THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 32, Ienue of November 16, pp. 16709-16713,1988 Q 1988 by The American Society for Biochemistry and Molecular Biology, Ine. Printed in U.S.A.

Tachyplesin, a Class of Antimicrobial Peptide from the Hemocytes of the Horseshoe Crab (Tach ypleus tridentatus) ISOLATION AND CHEMICAL STRUCTURE*

(Received for publication, April 15, 1988)

Takanori Nakamura, Hiromi Furunaka, Toshiyuki Miyata, Fuminori TokunagaS, Tatsushi MutaS, and Sadaaki Iwanagall From the Department of Bwlogy, Faculty of Science and the $Department of Molecular Biology, Graduate School of Medical Science, Kyushu University 33, Fukmka 812, Japan

Makoto Niwa From the Department of Bacteriology, Osaka City University Medical School, Osaka 545, Japan

Toshifumi Takao and Yasutsugu Shimonishi From the Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan

A cationic peptide, designated tachyplesin, was iso- lated from acid extracts of horseshoe crab (Tachypleus tridentatus) hemocyte debris. It consists of 17 residues and the structure determined by Edman degradation is:

I - 1 NHZ-K-W-C-F-R-V-C-Y-R-G-I-C-Y-R-R-C-R-CONE

The carbosyl-terminal end of this peptide was identi- fied as arginine a-amide, and the whole sequence in- cluding the a-amide was also confirmed by fast atom bombardment mass spectrometry, indicating a mass value of 2263. Tachyplesin inhibits growth of both Gram-negative and -positive bacteria at low concen- trations and formed a complex with bacterial lipopoly- saccharide. Tachyplesin seems likely to act as antimi- crobial peptide for self-defense in the horseshoe crab against invading microorganisms.

In 1982, a cationic protein that inhibits the limulus hemo- lymph coagulation system was found in hemocyte lysates from Japanese (Tachypleus tridentatus) and American (Limulus polyphemus) horseshoe crabs and named anti-lipopolysaccha- ride (LPS)’ factor (1-4). This protein of 102 residues specifi- cally inhibits the LPS-mediated activation of one of the clotting factors, factor C, the initiation factor in the limulus clotting cascade (5-7). Moreover, it has a strong antimicrobial effect on the growth of Gram-negative R-type bacteria and shows hemolytic activity on red blood cells sensitized with LPS (8).

In the course of the study, we found a new cationic peptide, tachyplesin, in hemocyte debris, which significantly inhibits

* This work was supported by a Grant-in-Aid for Scientific Re- search from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed. The abbreviations used are: LPS, lipopolysaccharide; Boc, N-tert-

butoxycarbonyl; pNA, p-nitroanilide; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; FAB, fast atom bombardment; factor C, ac- tivated factor C.

the LPS-mediated activation of factor C in a similar manner to anti-LPS factor. This paper deals with the isolation and structural elucidation of tachyplesin. The peptide consists of 17 residues and has a unique arginine a-amide at the COOH- terminal end.

MATERIALS AND METHODS AND RESULTS*

Amino Acid Sequence of Tachyplesin-Table I shows the amino acid composition of tachyplesin. It consists of 17 resi- dues including five arginine, but no acidic amino acids and hexosamines. The amino acid sequence of tachyplesin was elucidated by automated Edman degradation using native (10 nmol) and S-pyridylethylated (15 nmol) samples (Fig. M6 and Table MII). The COOH-terminal residue was predicted to be an arginine derivative by subtraction of the sequenced residues from the total in the molecule, since phenylthiohy- dantoin-arginine could not be identified.

