Download Mutants

Cutured Cell Lines with
Genetically Defined
Disorders of Glycosylation
Lecture 33
May 25, 2004
Jeff Esko
Overview

Utility of somatic cell mutants

Isolation of mutants

Mutants in N-glycan formation

Mutants in GPI anchor biosynthesis

Mutants in proteoglycan assembly

Application of glycosylation mutants
Background

Early studies in the 1950’s showed that
cells could be isolated from tissues and
cultured in vitro (in glass)

Mutants could be obtained and
phenotypes were stable

Techniques reserved for microbial
organisms could now be applied to
somatic cells
Advantages…..

Cultured cell lines can be propagated
indefinitely

Easily transfected and strong expression
systems available

Leads for making and understanding
organismal mutants

Mutants in glycoprotein, glycolipid, GPI
anchors, and proteoglycan assembly have
been isolated
…..and Disadvantages

Not always easy to obtain immortal lines for
study

Studies restricted to the phenotypes
exhibited by the selected cell line

Permanent lines are often aneuploid and
dedifferentiate….or differentiate
uncontrollably
Types of Mutants
b3
Normal
Ablate a
transferase

Loss of function
mutants usually lack a
transferase...

…but could also be due
to loss or gain of a
factor that regulates
expression
Types of Mutants
b3
Activate a latent
transferase
b3
Overexpress a
transferase

Gain of function of
mutants can manifest
quantitative or
qualitative changes

Transfection of cells
can cause gain or loss
of biological activity
Induction of Mutants

Spontaneous mutation rates are very low
(10-7/generation)

Mutagenesis increases mutation rates
several orders of magnitude

Sex-linked traits and hemizygosity in
aneuploid strains makes it easier to detect
the recessive phenotype

Need selection or enrichment to find rare
glycosylation defects
Enrichment Strategies
Resistance to cytotoxins that
bind to glycans
– Plant lectins
– Antibody conjugates with
a toxin
– Any CRD-toxin conjugate
– Anti-carbohydrate
antibody and complement
– Radiation suicide
Enrichment Strategies
1
10
2
10
3
10
Fluorescence
4
10
Cell sorting
- Bind fluorescent
protein with selectivity
for a cell surface
glycan
- Sort individual cells by
fluorescence intensity
Enrichment Strategies
Panning
– Coat a plate with an
adhesive protein that
binds to a glycan
– Collect adherent cells
or non-adherent cells
bFGF bFGF bFGF bFGF bFGF bFGF
Panning
bFGF bFGF bFGF bFGF bFGF bFGF
Replica Plating and Colony Screening
Mutant Characterization

Cell hybridization - Recessive/Dominance
testing

Complementation tests

Examine glycan composition

Determine missing enzyme activity or other
deficiency

Complement by cDNA transfer
Reverse Genetics
Homologous recombination to introduce inactive alleles
0.3 kb
RI
BgI
Sca Bam
RI
Bam
Mouse EXT1
gene
Exon 1
MC1tk
Lacz
0.9 kb
1kb
PGKneo
2.2 kb
Deletion
targeting vector
Homologous recombination
Bam
RI
RI
Lacz
5' probe
Bam
PGKneo
Recombinant
mutant allele
3' probe
Reverse Genetics
 …or can derive cell lines from knockout mice
— Fibroblasts and other cell types readily
propagated for 50 or so doublings
—“Immortalize” cells
- T-antigens, myc, ras, other oncogenes
- Telomerase
 ...siRNA and RNAi (interference) in cells
(epigenetic)
Strain
Lec32 (CHO)
Biochemical Defect
CMP-Sia synthetase
Glycosylation Phenotype
Reduced sialic acid
Lec2 (CHO)
CMP-Sia transporter
Lec8 (CHO)
UDP-Gal transporter
Reduced Sialic acid; N- and Olinked chains terminate in Gal
Reduced Gal; Chains terminate
in GlcNAc
Lec13 (CHO)
GDP-Man 2,4dehydratase
Reduced Fuc residues
ldlD (CHO)
UDP-Glc/UDP-Gal
UDPGlcNAc/UDP-GalNAc
4-epimerase
N-linked chains reduced in Gal,
and terminate in GlcNAc; Olinked chains and chondroitin
sulfate not present in the
absence of added GalNAc;
GAG deficient in the absence
of Gal
D33W25-1
(MDAY-D2)
SAP (CHO)
Emeg32
Activation of CMPNeu5Ac hydroxylase
Terminate in Neu5Gc
Inactivation of GlcN-6-P
acetyltransferase
Decreased O-GlcNAc on
cytosolic proteins
Pleiotropic Mutation in an Epimerase
From Diet or Salvage
N-linked
O-linked
GAG Linkage
UDP-
UDP-
UDP-
UDP-
GPI anchors
-Serine
O-linked
N-linked
Chondroitin
From Diet or Salvage
2
High-Mannose
Hybrid
Complex
3
3
2
2
2
3
3
2
6
b2
b2
6
 -mannosidase II
GlcNAc-TI
b4
GlcNAc-TII
b4
Asn X Ser/Thr
6
Asn
Asn
Asn
Asn
Asn
New
-mannosidase



