Strict specificity for high-mannose type N-glycans and primary structure of a red alga Eucheuma serra lectin

Abstract

We have elucidated the carbohydrate-binding profile of a non-monosaccharide-binding lectin named Eucheuma serra lectin (ESA)-2 from the red alga Eucheuma serra using a lectin-immobilized column and a centrifugal ultrafiltration-high performance liquid chromatography method with a variety of fluorescence-labeled oligosaccharides. In both methods, ESA-2 exclusively bound with high-mannose type (HM) N-glycans, but not with any of other N-glycans including complex type, hybrid type and core pentasaccharides, and oligosaccharides from glycolipids. These findings indicate that ESA-2 recognizes the branched oligomannosides of the N-glycans. However, ESA-2 did not bind with any of the free oligomannoses examined that are constituents of the branched oligomannosides implying that the portion of the core N-acetyl-d-glucosamine (GlcNAc) residue(s) of the N-glycans is also essential for binding. Thus, the algal lectin was strictly specific for HM N-glycans and recognized the extended carbohydrate structure with a minimum size of the pentasaccharide, Man(α1-3)Man(α1-6)Man (β1-4)GlcNAc(β1-4) GlcNAc. Kinetic analysis of binding with a HM heptasaccharide (M5) showed that ESA-2 has four carbohydrate-binding sites per polypeptide with a high association constant of 1.6 × 108 M-1. Sequence analysis, by a combination of Edman degradation and mass analyses of the intact protein and of peptides produced by its enzymic digestions, showed that ESA-2 is composed of 268 amino acids (molecular weight 27950) with four tandemly repeated domains of 67 amino acids. The number of repeats coincided with the number of carbohydrate-binding sites in the monomeric molecule. Surprisingly, the marine algal lectin was homologous to hemagglutinin from the soil bacterium Myxococcus xanthus. © The Author 2007. Published by Oxford University Press. All rights reserved.