhibition of 55% achieved at 100 mM (Figure 22).
Interestingly, fucose (6-deoxy-L-galactose) demonstrated near identical reactivity
compared to galactose at each concentration (Figures 22 and 23). At 5, 12 and 25 mM
identical inhibitory activity was observed, however at 50, 75 and 100 mM a slightly
decreased inhibition was obtained. At concentrations of 50 mM and greater, fucose was
able to inhibit the lectin-ligand binding to greater than 90% however unlike galactose,
fucose was unable to completely inhibit the CGL-ligand binding reaction (Figures 22
and 23).
86
CGL-Ligand Monosaccharide Inhibition
Percent Inhibition
100
80
60
Fucose
40
Glucose
20
0
0
20
40
60
80
100
Concentration (mM)
Figure 22 – Monosaccharide inhibition of the enzyme-linked lectin assay for CGL
ligand. Xenopus laevis egg jelly, 500 ng carbohydrate, was adsorbed to a microtiter
plate and incubated with 75 ng biotinylated CGL in the presence of the indicated
concentrations of monosaccharide. Each point represents the average of triplicate
assays.
87
CGL-Ligand Monosaccharide Inhibition
Percent Inhibition
100
80
Galactose
60
Fucose
40
Glucosamine
20
0
0
20
40
60
80
100
Concentration (mM)
Figure 23 – Monosaccharide inhibition of the enzyme-linked lectin assay for CGL
ligand. Xenopus laevis egg jelly, 500 ng carbohydrate, was adsorbed to a microtiter
plate and incubated with 75 ng biotinylated CGL in the presence of the indicated
concentrations of monosaccharide. Each point represents the average of triplicate
assays.
88
Previous research has also demonstrated that the galactose containing
disaccharides lactose and melibiose were potent inhibitors of the lectin-ligand binding
interaction so these were also examined. Figure 24 illustrates the percentage of CGL
binding that was inhibited when incubated with the disaccharides lactose, melibiose and
sucrose (a suspected non-inhibitor). Both lactose and melibiose elicited a greater
inhibitory effect than galactose at concentrations of 5, 12 and 25 mM. Increasing
concentrations of lactose and melibiose resulted in increased inhibition of CGL binding
to the adsorbed egg jelly. Both lactose and melibiose reached a 50% level of inhibition
at approximately 5 mM and obtained maximal inhibition of 100% at 75 and 100 mM,
respectively (Figure 24). Although lactose and melibiose displayed a greater inhibitory
response at lower concentrations relative to galactose, ultimately all three compounds
obtained maximal inhibition at or near 75 mM. Sucrose achieved ~15% inhibition at 12
mM, however increasing the concentration of this disaccharide up to 100 mM did not
result in a significant increase (< 20%) (Figure 24).
89
CGL-Ligand Disaccharide Inhibition
Percent Inhibition
100
80
Lactose
60
Melibiose
40
Sucrose
20
0
0
20
40
60
80
100
Concentration (mM)
Figure 24 – Disaccharide inhibition of the enzyme-linked lectin assay for CGL ligand.
Xenopus laevis egg jelly, 500 ng carbohydrate, was adsorbed to a microtiter plate and
incubated with 75 ng biotinylated CGL in the presence of the indicated concentrations
of disaccharide. Each point represents the average of triplicate assays.
90
The disaccharides maltose and lactulose were also tested and were found to
possess an intermediate level of inhibitory activity (Figure 25). Increasing
concentrations of both maltose and lactulose resulted in increased inhibition of CGL
binding to the adsorbed egg jelly with a maximal inhibition achieved at ~50 and ~75%
at 100 mM, respectively. Increasing the concentrations of these disaccharides from 5
mM out to 100 mM resulted in a relatively dose-dependent increase in inhibitory
activity, however inhibitory activity reached an upper limit near 75 mM for both
disaccharides (Figure 25).
91
CGL-Ligand Disaccharide Inhibition
Percent Inhibition
100
80
Lactose
60
Lactulose
40
Maltose
20
0
0
20
40
60
80
100
Concentration (mM)
Figure 25 – Disaccharide inhibition of the enzyme-linked lectin assay for CGL ligand.
Xenopus laevis egg jelly, 500 ng carbohydrate, was adsorbed to a microtiter plate and
incubated with 75 ng biotinylated CGL in the presence of the indicated concentrations
of disaccharide. Each point represents the average of triplicate assays.
