Purification and Characterization of Mannan‐Binding Protein from Mouse Serum

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Abstract
Mouse mannan-binding protein (MBP) was identified in serum by its Ca2+ -dependent binding to mannan. On gel permeation chromatography, the protein eluted corresponding to a molecular weight of approximately 750 kDa. Analysed on SDS-PAGE under reducing conditions, the polypeptide showed an apparent molecular weight of 28 kDa, while several high molecular weight bands were seen under non-reducing conditions. The presence of collagen-like domains within the molecule was indicated by a high glycine content (14.9%) and substantiated by sensitivity to collagenase. Rabbit anti-mouse MBP antisera were raised. The concentration of MBP in serum from normal mice was measured by rocket immunoelectrophoresis and found to be from below 1 μg/ml to 100 μg/ml (average 50 μg/ml, n= 60). The binding of mouse MBP to mannan could be inhibited by mono- and disaccharides in the following order of potency: L-fucose > D-mannose > N-acetyl-D-glucosamine > maltose > D-mannoheptulose > D-glucose > N-acetyl-D-mannosamine ≫ lactose > D-galactose ≫ N-acetyl-D-galactosamine. Mouse MBP was shown to activate the classical complement cascade after binding to mannan. The sequence of 14 NH2- terminal amino acid residues of the molecule showed 93% identity to rat MBP-A and complete identity to the translated cDNA sequences for mouse MBP-A and mouse Ra-reactive factor component P28b (RaRF P28b) published previously. The amino acid composition of mouse MBP showed a high degree of homology to MBPs from other species and mouse RaRF P28b.