Purification, subunit characterization and ultrastructure of three soluble bovine lectins: conglutinin, mannose-binding protein and the pentraxin serum amyloid P-component.
Conglutinin and mannose-binding protein (MBP) are members of the C-type lectins which are widely present in mammalian plasma. Serum amyloid P-component (SAP) is a member of the pentraxin family with lectin properties. A scheme for the partial purification of all three lectins by carbohydrate affinity chromatography and selective elution was developed. The purification was monitored by SDS-PAGE, Western blotting and electron microscopy. Binding of the lectins to Sephadex-iC3b, their collagenase sensitivity, and the size and antibody reactivity of their subunits was investigated. The demonstration, by SDS-PAGE, of 25-kDa subunits, which were unaffected by collagenase treatment but bound to Sephadex-iC3b and antibodies to human SAP, indicated the existence of bovine SAP. Bovine conglutinin (BK) also showed calcium-dependent binding to Sephadex-iC3b, whereas bovine MBP did not. The binding of BK was inhibitable with GlcNAc. A 3000-fold increase in BK activity (ELISA) was obtained in eluates from Sephadex-iC3b. SDS-PAGE analyses of BK and MBP revealed subunits with an Mr of 43 kDa and 30 kDa, respectively. These subunits were sensitive to collagenase treatment which reduced the Mr to 20 kDa. Electron micrographs revealed a prominent flexible tetramer molecule (diameter 96 nm) in the BK preparations, a predominantly hexameric structure (diameter 30 nm) in the MBP preparations, and single annular pentameric disc-like molecules (diameter 11 nm) in the SAP preparations.
Andersen
O
O
Department of Medical Microbiology, Odense University, Denmark.
Friis
P
P
Holm Nielsen
E
E
Vilsgaard
K
K
Leslie
R G
RG
Svehag
S E
SE
D000818:Animals; D015153:Blotting, Western; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002417:Cattle; D002846:Chromatography, Affinity; D037222:Collectins; D003179:Complement C3b / Q000378:metabolism; D037102:Lectins / Q000737:chemistry* / Q000648:ultrastructure; D046911:Macromolecular Substances; D037241:Mannose-Binding Lectins; D003012:Microbial Collagenase / Q000494:pharmacology; D008854:Microscopy, Electron; D008970:Molecular Weight; D000683:Serum Amyloid P-Component / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D012712:Serum Globulins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D002352:Carrier Proteins; D037222:Collectins; D037102:Lectins; D046911:Macromolecular Substances; D037241:Mannose-Binding Lectins; D000683:Serum Amyloid P-Component; D012712:Serum Globulins; C024014:conglutinin; D003179:Complement C3b; D003012:Microbial Collagenase