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Sangamo: ZFPTF

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Physiological state. No. of sites depends on 3-D structure of protein ... glycosylation states. in various mutants. 17. Umana et al. ... – PowerPoint PPT presentation

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Title: Sangamo: ZFPTF


1
Sangamo ZFP-TF
Zinc Finger DNA Binding Protein Transcription
Factor
2
Protein Glycosylation
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.
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.
Grabenhorst, E., Schlenke, P., Pohl, S., 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.
3
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4

Penta- saccharide common core

Diantennary With bisecting GlcNAc With
fucosylated core
Triantennary (also tetra-antennary)
Here, N-linked (to amide N of Asn in N-X-S or
N-X-T)
Substantial in size
Carbohydrates attached to loops or near termini
Also O-linked, to ser or thr (hydroxyl on side
chain)
5
Figure 7.28. Examples of O-linked
oligosaccharides O-linked oligosaccharides
usually consist of only a few carbohydrate
residues, which are added one sugar at a time.
6
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 afucosylated
7
neuraminic acid one of the sialic acids
both terms are used, confusedly
NAcNeu
Carboxyl (acid)
Glycerol moiety
mannose
Acetylated amino group
deoxy
8
Glycosylation pattern affects signaling,
for Delivery to the right cell receptor for
activity Clearance rate
Microheterogeneity Lots of isoforms, naturally
No apparent bottleneck in high-producing
cells 0.1 mg/l ? (amplify) ? 200 mg/l same
pattern
Insect cells (Baculovirus, high level transient
expression) Too simple a pattern compared to
human
Mouse and hamster cells similar to
human Hamster less heterogeneity
9
Genetic engineering of glycosylation to Modify
or enhance activity E.g. Better binding to a
receptor More specific binding Different binding
Also Antigenicity Clearance rate Decrease
microheterogeneity (for clinical application)
10
  • Modifying glycosylation
  • Add or subtract sites to your favorite protein
    (cis)
  • Change the general glycosylation phenotype of the
    host cell (trans)

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
11
2. Clone enzyme genesGlycosyl transferases,
mostlyAlso some synthetases (e.g., NAcNeu) Can
be complexe.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 protein.producing
permanent transfectants of BHK cells Works Get
both types of structures now, substantially (altho
ugh not exactly the same ratio as in human
cells).
12
Stanley Isolation of multiply mutated
glycosylation mutants. Lectins
carbohydrate-binding proteins Plant lectins used
mostly here (but occur widely) Sequential
selections, push - pull on resistance,
sensitivity Sensitivity because greater exposure
of underlying sugars in a transferase -
negative mutant Shows power of selection Shows
usefulness of complementation analysis via cell
hybridization Hybrid selection All lec-R
mutants are WGA (wheat germ agglutinin)
resistant (various degrees) pro- Tester
parent was single lec-R Gat- (req. glycine,
adenine and thymidine) Select in medium lacking
pro, GAT, and with /- WGA Complementing
hybrids will have regained sensitivity
WGA Mutants in the same gene will remain WGA
resistant (non-complementation) Could now be
used as a tabla rasa for introducing a series of
enzymes to build custom tailored
glyco-conjugates. Complicated though (order of
addition, location in the Golgi, etc. )
Potential targeting to carbohydrate-sensivie
recetors (e.g., liver asialoglycoprotein
receptor) clearance rate
Pam Stanley
13
transport to Golgi
5
Golgi
glucose
Exploits hypersenstivity to select against
certain phenotypes.
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15
Sequential mutagenesis and selections to isolate
mutliply-mutated glycosylation mutatnts
16
Predicted alteredglycosylation statesin various
mutants
17
Umana et al. Getting CHO cells to make more
bisected oligosaccharide in the Fc region of MAbs
to better activate antibody-dependent cellular
cytotoxicity (ADCC) GnT III glycosyltransferase
Methods Vectors (8) tTa neoR rat GnTIII
cDNAmychistag no introns that into tet
promoter vector Pur H-chain cDNAs (CMV bGH pA
SV40neo) synthetic leader L-chain cDNAs (CMV
bGH pA SV40neo) synthetic leader zeoR
Transfections (4) tTA neo, transient
tet-beta-gal, GnTIIIpur, HLzeo Westerns
Mass Spec, incl. enzyme digestions
sialidase peptide N-glycosidase F 4 vs. 5
hexoses??? ADCC (dye retention/release,
neuroblastoma cells)
18
Result ADCC efficiency followed proportion of
oligosaccharide with bisected sugar 15 ?
45 25 ? 50
Missing Zero bisection control CHO cells are
supposed to LACK GnTIII Westerns show 0 rat
GnTIII at 2000 ug/ml tetracycline Yet backgrounds
are high. OK for ADCC But Mass Spec data .
Try untransfected CHO? Westerns lying? ( lt30
ug.ml tet ? death too much enzyme)
Good example of enzyme engineering. Can still be
optimized.
Use a constitutive promoter, try different
version to find the best using ADCC as the
assay Check dependence on Ig production level.
19
Variably present
Transient transfection of GnTIII into tTA-bearing
CHO cells (western blot)
Permanent transfectant for tTA and GnTIII
20
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21
Mass spec products indicating the presence of the
bisecting NAcG (in dashed boxes)
22
ADCC assay
ADCC correlates with bisected complex content
Tet induction of GnTIII
No induction of GnTIII
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