Functional assays for the molecular chaperone cosmc.
Mucin type O-glycosylation involves sequential actions of several glycosyltransferases in the Golgi apparatus. Among those enzymes, a single gene product termed core 1 beta3-galactosyltransferase (T-synthase) in vertebrates is the key enzyme that converts the precursor Tn antigen GalNAcalpha1-Ser/Thr to the core 1 structure, Galbeta1-3GalNAcalpha1-Ser/Thr, also known as T antigen. This represents the most common structure within typical O-glycans of membrane and secreted glycoproteins. Formation of the active T-synthase requires that it interacts with Core 1 beta3Gal-T Specific Molecular Chaperone (Cosmc), which is a specific molecular chaperone in the endoplasmic reticulum (ER). T-synthase activity is commonly measured by its ability to transfer [3H]Gal from UDP-[3H]Gal to an artificial acceptor GalNAcalpha-1-O-phenyl to form [3H]Galbeta1-3GalNAcalpha-1-O-phenyl, which can then be isolated and quantified. Because the primary function of Cosmc is to form active T-synthase, the activity of Cosmc is assessed indirectly by its ability to promote formation of active T-synthase when it is coexpressed with T-synthase in cells lacking functional Cosmc. Such cells include insect cells, which constitutively lack Cosmc, and Cosmc-deficient mammalian cell lines. Cosmc is encoded by the X-linked Cosmc gene (Xq24 in human, Xc3 in mice), thus, acquired mutations in Cosmc, which have been observed in several human diseases, such as Tn syndrome and cancers, cause a loss of T-synthase, and expression of the Tn antigen. The methods described here allow the functional activities of such mutated Cosmc (mCosmc) to be measured and compared to wild-type (wtCosmc).
Ju
Tongzhong
T
Department of Biochemistry, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia, USA.
Cummings
Richard D
RD
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001681:Biological Assay / Q000379:methods*; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011134:Polysaccharides / Q000096:biosynthesis
C505369:C1GALT1C1 protein, human; D018832:Molecular Chaperones; D011134:Polysaccharides; D005700:Galactosyltransferases