ASABF, a Novel Cysteine-rich Antibacterial Peptide Isolated from the Nematode Ascaris suum
1996; Elsevier BV; Volume: 271; Issue: 48 Linguagem: Inglês
10.1074/jbc.271.48.30493
ISSN1083-351X
Autores Tópico(s)Insect Resistance and Genetics
ResumoPreviously, we reported antibacterial activity in the body fluid of the nematode Ascaris suum (Kato, Y. (1995) Zool. Sci. 12, 225-230). The antibacterial activity is due to a heat-stable and trypsin-sensitive molecule that was designated as ASABF (A. suum antibacterial factor). In the present study, the purification, determination of primary structure, and cDNA cloning of ASABF were carried out. The mature peptide of ASABF is a basic peptide consisting of 71 residues and containing four intramolecular disulfide bridges. The amino acid sequence of a precursor for ASABF, deduced from a cDNA clone, indicates that flanking peptides both at the N terminus and at the C terminus are eliminated by processing. ASABF exhibits potent antibacterial activity particularly against Gram-positive bacteria. ASABF has several features that resemble those of insect/arthropod defensins, whereas the statistical significance of the similarity is not observed on comparison of amino acid sequences. A search of data bases revealed ASABF homologues in Caenorhabditis elegans. Previously, we reported antibacterial activity in the body fluid of the nematode Ascaris suum (Kato, Y. (1995) Zool. Sci. 12, 225-230). The antibacterial activity is due to a heat-stable and trypsin-sensitive molecule that was designated as ASABF (A. suum antibacterial factor). In the present study, the purification, determination of primary structure, and cDNA cloning of ASABF were carried out. The mature peptide of ASABF is a basic peptide consisting of 71 residues and containing four intramolecular disulfide bridges. The amino acid sequence of a precursor for ASABF, deduced from a cDNA clone, indicates that flanking peptides both at the N terminus and at the C terminus are eliminated by processing. ASABF exhibits potent antibacterial activity particularly against Gram-positive bacteria. ASABF has several features that resemble those of insect/arthropod defensins, whereas the statistical significance of the similarity is not observed on comparison of amino acid sequences. A search of data bases revealed ASABF homologues in Caenorhabditis elegans. INTRODUCTIONAntimicrobial peptides originating from multicellular organisms have been discovered, mainly in arthropods including insects, vertebrates, and plants (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1497) Google Scholar). Interestingly, some antimicrobial peptides isolated from evolutionally distant origins are structurally similar. For example, defensins were originally found in mammalian neutrophil cells (2Zeya H.I. Spitznagel J.K. Science. 1963; 142: 1085-1087Crossref PubMed Scopus (112) Google Scholar). Insect/arthropod defensins, isolated from the body fluid of insects and other arthropods, show a certain degree of sequence similarity with mammalian defensins (3Lambert J. Keppi E. Dimarcq J-L. Wicker C. Reichhart J-M. Dunbar B. Lepage P. Dorsselaer A.V. Hoffmann J. Fothergill J. Hoffmann D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 262-266Crossref PubMed Scopus (251) Google Scholar). Both mammalian and insect defensins contain six cysteine residues contributing intramolecular disulfide bridges. Cecropins, linear and mostly helical antibacterial peptides without cysteine residues, were first detected in insects (4Steiner H. Hultmark D. Engstrom A. Bennich H. Boman H.G. Nature. 1981; 292: 246-248Crossref PubMed Scopus (1089) Google Scholar) and later isolated from porcine small intestine (5Lee J-Y. Boman A. Sun C. Andersson M. Jornvall H. Mutt V. Boman H.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9159-9162Crossref PubMed Scopus (375) Google Scholar). Plant defensins are antifungal peptides with eight cysteine residues (6Broekaert W.F. Terras F.R.G. Cammue B.P.A. Osborn R.W. Plant Physiol. (Bethesda). 