Heterogeneous nature of human complement factor B: an electrophoretic approach for the analysis of its oligosaccharide chain structure and its physiological breakdown products.
Factor B is a glycoprotein which plays an essential role in the alternative pathway of complement activation. It carries the proteolytic activity of the convertases, and its physiological breakdown products Ba and Bb have some effects on the cells of the immune system. Human factor B exhibits a microheterogeneity and five isoforms are present in serum. The nature and origin of the microheterogeneity was investigated by using electrophoretic techniques. Treatments of factor B with neuraminidase and glycopeptidase F show that this microheterogeneity is mainly due to differences in its sialic acid content, varying from seven to eleven residues per molecule, and resulting in different oligosaccharide structures. However, deglycosylated factor B reveals a residual, nonallotypic variation in the Bb region of the polypeptide backbone. We confirm the presence of four asparagine-linked oligosaccharide chains of the complex type in native factor B, two of which are located in the Ba fragment, and the two others in the Bb fragment. The prevalent isoform of the native protein carries two sialic acid residues per oligosaccharide chain. Biosynthesis experiments show that the microheterogeneity of secreted factor B from HepG2 cells is acquired during the processing of its glycans. However, in vitro-secreted factor B is more heterogeneous than the serum protein. We propose a structural model for the microheterogeneity of the native protein and its physiological fragments. We discuss as well the feasibility of electrophoretic techniques to deal with microheterogeneity analysis.
Institut National de la Santé et de la Recherche Médicale, U78, Bois-Guillaume, France.
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G
Garnier
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Davrinche
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Charlionet
D000581:Amidohydrolases; D011415:Complement Factor B / Q000378:metabolism*; D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004792:Enzyme Precursors / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D007536:Isomerism; D008956:Models, Chemical; D009439:Neuraminidase; D009844:Oligosaccharides / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011233:Precipitin Tests; D012794:Sialic Acids / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014508:Urea
D004792:Enzyme Precursors; D009844:Oligosaccharides; D012794:Sialic Acids; D014508:Urea; D009439:Neuraminidase; D011415:Complement Factor B; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase