UBC Theses and Dissertations
Investigating the expression of N-acetylglucosamine 6-0 sulfotransferases in myeloid cells and its implications on CD44 sulfation Tjew, Sie Lung
The sulfation of proteoglycans and glycoproteins has been implicated in many biological processes. Glycoproteins on high-walled endothelial venules present sialyl 6-sulfo Lewis x determinants that can mediate lymphocyte homing to lymph nodes by serving as ligands for L-selectin on circulating lymphocytes. CD44 represents a family of glycoproteins that is expressed on most cell types. Interactions between CD44 and hyaluronic acid (HA) have been implicated in many facets of the inflammatory response, including leukocyte recruitment. CD44-HA binding is tightly regulated by several mechanisms including changes in CD44 glycosylation. Recently, increased sulfation of CD44 was correlated with increased HA binding induced by the pro-inflammatory cytokine, tumour necrosis factor (TNF)α, in the myeloid cell line, SR91, and in human peripheral blood monocytes (PBM). Additional experiments determined 6-sulfo N-acetyllactosamine (LacNAc)/Lewis x carbohydrate epitopes, as defined by the monoclonal antibody AG107, were induced on N and O-linked glycans of CD44 by TNFα in SR91 cells. This led to the hypotheses that the induction of the AG107 epitope on CD44 was associated with increased expression of a specific N-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST) and that the AG107 epitope was involved in the inflammatory process by mediating CD44-ligand interactions. Semi-quantitative reverse-transcriptase polymerase chain reaction revealed that TNFα upregulated the expression of GlcNAc6ST-1 in SR91 cells and GlcNAc6ST-1 and -4 in human PBM. In addition, GlcNAc6ST-1 was implicated in the synthesis of the AG107 epitope on CD44. Transcripts for GlcNAc6STs were not increased in mouse macrophages and human M1 macrophages exposed to pro-inflammatory mediators. By contrast, GlcNAc6ST- 1 and GlcNAcGST-4 transcripts were upregulated in human M2 macrophages incubated with TNFα. However, the AG107 epitope was not detected in these cells. Overall, this work suggests that GlcNAc6ST-1 and GlcNAc6ST-4 are involved the inflammatory process by generating 6-sulfo LacNAc/Lewis x epitopes on CD44 of human PBM. However, the results presented herein do not support a role for the AG107 epitope in TNFα-induced HA binding or L-selectin mediated rolling. In addition, GlcNAc6ST- 1 and GlcNAc6ST-4 may also play a role in functions associated with human M2 macrophages, such as Th2-mediated immunity and wound healing, but not by generating the AG107 epitope.
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