1995; 108: 1353-1358Crossref PubMed Scopus (652) Google Scholar), and a homologue, drosomycin, was recently demonstrated in the fruit fly Drosophila melanogaster (7Fehlbaum P. Bulet P. Michaut L. Lagueux M. Broekaert W.F. Hetru C. Hoffmann J.A. J. Biol. Chem. 1994; 269: 33159-33163Abstract Full Text PDF PubMed Google Scholar).In addition, most immune proteins of insects, including antimicrobial peptides, are induced by bacterial challenge or wounding. The gene expression of these immune proteins is suggested to be regulated by transcription factors that resemble those controlling the genes for immunoglobulins and acute phase response proteins in vertebrates, e.g. NFκB. These results suggest that such regulatory systems are of evolutionally ancient origin, i.e. prior to the divergence of deuterostomes (e.g. vertebrates) from protostomes (e.g. insects) (8Ip Y.T. Reach M. Engstrom Y. Kadalayil L. Cai H. Gonzalez-Crespo S. Tatei K. Levine M. Cell. 1993; 19: 753-763Abstract Full Text PDF Scopus (382) Google Scholar).It is, therefore, possible to argue that some innate immune systems related to antimicrobial peptides may be evolutionally related. However, little has been experimentally studied on the early events in the evolution of the antimicrobial peptide-related defense systems. From this aspect, it is clearly important to explore how antimicrobial peptides and their gene regulation in lower invertebrates diverged during an ancient process of evolution. Although few fossil records are available, nematodes are thought to be of very ancient origin, at least comparable with the divergence time of the lines leading to vertebrates and to arthropods from an ancient group (9Vanfleteren J.R. Evers E.A.I.M. Van De Werken G. Van Beeumen J.J. Biochem. J. 1990; 271: 613-620Crossref PubMed Scopus (19) Google Scholar). The similarity of the antimicrobial peptide-related defense systems among evolutionally distant organisms, furthermore, encourages the application of model animals for studying the innate immunity. It has already been proposed that D. melanogaster may provide an excellent model for a molecular and genetic approach to innate immune reactions, including organisms other than insects (10Hultmark D. Trends Genet. 1993; 9: 178-183Abstract Full Text PDF PubMed Scopus (367) Google Scholar). Similarly, the nematode, Caenorhabditis elegans, can also be another candidate for a model.Parasitic nematodes in animal intestines can survive not only a hostile hydrolytic environment and host immune attacks but also a microbe-rich environment. Hence, the immune defenses against coliform microbes are essential for the parasites. We have already reported antibacterial, bacteriolytic, and agglutinating activities in the body fluid of the intestinal parasitic nematode, Ascaris suum (11Kato Y. Zool. Sci. 1995; 12: 225-230Crossref PubMed Scopus (14) Google Scholar). 1J. Moore, R. Parton, and M. W. Kennedy, personal communication. The antibacterial factor ASABF (A. suum antibacterial factor) is a heat-stable and trypsin-sensitive molecule, i.e. peptide/protein. In the present study, the purification, determination of primary structure, and cDNA cloning of ASABF were carried out. The results revealed that ASABF is a novel antibacterial peptide containing four intramolecular disulfide bridges and has several features similar to those of insect/arthropod defensins. ASABF homologues in C. elegans were, moreover, demonstrated by a computer-assisted search of data bases.MATERIALS AND METHODSNematodes and Collection of Body FluidAdult female A. suum were obtained from Tokyo Shibaura Zohki, Tokyo, Japan. The nematodes were kept at 4°C after isolation from pig small intestines, and body fluid was collected within 5 h as described previously (11Kato Y. Zool. Sci. 1995; 12: 225-230Crossref PubMed Scopus (14) Google Scholar). The collected body fluid was stored at −120°C.MicroorganismsBacterial strains, Micrococcus luteus (IFO12708), Bacillus subtilis (IFO3134), and Proteus vulgaris (IFO3851T), and fungal strains, Alternaria brassicicola (IFO31227) and Septoria tritci (IFO7347), were purchased from the Institute for Fermentation (IFO), Osaka, Japan. Escherichia coli (JM109) was purchased from Takara. Staphylococcus aureus (ATCC6538P) was a gift from Dr. Masanori Yamamoto. Trichoderma viride (MAFF236543) was obtained from the National Institute of Agrobiological Resources, Tsukuba, Japan.Antimicrobial AssayInhibition zone assay was performed for the anti-S. aureus assay, as described previously (11Kato Y. Zool. Sci. 1995; 12: 225-230Crossref PubMed Scopus (14) Google Scholar). Briefly, LB-agar plates (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) with small wells containing 105 colony-forming units/ml (final) logarithmic phase bacteria were prepared. Samples were poured into each well and incubated at 37°C