Transcript 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 Missingb1,6 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 GlcNAcGlcA GalGalXylSer 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