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