92
Collectively, the refined ELLA exhibited a modest background signal with
regard to the presumed non-inhibitory mono- and disaccharide controls (glucosamine
and sucrose). The effects of these controls elicited less than a 20% reduction in the
lectin-ligand binding interaction at 100 mM. Additional controls (glucose, maltose and
lactulose) elicited an intermediate level of inhibitory activity in the range of 50-75% at
100 mM levels. To discern specific from non-specific inhibition and to distinguish
whether or not the modest background signal elicited by these controls were an artifact
of the experiment, a 3% BSA treatment (inclusion in the binding media) was performed.
The BSA treatment yielded comparable CGL-ligand binding inhibition activity as did
the controls glucosamine and sucrose, eliciting a ~ 20% reduction in lectin-ligand
binding activity (data not shown). Therefore, the minor inhibitory response elicited by
both glucosamine and sucrose was deemed non-specific.
Taken together, both monosaccharide and disaccharide competitive binding
assay analysis revealed CGL binding specificities for fucose, galactose and galactose
containing disaccharides (lactose, melibiose and lactulose) (Figures 26 and 27). No
definitive anomeric specificity with regard to glycosidic linkages was discerned at the
disaccharide
level,
however.
The
disaccharide
melibiose
[α-
galactopyrasoyl(1,6)glucose] was slightly more inhibitory than galactose, however
lactose [β-galactopyransoyl(1,4)glucose] was significantly more inhibitory than
galactose and melibiose at 5, 12 and 25 mM. The disaccharide lactulose [βgalactopyransoy(1,4)fructose] was less inhibitory than galactose, lactose and melibiose
93
at all tested concentrations. Therefore, both 1-4 and 1-6 glycosidic disaccharide
linkages comprising galactose and glucose monomers were capable of inhibiting the
CGL lectin-ligand binding, and the β-derivative (lactose) appeared to be marginally
more effective than the α-derivative (melibiose) in inhibiting the CGL-ligand binding
interaction. Interestingly, the 1-4 glycosidic linkage between galactose and fructose
monomers, which constitute the disaccharide lactulose was significantly less inhibitory
than disaccharides comprised of galactose and glucose monomers (Figure 27).
94
6
CH2OH
5
O
H
4
OH
1
OH
3
2
H
OH
H
NH2
H
Galactose
Glucosamine
C-2
Position
H
O
H
H
CH3
H
OH
OH
OH
OH
Glucose
H
Fucose
Figure 26 – Monosaccharide structures illustrating the anomeric binding specificity of
the CGL in the lectin-ligand binding reaction, highlighting the importance of the C-2
position and orientation of various hydroxyl groups, notably carbon positions 2-4 within
these monosaccharide structures.
95
Lactulose
Gal(β1-4)Fru
Figure 27 – Disaccharide structures illustrating specific glycosidic linkages and
anomeric specificity of the CGL in the lectin-ligand binding reaction.
96
Competitive Plate Binding Assays Involving CGL Ligand Oligosaccharide Fractions
Initially, CGL ligand oligosaccharide inhibition analysis was performed utilizing
the whole oligosaccharide fractions released from the SPE cartridge in order to obtain a
relative semi-quantitative measurement of CGL ligand oligosaccharide inhibition
characteristics and whether or not these whole fractions possessed biological activity.
This analysis involved both neutral, acidic and a neutral and acidic combination in an
effort to relatively assess the inhibitory properties of these aggregate fractions (Figure
28). A relative comparison between these SPE fractions was performed by combining
two SPE fractions of eluted neutral CGL ligand oligosaccharides for one treatment.
Another treatment consisted of combining two SPE fractions of eluted acidic CGL
ligand oligosaccharides. The final treatment consisted of combining one SPE fraction of
neutral oligosaccharides and one SPE fraction of acidic oligosaccharides to assess if
there was a combined inhibitory effect. It is noteworthy to point out that amount of
neutral oligosaccharide mass contained in the SPE fractions is likely not comparable to
the amount of acidic oligosaccharide mass, thus equal proportions may not have been
incubated together. Therefore, this inhibitory analysis is relative and only semiquantitative.
97
100
75
50
25
SPE
Combination
SPE Acidics
SPE
Neutrals
SPE/HPLC
Treatment
Glucosamine
100 mM
Sucrose 100
mM
Galactose
100 mM
0
EGTA
Treatment
Percent Inhibition
SPE Released CGL Ligand Oligosaccharides
Experimental Condition
Figure 28 – Inhibition of the enzyme-linked lectin assay for whole SPE released CGL
ligand oligosaccharide fractions. Xenopus laevis egg jelly, 500 ng carbohydrate, was
adsorbed to a microtiter plate and incubated with 75 ng biotinylated CGL in the
presence of neutral, acidic and a combination of neutral and acidic CGL ligand
oligosaccharide fractions. All controls represent the average of triplicate assays.