for 18 h. Antibacterial activity was detected as clear zones around the wells after the incubation. The inhibition zone assay was also used for the antifungal assay. Fungi were inoculated on potato-dextrose agar plates (Difco). Wells cut at the edge of the developing fungal lawn received samples. The plates were incubated at 25°C for 72 h, and the formation of clear zones was observed.In order to determine the IC50, 2The abbreviations used are: IC5050% growth inhibitory concentrationHPLChigh pressure liquid chromatographyPCRpolymerase chain reactionSLspliced leaderntnucleotidekbpkilobase pair. the microdilution method described by Alvarez-Bravo et al. (13Alvarez-Bravo J. Kurata S. Natori S. Biochem. J. 1994; 302: 535-538Crossref PubMed Scopus (60) Google Scholar) was used with modification. Each bacterial strain in the logarithmic phase was suspended in 200 μl of optimum medium, containing a series of purified ASABF at a 2-fold increase in concentration. The optical density of the bacterial suspension was adjusted to 0.02 at 650 nm. The following media were used: LB medium for E. coli and S. aureus and IFO802 medium (10 g of polypeptone, 2 g of yeast extract, 1 g of MgSO4/7H2O, 1 liter of distilled water, pH 7.0) for B. subtilis, M. luteus, and P. vulgaris. The bacterial suspension was incubated at 37°C for E. coli and S. aureus and at 30°C for B. subtilis, M. luteus, and P. vulgaris. Twenty-four h after the incubation, the optical density of the bacterial suspension was measured at 650 nm.Purification of ASABFStep 1. Gel Permeation HPLCDefrosted body fluid was centrifuged at 20,000 × g for 10 min to remove debris. The supernatant was applied to a Superdex 75 HR 10/30 column (Pharmacia Biotech Inc.) connected to a Pharmacia fast protein liquid chromatography system. A modified saline solution (11Kato Y. Zool. Sci. 1995; 12: 225-230Crossref PubMed Scopus (14) Google Scholar) was used as the mobile phase at a flow rate of 0.5 ml/min. The elution pattern was monitored at 280 nm. A part of each fraction was directly subjected to inhibition zone assay, and the antibacterial activity against S. aureus was assessed. To estimate the molecular mass of ASABF, a series of standards was used: bovine serum albumin (67 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa), aprotinin (6.5 kDa), and cyanocobalamin (1.36 kDa). We repeated this step to purify a greater amount of ASABF.Step 2. Reversed-phase HPLCThe fractions exhibiting antibacterial activity, which were derived from 3-6 ml of parent body fluid, were applied to a Purecil C18 column (Millipore Corp.) connected to a Waters HPLC system. Two linear gradient elutions were employed after an elution with ultrapure water for 25 min: 0-40% acetonitrile over 40 min and 40-100% acetonitrile over 10 min. Both the ultrapure water and acetonitrile used as mobile phases contained 0.05% trifluoroacetic acid. The flow rate was constant at 1 ml/min at ambient temperature. The elution pattern was monitored at 225 nm. The fractions were vacuum-dried and then dissolved in ultrapure water again to test the antibacterial activity against S. aureus by inhibition zone assay. The purity of ASABF was tested by tricine/SDS-PAGE with 16% acrylamide gels (14Schagger H.J. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10442) Google Scholar). A series of standards were used to estimate the molecular mass of ASABF: myoglobin (16.9 kDa), myoglobin I-II (14.4 kDa), myoglobin I (8.2 kDa), myoglobin II (6.2 kDa), and myoglobin III (2.5 kDa). The proteins in the gels were visualized by a silver staining kit (Wako Pure Chemical Industries Ltd.).S-PyridylethylationTwenty to forty μg of purified ASABF was dissolved into 200 μl of 0.25 M Tris/HCl buffer, pH 8.5, containing 6 M guanidine hydrochloride, 1 M EDTA, and 0.1% 2-mercaptoethanol. The sample was flushed with nitrogen and incubated at 37°C for 2 h. Two μl of 4-vinylpyridine was added, and the sample was flushed with nitrogen again and incubated at room temperature for 2 h. S-Pyridylethylated ASABF was separated from the reagents by reversed-phase HPLC using a Sephacil C18 SC2.1/10 microbore column connected to a Pharmacia SMART system. Two linear gradient elutions were employed after an elution with ultrapure water for 3 min: 0-40% acetonitrile over 40 min and 40-100% acetonitrile over 5 min. Both the ultrapure water and acetonitrile used as mobile phases contained 0.05% trifluoroacetic acid. The flow