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 arthropodous peptide antibiotics, termed the tachyplesin family, and antibacterial protein, called anti-LPS factor, of which the former is located in the small (S) granules and the latter in the large (L) granules of the hemocytes. In our ongoing studies on granular components, we have identified here a novel defensin-like substance present in both L- and S-granules. This substance strongly inhibits the growth of Gram-negative and -positive bacteria, and fungi, such as Candida albicans. The isolated substance, tentatively termed "big defensin," consists of 79 amino acid residues, of which the COOH-terminal 37 residues have a sequence similar to those of mammalian neutrophil-derived defensins, especially rat defensin. Characterization of the disulfide motif in big defensin 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 the NH2-terminal hydrophobic sequence with 35 amino acid residues followed by the COOH-terminal cationic defensin portion. This amphipathic nature of big defensin seems likely to be associated with its potent antibacterial activity. Furthermore, antibacterial activities of the NH2-terminal hydrophobic region and the COOH-terminal defensin portion separated by tryptic digestion are significantly different: the former displays 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 possessing two functional domains with different antimicrobial activities.

Separation of large and small granules from horseshoe crab (Tachypleus tridentatus) hemocytes and characterization of their components.

We designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E.W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal Gly-Lys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.

cDNA and amino acid sequences of bovine tissue factor.

A cDNA encoding bovine tissue factor has been isolated from a lambda gt11 bovine adrenal cDNA library. The cDNA insert was 1877 base pairs with an open reading frame of 876 base pairs that encoded a presequence of 35 amino acids and a mature tissue factor of 257 amino acids. Bovine tissue factor had three potential N-glycosylation sites, four extracellular cysteine residues, a cytoplasmic cysteine residue, and one tripeptide tryptophan-lysine-serine motif. Identities of the amino acid sequences of the mature forms between the bovine tissue factor and each of human, mouse, and rabbit tissue factors were 70.4%, 57.2%, and 74.1%, respectively.