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Title: Analysis of cell surface glycoproteins by biotinylated concanavalin A
Author: Buckie, J. W.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1987
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Many of the immune effector functions of T-lymphocytes are mediated through cell-cell interactions involving membrane glycoproteins. During normal T-lymphocyte differentiation, many of these glycoproteins undergo changes in expression and structural modifications. To date, very little is known regarding altertions in the oligosaccharide groups of T-lymphocyte surface glycoproteins that accompany differentiation and maturation of these cells. For this purpose, a method for isolating glycoconjugates from the surface of intact cells was developed. This was achieved by treating acute lymphoblastic leukaemia cells of the AKR mouse with biotinylated concanavalin A (different degrees of biotinylation and a variety of biotinyl derivatives were examined), and retrieving the detergent solubilised biotinylated-Con A/glycoprotein complexes on immobilised stretavidin. From [35S]-methionine labelled cells, surface glycoproteins isolated by this procedure were analysed by two dimensional gel electrophoresis. This technique of 'indirect affinity chromatography' developed here with intact cells provides significant improvements for studying cell surface components compared to other methods which use isolated plasma membrane preparations. The indirect affinity chromatography procedure was also used to analyse the carbohydrate groups of the isolated Con A-binding glycoproteins labelled with [2-3H]-mannose, and resulted in the identification of a high mannose, endoglycosidase H sensitive oligosaccharide at the surface of the acute lymphoblastic leukaemia cells. The indirect affinity chromatography procedure was then applied in a comparative investigation of the difference between surface Con A-binding glycoproteins of quiescent and mitogen stimulated T-lymphpcytes. Using the two dimensional gel electrophoretic technique, changes in the expression of [35S]-methionine labelled Con A-binding glycoproteins were analysed. This sensitive technique allowed the detection of at least five major radiolabelled glycoproteins from stimulated T-lymphocytes that could not be detected in the profile of glycoproteins isolated from quiescent T-lymphocytes. The oligosaccharide groups of the Con A-binding, [2-3H]-mannose labelled glycoproteins isolated from quiescent and Con A-stimulated T-lymphocytes were compared. The predominant glycopeptide isolated from activated T-lymphocytes, using indirect affinity chromatography, was analysed by gel filtration following treatment with endo- and exoglycosidases and was shown to have a Man5G1cNAc2-structure. Since this oligosaccharide could not be isolated from the surface of quiescent T-lymphocytes the significance of its surface location in stimulated T-lymphocytes is discussed. This project shows that protein bound glycan patterns change with mitogenic activation of T-lymphocytes and strengthens the view that particular glycoproteins are differently glycosylated during stages of cellular development and these altered carbohydrate chains have different, specific functions. In an attempt to explain why Man5G1cNAc2- was synthesised by stimulated T-lymphocytes but not by quiescent T-lymphocytes, the enzyme G1cNAc-transferase I, for which this oligosaccharide is a substrate, was assayed in both types of cell. No conclusive answer was provided by this investigation, however, the hypothesis proposed in this dissertation, that changes in the activity of G1cNAc-transferase I with mitogenic stimulation result in the appearance of the Man5G1cNAc2 oligosaccharide at the surface of Con A-stimulated T-lymphocytes, is discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Protein bound glycon patterns