rate was constant at 0.1 ml/min at ambient temperature. The elution pattern was monitored at 225 and 280 nm. S-Pyridylethylated ASABF was eluted at 27% acetonitrile.Protease DigestionLysyl Endopeptidase DigestionTwenty to forty μg of purified ASABF or S-pyridylethylated ASABF was dissolved in 100 μl of 200 mM Tris/HCl buffer, pH 8.5, containing 8 M urea. The dissolved samples were diluted twice with ultrapure water, and 1 μg of lysylendopeptidase was added. The digestion was carried out at 37°C for 3 h.V8 Protease DigestionTwenty to forty μg of S-pyridylethylated ASABF was dissolved in 200 μl of 50 mM ammonium bicarbonate buffer (pH 7.0) containing 1 μg of S. aureus V8 protease and digested at 37°C for 18 h.The fragments derived by protease digestion were separated by reversed-phase HPLC as described for S-pyridylethylation.Determination of Amino Acid SequencePurified ASABF, S-pyridylethylated ASABF, and the fragments derived by protease digestion were lyophilized and subjected to automated sequence analysis using an Applied Biosystems Procise™ or a Beckman LF3000.Mass SpectrometryThe exact molecular mass of intact ASABF was determined by an ion spray ionization mass spectrometer (API 300 triple quadrupole mass spectrometer, Perkin-Elmer). The quadrupole was scanned over 500-2000 Da using a step size of 0.1 Da and a 1.0-ms dwell time/step. A matrix-assisted laser desorption ionization-time of flight mass spectrometry was used (Voyager™-RP, PerSeptive Biosystems) to determine the mass of the S-pyridylethylated fragment of ASABF, Arg68-Gly89.cDNA CloningA cDNA for ASABF was cloned using three-step PCR amplification.Step 1. Reverse Transcriptase-PCRThe poly(A)+ RNA isolated from the body walls of adult female A. suum, as described by Kuramochi et al. (15Kuramochi T. Hirawake H. Kojima S. Takamiya S. Furushima R. Aoki T. Komuniecki R. Kita K. Mol. Biochem. Parasitol. 1994; 68: 177-187Crossref PubMed Scopus (41) Google Scholar), was kindly given by Prof. Kiyoshi Kita, Tokyo University. Single-stranded cDNAs were synthesized from 0.3 μg of the poly(A)+ RNA and oligo(dT)20-M4 adaptor primer, 5′-GTTTTCCCAGTCACGAC(T)20-3′, using avian myeloblastosis virus reverse transcriptase. The cDNA coding Thr34-Arg43 was amplified by PCR using a set of degenerate primers: the sense primer (29-mer) whose sequence is deduced from Cys27-Gly33 with a designed 5′-flanking sequence, 5′-GCGCGCGCGTG(T/C)AA(A/G)TT(T/C)CA(A/G)AA(T/C)TG(T/C)GG-3′; and the antisense primer (27-mer) whose sequence is deduced from Pro44-Asp49 with designed 5′-flanking sequence, 5′-AGCAGCAGC(A/G)TC(A/G)CA(A/T/G/C)AC(A/G)CA(A/T/G/C)GT(A/T/G/C)GG-3′. All reagents used in this step were obtained from an RNA LA PCR kit (AMV) (Takara). Denaturation was carried out at 95°C for 6 min (first cycle) or 1 min (second and following cycles), annealing at 35°C (initial 10 cycles) or 45°C (following 30 cycles) for 1 min, and polymerization at 72°C for 1 min. The total number of cycles was 40. Only the product of expected size was found. This product was subcloned into pGEM-T vector (Promega) and sequenced by a dye terminator system (PRISM™, Applied Biosystems) with an automated DNA sequencer (373A, Applied Biosystems).Step 2. Amplification of 5′-End Using SL1 PrimerMost of the mRNAs in Ascaris lumbricoides are trans-spliced and acquire a common 22-nt SL1 sequence at the 5′-end (16Maroney P.A. Denker J.A. Darzynkiewicz E. Laneve R. Nilsen T.W. RNA. 1995; 1: 714-723PubMed Google Scholar). It is thus highly possible that cDNAs for ASABF contain the SL1 sequence. PCR was performed using a set of primers: the sense primer (22-mer) whose sequence is identical to the SL1 sequence, 5′-GGTTAAATTACCCAAGTTTGAG-3′; the antisense primer (23-mer) whose sequence is identical to that for Thr34-Arg43 revealed in "Step 1," 5′-CGACCTCCACGTTTCTCACAGTG-3′. All reagents used in this step were obtained from an LA PCR kit Ver.2 (Takara). Denaturation was carried out at 95°C for 6 min (first cycle) or 1 min (second and following cycles), annealing at 55°C for 1 min, and polymerization at 72°C for 1 min. Taq DNA polymerase was added during the first denaturation, i.e. a hot start mode. The cycle was repeated 30 times. The major product was shown to be of 0.2 kbp and was subcloned. The nucleotide sequence of the product was sequenced as described above. The nucleotide sequence deduced the putative signal sequence and the N-terminal region of the mature ASABF.Step 3: 