Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, Jeongeup
Sialic acid, N-acetylneuraminic acid (Neu5Ac), is a terminal sugar of oligosaccharides in glycoproteins, glycolipids, and polysaccharides. Glycan chains containing terminal Neu5Ac frequently play important roles as the receptors for the adhesion of bacteria to host tissues as an initial and essential step in the infection process. Lectins with carbohydrate binding specificity are powerful tools to analysis and isolation of glycoconjugates in organisms. The mushroom Polyporus squamosus (PSL) has isolectins, PSL1a and PSL1b, with specificities for sialylated glycoconjuages containing Neu5Acα2,6Galβ1,4GlcNAc/Glc. A recombinant PSL1a lectin was expressed in Escherichia coli as a fully active, soluble form, whereas a recombinant PSL1b lectin was obtained as aggregated from. In this study, to study the functional analysis of PSL1b isolectin, the lectin-coding gene was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 10 mg of recombinant lectin was purified per liter medium. Lectin blot analysis with ConA-lectin and endoglycosidase treatment revealed that the recombinant lectin was partly N-glycosylated and the 36 kDa lectin polypeptide contains an N-glycan which is absent in the 32 kDa polypeptide. Analysis of the glycans present in the recombinant lectin showed the most yeast abundant Man9-11 high-mannose structures. Glycan binding analysis showed that the recombinant PSL1b lectin interacts with sialoglycoconjugates containing α2,6-linked sialic acid. PSL1b will be a valuable lectin for detection of α2,6-linked sialic acid binding specificity compared with other sialic acid-binding lectins.
Acknowledgement
This work was supported by “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009783)” Rural Development Administration, KOREA