98
Qualitatively, the whole CGL ligand oligosaccharide inhibition analysis
revealed strong inhibition characteristics from the SPE derived neutral oligosaccharides,
eliciting a 95% inhibition of CGL binding to the adsorbed egg jelly. Conversely, the
acidic oligosaccharides displayed substantially less inhibitory properties, nevertheless,
elicited a 35% reduction in lectin-ligand binding. The combined neutral and acidic
oligosaccharide SPE fraction mixture did not achieve an intermediate value between the
two, but was higher than the acidic fraction alone (39% inhibition). Taken together, this
data indicates a prominent role of the neutral CGL ligand oligosaccharides in the
specificity for the CGL in preventing binding to its ligand partners contained in the
adsorbed egg jelly.
To further assess the biological role of the isolated and quantified CGL ligand
oligosaccharides derived from the CGL ligand, fractions 1, 2 and 3 (Figures 16 and 17)
were incubated with the native lectin-ligand interaction to determine whether any of
these fractions exhibited inhibitory effects. Initially, the molecular weight of each
fraction was estimated based on the HPLC chromatograms of the surrogate
oligosaccharide standards with respect to retention times. Fractions 1 and 2 possessed
retention times of 6.705 and 6.981 and thus were estimated to be an approximate degree
of polymerization (DP) equivalency of maltotriose (MW ~ 540 kD). Fraction 3 had an
observed retention time of 12.805 and thus was estimated to be an approximate DP
equivalency of maltohexaose (MW ~ 1080 kD).
99
Molecular weights of the CGL ligand oligosaccharide fractions were estimated
based on the retention times observed by the DP surrogate standards, however it is
notable to point out that branched structures containing the same number of monomer
subunits did not possess the same elution profiles. Branched structures such as LNFP II
(5 subunits) and LNH (6 subunits) displayed retention times equivalent to DP 3 and DP
5, respectively.
Once estimated molecular weights were established, the biological activity of
fractions 1, 2 and 3 were prepared and analyzed at identical micromolar concentrations
for a true comparative analysis to observe relative binding inhibition between the three
CGL ligand oligosaccharide fractions. In addition to mono- and disaccharide inhibition
controls (galactose, glucosamine and sucrose), a separate control was deemed necessary
to test whether there were any inhibitive compounds potentially in the isolated
oligosaccharides derived from the chemical release of the oligosaccharides and their
separation using the HPLC-CAD instrument. Thus, a mock sample (no glycoprotein)
was processed in an identical fashion from borohydride chemical release to HPLC-CAD
fraction collection and used in the ELLA (see materials and methods). No inhibitory
effect was noted for this mock sample control (Figure 29; labeled as SPE/HPLC
treatment)
All three ligand oligosaccharide fractions were prepared at 10 µM
concentrations (due to the limiting mass isolated from fraction 3) and separately
100
incorporated during the biotinylated CGL incubation as usual to determine if any of
these had an inhibitory effect on CGL binding (Figure 29). No inhibitory effects were
observed beyond that of the negative controls (e.g. glucosamine and sucrose) for any of
the CGL ligand oligosaccharide fractions at 10 µM concentrations.
CGL Ligand Oligosaccharide Comparative Analysis (10 uM)
100
75
50
25
ct
os
e
Tr
ea
tm
G
al
a
EG
TA
10
0
Su
m
M
cr
os
e
G
10
lu
co
0
m
sa
M
m
in
e
SP
10
E/
0
m
HP
M
LC
Tr
CG
ea
L
tm
Li
en
ga
t
nd
Fr
CG
ac
L
t io
Li
n
ga
1
nd
Fr
CG
ac
L
t io
Li
n
ga
2
nd
Fr
ca
t io
n
3
0
en
t
Percent Inhibition
101
Experimental Condition
Figure 29 – Inhibition of the enzyme-linked lectin assay for CGL ligand oligosaccharide
fractions 1, 2 and 3. Xenopus laevis egg jelly, 500 ng carbohydrate, was adsorbed to a
microtiter plate and incubated with 75 ng biotinylated CGL in the presence of 10 µM
concentrations of CGL ligand oligosaccharide fractions 1, 2 and 3. All controls
represent the average of triplicate assays.
102
Fractions 2 and 3 were subsequently analyzed at 45 µM concentrations while
fraction 3 was excluded due to being the limiting fraction based on mass (and thus was
consumed during the 10 µM comparative analysis). No significant inhibition was also
observed for the 45 µM inhibition assay for these two oligosaccharide fractions (Figure
30).
