Carbohydrate-binding protein 35. II. Analysis of the interaction of the recombinant polypeptide with saccharides

R. N. Knibbs, N. Agrwal, J. L. Wang, I. J. Goldstein

Research output: Contribution to journalArticle

79 Scopus citations

Abstract

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 β-linkage were capable of forming a precipitate with rCBP35. If the glycoprotein or glycoconjugate contained terminal Neu5Ac, or galactose in α-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 Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAc-BSA reacted more strongly than the disaccharide Galβ1,4GlcNAc-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 104 M-1. The binding site on the polypeptide appears to contain four subsites that recognize the sequence Galβ1,4GlcNAcβ1,Galβ1,X- . All disaccharides tested that contain a nonreducing β-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-β-D-galactoside itself is an extremely poor inhibitor.

Original languageEnglish (US)
Pages (from-to)14940-14947
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number20
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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