Identification of the COOH-terminal End of Tachyplesin- Since lysyl endopeptidase from Achromobacter lyticus specif- ically hydrolyzes peptide bonds on the carboxyl side of lysine and S-aminoethylcysteine residues, S-aminoethylated tachy- plesin was first digested with this enzyme to release the COOH-terminal residue. The digest was coupled with phen- ylkothiocyanate and the products analyzed by reversed-phase HPLC (Fig. M7). In addition to free lysine (as the phenyl- thiocarbamoyl derivative) released from the NH2-terminal end of tachyplesin, a peak with the same retention time as authentic arginine a-amide was found. When this unknown peak was collected, hydrolyzed with 5.7 M HC1 at 110 “C for 12 h, and again treated with phenylkothiocyanate, the result- ing material showed the same retention time as that of free phenylthiocarbamoyl-arginine on HPLC (as in Fig. M7). These results indicated that the COOH-terminal residue of tachyplesin is not arginine but arginine a-amide. The arginine a-amide residue was further confirmed by FAB mass spectro- metric analysis using native and S-aminoethylated tachyple- sins. Fig. M8 shows the FAB mass spectrum of native tachy-

Portions of this paper (including “Materials and Methods,” part of “Results,” Figs. M1-M10, and Tables MI-MIII) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

16709

16710 Tachyplesin, a Cationic Peptide in Limulus Hemocytes

TABLE I Amino acid composition of tachypksin Amino acid” Tachyplesin

residueslmlecule Glycine 1.2 (1) Half-cystineb 3.8 (4) Valine 1.0 (1) Isoleucine 0.9 (1) Tyrosine 1.8 (2) Phenylalanine 1.0 (1) Lysine 0.9 (1) Tryptophan‘ 1.0 (1) Arginine 4.8 (5)

Total 17 Calculated from extrapolated or average values estimated from

Determined as cysteic acid. Estimated on a 24-h hydrolysate with 3 M mercaptoethanesulfonic

24-, 48-, and 72-h hydrolysates in 5.7 M HCl.

acid.

TABLE I1 Antimicrobial activity of tachypksin

Strain Minimal inhibitory concentration

Gram(-) Salmonella typhimurium LT2(S) 3.1-6.3 S. typhimurium SL1102(Re) 0.8-1.6 Salmonella minnesota 1114W(S) 3.1-6.3 S. minnesota R595(Re) 3.1-12.5 E. coli K12 6.3-12.5

Staphylococcus aureus 209P 3.1 S. aureus ATCC 25923 6.3

Gram(+)

plesin. The observed mass value at m/z = 2263.0 (protonated form) corresponds to the theoretical value (2263.1) calculated from the amino acid sequence of tachyplesin with arginine a- amide at the COOH-terminal end, although the observed isotopic molecular ion distribution was not consistent with the theoretical. Within the molecular ion region of peptides containing cystine, a peak at two or more mass units than the molecular ion peak increases, because part of an intramolec- ular disulfide bond(s) suffers reductive cleavage during mass measurement (9). To further confirm the presence of an a- amide, the molecular weight of S-aminoethylated tachyplesin was examined. Fig. M9 shows the FAB mass spectrum of S- aminoethylated tachyplesin and the theoretical isotopic mo- lecular ion distribution calculated from the sequence with arginine a-amide at the COOH-terminal end. Both the ob- served mass value and isotopic molecular ion distribution are nearly identical to the the theoretical. This finding indicates that the molecular weight of S-aminoethylated tachyplesin is 2438.4, which agrees well with the theoretical value (2438.3) calculated from the sequence containing the COOH-terminal amide.

Assignment of Disulfide Linkage in Tachyplesin-No S- pyridylethylcysteine was found on amino acid analysis after treatment of tachyplesin with 4-vinylpyridine in the absence of dithiothreitol, although 4 residues of S-pyridylethylcy- steine/molecule were quantitated after S-alkylation. Thus, two disulfide linkages must be present in tachyplesin. The disulfides were identified from the amino acid compositions of the tryptic peptides derived from native tachyplesin. Fig. M10 shows the elution profile of a tryptic digest on a Cosmosil 5Cla-P column. The amino acid compositions of the two principal peptides are listed in Table MIII. Peptide T-1 is a pentapeptide (Lys-1 to Arg-5) joined to a dipeptide (Cys-16

to Arg a-amide-17). Peptide T-2 is a tetrapeptide (Val-6 to Arg-9) joined to a pentapeptide (Gly-10 to Arg-14). The results indicate the presence of disulfides between Cys-3 and Cys-16 and between Cys-7 and Cys-12.

