Molecular characterization of limulin, a sialic acid binding lectin from the hemolymph of the horseshoe crab, Limulus polyphemus

Abstract

The sialic acid binding lectin, limulin, was isolated by gel filtration and ion-exchange chromatography from the hemolymph of L. polyphemus. When the purified protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of b-mercaptoethanol, 2 major protein bands were observed. These 2 bands, subsequently contained carbohydrate as well, corresponded to MW of 25,000 and 27,000. Amino acid sequence analyses were performed on the intact protein and isolated cyanogen bromide fragments. Primary structural features noted in the aminoterminal region of limulin were the absence of histidine and alanine for the NH2-terminal 50 residues; the presence of 5 of the total 8 prolines of the molecule between positions 13 and 30; and a possible carbohydrate attachment site consisting of only the amino acids proline and serine between residues 13 and 19. The results of CNBr cleavage studies confirmed the presence of 2 methionine residues per subunit, at positions 25 and 58, respectively. No sequence heterogeneity was observed. While it is possible that limulin plays some role in the defense mechanisms of the horseshoe crab, there is no obvious sequence homology between this invertebrate lectin and vertebrate immunoglobulins.