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Protein glycosylation
Adds another layerof structure and specificity to proteins
Can enhance the function of a protein
Can extend the lifetime of a protein
Can help localize a protein within a cell
Can act as a specific antigen
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Two types of protein glycosylation
N-acetyl group
glucose
galactose
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1
1
2
3
Pentasaccharide
common
core
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=
=
All shown
Triantennary
here,
(also tetra-antennary)
N-linked
(to amide
N of Asn
in N-X-S
or N-X-T)
Carbohydrates
attached to exterior
loops or near termini
Sia= sialic acid (see below)
Diantennary
With bisecting GlcNAc
With fucosylated core
Substantial in size
Fucose
Also O-linked, to ser or thr
(hydroxyl on side chain); see below
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Enlargement for display
5
anomeric
carbon
Fisher
view
Chair view
Haworth view
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11
10
7
6
5
89
3
1 24
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Glucose
}
Gray = C
White = H
Red = O
C1
C6 (-CH2OH)
C5
Ring oxygen
Alpha or beta?
Polysaccharide formation
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down
H
H
or glycogen chain
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Examples of O-linked oligosaccharides O-linked oligosaccharides
usually consist of only a few carbohydrate residues, which are added
one sugar at a time.
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C4
glucose
Examples of other hexoses
C2
galactose
mannose
allose
What’s different from
glucose here?
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Metabolic intermediate
(Bacterial cell walls)
(Insect exoskeleton)
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Carbohydrate structure specific for:
Cell type
Physiological state
No. of sites depends on 3-D structure of protein
Structure at that site depends on the site
E.g., transferrin, from different cell types :
Cerebrospinal fluid (made in brain):
diantennary
asialoagalactofucosylated
bisecting GlcNAc
Blood (made in liver):
diantennary
NAcNeu (sialated= sialic acid)
afucosylated
Sialic acid structure: see next graphic
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neuraminic acid – one of the sialic acids = : both terms are used, confusedly
NAcNeu:
Carboxyl (acid)
Glycerol moiety
Mannose framework
Acetylated amino group
deoxy
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Glycosylation pattern affects signaling of proteins used therapeutically, for:
Delivery of the soluble glycoprotein drug to the right cell receptor for activity
Clearance rate
Microheterogeneity:
Lots of isoforms typically present
Glycosylation does not seem to represent a bottleneck in high-producing cells:
0.1 mg/l  (amplify)  200 mg/l = same pattern
Insect cells (Baculovirus, high level transient expression for production):
Too simple a pattern compared to human
Mouse and hamster cells: similar to human
Hamster:
less heterogeneity
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Genetic engineering of glycosylation to:
Modify or enhance activity
E.g.:
Better binding to a receptor
More specific binding
Different binding, in theory
Also:
Antigenicity
Clearance rate
Decrease microheterogeneity (for clinical application)
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Modifying glycosylation
1. Add or subtract sites to your favorite protein (cis)
1a. Subtract sites: Easy, change N or S or T to A by site-directed mutagenesis
1b. Add sites: Not so easy.
Consensus N-X-S does not work, e.g.:
Requires the insertion of a ~12 aa region encompassing a real N-glycosylation
site (6 suffices for O-linked)
Place on an end or on a loop (must know protein’s structure)
Works
2. Change the general glycosylation phenotype of the host cell (trans)
E.g., Pam Stanley: lectin-resistant mutants
Modifying glycosylation
1. Add or subtract sites to your favorite protein (cis)
2. Change the general glycosylation phenotype of the host cell (trans)
2. Clone enzyme genes:
Glycosyl transferases, mostly
Also some synthetases (e.g., NAcNeu synthetase)
Can be complex: e.g., 7 different fucosyl transferases (FTs),
with different (overlapping) substrate specificities
Simpler example: Hamster cells do only 2,3 sialylation.
Humans do 2,6 as well, via a 2,6-sialyl transferase (ST)
Experiment:
Over-express cloned human 2,6 ST, along with a substrate protein;
produce permanent transfectants of BHK cells (BHK = baby hamster kidney)
Get both types of structures now, substantially
(although not exactly the same ratio as in human cells).
