Binding of wheat germ agglutinin to extracellular network produced by cultured human fibroblasts.
The occurrence of diverse carbohydrate moieties on the cell surface and in the extracellular matrix, makes lectins the suitable probes to study the distribution of appropriate determinants produced in cell culture. Biotin-labelled wheat germ agglutinin (WGA) was used in microscopic and photometric detection of lectin binding to monolayer of human skin fibroblasts. The incubation of confluent fibroblast monolayer with labelled WGA reveals two principal patterns of binding of this lectin: to cell surface structures and, predominantly, to extracellular fibres; the alignment and density of extracellular network are not uniform. After binding of WGA to confluent culture, light microscopic analysis revealed the ubiquitous fibrillar network between and over cells, with some regions of increased compactness and altered orientation of fibrils. Binding to cell surfaces (manifested as specks) was predominant for the fibroblasts at the logarithmic phase of growth. N-acetylglucosamine (0.2 M) and native lectin (100 microg/ml) had a partial inhibitory effect on WGA binding to the extracellular network. Treatment with neuraminidase (0.1 unit/ml) of untreated or prefixed monolayers resulted in a significant decrease in WGA binding to fibrils (and increase in PNA binding), indicating that terminal sialic acid residues are mainly involved in the network-WGA interaction. Mild trypsinization (10 microg/ml) removed the target sites, which retained the ability to bind WGA, being spotted on hydrophobic Immobilon P paper; biotinylated lectin, bound to adsorbed glycopeptides, could be eluted and quantified in solid-phase inhibition assay.
Nizheradze
K A
KA
Department of Diabetology, Institute of Endocrinology and Metabolism, Kiev, Ukraine. kons.nizher@mailexcite.com
D000117:Acetylglucosamine / Q000494:pharmacology; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000494:pharmacology; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology; D009195:Peroxidase / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014909:Wheat Germ Agglutinins / Q000378:metabolism*
D006023:Glycoproteins; D037102:Lectins; D014909:Wheat Germ Agglutinins; D009195:Peroxidase; D009439:Neuraminidase; D014357:Trypsin; D019158:N-Acetylneuraminic Acid; D000117:Acetylglucosamine