Carbohydrate-binding motifs in a novel type lectin from the sea mussel Crenomytilus grayanus: Homology modeling study and site-specific mutagenesis

• Structure of the CGL lectin belonging to a novel lectin family was studied.
• Homology modeling was used to predict sugar-binding sites of CGL.
• CGL has three sugar-binding sites with conserved HPY(K)G motifs.
• Effects of mutations in three HPY(K)G motifs on CGL activity were studied.
• All three HPY(K)G motifs are involved in CGL-carbohydrate interactions.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) was shown to represent a novel family of lectins and to be characterized by three amino acid tandem repeats with high (up to 73%) sequence similarities to each other. We have used homology modeling approach to predict CGL sugar-binding sites. In silico analysis of CGL–GalNAc complexes showed that CGL contained three binding sites, each of which included conserved HPY(K)G motif. In silico substitutions of histidine, proline and glycine residues by alanine in the HPY(K)G motifs of the Sites 1–3 was shown to lead to loss of hydrogen bonds between His and GalNAc and to the increasing the calculated CGL–GalNAc binding energies.We have obtained recombinant CGL and used site-specific mutagenesis to experimentally examine the role of HPK(Y)G motifs in hemagglutinating and carbohydrate binding activities of CGL. Substitutions of histidine, proline and glycine residues by alanine in the HPYG motif of Site 1 and Site 2 was found to led to complete loss of CGL hemagglutinating and mucin-binding activities. The same mutations in HPKG motif of the Site 3 resulted in decreasing the mucin-binding activity in 6-folds in comparison with the wild type lectin. The mutagenesis and in silico analysis indicates the importance of the all three HPY(K)G motifs in the carbohydrate-binding and hemagglutinating activities of CGL.