J Biol Chem, Vol. 273, Issue 47, 30985-30994, November 20, 1998 In Vivo Specificity of Human 1,3/4Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans.
COEXPRESSION STUDIES FROM BHK-21 CELLS TOGETHER WITH HUMAN -TRACE PROTEIN Eckart
Grabenhorst , Manfred Nimtz , Júlia Costa§, and Harald S. Conradt ¶
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Isolate mutant mammalian cell lines deficient in specific glycosylation enzymes
Stanley: Isolation of multiply mutated glycosylation mutants by selecting lectin resistance
Lectins = carbohydrate-binding proteins
Plant lectins used mostly here (but occur widely in animals as well)
Sequential selections, push - pull on resistance, sensitivity
Resistance: enzyme deficiency  failure to add the sugar need for lectin binding
Sensitivity: failure to add a sugar produces greater exposure of underlying sugars
A transferase-negative mutant  better binding to the exposed sugar
Showed power of selection, usefulness of complementation via cell hybridization
Review: Nature Biotechnology 19, 913 - 917 (2001) ,
The bittersweet promise of glycobiology. Alan Dove
Pam Stanley
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Hybrid selection:
• All lec-R mutants were: WGA (wheat germ agglutinin) resistant (various degrees) &
pro- (required proline)
•
•
•
•
Tester parent was single lec-R + GAT- (req’d glycine, adenine and thymidine)
Select in medium lacking pro and GAT, and with +/- WGA
Complementing hybrids will have regained sensitivity to WGA
Mutants in the same gene will remain WGA resistant (non-complementation)
•
•
Potential: build a production cell line with all glycosyltrasnferases, etc. mutated out.
Could now be used as a tabla rasa (blank slate) introducing a series of enzymes to
build custom tailored glyco-conjugates. Complicated though (order of addition,
location in the Golgi, etc. )
Mostly not developed yet.
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Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999.
Engineered glycoforms of an antineuroblastoma IgG1 with optimized
antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17: 176-180.
Target here
(bisecting NAcG)
(NAcG =
N-acetyl-glucosamine here)
Presence of the bisecting NAcG enhances binding of T-cell receptor to the Fc region
of antibodies.
Binding is needed for ADCC.
Mouse and hamster cell lines used for commercial production lack the
glycosyltransferase needed for bisecting NAcG addition
A rat myeloma cell line does produce MAb with the bisecting NAcG.
Hypothesis: Expression of the rat enzyme in a CHO cell line will add a bisecting
NacG to the anti-neuroblastoma MAb produced by these cells. The modified MAb will
be a better mediator of ADCC.
Experiment: Clone the cDNA for this enzyme from the rat line and transfer it to CHO
cells, driven by an inducible tet promoter.
Check sugar structure of Mab (MS) and ADCC efficiency of the Mab (in vitro lysis).
ADCC
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TARGET CELL
Genentech
(Killer T-cell)
Commercial MAb injected as a therapeutic
T-cell surface receptor binds Fc region of
antibody molecule (Fc gammaR)
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Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999.
Engineered glycoforms of an antineuroblastoma IgG1 with optimized
antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17: 176-180.
Low tet, tet-off system,
= higher production
Neuroblastoma
cells + NK T-cells
+ antibody
Cytotoxicity
Yet lower tet, tet-off system,
= yet higher production
No tet, tet-off system,
= highest production
non-optimal
High tet, tet-off system,
= basal production
Anti-neuroblastoma anibody (ng/ml)
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Protein Glycosylation
Assigned:
Naoko Yamane-Ohnuki, et al.. Establishment of FUT8 knockout Chinese
hamster ovary cells: an ideal host cell line for producing completely
defucosylated antibodies with enhanced antibody-dependent cellular
cytotoxicity. Biotechnol Bioeng. 2004 Sep 5;87(5):614-22
Optional Update:
Kanda Y, Yamane-Ohnuki N, Sakai N, Yamano K, Nakano R, Inoue M, Misaka
H, Iida S, Wakitani M, Konno Y, Yano K, Shitara K, Hosoi S, Satoh
M. Comparison of cell lines for stable production of fucose-negative
antibodies with enhanced ADCC. Biotechnol Bioeng. 2006 Jul 5;94(4):680-8.