-mannosidase II deficient cells fail to make complex
type N-linked chains
Knock-outs in mice show that an alternate pathway
exists in many cells, but not in the one where the
somatic mutant was isolated
Mapping Functional Domains
These types of mutants picked up as hypomorphs,
i.e., strains with partial defects
Strain
Biochemical Defect
Lec1
GlcNAc-TI
Lec1a
GlcNAc-TI (Km defect
for both substrates)
Lec4a
GlcNAc-TV located in
the incorrect
compartment
Glycosylation Phenotype
Man5GlcNAc 2 accumulates on
glycoproteins
Reduced amounts of hybrid and
complex N-glycans
Missingb1,6 branch from Man1,6
arm in N-linked glycans
Gain of Function Mutants



Gain of function mutants arise from activation of a
latent gene (Dominant)
Some gain of function mutants could arise from
loss of a repressor (Recessive)
New phenotypes can reveal previously unknown
pathways
Strain
LEC11 (CHO)
Biochemical
Defect
3Fuc-T
LEC14(CHO)
LEC18(CHO)
LEC10 (CHO)
GlcNAc-TVII
GlcNAc-TVIII
GlcNAc-TIII
Glycosylation Phenotype
Terminal Le x , sLe x and V1M-2
Additional GlcNAc in core
Complex chains have the bisected
GlcNAc residue
GPI Anchor mutants


Mutational analysis of GPI anchor synthesis revealed
that multiple genes are needed to form several of the
linkages
These would not have been detected until the
enzyme was purified
Strain
A,C,H
J
E
B
F,K
Biochemical Defect
GlcNAc to PI transferase
GlcNAc N-deacetylase
Dol-P-Man synthase
Addition of 1,2 linked
Mannose
Ethanolamine-phosphate
addition reactions
Glycosylation Phenotype
Formation of GlcNAc-PI
Accumulates GlcNAc-PI
Additional GlcNAc in core
Man2GlcN-PI
Man3(Eth-P) 1-2GlcN-PI
Mutants in Proteoglycan Biosynthesis
Strain
Biochemical Defect
Phenotype
pgsA (CHO)
Xylosyltransferase
Defective heparan sulfate and ch ondroitin
sulfate formation
pgsB (CHO)
Galactosyltransferase I
Defective heparan sulfate and ch ondroitin
sulfate formation
pgsG (CHO)
Glucuronosyltransferase I
Defective heparan sulfate and ch ondroitin
sulfate formation
pgsD (CHO)
GlcA & GlcNAc
transferase
Heparan sulfate deficient & accumulates
chondroitin sulfate
ldlD (CHO)
UDP-Glc/UDP-Gal
UDP-GlcNAc/UDP-GalNAc
4-epimerase
Chondroitin sulfate not present in the
absence of added GalNAc; GAG deficient in
the absence of Gal
pgsC (CHO)
Sulfate transporter
Normal glycosaminoglycan biosynthesis
pgsE (CHO)
N-sulfotransferase
Undersulfated heparan sulfate
pgsF (CHO)
Heparan sulfate 2-Osulfotransferase
Defective 2-O-sulfation of heparan sulfate;
defective bFGF binding
Mouse LT A
cells
3-O-sulfotransferase-1
Defective antithrombin binding
CHO
6-O-sulfotransferase-1
Defective antithrombin binding
Glycosaminoglycan Mutants
Core Protein
6OSO3
D
IdoA GlcN  GlcA GlcNAcGlcA GalGalXylSer
G
2OSO3
F



B
A
SO3
E
Mutants in the linkage region depress both heparan
sulfate and chondroitin sulfate biosynthesis
Mutants in polymerization and N-sulfation define
bifunctional enzymes
Mutants in sulfation define multiple sulfotransferases
Mutants in Glycolipid and Mucin Assembly
Strain
GM-95
(B16 Melanoma)
ldlD (CHO)

Biochemical Defect
Glucosylceramide
Phenotype
No glycosphingolipids
UDP-Glc/UDP-Gal
UDP-GlcNAc/UDP-GalNAc
4-epimerase
O-linked chains not
present in the absence of
added GalNAc
Very few mutants have been identified in mucin
and glycolipid assembly
Glycosylation Mutants

Glycosylation mutants have been used in
hundreds of biological studies
– Protein sorting and secretion
– Viral assembly
– Cell adhesion

Easy to identify new genes by forward selection
of desired phenotype

With few exceptions, glycans are dispensable in
cell culture, but as you know they play critical
roles in development and normal physiology