Many glycoproteins have been identified as mediators of important physiological and pathological events. It has long been known that changes as small as a single residue in the amino acid sequence can have substantial functional effects. More recently, evidence has accumulated that modifications in the carbohydrate portions of glycoproteins, without alteration of the protein backbone, are also correlated with differences in function. We feel that characterization of carbohydrate moieties has been greatly impeded by the lack of simple methodology in laboratories without the expensive, specialized equipment and expertise for mass spectrometry, HPLC, etc. An immense literature has shown the usefulness of SDS-PAGE and immunoblot for separation and analysis of proteins, and of glycoproteins manipulated through their polypeptide backbones. In contrast, the literature on gel electroplioresis of oligosaccharides is scant. Reports of blotting from such gels indicate, at best, very limited success. We are unaware of any studies demonstrating simultaneous resolution of oligosaccharides and polypeptides, although these two species might well be the predicted products of analysis of a glycoprotein by selective enzymatic digestions. Our preliminary data outline novel conditions of SDS-PAGE which resolve free oligosaccharides and polypeptides simultaneously, and suggest the feasibility of a simple fluorescence method for detecting oligosaccharides of uncertain immunoreactivity. The research proposed in this application has two major goals: l) We would like to complete the development of the new methodology discussed above, with particular reference to establishment of conditions of immunoblot and/or fluorescence detection appropriate for free oligosaccharides. 2) We hope to use the novel methodology to characterize a glycoprotein which has been a long term research focus of this laboratory. Specifically, we plan to assess the cutaneous lymphocyte-associated antigen (CLA) carbohydrate modification of the P-selectin glycoprdtein ligand-l (PSGL-l) as fully as possible by simple methodology, and to isolate the carbohydrate for structural analysis by the more specialized methods noted above. PSGL-l, expressed by leukocytes, functions as an adhesion molecule for P-selectin on the vascular endothelium. Recent research indicates that the CIA carbohydrate modification of PSGL- l confers the capacity to bind E-selectin as well as P-selectin, and that this dual binding capacity is required for efficient migration of T cells into inflamed skin. Thus detailed knowledge of the biochemistry of CIA may well suggest possibilities for intervention in the T cell extravasation of chronic inflammation and of lymphoid tissue metastasis.
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