J. Biochem, 1996, Vol. 120, No. 6 1253-1260
© 1996 Japanese Biochemical Society
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Tachycitin, a Small Granular Component in Horseshoe Crab Hemocytes, Is an Antimicrobial Protein with Chitin-Binding Activity1
,2
*Department of Biology, Faculty of Science Fukuoka 812-81
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Fukuoka 812-81
Department of Bacteriology, School of Medicine, Iwate Medical University Morioka 020
Central Research Institute, Maruha Co., Ltd. Tsukuba 300-42
|the Institute for Protein Research, Osaka University Suita, Osaka 565
2To whom correspondence should be addressed at: Department of Biology, Faculty of Science, Kyuahu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-81. Tel and Fax: +81-92-642-2634, E mail: skswaacb{at}mbox.nc.kyushu-u.ac.jp
Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 58), with unknown functions. In the present study, we examined the biological properties and primary structure of a small granular component S2, named tachycitin. This component was purified from the acid extract of hemocyte debris by two steps of chromatography. The purified tachycitin was a single chain protein with an apparent Mr =8,500 on Tricine-SDS-polyacrylamide gel electrophoresis. Ultracentrifugation analysis revealed tachycitin to be present in monomer form in solution. Tachycitin inhibited the growth of both Gram-negative and -positive bacteria, and fungi, with a bacterial agglutinating property. Moreover, tachycitin and big defensin acted synergistically in antimicrobial activities. The amino acid sequence and intrachain disulfide bonds of tachycitin were determined by amino acid and sequence analyses of peptides produced by enzymatic cleavages. The mature tachycitin.achycitin consisted of 73 amino acid residues containing five disulfide bonds with no N-linked sugar. A cDNA coding for tachycitin was isolated from a hemocyte cDNA library. The open reading frame coded for an NH2-terminal signal sequence followed by the mature peptide and an extension sequence of -Gly-Arg-Lys at the COOH-terminus, which is a putative amidating signal. The COOH-terminal threonine amide released after digestion of tachycitin with lysylendopeptidase was identified. The NH2-terminal 28 residues tachycitin shows sequence homology to a part of chitin-binding regions found in antifungal chitin-binding peptides, chitin-binding lectins, and chitinases, all of which have been isolated from plants. Tachycitin showed a specific binding to chitin but did not bind with the polysaccharides cellulose, mannan, xylan, and laminarin. Tachycitin may represent new class of chitin-binding protein family in animals.
1This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EMBL Data Bank with an accession number D85756 [GenBank] .
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