To identify glycosylation-specific targets contributing to the interferon gamma-dependent upregulation of sialylated CD44v6 in certain types of intestinal epithelial cells

ABSTRACT: This project was done in collaboration with Dr. Richard Cummings to examine sialylation of CD44v6 and its role in clearance of neutrophils from inflamed intestinal epithelium. Specifically, we have employed a functional approach using membrane preparations from interferon gamma-stimulated intestinal epithelial cells to generate a monoclonal antibody, designated GM35, which blocks neutrophil transepithelial migration through the promotion of neutrophil adhesion at the apical surface of the intestinal epithelium. Protein biochemistry, sequencing, confocal microscopic analysis, and immunoprecipitation studies all identify the protein ligand for this antibody as CD44v6. However, selective inhibition of O- or N-linked glycosylation reveal that the antibody is specific for an O-linked glycotope and glycoarray analysis of the GM35 antibody by Core H of the Consortium for Functional Glycomics reveal that this antibody binds with high affinity and specificity to a carbohydrate epitope consistent in structure with sLeA. Inhibition of O-linked glycosylation attenuated both GM35 binding and its functional effects as did specific cleavage of sialic acid residues from the cell surface, using neuraminidase, although the functional effects of cleavage were smaller and harder to assess. CD44v6 has previously been studied as a marker of inflammation in Inflammatory Bowel Disease, and GM35 staining is clearly upregulated by interferon gamma in T84 and HT29 intestinal epithelial cells. The specific enzymes governing the sialylation of CD44v6 in intestinal epithelium have yet to be determined. Analysis of a panel of intestinal epithelial cells including T84, HT29, and Caco-2 cells revealed that cell-type specific regulation of both splicing and glycosylation is essential for the regulation of the expression of the GM35 epitope. And, forcible expression of CD44 in Caco-2 cells, which are negative for CD44v6 at baseline, results only in upregulation of previously expressed isoforms of CD44, but not in GM35 expression. Based on western blot and immunofluorescent staining, GM35 expression is greatest in interferon gamma-treated T84 cells. Thus, we analyzed RNA samples for the expression of glycosylation specific genes in interferon gamma-stimulated T84 cells relative to that of both non-stimulated T84 cells and GM35-negative Caco-2 cells in order to identify glycosylation-specific targets contributing to the interferon gamma-dependent upregulation of sialylated CD44v6 in intestinal epithelial cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Steven Head 

PROVIDER: E-GEOD-31937 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress