Protein glycosylation

1
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
2
Two types of protein glycosylation
N-acetyl group
glucose
galactose
3
2
1

Penta- saccharide common core

All shown here, N-linked (to amide N of Asn in
N-X-S or N-X-T)
Diantennary With bisecting GlcNAc With
fucosylated core
Triantennary (also tetra-antennary)
Substantial in size
Carbohydrates attached to exterior loops or near
termini
Fucose
Also O-linked, to ser or thr (hydroxyl on side
chain) see below
Sia sialic acid (see below)
4
Enlargement for display
5
anomeric carbon
Fisher view
Haworth view
Chair view
6
(No Transcript)
7
Glucose
Alpha or beta?
8
Polysaccharide formation
down
out
H
H
or glycogen chain
9
Examples of O-linked oligosaccharides O-linked
oligosaccharides usually consist of only a few
carbohydrate residues, which are added one sugar
at a time.
10
Examples of other hexoses
C2
C4
allose
glucose
galactose
mannose
Whats different from glucose here?
11
Metabolic intermediate
(Bacterial cell walls)
(Insect exoskeleton)
12
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 asialo- agalacto- fucosyla
ted bisecting GlcNAc
Blood (made in liver) diantennary NAcNeu
(sialated sialic acid) afucosylated
Sialic acid structure see next graphic
13
neuraminic acid one of the sialic acids
both terms are used, confusedly
NAcNeu
Carboxyl (acid)
Glycerol moiety
Mannose framework
Acetylated amino group
deoxy
14
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
15
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)
16

• Modifying glycosylation
• 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
  proteins structure)
• Works
• Change the general glycosylation phenotype of the
  host cell (trans)
• E.g., Pam Stanley lectin-resistant mutants

17

• Modifying glycosylation
• Add or subtract sites to your favorite protein
  (cis)
• Change the general glycosylation phenotype of the
  host cell (trans)

2. Clone enzyme genesGlycosyl transferases,
mostlyAlso 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) ExperimentOver-express cloned
human 2,6 ST, along with a substrate
proteinproduce 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
1,3/4-Fucosyltransferases 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
18
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
19

• Hybrid selection
• All lec-R mutants were WGA (wheat germ
  agglutinin) resistant (various degrees) pro-
  (required proline)
• Tester parent was single lec-R GAT- (reqd
  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.

20
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.
(NAcG N-acetyl-glucosamine here)
Target here (bisecting NAcG)
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).
21
ADCC
TARGET CELL
Genentech
(Killer T-cell)
Commercial MAb injected as a therapeutic
T-cell surface receptor binds Fc region of
antibody molecule (Fc gammaR)
22
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
Yet lower tet, tet-off system, yet higher
production
No tet, tet-off system, highest
production non-optimal
Neuroblastoma cells NK T-cells antibody
Cytotoxicity
High tet, tet-off system, basal production
Anti-neuroblastoma anibody (ng/ml)
23
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 587(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
594(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.
24
Hypothesis Fucose interferes with binding of the
T-cells 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
25
Double knock-out strategy for FUT8 an
alpha-1,6,fucosyl transferase
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
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 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 10s of thousands of PCRs performed to
screen for homologous recomb., using 96-well
plates
26
Double knockout evidence
After Cre treatment
Original KOd genes have a 1.5 kb
insertion (Southern blot)
mRNA has 200 nt deletion (RT-PCR)
27
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 -/-
28
Rituxan (retuximab, anti-CD20) produced in FUT
-/- cells does not contain fucose(HPLC analysis)
Digestion all the way to monosaccharides
Missing d - g
29
In ADCC, FUT8-/- anti-CD20 gtgt Rituxan
Binding to CD20 membranes FUT8-/- anti CD20
Rituxan
Anti-CD20 from a partially FUT-deficient rat cell
line
Fc-Receptor protein binding assay
Rat line
FUT-/-s
Complement-mediated cell toxicity is the same
for FUT8-/- and Rituxan
Rituxan commercial product, 98 fucosylated
30
Very laborious, but apparently a big
payoff. Better selection? Why not use the
fluorescent LCA to select for the FUT8 KOs along
with G418 resistance (double sequential
selection)?
31
Hans Henning von Horsten et al., Glycobiology
vol. 20 no. 12 pp. 16071618, 2010 Production of
non-fucosylated antibodies by co-expression
of heterologous GDP-6-deoxy-D-lyxo-4-hexulose
reductase (RMD)
Clone bacterial RMD cDNAConstruct mam. expn
vector Transfect into CHO cells making Herceptin
(anti EGF receptor) Deflects intermediate in
fuciose biosynthetic path
32
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
33
Binding assay to Fc receptor (ELISA-type assay)
About10-fold more effective
3 transfectants
WT
Antibody concentration (ng/ml)
ELISA Enzyme-linked immunosorbent assay
34
ADCC lysis assay vs. a HER2 breast carcinoma
cell line
About10-fold more effective
lysis
3 transfectants
WT
Concentration of anitbody