Purification and characterization of bovine mannan-binding protein

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Abstract
Bovine mannan-binding protein (bMBP) was observed in serum by its Ca2+ -dependent binding to mannan and by an Mrof 28 kDa under reducing conditions on sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS—PAGE). The lectin was isolated by precipitation with polyethyl-eneglycol (PEG), affinity chromatography on mannan—Sepharose eluted with EDTA, and absorption on Sepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies. Fractions containing the lectin were reapplied to mannan—Sepharose and eluted first with N-acetyl-D-glucosamine (GlcNAc) to remove conglutinin, and then with mannose to elute the 28 kDa lectin. Further purification was achieved by ion-exchange chromatography on Mono-Q and by man-nose-gradient elution from a mannan-Sepharose column. SDS—PAGE of the purified lectin showed three high molecular weight bands under non-reducing conditions. The reduced protein gave a single band of 28 kDa. On gel permeation chromatography under non-dissociating conditions, the protein emerged at a volume corresponding to Mr ∼750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine, and a high glycine content (17.7%), suggesting the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The N-terminal 26 amino acids showed 62%identity with human MBP, when three gaps were allowed in the alignment. The binding of bio-tinylated bMBP to solid-phase mannan was inhibited by carbohydrates in the following order of potency: fucose >mannose > ManNAc > GlcNAc >glucose >> maltose, galactose, lactose, GalNAc. Previously, a 45 kDa bovine protein has been tentatively identified as bovine MBP (Kawasaki, N., Kawasaki, T. and Yamashina, I. (1985) J. Biochem., 98, 1309–1320). We conclude that the 28 kDa lectin which we have described is more likely to represent the bovine analogue of rat and human MBP.

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