Sialylated oligosaccharides play pivotal roles in a variety of important biological processes, including inflammation, B cell maturation and activation, virus infection, migration of embryonic and cancer cells, and maintenance of glycoproteins in circulation. The overall goal of this research program is to understand the mechanisms that control the biosynthesis of sialylated oligosaccharides. In this research proposal the applicant will investigate mechanisms that control the activity of the alpha 2,6-sialyltransferase of Asn-linked glycosylation (ST6Gal I, ST). These include {1} enzyme compartmentation in specific Golgi cisternae, {2} differential expression of two enzyme isoforms that differ in catalytic activity, cellular localization and turnover, and {3} formation of inactive enzyme dimers. Three hypotheses will be tested. Hypothesis I: The specific Golgi compartmentation of the ST involves not only its transmembrane region, but also its lumenal stem and catalytic domains, and these regions mediate an oligomerization event that leads to Golgi retention. To test this hypothesis the applicant will determine which stem sequences are required for Golgi retention, investigate the role of the ST catalytic domain in Golgi retention by EM localization of two enzyme isoforms that differ by a single amino acid in this region, and determine whether Golgi retention correlates with ST oligomerization. Hypothesis II: The differential expression of two ST isoforms profoundly impacts the ability of specific cells and tissues to sialylate glycoproteins. To test this hypothesis the applicant will determine the distribution of these two ST isoforms in different tissues and cell types and investigate the qualitative and quantitative differences in isoform activity in vitro and in vivo. Hypothesis III: High levels of ST expression lead to the formation of inactive ST homodimers which are formed by disulfide bonds between cysteine residues in the enzyme's donor binding site. To test this hypothesis the applicant will determine where the ST dimer is formed, if dimerization increases turnover rate, which cysteine residues are involved in dimerization, and what conditions favor dimerization. The information gained from this investigation will not only further our understanding of the biosynthesis of sialylated oligosaccharides and how this process can be altered during cancer and disease, but also allow the applicant to apply what is learned to enhancing and developing a variety of carbohydrate-based therapeutic strategies.
| Zhao, Weihan; Chen, Tung-Ling L; Vertel, Barbara M et al. (2006) The CMP-sialic acid transporter is localized in the medial-trans Golgi and possesses two specific endoplasmic reticulum export motifs in its carboxyl-terminal cytoplasmic tail. J Biol Chem 281:31106-18 |
| Fenteany, Fiona H; Colley, Karen J (2005) Multiple signals are required for alpha2,6-sialyltransferase (ST6Gal I) oligomerization and Golgi localization. J Biol Chem 280:5423-9 |
| Close, Brett E; Mendiratta, Shalu Shiv; Geiger, Kristin M et al. (2003) The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II. J Biol Chem 278:30796-805 |
