Overview

1. Cutured Cell Lines with Genetically Defined Disorders of GlycosylationLecture 33May 25, 2004Jeff Esko

2. 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

3. 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

4. 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

5. …..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

6. b3 Ablate a transferase 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

7. b3 b3 Activate a latent transferase Overexpress a transferase 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

8. 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

9. 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

10. 1 2 3 4 10 10 10 10 Fluorescence Enrichment Strategies Cell sorting - Bind fluorescent protein with selectivity for a cell surface glycan - Sort individual cells by fluorescence intensity

11. 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

12. Replica Plating and Colony Screening

13. Mutant Characterization • Cell hybridization - Recessive/Dominance testing • Complementation tests • Examine glycan composition • Determine missing enzyme activity or other deficiency • Complement by cDNA transfer

14. Reverse Genetics • Homologous recombination to introduce inactive alleles

15. 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)

17. UDP- UDP- UDP- UDP- -Serine Pleiotropic Mutation in an Epimerase From Diet or Salvage N-linked O-linked GAG Linkage GPI anchors O-linked N-linked Chondroitin From Diet or Salvage

18. 2 a High-Mannose Hybrid Complex 3 a 3 a 2 2 2 a a a 2 b 2 3 6 a 2 a a b 3 6 a a a GlcNAc-TI -mannosidase II GlcNAc-TII 4 b 4 b 6 a A s n A s n A s n X S e r / T h r A s n A s n A s n New -mannosidase 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

19. Mapping Functional Domains These types of mutants picked up as hypomorphs, i.e., strains with partial defects

20. 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

21. 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

22. Mutants in Proteoglycan Biosynthesis

23. S O 2 O S O 3 3 Glycosaminoglycan Mutants C o r e P r o t e i n 6 O S O 3 D ˛ ˛ ˛ ˛ ˛ ˛ ˛ I d o A G l c N G l c A G l c N A c G l c A G a l˛ G a l X y l S e r G B A F 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

24. Mutants in Glycolipid and Mucin Assembly • Very few mutants have been identified in mucin and glycolipid assembly

25. 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