Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity

Shun-Ichiro Kawabata, Ranko Nagayama, Michimasa Hirata, Takeshi Shigenaga, Kishan Lal Agarwala, Tetsu Saito, Junko Cho, Hiroshi Nakajima, Toshio Takagi, Sadaaki Iwanaga

Research output: Contribution to journalArticle

109 Citations (Scopus)

Abstract

Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), 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 M(r) = 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 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 of 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 a new class of chitin-binding protein family in animals.

Original languageEnglish
Pages (from-to)1253-1260
Number of pages8
JournalJournal of biochemistry
Volume120
Issue number6
DOIs
Publication statusPublished - Jan 1 1996

Fingerprint

Horseshoe Crabs
Hemocytes
Chitin
Defensins
Proteins
Amino Acids
Disulfides
Peptides
Mannans
S 6
Xylans
Chitinases
Ultracentrifugation
Protein Sequence Analysis
Gram-Positive Bacteria
Threonine
Sequence Homology
Protein Sorting Signals
Chromatography
Electrophoresis

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity. / Kawabata, Shun-Ichiro; Nagayama, Ranko; Hirata, Michimasa; Shigenaga, Takeshi; Lal Agarwala, Kishan; Saito, Tetsu; Cho, Junko; Nakajima, Hiroshi; Takagi, Toshio; Iwanaga, Sadaaki.

In: Journal of biochemistry, Vol. 120, No. 6, 01.01.1996, p. 1253-1260.

Research output: Contribution to journalArticle

Kawabata, S-I, Nagayama, R, Hirata, M, Shigenaga, T, Lal Agarwala, K, Saito, T, Cho, J, Nakajima, H, Takagi, T & Iwanaga, S 1996, 'Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity', Journal of biochemistry, vol. 120, no. 6, pp. 1253-1260. https://doi.org/10.1093/oxfordjournals.jbchem.a021549
Kawabata, Shun-Ichiro ; Nagayama, Ranko ; Hirata, Michimasa ; Shigenaga, Takeshi ; Lal Agarwala, Kishan ; Saito, Tetsu ; Cho, Junko ; Nakajima, Hiroshi ; Takagi, Toshio ; Iwanaga, Sadaaki. / Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity. In: Journal of biochemistry. 1996 ; Vol. 120, No. 6. pp. 1253-1260.
@article{e1bf53a48a634d2a943de510087ebb77,
title = "Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity",
abstract = "Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), 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 M(r) = 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 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 of 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 a new class of chitin-binding protein family in animals.",
author = "Shun-Ichiro Kawabata and Ranko Nagayama and Michimasa Hirata and Takeshi Shigenaga and {Lal Agarwala}, Kishan and Tetsu Saito and Junko Cho and Hiroshi Nakajima and Toshio Takagi and Sadaaki Iwanaga",
year = "1996",
month = "1",
day = "1",
doi = "10.1093/oxfordjournals.jbchem.a021549",
language = "English",
volume = "120",
pages = "1253--1260",
journal = "Journal of Biochemistry",
issn = "0021-924X",
publisher = "Oxford University Press",
number = "6",

}

TY - JOUR

T1 - Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity

AU - Kawabata, Shun-Ichiro

AU - Nagayama, Ranko

AU - Hirata, Michimasa

AU - Shigenaga, Takeshi

AU - Lal Agarwala, Kishan

AU - Saito, Tetsu

AU - Cho, Junko

AU - Nakajima, Hiroshi

AU - Takagi, Toshio

AU - Iwanaga, Sadaaki

PY - 1996/1/1

Y1 - 1996/1/1

N2 - Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), 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 M(r) = 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 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 of 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 a new class of chitin-binding protein family in animals.

AB - Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), 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 M(r) = 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 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 of 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 a new class of chitin-binding protein family in animals.

UR - http://www.scopus.com/inward/record.url?scp=12644287510&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=12644287510&partnerID=8YFLogxK

U2 - 10.1093/oxfordjournals.jbchem.a021549

DO - 10.1093/oxfordjournals.jbchem.a021549

M3 - Article

C2 - 9010778

AN - SCOPUS:12644287510

VL - 120

SP - 1253

EP - 1260

JO - Journal of Biochemistry

JF - Journal of Biochemistry

SN - 0021-924X

IS - 6

ER -