Biochemical and ultrastructural studies of the C-type lectin bovine conglutinin.
The aim of this study was to correlate the supramolecular organization of conglutinin (BK) with its primary and tertiary structure and to gain more knowledge of functionally important regions of the molecule. BK analyzed by SDS-PAGE under standard reducing conditions (40 mM DTT) showed a major band at 43 kDa and weaker bands at 86 and 180 kDa. In contrast, reduction with 6-50 mM L-cysteine resulted in 37-kDa subunits indicating the presence of intrachain disulfide bonds within this subunit. Hydroxylamine treatment indicated presence of ester bonds in the 86- and 180-kDa subunits. Collagenase digestion and SDS-PAGE under reducing and nonreducing conditions resulted in bands of 20 and 15 kDa, respectively, indicating the presence of intrachain, rather than interchain, disulfide bonds in the carboxy terminus. Deglycosylation and glycan differentiation analysis of BK revealed the presence of O-linked glycans of GalNAc and alpha (2-3) linked sialic acid type, whereas no N-linked glycans were demonstrated. Binding experiments with GlcNAc-gold suggested that multivalency is required for carbohydrate binding to BK. Electron microscopy showed mostly tetramers, 96 nm in diameter, but also mono-, di-, and trimers were seen. The tetramers consisted of 40-nm strands, each with a peripheral globular head composed of subunits and connected to a common central lobe built from four ring-formed structures. The strands occasionally showed two bends, one close to the central lobe and another 25 nm from the lobe. These bends most likely correspond to the interrupted Gly-Xaa-Yaa repeats at residues 38 and 123.
Andersen
O
O
Department of Medical Microbiology, Odense University, Denmark.
Nielsen
E H
EH
Storgaard
P
P
Højrup
P
P
Friis
P
P
Leslie
G
G
Svehag
S E
SE
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D017364:Collagenases; D037222:Collectins; D006031:Glycosylation; D019811:Hydroxylamine; D006898:Hydroxylamines; D037102:Lectins / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012712:Serum Globulins / Q000737:chemistry* / Q000648:ultrastructure
D037222:Collectins; D006898:Hydroxylamines; D037102:Lectins; D012712:Serum Globulins; C024014:conglutinin; D019811:Hydroxylamine; D017364:Collagenases