CARBOHYDRATE-BINDING PROTEIN-35 .2. ANALYSIS OF THE INTERACTION OF THE RECOMBINANT POLYPEPTIDE WITH SACCHARIDES

The carbohydrate binding specificity of recombinant carbohydrate-bindi ng protein 35 (rCBP35) has been investigated by quantitative precipita tion using a series of glycoproteins and carbohydrate-protein conjugat es and by inhibition of precipitation using well defined carbohydrate haptens. Synthetic glycoconjugates and glycoproteins containing termin al nonreducing galactosyl units in beta-linkage were capable of formin g a precipitate with rCBP35. If the glycoprotein or glycoconjugate con tained terminal Neu5Ac, or galactose in alpha-linkage, precipitate for mation was not observed. We also found that murine laminin, which cont ains polylactosamine structures, reacted more strongly than did bovine fetuin. Using carbohydrate-bovine serum albumin (BSA) glycoconjugates , we found that the tetrasaccharide Galbeta1,4GlcNAcbeta1,3Galbeta1,4- GlcNAc-BSA reacted more strongly than the disaccharide Galbeta1,4GlcNA c-BSA conjugate, suggesting that the binding site accommodates carbohy drate ligands greater in size than a disaccharide. Equilibrium dialysi s experiments using [H-3]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 co ntain four subsites that recognize the sequence Galbeta1,4GlcNAcbeta1, Galbeta1,X-. All disaccharides tested that contain a nonreducing beta- galactosyl unit behaved as inhibitors of precipitation at approximatel y the same concentration, suggesting that the reducing position of the tetrasaccharide does not play an important role in the specific bindi ng to the fourth subsite. The reducing sugar may serve to hold the sac charide in a tunnel like binding pocket since methyl-beta-D-galactosid e itself is an extremely poor inhibitor.