103
100
75
50
25
2
Fr
ac
t io
n
1
Li
L
CG
CG
L
Li
ga
nd
Fr
ac
t io
n
en
t
ga
nd
Tr
ea
tm
M
HP
LC
SP
E/
G
lu
co
sa
m
in
e
10
0
10
0
m
m
m
M
Su
cr
os
e
10
0
ct
os
e
G
al
a
Tr
ea
tm
EG
TA
M
0
en
t
Percent Inhibition
CGL Ligand Oligosaccharide Comparative Analysis (45 uM)
Experimental Condition
Figure 30 – Inhibition of the enzyme-linked lectin assay for CGL ligand oligosaccharide
fractions 1 and 2. Xenopus laevis egg jelly, 500 ng carbohydrate, was adsorbed to a
microtiter plate and incubated with 75 ng biotinylated CGL in the presence of 45 µM
concentrations of CGL ligand oligosaccharide fractions 1 and 2. All controls represent
the average of triplicate assays.
104
DISCUSSION
The block to polyspermic fertilization in Xenopus laevis is mediated by a
calcium-dependent, galactose specific binding reaction between a lectin derived from
the cortical granules and its ligand partners located in the immediate surroundings
within the egg extracellular matrix. The cortical granule lectin (CGL) ligands have been
shown to possess O-linked oligosaccharides as the functional moieties in binding
activity with binding specificity for the CGL in producing the functional block to
polyspermy. In the current work, an HPLC based oligosaccharide isolation and
quantification methodology was developed in an attempt to directly isolate and
accurately quantify functionally relevant ligand O-linked oligosaccharides. Isolation
and quantification of specific oligosaccharide constituents derived from the CGL
ligands were subsequently analyzed via a refined competitive enzyme-linked lectin
assay (ELLA) in an attempt to assess their biological activity. Elucidation of the CGL
ligand oligosaccharide constituents and determining whether they can bind to the CGL
will likely advance our understanding of fertilization and cell-cell binding interactions
involving the CGL and the eglectin family of CGL-like proteins since the binding
properties and biological role appear to be evolutionarily conserved. This work was
centered on contributing to a more definitive molecular mechanism and role in the
block to polyspermic fertilization involving the lectin-ligand binding interaction.
105
The current work has substantially expanded on the utility of the HPLC-CAD
device for the detection, quantification and isolation of underivatized biologically
derived oligosaccharides for subsequent biological assays. The isolation and
quantification HPLC-CAD method presented in the results section demonstrated
excellent sensitivity in the analysis of oligosaccharides. This detection method has
successfully demonstrated sensitivity in the picomolar range or mass detection limits of
0.3 to 0.7 ng with regard to oligosaccharides under optimized conditions. In
comparison, both UV and oligosaccharide derivatization procedures have demonstrated
sensitivities in the picomolar range as well, however the HPLC-CAD method was
demonstrated to be approximately five times more sensitive than conventional UV
absorbance detection. Conversely, the HPLC-CAD was demonstrated to be
approximately ten times less sensitive than derivatization detection with regard to
oligosaccharides [30].
This developed HPLC-CAD system provided a universal detection method
along with greater sensitivity than previous HPLC-detection based methods. Also, since
the detection method is universal (non-volatile charged particle detection),
quantification was demonstrated to be possible without exact oligosaccharide standards
[20,21,23]. This developed HPLC-CAD quantification methodology has proven to be
superior to conventional HPLC-detection based methods in its ability to quantify and
isolate underivitized biologically relevant
oligosaccharides. This circumvents
106
previously encountered limitations with current derivitization methodologies since
removal of these derivitization agents is required for subsequent biological assays.
With regard to the HPLC-CAD quantitative work, a non-linear calibration
scheme was generated and utilized as a result of surrogate oligosaccharide standards not
displaying a true linear response. Therefore, ‘A’ and ‘b’ terms derived from power-fit
calibration equations were employed as appropriate calibration terms for the
quantification of oligosaccharides. The surrogate oligosaccharide standards displayed
definitive trends in ‘A’ and ‘b’ terms as a function of retention time, allowing the
prediction of calibration parameters for test and unknown oligosaccharides. Therefore,
based on observed retention time, ‘A’ and ‘b’ values are established and along with
peak area, concentration of an unknown oligosaccharide was determined.
Interestingly, surrogate oligosaccharide standards maltohexaose (DP 7) and
maltooctaose (DP 8) displayed a significantly decreased response compared to the other
standards, glucose though maltohexaose and maltosyl-β-cyclodextrin in the power-fit
concentration to peak area relationship. Additionally, both DP 7 and DP 8 displayed
significant deviations from both ‘A’ and ‘b’ calibration trend lines as well, indicating
that these standards may be somewhat suspect with regard to purity considering the
discontinuous nature in response at the latter portions of these calibration curves
involving these larger DP surrogate standards. Furthermore, maltooctaose was the only
surrogate oligosaccharide standard that displayed an absolute average percent error in
107
excess of 5%, suggesting that purity may be an issue as indicated by consistently
decreased response by the HPLC-CAD instrument.
