(1) Effects of N-glycosylation on the activity and trafficking of GlcNAc-6-sulfotransferase 1 and (2) Metabolic incorporation of a photosugar into cellular glycolipids

Oligosaccharides covalently linked to proteins or lipids, also known as glycans, mediate interactions of cells with their environments, including cell-pathogen interactions, cell-cell interactions, and cell-toxin interactions. These heterogeneous sugars are synthesized in the secretory pathway of eukaryotic cells in a stepwise fashion by enzymes called glycosyltransferases.;Glycans mediate many cellular interactions, including those between a glycolipid and a protein. The Kohler lab recently developed photoactivatable sugars that have been successfully incorporated into cellular glycoproteins in cells; however, it was unknown whether these sugars could be incorporated into glycolipids in cells. The goal of this part of my thesis was to demonstrate the metabolic incorporation of photocrosslinking sugars into gangliosides. I used two general approaches: (1) testing whether photosugar sialic acids incorporated into gangliosides in cells can bind with known ganglioside binding partners and (2) demonstrating that these photo-gangliosides are able to crosslink with a protein binding partner. In order to accomplish these goals, I began experimenting with the K20 sublines of the human Burkitt's lymphoma BJAB cell line, because BJAB K20 cells lack UDP-GlcNAc-2-epimerase, and are thus unable to synthesize sialic acid. These cells may be grown in serum-free media to ensure that sialic acid taken up from the media comes from sialic acids added to the media, rather than from sialic acids in the serum. The parent BJAB cell line manufactures GM1, among other gangliosides. Thus, upon addition of sialic acid to K20 cells, we expected that the biosynthesis of sialic acid containing glycolipids would be reconstituted, and that the cells would be able to bind the cholera toxin B subunit. Upon addition of exogenous sialic acid (NeuAc, SiaDAz) or an analog of its precursor ManNAc (ManDAz) to BJAB K20 cells maintained without serum, this interaction should be reconstituted and monitored using flow cytometry. After initial promising results where I titrated the amount of FITC labeled cholera toxin B subunit used in the experiment, I obtained several confusing results, where cholera toxin appeared to bind with higher affinity to BJAB K20 cells grown in serum-free medium, rather than to cells with either added sialic acid or to K88 cells. I tested the hypothesis that the toxin was binding to BSA used in the blocking solution but found that this did not appear to be the case. Rather, cell lines lacking the UDP-GlcNAc 2-epimerase may be unable to synthesize gangliosides due to concurrent downregulation of ganglioside biosynthetic enzymes. This downregulation is a likely cause of some of the unexpected binding events. In a continued effort to determine whether photosugars were incorporated into glycolipids in cells, I began an effort to determine if photosugars are incorporated in glycolipids in 3T3-L1 murine adipocytes and if the resulting photo-ganglioside GM3 is competent to interact with the insulin receptor. I was able to confirm the ganglioside content of these cells, by isolating the total lipid extract and analyzing it by TLC, and to immunoprecipitate insulin receptor from differentiated 3T3-L1 cells. Further experiments will be required to determine whether the photosugars are being incorporated into gangliosides in these cells, and whether they are able to crosslink to the insulin receptor.;Glycans may be modified with functional groups other than sugars, such as sulfate. N-acetylglucosamine-6-sulfotransferase-1 (GlcNAc6ST-1) is a Golgi-resident glycoprotein that is responsible for sulfation of the L-selectin ligand on endothelial cells. Here we report the sites at which GlcNAc6ST-1 is modified with N-linked glycans and the effects that each glycan has on enzyme activity, specificity, and trafficking. We determined that glycans are added at three of four potential N-linked glycosylation sites: N196, N410, and N428. The N428 glycan is required for production of sulfated cell surface glycans: cells expressing a mutant enzyme lacking this glycan were unable to sulfate the sialyl Lewis X tetrasaccharide or the lower branch of the core 1 O-linked glycan. The N196 and N410 glycans differentially affect sulfation of two different substrates: cells that express an enzyme lacking the N410 glycan are able to sulfate the sialyl Lewis X substrate, but do not produce the sulfated peripheral lymph node addressin epitope; cells that express an enzyme lacking the N196 glycan are able to produce both sulfated epitopes, but sulfate sialyl Lewis X at only low levels. Our data also suggest a role for the N410 in the process that results in recognition of incorrectly folded proteins. Glycans' effects on enzyme activity may be mediated, in part, by changes in enzyme localization. While most mutants that lacked glycans localized normally within the Golgi, the N428A mutant and a mutant lacking all glycans were also found to localize ectopically. Altered trafficking of mutants may be associated with the mechanisms by which misglycosylated enzymes are degraded.