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Characterization of mouse CD43 recombinant proteins secreted by EL4, CTL2c, CTLL and NSF60 cells

University of British Columbia

Leukosialin (CD43) is a heavily glycosylated mucin-type acidic cell surface protein, carrying 70-80 O-glycans. The molecular weight heterogeneity of both human and murine CD43 glycoforms are due to the variation in their O-linked glycans. There are two major CD43 glycoforms. Resting T cells express predominantly a 115 kDa glycoform of CD43, carrying mainly tetrasaccharide side chains, whereas activated T cells carry mainly hexasaccharide side chains and express CD43 as a 135 kDa glycoform. The activity of (31- 6GlcNAc transferase (C2GnT) has been shown to be correlated with the expression of CD43 135 kDa. Since the function of the two major CD43 glycoforms is not clear, CD43 chimeric proteins have been produced to identify potential ligands of CD43 and to study its role(s) in the immune response. The recombinant chimeric glycoprotein comprises the extracellular domain of murine CD43 (mCD43) and part of human IgGl (hlgG) including the hinge, CH2 and CH3 domains. The cDNA encoding the mCD43-hIgG chimeric molecule was subcloned into two expression vectors which are driven by either metallothionein or SRa promoter. Both vectors were transfected into three T cell lines: EL4, CTL2c, CTLL and myeloid cell line, NSF60. EL4 cells express exclusively CD43 115 kDa which is specifically recognized by the monoclonal antibody (mAb), S7 whereas CTL2c and CTLL cells express exclusively CD43 135 kDa which is specifically recognized by mAb 1B11. NSF60 cells express CD43 glycoforms which are detected by S7 and IB 11. Western blotting analysis demonstrated that the anti-CD43 antibody reactivity of the chimeras secreted by transfected EL4, CTL2c, CTLL and NSF60 cells was identical to the cell surface CD43. The EL4 chimera was recognized by mAb S7, while the CTL2c and CTLL chimeras were recognized by mAb 1B11. NSF60 cells secreted a chimera which was reactive with both antibodies. These results suggest that the mCD43-hIgG chimera secreted by transfected EL4 cells predominantly carried tetrasaccharide cores whereas the chimeras secreted by transfected CTL2c and CTLL cells predominantly carried hexasaccharide cores. NSF60 chimeric protein had tetrasaccharide and hexasaccharide structures. The four chimeras had a lOkDa higher MW than CD43 expressed on the surface of its corresponding cells. This 10 kDa difference in MW can be fully explained by the replacement of the transmembrane and cytoplasmic domains of CD43 with pools of the human IgG constant domain. This further indicated that chimeras have similar or identical glycosylation as cell surface CD43. Non-reducing SDS-PAGE showed that all four chimeras were as expected secreted as dimers. Precipitation using Jacalin-sepharose specific for tetrasaccharide cores, demonstrated that CD43 115 kDa expressed on EL4 cells and its corresponding 125 kDa chimera were efficiently precipitated, while the other three chimeric proteins were less reactive with Jacalin sepharose. This result supported that the O-glycans expressed on the chimeric proteins were identical to those expressed on CD43. This study has successfully established novel tools for the search of CD43 glycoforms specific ligands.

Thesis/Dissertation

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2009-03-09

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http://hdl.handle.net/2429/5764

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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