Purification and characterization of a bovine serum lectin (CL-43) with structural homology to conglutinin and SP-D and carbohydrate specificity similar to mannan-binding protein.
A previously undescribed bovine serum lectin (designated CL-43) was identified by its Ca(2+)-dependent binding to mannan and by its molecular mass of 43 kDa under reducing conditions on SDS-PAGE. The lectin was isolated by polyethylene glycol precipitation, affinity chromatography on mannan-Sepharose (followed by elution with EDTA), and absorption on Sepharose-4B-coupled rabbit anti-bovine Ig (to remove anti-mannan antibodies). Fractions containing the lectin were reapplied to mannan-Sepharose. Bound conglutinin was eluted with GlcNAc, and then the 43-kDa lectin, together with mannan-binding protein (MBP), was eluted with mannose. The 43-kDa lectin was separated from MBP by ion exchange chromatography on Mono-Q. On SDS-PAGE under nonreducing conditions the lectin showed a molecular mass of 120 kDa. On gel chromatography under nondissociating conditions the protein was eluted at a volume corresponding to a molecular mass of approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine and a high content of glycine (24.3%) indicating the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The designation CL-43 was chosen since this molecule appears to belong to the collectins, i.e. proteins with collagen structure and lectin activity. The N-terminal sequence (27 amino acids) showed 56% identity with bovine SP-D and 44% identity to bovine conglutinin. An inhibition assay with biotinylated CL-43, using solid-phase mannan as ligand, revealed the following carbohydrate inhibition pattern: mannose and ManNAc > fucose > GlcNAc > glucose and maltose > galactose > lactose >> GalNAc. We conclude that CL-43 is a circulating lectin, with structural similarities to bovine conglutinin and SP-D, and a ligand binding profile resembling that of MBP.
Holmskov
U
U
Department of Medical Microbiology, University of Odense, Denmark.
Teisner
B
B
Willis
A C
AC
Reid
K B
KB
Jensenius
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002241:Carbohydrates / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry*; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D037222:Collectins; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D037102:Lectins / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012712:Serum Globulins / Q000737:chemistry*
D002241:Carbohydrates; D002352:Carrier Proteins; D037222:Collectins; D006023:Glycoproteins; D037102:Lectins; D008351:Mannans; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D012712:Serum Globulins; C024014:conglutinin; C086954:collectin-43 protein, bovine