Abstract
A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on beta-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 alpha(2)-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 beta-D-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acalpha2, 6Galbeta1,4Glc/GlcNAc (6'-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for alpha2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.
Highlights
Lectins are proteins, other than antibodies and enzymes, that bind and reversibly to carbohydrates, resulting in cell agglutination or precipitation of glycoconjugates [1]
There has been a growing interest in fungal lectins, largely due to the discovery that some of these lectins exhibit antitumor activities, e.g. Volvariella volvacea lectin shows antitumor activity against sarcoma S-180 cells [3], Grifola frondosa lectin is cytotoxic to Hela cells [4], Agaricus bisporus lectin possesses antiproliferation activities against human colon cancer cell lines HT29, breast cancer cell lines MCF-7 [5], and Tricholoma mongolicum lectin inhibits mouse mastocytoma P815 cells in vitro and sarcoma S-180 cells in vivo [6]
Apart from the lectin from A. bisporus, which binds to Gal1,3GalNAc␣-Ser/Thr (T-disaccharide) [10], the detailed carbohydrate specificities of these fungal lectins have not been investigated in depth
Summary
Lectins are proteins (or glycoproteins), other than antibodies and enzymes, that bind and reversibly to carbohydrates, resulting in cell agglutination or precipitation of glycoconjugates [1]. They are ubiquitous in the biosphere, having been found in viruses, bacteria, fungi, plants, and animals [2]. Lectins of known specificity recognizing sialic acid serve as valuable reagents in glycobiological research They can be employed for the detection and preliminary characterization of sialic acid-containing glycoconjugates on the surface of cells and for assaying the incorporation of sialic acid into complex carbohydrates in biosynthetic studies. We report the purification and characterization of Polyporus squamosus lectin (designated PSA), a Neu5Ac␣2– 6Gal1– 4Glc/GlcNAc-specific lectin present in the carpophores (fruiting bodies) of this member of the Polyporaceae family
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