Collectively, the developed HPLC-CAD quantitative work involving surrogate,
short chain and biological test oligosaccharide structures were accurately quantified to
within less than a 19% average error with excellent reproducibility. Employing the nonlinear calibration scheme, all surrogate oligosaccharide standards (glucose through
maltooctaose and maltosyl-β-cyclodextrin) were accurately quantified to within a 5%
error, with the exception of maltooctaose, which displayed an average percent error of 10.7%. There were no correlative relationships among degree of polymerization and
average percent error or standard deviation. The developed HPLC-CAD methodology
appeared to possess an impartial response in the quantification of these surrogate
oligosaccharide standards. It is noteworthy to point out that maltosyl-β-cyclodextrin
was utilized as an additional surrogate oligosaccharide standard despite its structural
dissimilarity to the other glucose oligomers. This was deemed necessary in order to
extend the quantification calibration curves to accommodate the quantification of the
larger biological test oligosaccharide structures. Although it would have been preferred,
a linear glucose oligomer possessing a degree of polymerization (DP) of 9 was not
commercially available therefore this compound was selected in an effort to substitute
for the projected retention time that a DP 9 would possess.
108
The initial test oligosaccharides (isomaltotriose, stachyose, raffinose and
melezitose) were all accurately quantified to within less than an absolute average
percent error of 11%. Stachyose and melezitose displayed comparable responses and
variations of 1.58 +/- 13.7 and -3.81 +/- 11.9, respectively. The variations between both
of these compounds spanned both negative and positive territories and thus the
responses were not consistently clustered in either direction, suggesting some intrinsic
variability in response by the HPLC-CAD instrument. Isomaltotriose and raffinose on
the other hand displayed comparable responses and variations of -10.8 +/- 7.07 and 10.2 +/- 8.18, respectively. The response of both of these compounds consistently
resulted in negative values and thus the variations were clustered in negative territory.
Considering these two compounds always elicited a negative response in quantification
from the HPLC-CAD instrument, this may signify a decrease in water retention by these
compounds, which may have affected nebulization efficiency in relation to
oligosaccharide
standards,
thus
underestimating
the
quantification
of
these
oligosaccharides [31]. Despite the observed intrinsic variability in HPLC-CAD response
of these initial test oligosaccharides, low average percent errors along with a defined
range of variability in response demonstrated potential for the quantification of more
complex oligosaccharides translatable to functional moieties found on the CGL ligand
oligosaccharide moieties.
Biological test oligosaccharide standards of varying complexity, representative
of oligosaccharide moieties found on glycoproteins displayed a modest overall increase
109
in average percent error and variation in relation to both the surrogate and initial test
oligosaccharides. There were no correlative relationships between core oligosaccharides
(MAN-3 and LNH) and complex high-mannose type oligosaccharides (NA2 and NA4)
with regard to absolute average percent errors (7.31 vs. 6.99), however variation in
response appeared to increase as complexity increased (4.1 vs. 11.6). Additionally,
structures containing residues found to exist in the CGL ligand oligosaccharides by
NMR
and
MS
[19,21,22]
such
as
core
glycoprotein
oligosaccharide
N-
acetylglucosamine structures with terminal fucose residues (LDFT), and Nacetylglucosamine structures with terminal fucose and galactose residues (LNFP II),
displayed very low average percent errors with excellent reproducibility (6.63 +/- 8.51
and 3.46 +/- 3.69, respectively). Mannopentaose, a five-chain high-mannose structure
fragment commonly found among N-linked oligosaccharides displayed excellent
reproducibility, however the average percent error was relatively high (-14.3 +/- 3.78).
Notably, both NA2 and NA4 displayed the greatest range in variance 18.4 +/- 7.37 and 4.42 +/- 15.9, respectively. Therefore, there appears to be a correlation between
quantification of larger and more complex oligosaccharides and an increase in response
variability, but not necessarily absolute average percent error.
The overall variability in response with regard to the HPLC-CAD quantitative
analysis results may be attributed to a variety of reasons involving but not limited to
experimental errors associated with sample preparation, purity, transferring, solubility
issues and/or obtaining standards from different manufacturers, in which often may not
110
be accurate with amounts provided. Additionally, a significant contributor to possibly
explain some of the differences in response and thus quantification errors of specific
compounds may be due to differences in water retention or hydrates. It has be
postulated that water retention by some oligosaccharides and/or an increase in the
percent water during the HPLC gradients for later eluting compounds, may negatively
affect nebulization efficiency in producing optimal charged droplets for detection.
