PRIMARY STRUCTURE OF ANTI-LIPOPOLYSACCHARIDE FACTOR ISOLATED FROM AMERICAN HORSESHOE CRAB

Abstract

In 1982, a protein component that inhibits the limulus coagulation cascade was found in the hemocyte lysates from Japanese and American horseshoe crabs and named anti-lipopolysaccharide (LPS) factor. This protein specifically inhibited the LPS-mediated activation of limulus factor C and had a strong anti-bacterial effect on the growth of Gram-negative R-type bacteria. Moreover, it had a hemolytic activity on the red blood cells sensitized with LPS.In the present study, the complete amino acid sequence of anti-LPS factor purified from the Limulus (L) polyphemus hemocytes was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, clostripain, and Staphylococcus aureus V8 protease. Upon sequencing the peptides by the automated Edman method, the following primary structure was obtained:During the sequence analysis, two species of the protein, which differed from each other at one locus, were found and characterized. L. polyphemus anti-LPS factor was a basic protein consisting of a single polypeptide chain of 101 residues and a calculated molecular weight of 11,786 or 11,800. The hydrophobic NH2-terminal sequence and positive charges found in the disulfide loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein (J. Aketagawa, et al. (1986) J. Biol. Chem. 261, 7357-7365), and the sequence similar to that observed in the EF-hand structure was found to contain in the COOH-terminal portions of these proteins, although its function is unknown.