3′ Rapid Amplification of cDNA EndsTo determine the sequence of a full-length cDNA for ASABF, 3′ rapid amplification of cDNA ends was carried out using a set of primers: the sense primer (35-mer) whose sequence is identical to the 5′ untranslated region revealed in "Step 2," 5′-GATATTCAGCAAAAAAGACAAAACTACTGTCGACC-3′; and M13M4 primer (17-mer) as an antisense primer, 5′-GTTTTCCCAGTCACGAC-3′. PCR conditions were identical to those described under "Step 2." Major products were found to be of 0.6 and 0.25 kbp. Their sequences revealed that the product of 0.6 kbp was the full-length cDNA for ASABF, except for the SL1 sequence.Computer-assisted Sequence AnalysisStandard sequence analyses were performed using Genetyx-Mac Ver. 7.3 (Software Development, Tokyo, Japan). The MPsrch™ (Smith-Waterman algorithm, University of Edinburgh, U. K.) was used for searching the nucleic acid data bases at DDBJ, GenBank, and EBI Data Bank and the protein data bases at Swiss-Prot, Protein Information Resource, GenPept, and Protein Data Bank via the on-line E-mail server of the DNA Information and Stock Center, Tsukuba, Japan. Furthermore, the cDNA catalogue of C. elegans, including unpublished data, was searched using the BLAST algorithm (17Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69141) Google Scholar) through the kindness of Prof. Yuji Kohara (National Institute of Genetics, Mishima, Japan). The statistical significance of sequence similarity was estimated by a jumbling test (18Schwartz, R. M., Dayhoff, M. O., (1978) Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, pp. 353–358, National Biomedical Research Foundation, Washington, D. C..Google Scholar) using the program employed by Nagata et al. (19Nagata A. Suzuki Y. Igarashi M. Eguchi N. Toh H. Urade Y. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4020-4024Crossref PubMed Scopus (174) Google Scholar). The criteria described by Doolittle (20Doolittle R.F. Methods Enzymol. 1990; 183: 99-110Crossref PubMed Scopus (72) Google Scholar) were used to evaluate the score of the jumbling test.DISCUSSIONThis paper describes the purification, primary structure, and cDNA cloning of the novel antibacterial peptide ASABF discovered in the body fluid of the nematode A. suum. ASABF has been confirmed as a peptide, and it is thus strongly suggested that antibacterial peptides contribute to the immune defense of the ancient animal nematodes. Mature ASABF is a basic 71-residue peptide containing eight cysteines engaged in intramolecular disulfide bridges. To the best of our knowledge, this is the first report on the structure of an antibacterial protein in a nematode.In some trials, a protein exhibiting weak antibacterial activity was eluted at higher acetonitrile concentration than that of ASABF, by reversed-phase HPLC in step 2 of the purification. The IC50 of this minor antibacterial protein against S. aureus was estimated to be 200 μg/ml, which is much higher than that of ASABF. The N-terminal 19-amino acid sequence of this peptide was completely identical to that of ABA-1 (21Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar). ABA-1 is known to be the most abundant protein in the body fluid of A. suum and binds fatty acids at high affinities. We separated ABA-1 by gel permeation HPLC using a Superdex 75 HR 10/30 column. This partially purified ABA-1 was subjected to reversed-phase HPLC. Neither the partially purified ABA-1 nor any fractions separated by reversed-phase HPLC exhibited antibacterial activity against S. aureus and M. luteus. We speculate that the minor antibacterial activity might be attributed to a protein similar to ABA-1 or a degraded ABA-1. However, further analyses could not be carried out because this minor antibacterial activity was not always observed. There is no evidence to indicate other antibacterial factors in the body fluid. Furthermore, the antibacterial spectrum of ASABF is in good agreement with that of the body fluid (11Kato Y. Zool. Sci. 1995; 12: 225-230Crossref PubMed Scopus (14) Google Scholar). 