Glycosphingolipids (GSL) function as tumor markers, modulators of growth factor acceptor activity, blood group antigens, and receptors for toxins and microbes. In order to better understand how GSL performed these functions, our knowledge of the cell metabolism, trafficking, and distribution must be increased. The applicant has recently defined these parameters for the endogenous GSL of Chinese hamster ovary (CHO) cells and one of its glycosylation mutants. The applicant proposes to extend these studies to members of three other GSL synthetic pathways: gangliotriosylceramide (GA2) of the asialo series, ganglioside (GM2) of the ganglio A series, and galactosylceramide (GAL CER). Experiments will be performed with cells that express these GSL endogenously, as well as CHO cells that have been transfected with cloned glycosyltransferase cDNAs responsible for synthesis of the respective GSL; namely, N-acetylgalactosaminyltransferase (GalNAc-T) for synthesizing GA2, and GM2, and ceramide UDP-galactosyltransferase for GAL CER synthesis. All studies will be completed in transfected CHO cells and in control, parental CHO cells, to avoid complications arising from cell type-differences in GSL trafficking.
Four specific aims are proposed with each set of cells. These involve i.) determining kinetics of synthesis and turnover of GSL, ii.) determining the rate and extent to which these GSL reach plasma membranes, iii.) determination of trafficking of these GSL to the plasma membrane under conditions known to diminish or terminate Golgi trafficking, iv.) localize the GalNAc-T and ceramide UDP galactosyltransferase using immunoelectron microscopy, in order to correlate physical localization of the glycosyltransferases with biochemical results obtained with the first three specific aims. An additional fifth specific aim will utilize a cloned ceramide UDP galactosyltransferase cDNA to directly determine, in a genetic system, if galactosylceramide can operate as a receptor for the AIDS virus, alternative to the CD4-dependent HIV infection pathway.
| Li, J; Yen, T Y; Allende, M L et al. (2000) Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains. J Biol Chem 275:41476-86 |
| Allende, M L; Li, J; Darling, D S et al. (2000) Evidence supporting a late Golgi location for lactosylceramide to ganglioside GM3 conversion. Glycobiology 10:1025-32 |
