Separation of large and small granules from horseshoe crab (Tachypleus tridentatus) hemocytes and characterization of their components1

Takeshi Shigenaga, Yoko Takayenoki, Shoko Kawasaki, Noriaki Seki, Tatsushi Muta, Yoshihiro Toh, Akio Ito, Sadaaki Iwanaga

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40 Citations (Scopus)

Abstract

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 kOa 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 Chenu 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesinsI 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 inL-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.

Original languageEnglish
Pages (from-to)307-316
Number of pages10
JournalJournal of biochemistry
Volume114
Issue number3
DOIs
Publication statusPublished - 1993 Sep

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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