3Y. Kato, unpublished data. It is, therefore, suggested that ASABF is the major antibacterial molecule in the body fluid of A. suum.The cDNA cloning studies reveal that mature ASABF is processed from a 93-residue precursor. A 4-residue peptide, Arg90-Ser93, is thought to be removed in addition to the elimination of a putative signal peptide by the processing. The exact mechanism of the processing remains to be elucidated. It is noteworthy that the 4-residue peptide includes a dibasic cleavage site Arg90-Arg91 (22Barr P.J. Cell. 1991; 66: 1-3Abstract Full Text PDF PubMed Scopus (553) Google Scholar, 23Bulet P. Dimarcq J-L. Hetru C. Lagueux M. Charlet M. Hegy G. Dorsselaer A.V. Hoffmann J.A. J. Biol. Chem. 1993; 268: 14893-14897Abstract Full Text PDF PubMed Google Scholar).Several types of antimicrobial peptides containing cysteine residues have been reported. Insect/arthropod defensins are antibacterial peptides containing six cysteine residues engaged in intramolecular disulfide bridges (24Chernysh S. Cociancich S. Briand J-P. Hetru C. Bulet P. J. Insect Physiol. 1996; 42: 81-89Crossref Scopus (70) Google Scholar). We found similarity between ASABF and insect/arthropod defensins in several features. Both of them are cationic peptides and more effective against Gram-positive bacteria than Gram-negative bacteria. Insect/arthropod defensins have a consensus sequence, Cys1-[…]-Cys2-Xaa-Xaa-Xaa-Cys3-[…]-Gly-Xaa-Cys4-[…]-Cys5-Xaa-Cys6 (25Cociancich S. Goyffon M. Bontems F. Bulet P. Bouet F. Menez A. Hoffmann J. Biochem. Biophys. Res. Commun. 1993; 194: 17-22Crossref PubMed Scopus (140) Google Scholar). This consensus sequence is highly conservative among insect/arthropod defensins, except for the Gly between Cys3 and Cys4 of sapecin B (26Yamada K. Natori S. Biochem. J. 1993; 291: 275-279Crossref PubMed Scopus (68) Google Scholar). We can find the consensus sequence in the sequence of mature ASABF ignoring Cys50 and Cys69. The intramolecular disulfide bridges are essential for the antibacterial activity of the insect defensin, sapecin (27Kazuhara T. Nakajima Y. Matsuyama K. Natori S. J. Biochem. (Tokyo). 1990; 107: 514-518Crossref PubMed Scopus (47) Google Scholar). The antibacterial activity of ASABF was also lost by S-pyridylethylation. In contrast to these similar features, some other data do not support the belief that ASABF is a member of the insect/arthropod defensin family. A computer-assisted multiple alignment suggests that tenecin (28Moon H.J. Lee S.Y. Kurata S. Natori S. Lee B.L. J. Biochem. (Tokyo). 1994; 116: 53-58Crossref PubMed Scopus (80) Google Scholar) is the insect/arthropod defensin most similar to ASABF (Fig. 3). The homology between tenecin and ASABF is 25% identity and 52% similarity in the optimum region corresponding to Ser24-Cys66 of ASABF. The statistical significance of the sequence similarity was evaluated by a jumbling test. The normalized alignment score was estimated to be 3.82 of the standard deviation, and its evaluation is "marginal" (20Doolittle R.F. Methods Enzymol. 1990; 183: 99-110Crossref PubMed Scopus (72) Google Scholar). All of the normalized alignment scores between ASABF and other insect/arthropod defensins are >3.0 of the standard deviation, i.e. "improbably significant," except for sapecin B (3.01 of the standard deviation). 4The jumbling test was carried out for the insect/arthropod defensins listed in 24Chernysh S. Cociancich S. Briand J-P. Hetru C. Bulet P. J. Insect Physiol. 1996; 42: 81-89Crossref Scopus (70) Google Scholar. In conclusion, the significant sequence similarity is not verified, whereas some similar features are observed between ASABF and insect/arthropod defensins. Moreover, ASABF contains eight cysteine residues, whereas the number of cysteine residues is six, without exception, in insect/arthropod defensins (24Chernysh S. Cociancich S. Briand J-P. Hetru C. Bulet P. J. Insect Physiol. 1996; 42: 81-89Crossref Scopus (70) Google Scholar). We thus propose to classify ASABF into a novel group of antibacterial proteins in the present situation. However, we are not rejecting the proposal that ASABF and insect/arthropod defensins are possibly related by common ancestry. Are they evolutionally related? This question is curious from the aspect of a search for the origin of cysteine-rich antibacterial peptides. Insect/arthropod defensins show a certain degree of sequence similarity with mammalian defensins as mentioned in the Introduction. It is unclear whether these antibacterial peptides diverged from a common ancestor molecule. The discovery of ASABF, however, suggests that the cysteine-rich antibacterial peptides could be very ancient in origin. Further studies on antibacterial proteins in lower invertebrates should elucidate the evolutional relationship among ASABF, insect/arthropod defensins, and mammalian defensins. In addition, antifungal peptides containing eight cysteine residues have been also reported, i.e. plant defensins and drosomycin (see Introduction). ASABF exhibits no potent antifungal activity and no significant sequence similarity to these antifungal peptides. However, allowing for several gaps, the array consisting of eight cysteine residues seems to be arranged in a similar pattern between ASABF and these antifungal peptides. Interestingly, it has been suggested that structural and functional properties of plant defensins resemble those of insect and mammalian defensins (6Broekaert W.F. Terras F.R.G. Cammue B.P.A. Osborn R.W. Plant Physiol. (Bethesda). 