HUMORAL AND HEMOCYTE-ASSOCIATED LECTINS FROM THE BLUE CRAB CALLINECTES SAPIDUS: SEROLOGICAL AND BIOCHEMICAL PROPERTIES (AGGLUTININ, CARBOHYDRATE RECOGNITION, LECTIN SPECIFICITY)

This study was designed to determine the presence and distribution of the carbohydrate binding proteins in Callinectes sapidus serum and hemocytes, describe them by biochemical and serological characteristics, and attempt to obtain insight into their biological role. C. sapidus serum and the microsomal preparation isolated from disrupted hemocytes agglutinated a panel of vertebrate erythrocytes. Crossed absorption experiments suggested the presence of multiple specific lectins in serum. The microsomal preparation showed a 35-fold increase in specific activity when compared to the hemocyte lysate suggesting that hemocyte lectins are membrane-associated. Two humoral lectins were purified by passing the 35% ammonium sulfate cut of C. sapidus serum through an affinity matrix of colominic acid (-NeuAc-(alpha)-2-8-NeuAc-) bound to epoxy-activated Sepharose. The first lectin (CSL-I) could be eluted with G1cNAc or EDTA and the second (CSL-II) with NeuAc or EDTA. The two subunits of CSL-I were calculated to have M(,r) of 37,000 and 38,000, while the two subunits of CSL-II had M(,r) of 31,000 and 33,000. Isoelectric focusing patterns of reduced and alkylated lectins demonstrated that CSL-I consisted of two major bands, pI of 7.2 and 7.1; CSL-II pattern consisted of thirteen major bands with a pI range of 7.2 to 4.8.; The specificity of the first lectin, CSL-I, has been determined through hemagglutination-inhibition experiments to be for N-acylamino compounds. Through an analysis of the structures of the saccharide and non-saccharide inhibitors it appears that CSL-I contains a small combining site for the N-acetyl group alone, giving this lectin novel properties. Although the combining site is small, CSL-I agglutinates RBC in a manner similar to other lectins which bind N-acylamino sugars. Vibrio parahemolyticus, a gram negative baterium, is a pathogen to C. sapidus and is also a causative agent in human gastroenteritis. Eleven serotypes of V. parahemolyticus with different K and O antigens were tested to examine whether serum and CSL-I would agglutinate them. Serum agglutinated ten serotypes while CSL-I agglutinated three (04:K11;04:K13;04:K63). The 04 antigen contains G1cNAc and serotypes agglutinated by CSL-I were inhibited by N-acetylated acids and amines.