The CGL ligands contain both neutral and acidic oligosaccharides attached to
their polypeptide backbone, however the current work was focused on solely analyzing
the neutral oligosaccharides. A variety of reasons limited this research to strictly
elucidating neutral CGL ligand oligosaccharides as opposed to the collective charged
and neutral species in a quantitative manner. Firstly, the HPLC-CAD quantification
methodology was originally exploratory in its nascent stages in an effort to demonstrate
proof of concept. This was achieved utilizing only neutral surrogate oligosaccharide
standards to demonstrate its application towards biologically derived oligosaccharides.
Secondly, the analytical Prevail Carbohydrate ES HPLC column employed for this
initial work may not be optimal for the separation of oligosaccharides spanning both
neutral and acidic glycans. Thus, an additional methodology involving less available
charged surrogate and charged biological test oligosaccharides would have been needed
to be developed on a potentially different HPLC column that was robust enough to
withstand the acidic modifier needed to elute charged oligosaccharide species. Thirdly,
it was largely unknown how much oligosaccharide mass could be processed for
111
isolation following quantification analysis utilizing a flow splitter and using only
analytical HPLC columns. Therefore, time and resources were invested to develop and
optimize this isolation and quantification methodology and demonstrate its application
for future applications.
X. laevis O-linked oligosaccharide HPLC profiles were optimized for initial
separation and isolation with a Hypercarb PGC HPLC column. This was due to the
robustness of the column with its ability to process greater injection volumes while the
different mechanism of separation yielded better selectivity of structurally similar
compounds and better resolution as opposed to the Prevail Carbohydrate ES HPLC
column. Additionally, the Hypercarb PGC column provided a high-resolving HPLC
separation of the CGL ligand oligosaccharide mixture. Therefore, the Prevail
Carbohydrate ES HPLC column was utilized for quantification work and secondary
purification only.
Although the separation mechanism between the Prevail Carbohydrate ES
HPLC and the Hypercarb PGC HPLC column differ significantly, the chromatographic
profiles are reasonably comparable with regard to the number of major oligosaccharide
peaks and their abundance. Both profiles display four major oligosaccharide peaks,
however a specific major oligosaccharide peak observed on the Prevail column is not
correlative to a specific major oligosaccharide peak observed on the Hypercarb column
as a result in their differences in separation/selectivity mechanisms. In comparison, the
112
Prevail analysis demonstrated the presence of approximately 17 oligosaccharide peaks
whereas the Hypercarb analysis demonstrated the presence of approximately 20
oligosaccharide peaks. The differences observed between the two HPLC profiles with
regard to number of peaks may be attributed to unresolved peaks co-migrating in the
chromatograms. Taken together, these HPLC profiles illustrate the abundance and
complexity of CGL ligand oligosaccharides consistent with previous NMR and MS
studies [18,21,22].
Hypercarb PGC X. laevis HPLC profiles indicate the variety and complexity of
the neutral oligosaccharide moieties released from the CGL ligand polypeptide
backbone. The X. laevis chromatograms presented here suggests that there is an
abundant amount of oligosaccharide species possessing different structural properties
which are all in the molecular size range of trisaccharides to octasaccharides, which
corroborates previous NMR/MS analyses [18,21,22]. The X. laevis chromatograms
highlighted approximately five major peaks which were targeted for isolation and
quantification for subsequent ELLA analysis. Two of the major oligosaccharide peaks
initially targeted could not be resolved to homogeneity with a single separation. An
attempt at a secondary purification resulted in multiple peaks on the Prevail
Carbohydrate ES HPLC column, therefore adequate mass for subsequent biological
assays was unattainable from these fractions. As a consequence, attempts in isolation of
these peaks were abandoned.
113
Despite the obstacles in obtaining single purified oligosaccharide components
for two of the isolated oligosaccharide peaks, the successful isolation and quantification
was attainable with three other major oligosaccharide peaks. These three peaks
illustrated the presence of one predominate oligosaccharide component free of any
significant interfering oligosaccharides when measured by both the CAD and the UV
signals. Employing a blank subtraction method, all three fractions were ultimately
determined to be greater than 90% purity. These three fractions were subsequently
equilibrated to a molarity equivalency for an ELLA comparative analysis.
