Sangamo: ZFPTF

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Sangamo ZFP-TF
Zinc Finger DNA Binding Protein Transcription
Factor
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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.
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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)
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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.
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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 asialo agalacto fucosyla
ted bisecting GlcNAc Blood (made in
liver) diantennary NAcNeu afucosylated
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neuraminic acid one of the sialic acids
both terms are used, confusedly
NAcNeu
Carboxyl (acid)
Glycerol moiety
mannose
Acetylated amino group
deoxy
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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
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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
Also Antigenicity Clearance rate Decrease
microheterogeneity (for clinical application)
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• 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
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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).
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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
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transport to Golgi
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Golgi
glucose
Exploits hypersenstivity to select against
certain phenotypes.
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Sequential mutagenesis and selections to isolate
mutliply-mutated glycosylation mutatnts
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Predicted alteredglycosylation statesin various
mutants
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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)
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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.
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Variably present
Transient transfection of GnTIII into tTA-bearing
CHO cells (western blot)
Permanent transfectant for tTA and GnTIII
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Mass spec products indicating the presence of the
bisecting NAcG (in dashed boxes)
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ADCC assay
ADCC correlates with bisected complex content
Tet induction of GnTIII
No induction of GnTIII