1995; 108: 1353-1358Crossref PubMed Scopus (652) Google Scholar). Revealing the relationship between ASABF and these antifungal peptides might be another key to studying the evolutional relationship among cysteine-rich antimicrobial peptides.One of our goals is to introduce the nematode C. elegans as a model animal for investigation on innate immunity, as mentioned in the Introduction. From this aspect, it is very curious regarding whether ASABF homologues exist in C. elegans. A cDNA catalogue by Prof. Yuji Kohara was searched. BLAST data base searches revealed significant sequence identity with a deduced protein from the cDNA sequence, yk150c7 (Fig. 4). In the optimum region corresponding to Leu33-Gly72 of ASABF, yk150c7 exhibits 42% identity and 57% similarity with a normalized alignment score of 5.61 of the standard deviation, i.e. "probably significant." Furthermore, nucleic acid data bases and protein data bases were searched. The protein deduced from the putative gene, T22H6.5, was found to be a protein most similar to both ASABF and yk150c7 by the MPsrch™ data base search (Fig. 4). In the optimum region corresponding to Leu40-Cys69 of ASABF, T22H6.5 exhibits 39.4% identity and 54.5% similarity with a normalized alignment score of 4.61 of the standard deviation. T22H6.5 is also similar to yk150c7 with 48.6% identity and 67.6% similarity in the optimum region corresponding to Phe38-Cys74 of T22H6.5. T22H6.5 contains nine cysteine residues, and one of the cysteines is found in the highly hydrophobic putative signal sequence at the N-terminal region. The array consisting of eight other cysteine residues is similar to that of ASABF. It is noteworthy that the highly similar region among ASABF, yk150c7, and T22H6.5 is almost identical to the region overlapping insect/arthropod defensins (Fig. 3, Fig. 4). The function of the deduced proteins from yk150c7 and T22H6.5 has been unknown and is not predicted. Further experimental analyses are necessary to confirm the function of these ASABF homologues in C. elegans.Fig. 4Alignment of ASABF and the homologues in C. elegans. The conserved arrays consisting of eight cysteine residues are marked with asterisks. Identical residues are dark-stippled, and similar residues are light-stippled.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To date, a number of antimicrobial proteins were isolated from multicellular animals. Most of them are, however, derived from higher animals, e.g. vertebrates and arthropods. The higher animals seem to develop characteristic defense systems, e.g. B- and T-cell-based adaptive immunity in vertebrates and prophenoloxidase cascades in arthropods, overlaying primitive immunity as described previously (11Kato Y. Zool. Sci. 1995; 12: 225-230Crossref PubMed Scopus (14) Google Scholar). Studying the immune defense of lower invertebrates, such as nematodes, could be a way to isolate the primitive systems from these additional systems. The present work was carried out as the initial step of this project. INTRODUCTIONAntimicrobial peptides originating from multicellular organisms have been discovered, mainly in arthropods including insects, vertebrates, and plants (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1497) Google Scholar). Interestingly, some antimicrobial peptides isolated from evolutionally distant origins are structurally similar. For example, defensins were originally found in mammalian neutrophil cells (2Zeya H.I. Spitznagel J.K. Science. 