Purification and characterization of mannan-binding protein from mouse serum.
Mouse mannan-binding protein (MBP) was identified in serum by its Ca(2+)-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 microgram/ml to 100 micrograms/ml (average 50 micrograms/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.
Holt
P
P
Department of Medical Microbiology, University of Odense, Denmark.
Holmskov
U
U
Thiel
S
S
Teisner
B
B
Højrup
P
P
Jensenius
J C
JC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001797:Blood Protein Electrophoresis; D002352:Carrier Proteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D037222:Collectins; D003171:Complement Pathway, Classical; D004187:Disaccharides / Q000494:pharmacology; D008351:Mannans / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000494:pharmacology
D000596:Amino Acids; D002352:Carrier Proteins; D037222:Collectins; D004187:Disaccharides; D008351:Mannans; D009005:Monosaccharides