| Popis: |
Carbohydrate residues on many cell types are customarily masked by strong anions such as sulfate (1). These terminal structures are significant in cell function (2); for example, they are involved in immunogenicity, in binding of the underlying sugars to specific antibodies or to lectins, and in secretory phenomena (3,4). In addition, sulfates protect the underlying sugars from degradation by glycosidases (5). Such sugars can be exposed by chemical or enzymatic methods that remove the anionic structures (6). Our interest is based upon earlier biochemical findings that adhesive proteoglycans (AP) derived from the marine sponge Microciona prolijku are highly sulfated, as are the surfaces of the sponge cells (7). The following histochemical experiments, using lectins with affinities for specific sugars, illustrate masking of cell-surface carbohydrates. Chemically dissociated sponge cells were pelleted, fixed in formalin, embedded in paraffin, and sectioned at 5 pm. Lectin histochemistry (8) was carried out with the following horseradish peroxidase-conjugated (HRP) lectins: soybean agglutinin (SBA) with specificity for N-aCCtyl-D-galaCtOSaminC (GalNAc), wheat germ agglutinin (WGA) for N-acetyl-glucosamine (GlcNAc), peanut agglutinin (PNA) for /3-galactose (Gal), Ulex ewopaeus agglutinin (UEAI) for cr-L-fucose (Fuc), concanavalin A (Con A) for a-mannose (man), and Limuluspolyphemus agglutinin (LPA) for sialic acid (SA). Controls consisted of substitution of phosphate-buffered saline for lectins and use of appropriate sugars to inhibit lectin reactivity. Desulfation of cell-surface glycoconjugates in sections was carried out with 1% HCl in methanol at 62.5”C for 4 h prior to lectin staining. Methanol control sections were prepared under similar conditions. Immunohistochemical staining with Block 2 monoclonal antibody (MoAb) followed by HRP secondary antibody was used to define sulfated epitope (6, 9). The results demonstrate that chemical desulfation enhances lectin staining in the case of SBA (Fig. la,b), as well as WGA |