Review:
Grabenhorst, E., Schlenke, P., Pohl,., Nimtz, M., and Conradt, H.S. 1999.
Genetic engineering of recombinant glycoproteins and the glycosylation
pathway in mammalian host cells. Glycoconj J 16: 81-97.
Background:
Stanley, P. 1989. Chinese hamster ovary cell mutants with multiple glycosylation
defects for production of glycoproteins with minimal carbohydrate heterogeneity.
Mol Cell Biol 9: 377-383.
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Biotechnol Bioeng. 2004 Sep 5;87(5):614-22
Hypothesis:
Fucose interferes with binding of the T-cell’s Fcgamma3 receptor to the Fc region
of an antibody molecule.
Elimination of fucose from produced MAbs will increase ADCC.
Create a mutant CHO cells (starting with amplifiable dhfr- cells) in which the fucose
transferase (biosynthesis) genes have been knocked out.
All mAbs produced in these mutant cells will be better at promoting ADCC
Double knock-out strategy for FUT8 an alpha-1,6,fucosyl transferase
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Little sequence data available for Chinese hamster
Isolate CHO cDNA using mouse sequence data for primers
Use CHO cDNA to isolate CHO genomic fragments from a commercial lambda library
K.O. exon 1 translation start region
Homology regions
DT= diphtheria toxin gene,
Kills if integrated via
non-homologous recombination
For hemizygote:
Select for G418 resistance,
Screen by PCR for homologous recomb.
108 cells  45,000 colonies 40 false
recombinants (extension-duplications) + 1 true
recombinant
Step 2 for homozygote,
select for Pur-resistance
Lox sites
1.6X10870,000 screened 
10 double KO homozygotes.
Remove drug resis. genes by
transient transfection with Cre
Recombinase. Exon 1 suffers a
200 nt deletion
Note: 10’s of thousands of PCRs performed to screen for homologous recomb., using 96-well plates
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Double knockout evidence
After Cre
treatment
Original KO’d genes have a 1.5 kb insertion
(Southern blot)
mRNA has 200 nt deletion
(RT-PCR)
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Use of a fluoresceinated lentil lectin (LCA) that binds fucose oligosaccharides to
demonstrate lack of fucosylation in glycosylated proteins in the FUT8 -/- cells
Control background
fluorescence
(FL-anti avidin)
FUT8 +/+
FUT8 +/Surprising: CHO cells
do not have excess
fucosylation capacity
FUT8 -/-
Rituxan (retuximab, anti-CD20) produced in FUT -/- cells does not contain fucose
(HPLC analysis)
Digestion all the way to monosaccharides
Missing d - g
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Binding to CD20 membranes
In ADCC, FUT8-/- anti-CD20 >> Rituxan 29
FUT8-/- anti CD20 = Rituxan
Anti-CD20
from a partially
FUT-deficient
rat cell line
Fc-Receptor
protein
binding assay
Rat line
Complement-mediated
cell toxicity is
the same for FUT8-/- and
Rituxan
FUT-/-’s
Rituxan = commercial product,
98% fucosylated
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Very laborious, but apparently a big payoff.
Better selection?:
Why not use the fluorescent LCA to select for the FUT8 KO’s along with G418
resistance (double sequential selection)?
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Hans Henning von Horsten et al., Glycobiology vol. 20 no. 12 pp. 1607–1618, 2010
Production of non-fucosylated antibodies by co-expression of
heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD)
Clone bacterial RMD cDNA
Construct mam. expn vector
Transfect into CHO cells
making Herceptin (anti EGF
receptor)
Deflects intermediate in
fuciose biosynthetic path
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Select for G418 resistance, screen for lack of fucose.
WT CHO cells
One of 3 clones:
No fucose in transfectant glycoproteins
Also absent by MS
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Binding assay to Fc receptor (ELISA-type assay)
About10-fold more effective
3 transfectants
WT
Antibody concentration (ng/ml)
ELISA = Enzyme-linked immunosorbent assay
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ADCC lysis assay vs. a HER2+ breast carcinoma cell line
% lysis
About10-fold more effective
3 transfectants
WT
Concentration of anitbody