Identification of N-acetylglucosamine binding residues in Griffonia simplicifolia lectin II

Abstract

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp 88, Glu 108, Tyr 134, Asn 136, Leu 226 and Gln 227) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp 88, Tyr 134 or Asn 136 eliminated GlcNAc binding activity by GS-II, while those at Glut 108, Leu 226 or Gln 227 did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr 134 eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu 226 and Gln 227 did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.