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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

2Two types of protein glycosylation

glucose galactose

N-acetyl group

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Triantennary(also tetra-antennary)

DiantennaryWith bisecting GlcNAcWith fucosylated core

Carbohydrates attached to exterior loops or near termini

Substantial in size

All shown here,N-linked (to amide N of Asnin N-X-S or N-X-T)

Also O-linked, to ser or thr(hydroxyl on side chain); see below

=

=Penta-

saccharidecommon

core

Fucose

Sia= sialic acid (see below)

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3

1 2

4Enlargement for display

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anomeric carbon

Haworth view

Fisher view

Chair view

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1 234567891011

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Glucose

}Gray = CWhite = HRed = O

Ring oxygen

C6 (-CH2OH)

C5

C1

hydr

oxyl

Alpha or beta?

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or glycogen chain

down

out

H

H

Polysaccharide formation

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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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C2

glucose galactose mannose

C4

What’s different from glucose here?

Examples of other hexoses

allose

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(Insect exoskeleton)

(Bacterial cell walls)Metabolic intermediate

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Carbohydrate structure specific for:

Cell typePhysiological stateNo. of sites depends on 3-D structure of proteinStructure at that site depends on the site

E.g., transferrin, from different cell types :

Cerebrospinal fluid (made in brain):diantennaryasialo-agalacto-fucosylatedbisecting GlcNAc

Sialic acid structure: see next graphic

Blood (made in liver):diantennary NAcNeu (sialated= sialic acid)

afucosylated

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neuraminic acid – one of the sialic acids = : both terms are used, confusedly

Mannose framework

Carboxyl (acid)

Acetylated amino group

Glycerol moiety

deoxy

NAcNeu:

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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 activityClearance 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 humanHamster: less heterogeneity

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Genetic engineering of glycosylation to:

Modify or enhance activity

E.g.:

Better binding to a receptorMore specific bindingDifferent binding, in theory

Also:

AntigenicityClearance rateDecrease 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

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2. Clone enzyme genes:Glycosyl 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)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).

Modifying glycosylation

1. Add or subtract sites to your favorite protein (cis)2. Change the general glycosylation phenotype of the host cell (trans)

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 ¶

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Stanley: Isolation of multiply mutated glycosylation mutants by selecting lectin resistanceLectins = carbohydrate-binding proteinsPlant 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

Pam Stanley

Isolate mutant mammalian cell lines deficient in specific glycosylation enzymes

Review: Nature Biotechnology  19, 913 - 917 (2001) , The bittersweet promise of glycobiology. Alan Dove

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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.

20Umana, 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)

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 additionA 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).

(NAcG = N-acetyl-glucosamine here)

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Genentech(Killer T-cell)

T-cell surface receptor binds Fc region of antibody molecule (Fc gammaR)

Commercial MAb injected as a therapeutic

TARGET CELL

ADCC

22Umana, 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.

Cyt

otox

icity

Anti-neuroblastoma anibody (ng/ml)

High tet, tet-off system, = basal production

Low tet, tet-off system, = higher production

Yet lower tet, tet-off system, = yet higher production

No tet, tet-off system, = highest productionnon-optimalNeuroblastoma

cells + NK T-cells + antibody

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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.

Protein Glycosylation

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

Assigned:

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.

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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

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Homology regions

K.O. exon 1 translation start region

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-resistance1.6X10870,000 screened 10 double KO homozygotes.

Remove drug resis. genes bytransient transfection with Cre Recombinase. Exon 1 suffers a 200 nt deletion

Lox sites

Double knock-out strategy for FUT8 an alpha-1,6,fucosyl transferase

Note: 10’s of thousands of PCRs performed to screen for homologous recomb., using 96-well plates

Little sequence data available for Chinese hamsterIsolate CHO cDNA using mouse sequence data for primersUse CHO cDNA to isolate CHO genomic fragments from a commercial lambda library

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Double knockout evidence

mRNA has 200 nt deletion(RT-PCR)

Original KO’d genes have a 1.5 kb insertion(Southern blot)

After Cre treatment

27Use 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 +/-

FUT8 -/-

Surprising: CHO cells do not have excess fucosylation capacity

28Rituxan (retuximab, anti-CD20) produced in FUT -/- cells does not contain fucose(HPLC analysis)

Digestion all the way to monosaccharides

Missing d - g

29Binding to CD20 membranes

FUT8-/- anti CD20 = Rituxan

Complement-mediated cell toxicity is the same for FUT8-/- and Rituxan

In ADCC, FUT8-/- anti-CD20 >> Rituxan

Anti-CD20 from a partially FUT-deficient rat cell line

FUT-/-’sRat line

Rituxan = commercial product, 98% fucosylated

Fc-Receptor protein binding assay

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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, 2010Production of non-fucosylated antibodies by co-expression ofheterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD)

Clone bacterial RMD cDNAConstruct mam. expn vectorTransfect into CHO cells making Herceptin (anti EGF receptor)Deflects intermediate in fuciose biosynthetic path

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Also absent by MS

No fucose in transfectant glycoproteins

Select for G418 resistance, screen for lack of fucose.

One of 3 clones:WT CHO cells

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Binding assay to Fc receptor (ELISA-type assay)

ELISA = Enzyme-linked immunosorbent assay

About10-fold more effective

WT

3 transfectants

Antibody concentration (ng/ml)

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ADCC lysis assay vs. a HER2+ breast carcinoma cell line

Concentration of anitbody

% ly

sis

About10-fold more effective

WT

3 transfectants