Binding of sugar ligands to Ca(2+)-dependent animal lectins. II. Generation of high-affinity galactose binding by site-directed mutagenesis.

S.T Iobst,K Drickamer

doi:10.1016/s0021-9258(17)40709-5

Abstract

Changes have been introduced into the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein by site-directed mutagenesis to model the binding sites of homologous galactose-binding CRDs. Binding assays reveal that galactose-binding activity nearly identical to that of the CRD from the asialoglycoprotein receptor can be introduced into the mannose-binding site by 3 single amino acid changes and insertion of a segment of 5 amino acids. Separate changes are required to establish high-affinity binding to galactose and create high selectivity by exclusion of mannose from the binding site. The mutagenesis studies and NMR analysis of sugar-CRD titrations demonstrate that an important component of high-affinity galactose binding is interaction between the B face of the sugar and tryptophan. The binding properties of the C-type CRD from the cartilage proteoglycan, aggrecan, can also be modeled based on the mannose-binding CRD frame-work. This lower affinity binding site involves stacking of a phenylalanine residue against the sugar ligand.

Highlights

Ent carbohydrate-recognition domain (CRD) of rat se- the relative affinities of the different collectins for rum mannose-binding protein by site-directed mutagethne-se sugars varies widely [5, 6].In contrast, the asialoglycoesis to model the bindinsgites of homologous galactose- protein receptor and related lectins preferentially bindgalacbindingCRDs.Bindingassaysrevealthatgalactosetose and N-acetylgalactosamine [7]
Acid carbohydrate-recognition domains (CRDs)‘ [1].Based on the crystal structure of the C-type CRD from rat serum mannose-binding protein [2], it seems likely that all the CRDs have similar overall three-dimensional folds, since the 32 conserved amino acid residues characteristicof all of these CRDs form two disulfide bonds, Ca2+-binding sites, and the hydrophobic core which together determine the structureof the CRD
Given their proximity to the mannose- charides show that selectivity of the QPDW mutant for galacbinding site, certain of these changes were selected as candi- tose over mannose is not comparable to the natural galactosedates for incorporation inttohe mannose-binding CRD. selective CRD, and isnot significantly different from the QPD

Summary

11. GENERATION OF HIGH-AFFINITY GALACTOSE BINDING BY SITE-DIRECTED MUTAGENESIS*

(Received for publication, February 8, 1994, and in revised form, March 31, 1994). From the Department of Biochemistry and Molecular Biophysics, Columbia University,New York, New York 10032. Acid carbohydrate-recognition domains (CRDs)‘ [1].Based on the crystal structure of the C-type CRD from rat serum mannose-binding protein [2], it seems likely that all the CRDs have similar overall three-dimensional folds, since the 32 conserved amino acid residues characteristicof all of these CRDs form two disulfide bonds, Ca2+-binding sites, and the hydrophobic core which together determine the structureof the CRD. NMR procedures were exactly as for mannose-binding mutants [11]

AND DISCUSSION

Recipient of a Faculty Salary Awardfrom the American Cancer

B WTATIOWS I N QPDUG BACKGROUND NcoIAM IR saISelJAI

PROTEIN

A MUTANT QPDW n

A MUTANT QPDWG