Carbohydrate-binding protein 35. II. Analysis of the interaction of the recombinant polypeptide with saccharides.
The carbohydrate binding specificity of recombinant carbohydrate-binding protein 35 (rCBP35) has been investigated by quantitative precipitation using a series of glycoproteins and carbohydrate-protein conjugates and by inhibition of precipitation using well defined carbohydrate haptens. Synthetic glycoconjugates and glycoproteins containing terminal nonreducing galactosyl units in beta-linkage were capable of forming a precipitate with rCBP35. If the glycoprotein or glycoconjugate contained terminal Neu5Ac, or galactose in alpha-linkage, precipitate formation was not observed. We also found that murine laminin, which contains polylactosamine structures, reacted more strongly than did bovine fetuin. Using carbohydrate-bovine serum albumin (BSA) glycoconjugates, we found that the tetrasaccharide Gal beta 1, 4GlcNAc beta 1, 3Gal beta 1,4-GlcNAc-BSA reacted more strongly than the disaccharide Gal beta 1, 4GlcNAc-BSA conjugate, suggesting that the binding site accommodates carbohydrate ligands greater in size than a disaccharide. Equilibrium dialysis experiments using [3H]lactose showed that rCBP35 binds 1 mol (n = 0.84) of lactose/30,000 g atoms of protein, with an affinity constant of 2.07 x 10(4) M-1. The binding site on the polypeptide appears to contain four subsites that recognize the sequence Gal beta 1,4GlcNAc beta 1, Gal beta 1,X-. All disaccharides tested that contain a nonreducing beta-galactosyl unit behaved as inhibitors of precipitation at approximately the same concentration, suggesting that the reducing position of the tetrasaccharide does not play an important role in the specific binding to the fourth subsite. The reducing sugar may serve to hold the saccharide in a tunnel like binding pocket since methyl-beta-D-galactoside itself is an extremely poor inhibitor.
Knibbs
R N
RN
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Agrwal
N
N
Wang
J L
JL
Goldstein
I J
IJ
D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism*; D001665:Binding Sites; D002240:Carbohydrate Sequence; D037502:Galectin 3; D006001:Glycoconjugates / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006241:Haptens / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism
D000943:Antigens, Differentiation; D037502:Galectin 3; D006001:Glycoconjugates; D006023:Glycoproteins; D006241:Haptens; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine