A NOVEL BIG DEFENSIN IDENTIFIED IN HORSESHOE-CRAB HEMOCYTES - ISOLATION, AMINO-ACID-SEQUENCE, AND ANTIBACTERIAL ACTIVITY

Hemocytes of the horseshoe crab (limulus) contain a family of arthropo dous peptide antibiotics, termed the tachyplesin family, and antibacte rial protein, called anti-LPS factor, of which the former is located i n the small (S) granules and the latter in the large (L) granules of t he hemocytes, In our ongoing studies on granular components, we have i dentified here a novel defensin-like substance present in both L- and S-granules, This substance strongly inhibits the growth of Gram-negati ve and -positive bacteria, and fungi, such as Candida albicans, The is olated substance, tentatively termed ''big defensin,'' consists of 79 amino acid residues, of which the COOH-terminal 37 residues have a seq uence similar to those of mammalian neutrophil-derived defensins, espe cially rat defensin, Characterization of the disulfide motif in big de fensin indicated that the disulfide array is identical to that of beta -defensins from bovine neutrophils, One clear structural difference is that the limulus hemocyte-derived big defensin has an extension of th e NH2-terminal hydrophobic sequence with 35 amino acid residues follow ed by the COOH-terminal cationic defensin portion, This amphipathic na ture of big defensin seems likely to be associated with its potent ant ibacterial activity, Furthermore, antibacterial activities of the NH2- terminal hydrophobic region and the COOH-terminal defensin portion sep arated by tryptic digestion are significantly different: the former di splays a more potent activity against Gram-positive bacteria, whereas the latter is more potent against Gram-negative bacteria, Big defensin , therefore, may prove to represent a new class of defensin family pos sessing two functional domains with different antimicrobial activities .