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Analysis of components responsible for the cellular localization and adhesive properties of CD44

University of British Columbia

CD44 is a widely expressed cell adhesion molecule that binds the extracellular matrix component, hyaluronan in a tightly regulated manner. As a molecule involved in adhesion and cell migration, CD44 has been shown to interact with elements of the cytoskeleton via its cytoplasmic domain. A significant portion of CD44 is Triton X-100 insoluble in fibroblasts and this has been interpreted to reflect associations with the actin cytoskeleton. However, the detergent insolubility of CD44 in NIH 3T3 cells cannot be attributed to an interaction with the cytoskeleton since CD44 lacking a cytoplasmic tail remains insoluble after Triton X-100 extraction. Instead, after equilibrium density gradient centrifugation on sucrose, a proportion of CD44 was found in the low density lipid fractions, indicating that CD44 insolubility in NIH 3T3 cells is due to an association with Triton X-100-insoluble lipids. Furthermore, the migration of CD44 to the low density fraction is dependent on the transmembrane domain of CD44, not the cytoplasmic domain. Previous studies have indicated that the CD44-hyaluronan interaction is affected by changes in the glycosylation state of CD44. Murine L cell variants defective in glycosaminoglycan synthesis and oligosaccharide processing were used to assess the effects of these modifications on CD44-mediated hyaluronan binding. Analysis of constitutive and antibody-induced hyaluronan binding ability in these cells indicated that sulfation, and in particular, modification by chondroitin sulfate is required for inducible hyaluronan binding in L cells. In the absence of fully processed oligosaccharides, chondroitin sulfate is not essential for hyaluronan binding, indicating that the effect of chondroitin sulfate is dependent on the glycosylation state of the cell. Thus, in addition to glycosylation, chondroitin sulfate biosynthesis is an important post-translational modification that can affect CD44-mediated hyaluronan binding. CD44-hyaluronan interactions have been been implicated in leukocyte rolling and extravasation at inflammatory sites. CD44 is normally expressed on leukocytes in an inactive state that cannot bind hyaluronan but can be converted to an active state upon activation by antigen or cytokines. Here it is demonstrated that the pro-inflammatory cytokine tumour necrosis factor α, but not interferon γ, could convert CD44 from an inactive to an active hyaluronan binding form by inducing the sulfation of CD44 on the SR91 myeloid cell line. This post-translational modification was required for CD44-mediated binding to hyaluronan and to the vascular endothelial cell line, SVEC4-10. Tumour necrosis factor α-inducible hyaluronan binding was also observed on peripheral blood mononuclear cells, especially on CD14 positive monocytes and hyaluronan binding appeared to be at least partially sulfation dependent. In addition, monocyte-specific pro-inflammatory chemokines macrophage inflamatory protein-1α and monocyte chemoattractant protein-1 could induce hyaluronan binding in a subpopulation of peripheral blood mononuclear cells although it could not be determined definitively that the induction was sulfation dependent. Taken together, sulfation may be a means of regulating CD44-mediated adhesion, both in leukocytes at inflammatory sites as well as in fibroblasts to facilitate cell migration.

Thesis/Dissertation

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2009-07-15

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http://hdl.handle.net/2429/10818

Microbiology and Immunology

University of British Columbia

UBCV

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