Primary Structure of Anti-Lipopolysaccharide Factor from American Horseshoe Crab, Limulus polyphemus1

The complete amino acid sequence of anti-lipopolysaccharide (LPS) factor purified from the hemocytes lysate of the American horseshoe crab, Limulus polyphemus , was determined by characterization of the NH ₂ -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: ––S–S–– DGIWTQLIFTL $VKN$ NLATLWQSGDFQDHECHYRIKPTFRRLKWKYKGKFWCP SWTSITGRATKSSRSGAVEHSVRNFVGQAKSSGLITQRQAEQFISQYN 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 NH ₂ -terminal sequence and the clustering of positive charges found in the disulfied loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein, 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 iss unknown.