The long range objective of this proposal is to understand the mode of action and utility of disaccharide-based primers for altering glycosylation in cells. Glycoconjugates play important roles in inflammation, tumor cell migration, and infection. Thus, methods to alter glycosylation may provide new therapeutic agents to treat human disease. To achieve these long-range goals, we propose a series of experiments to test glycoside primers as inhibitors of oligosaccharide biosynthesis. These compounds act like natural biosynthetic intermediates and divert the assembly of oligosaccharides from endogenous glycoproteins to the primers, which are then secreted by the cells. Preliminary studies show that acceptilated Galbeta1-4GlcNAcbeta-O-naphthalenemethanol (AcLacNAc-NM) and acceptilated GlcNAcbeta1-3Galbeta-O-naphthalene-methanol (acGlcNAcGal-NM) permeate cell membranes, prime oligosaccharide synthesis, and alter the expression of important carbohydrates involved in inflammation, such as sialyl Lewis X (sLex). The five year goals include the following studies: 1. Synthesize new disaccharide primers and analogs: AcLacNAc-NM and AcGlcNAcGal-NM prime oligosaccharides and inhibit the expression of sLex when fed to F9, HL-60 and U-937 cells in culture. To study the generality of priming and inhibition of glycoprotein synthesis by disaccharides, the applicant will make acceptilated Galbeta1-3GalNAcalpha-O-naphthalenamethanol, acceptilated GlcNAcbeta1-6GalNAcalpha-O-naphthalenemethanol, and acceptilated GlcNAcbeta1-6Galbeta-O-naphthalenemethanol, which represent subunits of O-linked polylactosaminoglycan chains. The applicant also will prepare analogs in which key hydroxyl groups are missing or methylated as potential inhibitor. 2. Study priming activity and inhibition of glycoconjugate formation: When fed to cells, AcLacNAc-NM and AcGlcNAcGal-NM primed a number of oligosaccharide products. The applicant plans to study how cells activate the acceptilated primers and if different blocking strategies affect uptake and priming. Analytical work will establish the structure of the oligosaccharides made on the primers and whether truncated oligosaccharides accumulate on endogenous glycoproteins. The applicant will examine PSGL-1, the major glycoprotein on HL-60 cells that mediates adhesion to E- and P-selectins. If any of the methylated or deoxygenated disaccharide analogs inhibit glycosylation, the applicant will examine their action on specific enzymatic targets in vitro. 3. Examine adhesion of leukocytes in vitro and in vivo: Treating HL-60 and U-037 cells with AcLacNAc-NM and AcGlcNAcGal-NM inhibits static adhesion to TNF-alpha activated human umbilical vein endothelial cells. The applicant plans to study the effect of active compounds under dynamic flow condition in vitro. Active compounds also will be tested in a mouse inflammatory model that measures infiltration of leukocytes into the peritoneum in response to irritation. A second model involves measuring the anti-arthritic activity of disaccharides in mice immunized with bovine Type II collagen or cartilage proteoglycan.
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