Purification and characterization of bovine mannan-binding protein.
Bovine mannan-binding protein (bMBP) was observed in serum by its Ca(2+)-dependent binding to mannan and by an M(r) of 28 kDa under reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The lectin was isolated by precipitation with polyethyleneglycol (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 mannose-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 M(r) approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Holmskov
U
U
Department of Medical Microbiology, University of Odense, Denmark.
Holt
P
P
Reid
K B
KB
Willis
A C
AC
Teisner
B
B
Jensenius
J C
JC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002845:Chromatography; D017364:Collagenases / Q000378:metabolism; D037222:Collectins; D004591:Electrophoresis, Polyacrylamide Gel; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight
D000596:Amino Acids; D002241:Carbohydrates; D002352:Carrier Proteins; D037222:Collectins; D008351:Mannans; D017364:Collagenases