Antimicrobial Activity of Tachyplesin-Minimal inhibitory concentraitons of tachyplesin against several bacterial strains were determined by the microplate culture method as de- scribed under “Materials and Methods.” As summarized in Table 11, tachyplesin displays potent antimicrobial activity against several strains of microorganisms. It is effective against not only rough and smooth strains of Gram-negative but also Gram-positive cells, such as Staphylococcus species. In the presence of tachyplesin at 3.5 pg/ml Salmonella strains irreversibly lose viability (data not shown). This antimicrobial potency is comparable to that of anti-LPS factor previously reported (8).

DISCUSSION

The hemocytes circulating in limulus hemolymph are highly sensitive to Gram-negative bacterial endotoxins (10, 11). Ex- posure of hemocytes to LPS results in activation of the intracellular clotting system, which participates both in he- mostasis and in defense against invading microorganisms. Our recent biochemical studies indicate that hemocytes con- tain at least three serine protease zymogens closely associated with the clotting system, in addition to a clottable protein, coagulogen (5, 6, 12-15). Among these proteases, factor C is an LPS-sensitive protease zymogen and participates in the initiation reaction triggered by endotoxin (5-7). The cationic peptide tachyplesin from hemocytes strongly inhibits the LPS-mediated activation of the zymogen factor C. Tachyple- sin appears to exist abundantly in hemocyte debris, suggesting that it is one of the major cationic components of the hemo- cyte membrane. This cationic peptide is also able to bind to Escherichia coli-type LPS (16) and to neutralize the factor C- activating activity of LPS in a manner similar to that of anti- LPS factor (1-4). Moreover, the peptide displays antimicro- bial activity against both Gram-negative and -positive bacte- ria (Table 11). Therefore, tachyplesin seems very likely to act as an antimicrobial peptide for the defense of horseshoe crabs against microbial infections.

Tachyplesin is highly stable at low pH and high tempera- ture, since the LPS-binding ability of tachyplesin is not affected even in the 0.1% trifluoroacetic acid used for HPLC and by heat treatment in neutral pH buffer at 100 “C for 30 min (data not shown). This stability seems to be due to the rigid structure imposed by the two disulfide linkages. It is also of interest that tachyplesin shows a characteristic structure with three tandem repeats of a tetrapeptide sequence, namely hydrophobic amino acid-Cys-hydrophobic amino acid-Arg, indicating that its amphipathic nature is closely associated with biological activity (Fig. 1). The COOH-terminal residue

10

1

FIG. 1. Repeated tetrapeptide sequence found in tachyple- sin. Three tandem repeats of tetrapeptides consisting of hydrophobic amino acid-half-cystine-hydrophobic amino acid-arginine are indi- cated by stippling.

Tachyplesin, a Cationic Peptide in Limulus Hemocytes 16711

of tachyplesin is arginine a-amide, as identified by chemical and FAB mass spectrometric analyses. Naturally occurring peptides containing arginine a-amide at the COOH termini have been reported in a scorpion polypeptide toxin (17) and sarcotoxins from Sarcophugu peregrinu (18). The amino acid sequence of tachyplesin was subjected to a computer-assisted search for homology with known sequences using the data base "PRF/SEQDB" (Protein Research Foundation, Osaka, Japan). However, there are no sequence similarities with any other cationic polypeptides so far reported.

Acknowledgments-We wish to express our thanks to Chizuko Takabayashi-Sueyoshi and Satsuki Kajiyama for amino acid and sequence analyses, and also to He Hua for her technical assistance. We also thank Mizumo Akiyoshi and Nobuko Ueno for typing the manuscript.

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16712 Tachyplesin, a Cationic Peptide in Limulus Hemocytes

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Tachyplesin, a Cationic Peptide in Limulus Hemocytes 16713