The refined ELLA, adopted from previous work by Hedrick et al., demonstrated
greater sensitivity than previous assays with regard to monosaccharide and disaccharide
inhibition analysis of the lectin-ligand binding interaction [11]. The refined ELLA
demonstrated that 75 mM galactose was sufficient to completely inhibit the lectinligand binding interaction. Previously, the most sensitive ELLA reported by Quill and
Hedrick required 150 mM galactose to completely inhibit the lectin-ligand binding
interaction [11]. Additionally, the sensitivity is greater than that reported by Nishihara
et al., which demonstrated that 100 mM galactose only inhibited the lectin-ligand
reaction by 75%, utilizing a CGL-J1 precipitin reaction assay [9]. Additional analysis
by Nishihara and co-workers utilizing the disaccharide inhibitors lactose and melibiose
exhibited significantly less sensitivity than the refined ELLA as well. Specifically with
regard to lactose, the refined ELLA demonstrated inhibition activity of 85 and 95% at
25 and 50 mM, respectively, whereas Nishihara et al., achieved inhibition activity of 50
114
and 75% at the two corresponding concentrations. It is noteworthy to point out that both
assays achieved total inhibition with lactose at 100 mM. With regard to melibiose, the
refined ELLA demonstrated inhibition activity of 80 and 90% at 25 and 50 mM,
respectively, whereas Nishihara et al., achieved inhibition activity of 0 and 50% at the
two corresponding concentrations. Therefore, specifically at lower concentrations of
selected inhibitors, the refined ELLA was demonstrated to be substantially more
sensitive with regard to the specific inhibitors galactose, lactose and melibiose.
Some discrepancies in the developed ELLA did arise with regard to glucose and
lactulose in comparison to work by Nishihara et al., which may be a result of the
enhanced sensitivity of the developed assay as observed with melibiose. At 100 mM
concentrations, Nishihara and co-workers reported that no inhibitory activity was
observed with glucose or lactulose during the CGL-J1 precipitin reaction assay. In
contrast, the data reported here demonstrates that these two compounds possess some
intermediate inhibitory activity, thus suggesting these compounds may possess a weak
affinity towards the CGL binding pocket. This weak affinity may not have been
detectable with previous assays as a result of the decreased sensitivity.
Interestingly, the refined ELLA demonstrated that the monosaccharide fucose
displayed nearly identical inhibitory activity to that of the well established inhibitor,
galactose. Previous MS and NMR analysis of both neutral and acidic oligosaccharides
derived from whole egg jelly and specifically from the combined J1 and J2 segments
115
revealed that terminal fucose residues are vastly abundant throughout these discerned
oligosaccharide structures and thus may be a biologically relevant structure recognized
by the CGL. Additionally, the ligand O-linked oligosaccharide structures responsible
for ligand activity contained terminal galactose residues associated with fucose and
sulfate residues [11,13,18,22,25].
The biological significance of these terminal fucose residues attached to the
CGL ligand has been partially investigated by Quill and Hedrick via ELLA analysis
[11]. Treatment of purified CGL ligand with α-fucosidase to remove terminal fucose
residues revealed that no significant decrease in lectin-ligand binding was obtained.
However, in this treatment the fucosidase utilized may not have released the relevant
fucose residues since these enzymes are highly specific. Also, there is a possibility that
the reactions may not have been taken to completion and thus not released all the fucose
residues. Therefore, the data presented by Quill and Hedrick suggests that from a
biological standpoint, fucose residues may possess a limited ability to act independently
in eliciting a binding interaction/specificity for the CGL since the removal of these
fucose residues did not significantly reduce binding as measured by the ELLA.
However, considering the in-vitro ELLA assay data demonstrated here, it is apparent
that fucose is a strong and specific inhibitor. Therefore, biologically these fucose
structures may act in concert with galactose within the CGL ligand oligosaccharide
moieties in order to capture and bind to the CGL in producing the block to polyspermy.
116
Thus, the evidence from these studies suggests that fucose is likely involved in the
oligosaccharide chains that participate in lectin-ligand binding.
The ELLA data obtained in the current work suggests that the CGL possesses
anomeric binding specificity for galactose, fucose and galactose containing
disaccharides lactose and melibiose. Collectively, both 1-4 and 1-6 glycosidic
disaccharide linkages comprising galactose and glucose monomers were capable of
inhibiting the CGL lectin-ligand binding, however the β-derivative (lactose) appeared to
be marginally more effective than the α-derivative (melibiose) in inhibitive properties.
Additionally, configurations of hydroxyl groups at carbons 2-4 of galactose were
demonstrated to be central to the observed inhibitory properties. Sugars that differ from
that of galactose in specific orientation of the hydroxyl groups at carbon positions 2-4
displayed substantially decreased or insignificant inhibition characteristics (e.g.
lactulose).