1963; 142: 1085-1087Crossref PubMed Scopus (112) Google Scholar). Insect/arthropod defensins, isolated from the body fluid of insects and other arthropods, show a certain degree of sequence similarity with mammalian defensins (3Lambert J. Keppi E. Dimarcq J-L. Wicker C. Reichhart J-M. Dunbar B. Lepage P. Dorsselaer A.V. Hoffmann J. Fothergill J. Hoffmann D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 262-266Crossref PubMed Scopus (251) Google Scholar). Both mammalian and insect defensins contain six cysteine residues contributing intramolecular disulfide bridges. Cecropins, linear and mostly helical antibacterial peptides without cysteine residues, were first detected in insects (4Steiner H. Hultmark D. Engstrom A. Bennich H. Boman H.G. Nature. 1981; 292: 246-248Crossref PubMed Scopus (1089) Google Scholar) and later isolated from porcine small intestine (5Lee J-Y. Boman A. Sun C. Andersson M. Jornvall H. Mutt V. Boman H.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9159-9162Crossref PubMed Scopus (375) Google Scholar). Plant defensins are antifungal peptides with eight cysteine residues (6Broekaert W.F. Terras F.R.G. Cammue B.P.A. Osborn R.W. Plant Physiol. (Bethesda). 1995; 108: 1353-1358Crossref PubMed Scopus (652) Google Scholar), and a homologue, drosomycin, was recently demonstrated in the fruit fly Drosophila melanogaster (7Fehlbaum P. Bulet P. Michaut L. Lagueux M. Broekaert W.F. Hetru C. Hoffmann J.A. J. Biol. Chem. 1994; 269: 33159-33163Abstract Full Text PDF PubMed Google Scholar).In addition, most immune proteins of insects, including antimicrobial peptides, are induced by bacterial challenge or wounding. The gene expression of these immune proteins is suggested to be regulated by transcription factors that resemble those controlling the genes for immunoglobulins and acute phase response proteins in vertebrates, e.g. NFκB. These results suggest that such regulatory systems are of evolutionally ancient origin, i.e. prior to the divergence of deuterostomes (e.g. vertebrates) from protostomes (e.g. insects) (8Ip Y.T. Reach M. Engstrom Y. Kadalayil L. Cai H. Gonzalez-Crespo S. Tatei K. Levine M. Cell. 1993; 19: 753-763Abstract Full Text PDF Scopus (382) Google Scholar).It is, therefore, possible to argue that some innate immune systems related to antimicrobial peptides may be evolutionally related. However, little has been experimentally studied on the early events in the evolution of the antimicrobial peptide-related defense systems. From this aspect, it is clearly important to explore how antimicrobial peptides and their gene regulation in lower invertebrates diverged during an ancient process of evolution. Although few fossil records are available, nematodes are thought to be of very ancient origin, at least comparable with the divergence time of the lines leading to vertebrates and to arthropods from an ancient group (9Vanfleteren J.R. Evers E.A.I.M. Van De Werken G. Van Beeumen J.J. Biochem. J. 1990; 271: 613-620Crossref PubMed Scopus (19) Google Scholar). The similarity of the antimicrobial peptide-related defense systems among evolutionally distant organisms, furthermore, encourages the application of model animals for studying the innate immunity. It has already been proposed that D. melanogaster may provide an excellent model for a molecular and genetic approach to innate immune reactions, including organisms other than insects (10Hultmark D. Trends Genet. 1993; 9: 178-183Abstract Full Text PDF PubMed Scopus (367) Google Scholar). Similarly, the nematode, Caenorhabditis elegans, can also be another candidate for a model.Parasitic nematodes in animal intestines can survive not only a hostile hydrolytic environment and host immune attacks but also a microbe-rich environment. Hence, the immune defenses against coliform microbes are essential for the parasites. We have already reported antibacterial, bacteriolytic, and agglutinating activities in the body fluid of the intestinal parasitic nematode, Ascaris suum (11Kato Y. Zool. Sci. 1995; 12: 225-230Crossref PubMed Scopus (14) Google Scholar). 1J. Moore, R. Parton, and M. W. Kennedy, personal communication. The antibacterial factor ASABF (A. suum antibacterial factor) is a heat-stable and trypsin-sensitive molecule, i.e. peptide/protein. In the present study, the purification, determination of primary structure, and cDNA cloning of ASABF were carried out. The results revealed that ASABF is a novel antibacterial peptide containing four intramolecular disulfide bridges and has several features similar to those of insect/arthropod defensins. ASABF homologues in C. elegans were, moreover, demonstrated by a computer-assisted search of data bases.
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