Purification and Characterization of a Neu5Acα2–6Galβ1–4Glc/GlcNAc-specific Lectin from the Fruiting Body of the Polypore Mushroom Polyporus squamosus

• Published in: The Journal of biological chemistry Vol. 275; no. 14; pp. 10623 - 10629
• Main Authors: Mo, Hanqing, Winter, Harry C., Goldstein, Irwin J.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 07.04.2000
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.14.10623

A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on β-d-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human α2-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches,i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds β-d-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acα2,6Galβ1,4Glc/GlcNAc (6′-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for α2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.