In addition to the observed anomeric specificity of the CGL, MS and NMR
analysis revealed that the combined J1 and J2 layers contained all the sulfated species
within the egg jelly extract and the CGL ligands themselves possess sulfated
oligosaccharides. Along with previous studies, the incorporation of a calcium-specific
chelating agent (EGTA) resulted in complete inhibition of the lectin-ligand binding
interaction. Thus, these sulfated oligosaccharides may be essential for interactions with
calcium so as to facilitate binding, whereas terminal galactose and fucose residues may
117
serve in the molecular recognition and physical binding/capture of the CGL.
Collectively, fucose appears to be underappreciated as a critical constituent involved in
the lectin-ligand binding reaction.
The comparative analysis of the isolated and quantified CGL ligand
oligosaccharides at concentrations of 10 and 45 µM were unable to elicit an inhibitory
response of the lectin-ligand binding interaction. Conversely, a semi-quantitative
assessment of whole CGL ligand oligosaccharide fractions released from the SPE
cartridge possessed strong inhibition properties. Whole SPE neutral CGL ligand
oligosaccharide fractions demonstrated strong lectin-ligand inhibition characteristics
eliciting a 95% reduction in binding. The acidic CGL ligand SPE released
oligosaccharides elicited substantially decreased inhibition properties, while the
combined neutral and acidic oligosaccharide treatment yielded a modest increase over
that of the acidic oligosaccharides.
Molecular weights of the CGL ligand oligosaccharide fractions were estimated
based on the retention times observed by the DP surrogate standards, however it is
notable to point out that branched structures containing the same number of monomer
subunits did not possess the same elution profiles. Branched structures such as LNFP II
(5 subunits) and LNH (6 subunits) displayed retention times equivalent to DP 3 and DP
5, respectively. Therefore, the estimated molecular weights of the CGL ligand
oligosaccharides may have been underestimated since branched structures appear to
118
interact with the stationary phase with less surface area and thus display a decrease in
retention times compared to their monomeric DP equivalent counterparts.
A number of hypotheses arise as a result of the ELLA analysis with regard to the
isolated and quantified CGL ligand oligosaccharides as well as the SPE released whole
fractions. From the data presented here, it may be possible that the CGL ligand
polypeptide backbone may be essential in maintaining the three-dimensional integrity of
the CGL ligand glycoprotein and thus be integral in displaying a specific orientation of
the oligosaccharide moieties since lectins are known to bind with spatial specificities.
Such an orientation may be central to the CGL-ligand binding, where one
oligosaccharide moiety may not necessarily be dominant. Conversely, the collective
orientation of oligosaccharides dictated by the polypeptide backbone may act in concert
to produce the binding specificity and affinity via multiple binding interactions
(multivalancy) to its CGL partner in producing the functional block to polyspermy. It is
a possibility that chemical and HPLC separation treatments following the SPE
procedure may have resulted in some loss of biological activity. However, since the
SPE fractions (neutral and acidic) elicited significant inhibitory activity (i.e. 95% for
neutral fraction), it can be assumed that the released oligosaccharides have retained
their biological activity.
In this study, there was no conclusive evidence of any isolated oligosaccharide
fraction possessing an inhibitory effect on lectin-ligand binding. This is likely due to an
119
inability of the developed methodology to yield large enough quantities to assess
binding inhibition in a dose-response manner. The CGL oligosaccharide mass obtained
was only adequate enough to produce micromolar concentrations or a 1000-fold less
concentrated samples than all of the analyzed mono- and disaccharide inhibitors. It may
have also been possible that strong interacting CGL ligand oligosaccharides were not
isolated, thus it is likely necessary to employ semi-preparative or preparative sized
HPLC columns in order to enable the purification of enough of the oligosaccharide
fractions to observe an inhibitory effect in the binding assays developed. However,
compelling evidence suggests that at perhaps greater concentrations, specific CGL
ligand oligosaccharides may have a preferential role in CGL binding as observed with
the whole SPE fraction inhibition analysis (i.e. neutral fraction). Therefore, future work
should involve a preparative HPLC column for acquiring adequate oligosaccharide
mass for subsequent biological assays.
In summary, this research has successfully developed an oligosaccharide
isolation and quantification methodology applicable to biologically derived glycans.
This developed methodology circumvents previously encountered complications in
utilizing derivatization agents while maintaining detection limits at the picomolar level.
This methodology can ultimately be applied to a wide range of neutral oligosaccharide
containing glycoproteins. This work has also expanded on the lectin-ligand ELLA
analysis via developing a more sensitive assay to assess biological activity of selected
compounds. It was found that whole neutral oligosaccharide fractions derived from the
120
ligands appears to inhibit binding more strongly than the acidic oligosaccharide
fractions. And finally, further specificity of the CGL-ligand binding has been deduced
from the refined ELLA analyses, specifically with regard to fucose monomers.
121
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