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FinC@  g4bdbd t p|p< <4BdBd- pg8g4pdpd4  p@ pp<4!d!d- pg8uʚ;2Nʚ;<4dddd- puʚ;<___PPT9 @  h___PPT2001D<4X? %A  mCutured Cell Lines with Genetically Defined Disorders of Glycosylation Lecture 33 May 25, 2004 Jeff Esko .nG(#b  OverviewUtility 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"PlAi  BackgroundEarly 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 $PlAi Advantages& ..Cultured cell lines can be propagated indefinitely Easily transfected and strong expression systems available Leads for making and understanding organismal mutants Mutants in glycoprotein, glycolipid, GPI anchors, and proteoglycan assembly have been isolated$PlAi&(& ..and DisadvantagesNot 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$PlAi0Types of MutantsLoss of function mutants usually lack a transferase... & but could also be due to loss or gain of a factor that regulates expression$PlAic 'Types of MutantsGain of function of mutants can manifest quantitative or qualitative changes Transfection of cells can cause gain or loss of biological activity$PlAiInduction of Mutants.Spontaneous mutation rates are very low (10-7/generation) Mutagenesis increases mutation rates several orders of magnitude Sex-linked traits and hemizygosity in aneuploid strains makes it easier to detect the recessive phenotype Need selection or enrichment to find rare glycosylation defects2/ lA25Enrichment StrategiesResistance to cytotoxins that bind to glycans Plant lectins Antibody conjugates with a toxin Any CRD-toxin conjugate Anti-carbohydrate antibody and complement Radiation suicide:.lAH A.Enrichment StrategiesCell sorting - Bind fluorescent protein with selectivity for a cell surface glycan - Sort individual cells by fluorescence intensityl)Enrichment StrategiesqPanning Coat a plate with an adhesive protein that binds to a glycan Collect adherent cells or non-adherent cells,jj+$Replica Plating and Colony Screening % Mutant CharacterizationCell hybridization - Recessive/Dominance testing Complementation tests Examine glycan composition Determine missing enzyme activity or other deficiency Complement by cDNA transfer lA2$Reverse Genetics6Homologous recombination to introduce inactive alleles&7M6-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)v.M_  6 9M._69     $Pleiotropic Mutation in an Epimerase   a-mannosidase II deficient cells fail to make complex type N-linked chains Knock-outs in mice show that an alternate pathway exists in many cells, but not in the one where the somatic mutant was isolated,lA Mapping Functional DomainsRThese types of mutants picked up as hypomorphs, i.e., strains with partial defects SlAS:Gain of Function MutantsGain of function mutants arise from activation of a latent gene (Dominant) Some gain of function mutants could arise from loss of a repressor (Recessive) New phenotypes can reveal previously unknown pathways lAGPI Anchor mutantsMutational analysis of GPI anchor synthesis revealed that multiple genes are needed to form several of the linkages These would not have been detected until the enzyme was purified4tlAAlA$Mutants in Proteoglycan BiosynthesisGlycosaminoglycan MutantsMutants 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 lA8(Mutants in Glycolipid and Mucin AssemblyFVery few mutants have been identified in mucin and glycolipid assembly GlAGGlycosylation MutantsRGlycosylation 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 physiologyVG lA2;@ A lA2G;/ !"#(*, . / 0 1 23456;<>?@APsx,,  c yg(HH(d'h  ` ̙33` ` ff3333f` 333MMM` f` f` 3>?" dZ@v? 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