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Cutured Cell Lines with Genetically Defined Disorders
of Glycosylation
Lecture 33
May 25, 2004Jeff 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
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
Background
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
Advantages…..
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
…..and Disadvantages
Types of Mutants
Loss of function mutants usually lack a transferase...
…but could also be due to loss or gain of a factor that regulates expression
Normal Ablate atransferase
3
Types of Mutants
Gain of function of mutants can manifest quantitative or qualitative changes
Transfection of cells can cause gain or loss of biological activity
Activate a latenttransferase
3 3
Overexpress atransferase
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
Cytotoxin
Enrichment Strategies
Cell sorting
- Bind fluorescent protein with selectivity for a cell surface glycan
- Sort individual cells by fluorescence intensity
101 102 103 104
Fluorescence
Enrichment Strategies
Panning
–Coat a plate with an adhesive protein that binds to a glycan
–Collect adherent cells or non-adherent cells
bFGF bFGF bFGFbFGFbFGF bFGF
bFGF
Panning
bFGF bFGFbFGFbFGF bFGF
PlateMutagenized
CHO Cells
Enrich forMutants
ReplicatedColonies
35SO4Incorporation
125I-bFGFBinding
Autoradiography Mutants
ReplicaPlate
Survivors
Replica Plating and ScreeningReplica 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
Homologous recombination
0.3 kb
Deletion targeting vector
Recombinantmutant allele
Bam
Exon 1
Mouse EXT1gene
RIBamScaBgIRI
1kb
2.2 kbMC1tk Lacz PGKneo
0.9 kb
Lacz PGKneo
BamRI Bam RI
5' probe 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 Biochemical Defect Glycosylation Phenotype
Lec32 (CHO) CMP-Sia synthetase Reduced sialic acid
Lec2 (CHO) CMP-Sia transporter Reduced Sialic acid; N- and O-linked chains terminate in Gal
Lec8 (CHO) UDP-Gal transporter Reduced Gal; Chains terminatein GlcNAc
Lec13 (CHO) GDP-Man 2,4-dehydratase
Reduced Fuc residues
ldlD (CHO) UDP-Glc/UDP-Gal
UDPGlcNAc/UDP-GalNAc
4-epimerase
N-linked chains reduced in Gal,and terminate in GlcNAc; O-linked chains and chondroitinsulfate not present in theabsence of added GalNAc;GAG deficient in the absenceof Gal
D33W25-1(MDAY-D2)SAP (CHO)
Activation of CMP-Neu5Ac hydroxylase
Terminate in Neu5Gc
Emeg32 Inactivation of GlcN-6-Pacetyltransferase
Decreased O-GlcNAc oncytosolic proteins
Pleiotropic Mutation in an Epimerase
UDP-
UDP-
UDP-
UDP-
From Diet or Salvage
From Diet or Salvage
N-linked
O-linked
GAG Linkage
GPI anchors
-Serine
Chondroitin
O-linked
N-linked
-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
-mannosidase II
22
GlcNAc-TIIGlcNAc-TI
6
AsnAsnAsn Asn Asn
2
3
3
2
2
3
4
4
63
2 2
6
Asn X Ser/Thr
High-Mannose Hybrid Complex
New -mannosidase
Mapping Functional Domains
These types of mutants picked up as hypomorphs, i.e., strains with partial defects
Strain Biochemical Defect Glycosylation Phenotype
Lec1 GlcNAc-TI Man5GlcNAc2 accumulates onglycoproteins
Lec1a GlcNAc-TI (Km defectfor both substrates)
Reduced amounts of hybrid andcomplex N-glycans
Lec4a GlcNAc-TV located inthe incorrectcompartment
Missing 1,6 branch from 1,6Man - 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 BiochemicalDefect
Glycosylation Phenotype
LEC11 (CHO) 3 -Fuc T Terminal Lex, sLex 1 -2and V M
14( )LEC CHO18( )LEC CHO
-GlcNAc TVII-GlcNAc TVIII
Additional GlcNAc in core
10 ( )LEC CHO -GlcNAc TIII 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 Biochemical Defect Glycosylation Phenotype
A,C,H GlcNAc to PI transferase Formation of GlcNAc-PIJ GlcNAc N-deacetylase Accumulates GlcNAc-PIE Dol-P-Man synthase Additional GlcNAc in coreB Addition of 1,2 linked
MannoseMan2 -GlcN PI
,F K -Ethanolamine phosphate addition reactions
Man3( - )Eth P 1-2 -GlcN PI
Mutants in Proteoglycan Biosynthesis
Strain Biochemical Defect Phenotype
pgsA (CHO) Xylosyltransferase Defective heparan sulfate and chondroitinsulfate formation
pgsB (CHO) Galactosyltransferase I Defective heparan sulfate and chondroitinsulfate formation
pgsG (CHO) Glucuronosyltransferase I Defective heparan sulfate and chondroitinsulfate formation
pgsD (CHO) GlcA & GlcNActransferase
Heparan sulfate deficient & accumulateschondroitin sulfate
ldlD (CHO) UDP-Glc/UDP-GalUDP-GlcNAc/UDP-GalNAc4-epimerase
Chondroitin sulfate not present in theabsence of added GalNAc; GAG deficient inthe absence of Gal
pgsC (CHO) Sulfate transporter Normal glycosaminoglycan biosynthesis
pgsE (CHO) N-sulfotransferase Undersulfated heparan sulfate
pgsF (CHO) Heparan sulfate 2-O-sulfotransferase
Defective 2-O-sulfation of heparan sulfate;defective bFGF binding
Mouse LTAcells
3-O-sulfotransferase-1 Defective antithrombin binding
CHO 6-O-sulfotransferase-1 Defective antithrombin binding
Glycosaminoglycan Mutants
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
SO32OSO3
Core Protein
IdoA ˛ GlcN GlcA ˛ GlcNAc ˛ GlcA ˛ Ga l˛ Gal ˛ Ser
6OSO3
F E
D
B A
Xyl ˛G
˛
Mutants in Glycolipid and Mucin Assembly
Very few mutants have been identified in mucin and glycolipid assembly
Strain Biochemical Defect Phenotype
GM-95
(B16 Melanoma)
Glucosylceramide No glycosphingolipids
ldlD (CHO) UDP-Glc/UDP-GalUDP-GlcNAc/UDP-GalNAc4-epimerase
O-linked chains notpresent in the absence ofadded GalNAc
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