ABSTRACT
A lectin was purified from the hemolymph of the freshwater Indian gastropod Belamyia bengalensis. The purification involved successive ion-exchange chromatography on Resource Q and gel filtration on Superose 12 column in FPLC system. Homogeneity of the protein was confirmed by polyacrylamide gel electrophoresis. Belamyia bengalensis lectin (BBL) was a monomeric protein with a molecular weight of 33 kDa as demonstrated by gel filtration and SDS-PAGE. It is a glycoprotein containing 6% total sugar and its activity is highly dependent on Ca(2+). BBL agglutinated human erythrocytes and is a blood group non-specific lectin. It agglutinated animal erythrocytes also. Hapten inhibition studies indicated that BBL shows binding specificity only for N-acetyl-D-glucosamine and N-acetyl-D-galactosamine at a high concentration among the mono- and oligosaccharides tested. Among the glycoproteins used for hemagglutination-inhibition assay, porcine submaxillary mucin was found to be the best inhibitor. Chemical modification studies indicated that Lys, Arg, and Trp are essential for the sugar-binding activity of BBL. Circular dichroism spectra revealed high content of alpha-helical structure in the lectin. BBL is a potent mitogen as it stimulated the T-lymphocyte proliferation, specifically the Th1 subset.
Subject(s)
Lectins/metabolism , Lectins/pharmacology , Mitogens/pharmacology , Mucins/metabolism , Snails/chemistry , Amino Acids/chemistry , Amino Acids/metabolism , Animals , Blotting, Western , Cations, Divalent/pharmacology , Cell Division/drug effects , Cell Line , Galactosamine/analogs & derivatives , Galactosamine/metabolism , Galactosamine/pharmacology , Glucosamine/analogs & derivatives , Glucosamine/metabolism , Glucosamine/pharmacology , Glycoproteins/metabolism , Glycoproteins/pharmacology , Hemagglutination/drug effects , Hemagglutination/physiology , Hemolymph/chemistry , Hydrogen-Ion Concentration , Lectins/chemistry , Lectins/isolation & purification , Mice , Mitogens/isolation & purification , Mitogens/metabolism , Protein Binding , Protein Structure, Secondary , Substrate SpecificityABSTRACT
An agglutinin, a monomeric glycoprotein with a molecular mass of about 6.5 kDa and containing 18% sugar has been purified to an apparent homogeneity from a 21 days old culture filtrate of an anthropophilic dermatophyte Tricophyton rubrum. It is a human blood group non-specific agglutinin which also agglutinates animal erythrocytes and Ehrlich ascites carcinoma and Sarcoma-180 cells. It is thermally stable and exhibits maximum activity at pH 8. Amino acid analysis shows a significant amount of glycine, with no cysteine. Glycoproteins inhibited the hemagglutination of the agglutinin, but not the simple sugars, including sialic acid. Fetuin is the most potent inhibitor among the glycoproteins tested. This inhibition gives a hint to binding with Galbeta1-3GalNAc or Galbeta1-4GlcNAc residue containing sialic acid at the terminal position with alpha 2-6 or alpha 2-3 linkage.
