Application of a lectin from the mushroom Polysporus squamosus for the histochemical detection of the NeuAc alpha 2,6Gal beta 1,4Glc/GlcNAc sequence of N-linked oligosaccharides: a comparison with the Sambucus nigra lectin

The lectin from the mushroom Polysporus squamosus (PSL) has an extended car bohydrate combining site, which exhibits a high specificity and affinity to ward the NeuAc5 alpha2,6Gal beta1,4Glc/GlcNAc trisaccharide sequence of asp aragine-linked oligosaccharides. Therefore, PSL should be a superior reagen t to the lectin from Sambucus nigra (SNA), which does not discriminate betw een alpha2,6-linked NeuAc5 present either in asparagine or serine/threonine -linked oligosaccharides. We have prepared a dioxigenin-conjugated PSL and applied it for histochemistry and blotting. We observed a more restricted s taining pattern by PSL as compared to SNA in paraffin sections from differe nt rat organs. Pretreatment of sections with N-glycanase F abolished PSL st aining indicating that it interacts only with asparagine-linked oligosaccha rides. Furthermore, PSL staining was neuraminidase sensitive. In contrast, SNA staining was only partially sensitive to N-glycanase F pretreatment dem onstrating that it was in part due to alpha ,2,6-linked NeuAc5 present in s erine/threonine-linked oligosaccharides. The most striking observation in t his regard was that PSL, in contrast to SNA, did not stain the mucus of she ep submandibular gland, which is extremely rich in serine/threonine-linked Neu5Ac alpha2,6N-acetylgalactosamine. Furthermore, in some tissues neuramin idase pretreatment resulted in increased intensity of SNA staining probably due to binding to exposed terminal N-acetylgalactosamine residues. Collect ively, these results indicate that PSL is a useful tool for the histochemic al detection of alpha2,6-linked NeuAc5 in asparagine-linked oligosaccharide s.