cDNA cloning of halocidin and a new antimicrobial peptide derived from the N-terminus of Ci-META4

Halocidin is an antimicrobial peptide, which is isolated from hemocytes from the tunicate, Halocynthia aurantium. In this study, we cloned the full-length cDNA of halocidin from pharyngeal tissue, using a combination of RT-PCR and 5′-RACE-PCR. The observed cDNA structure indicated that halocidin is synthesized as a 10.37 kDa prepropeptide. Based on the cDNA structure and the known amino acid sequence of the mature peptide, it was concluded that the precursor of halocidin contains a 21-residue signal peptide, followed by the 18 residues of the mature peptide, and a 56-residue anionic C-terminal extension, which is removed later on in the process. The signal sequence of halocidin exhibited a high degree of similarity with the corresponding portion of the Ci-META4 protein, which had been previously discovered in the coelomic cells of another tunicate, Ciona intestinalis, and is considered to play a role in metamorphosis. However, in several respects, the cDNA structure of Ci-META4 suggested that it might constitute a precursor for an antimicrobial peptide. Thus, we prepared a synthetic peptide, which was comprised of 19 N-terminal amino acid residues in the predicted mature region of Ci-META4, and tested it with regard to its antimicrobial activity. As a result, we confirmed that the synthetic peptide exhibited potent antimicrobial activity against Gram (+) and (−) bacteria, while evidencing no hemolytic activity toward human erythrocytes.     

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.     

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.     

A novel antimicrobial peptide from skin secretions of the earthworm, Pheretima guillelmi (Michaelsen)

A novel lumbricin-like antimicrobial peptide named lumbricin-PG was isolated from skin secretions of the earthworm, Pheretima guillelmi (Michaelsen), using a procedure of one step Sephadex G-50 gel filtration and one step C₈ reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as FSRYARMRDSRPWSDRKNNYSGPQFTYPPEKAPPEKLIKWNN EGSPIFEMPAEGGHIEP by Edman degradation combined with cDNA cloning and mass spectrometry analysis. The cDNA encoding lumbricin-PG was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 73 amino acid residues including a mature lumbricin-PG and predicted signal peptide. It showed similarity with lumbricin antimicrobial peptide from the earthworm, Lumbricus rubellus by BLAST search. Purified lumbricin-PG exerted potential antimicrobial activities against bacteria and fungi; it showed weak hemolysis activity against human and rabbit red cells.     

Activation and maturation mechanisms of boar acrosin zymogen based on the deduced primary structure

We have isolated cDNA clones encoding boar acrosin, a serine protease participating in the initial stage of fertilization, from boar testis lambda gt11 cDNA libraries. Nucleotide sequencing of the overlapping clones indicates that the composite cDNA inserts contain 1,391 base pairs coding for a 5'-untranslated region, an open reading frame, a stop codon, a 3'-untranslated region, and a poly(A)+ tail. A polyadenylation signal, AATAAA, is located 33 bases upstream from the start of the poly(A)+ tail. The amino acid sequence deduced from the cDNAs shows that boar acrosin is initially synthesized as a prepro-protein with a 16-residue signal peptide at the NH2 terminus. This signal sequence is followed by a 399-residue sequence corresponding to the acrosin zymogen. COOH-terminal sequence analysis of boar sperm 55-kDa proacrosin and its processed forms indicates that the mature acrosin molecule contains 322 amino acid residues in two polypeptide chains, a 23-residue light chain and a 299-residue heavy chain, with a combined molecular mass of 35,735 Da, and that the 55-kDa proacrosin molecule has 14-, 18-, and 43-residue segments as COOH-terminal extensions that are removed during proacrosin maturation. The COOH-terminal 43-residue segment is rich in proline residues, including an unusual repeat of 23 consecutive prolines. The deduced amino acid sequence of boar acrosin shows a high degree of identity with major portions of other serine proteases, including the active site region and the location of cysteine residues. We conclude that boar acrosin is synthesized as a single-chain polypeptide with the regions corresponding to the light and heavy chains covalently connected by two disulfide bonds, and that the single-chain molecule is autoactivated by cleavage of the Arg23-Val24 bond after removal of the COOH-terminal 14-residue segment, resulting in the formation of the light and heavy chains. This two-chain molecule is then converted to the mature enzyme by removal of the COOH-terminal 18- and 43-residue segments.     

Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus.

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68,993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme.     

Structure and function of papiliocin with antimicrobial and anti-inflammatory activities isolated from the swallowtail butterfly, Papilio xuthus

Papiliocin is a novel 37-residue cecropin-like peptide isolated recently from the swallowtail butterfly, Papilio xuthus. With the aim of identifying a potent antimicrobial peptide, we tested papiliocin in a variety of biological and biophysical assays, demonstrating that the peptide possesses very low cytotoxicity against mammalian cells and high bacterial cell selectivity, particularly against Gram-negative bacteria as well as high anti-inflammatory activity. Using LPS-stimulated macrophage RAW264.7 cells, we found that papiliocin exerted its anti-inflammatory activities by inhibiting nitric oxide (NO) production and secretion of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, producing effects comparable with those of the antimicrobial peptide LL-37. We also showed that the innate defense response mechanisms engaged by papiliocin involve Toll-like receptor pathways that culminate in the nuclear translocation of NF-κB. Fluorescent dye leakage experiments showed that papiliocin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of papiliocin in 300 mm dodecylphosphocholine micelles by NMR spectroscopy, showing that papiliocin has an α-helical structure from Lys(3) to Lys(21) and from Ala(25) to Val(36), linked by a hinge region. Interactions between the papiliocin and LPS studied using tryptophan blue-shift data, and saturation transfer difference-NMR experiments revealed that Trp(2) and Phe(5) at the N-terminal helix play an important role in attracting papiliocin to the cell membrane of Gram-negative bacteria. In conclusion, we have demonstrated that papiliocin is a potent peptide antibiotic with both anti-inflammatory and antibacterial activities, and we have laid the groundwork for future studies of its mechanism of action.     

Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.     

景东湍蛙抗菌肽jindongenin-1d的分析和活性测定

新型抗菌肽研究有助于解决细菌对抗生素的耐药性问题。本研究用SMART技术构建了景东湍蛙Amolops jingdongensis皮肤的全长cDNA文库。通过单克隆和测序获得一个抗菌肽cDNA序列,序列比对结果表明其属于jindongenin-1家族,命名为jindongenin-1d。其cDNA序列全长321bp,编码含66个氨基酸残基的多肽。该多肽包括1个信号肽和1个前肽序列。成熟jindongenin-1d多肽包含24个氨基酸残基,理论分子量为2 709.38,等电点为9.24。对人工合成的jindongenin-1d蛋白进行了抗菌和溶血活性分析,结果表明jindongenin-1d对所选的革兰氏阴性菌、革兰氏阳性菌和真菌均有显著抑制作用,同时有弱溶血活性。本研究结果有助于进一步了解两栖动物皮肤分泌物活性物质的多态性和新型抗感染药物的设计。     

Structure of soybean Kunitz trypsin inhibitor mRNA determined from cDNA by using oligodeoxynucleotide primers

Oligodeoxynucleotides complementary to the deduced mRNA sequence of soybean Kunitz trypsin inhibitor (KTI) were used to prime the synthesis of cDNA from soybean cotyledon total poly(A) RNA. The primed cDNA was used to select clones from a Glycine max cotyledon cDNA library. Two out of twelve hybridizing clones were shown to contain KTI cDNA. The nucleotide sequence of one clone, pSTI 9-2, was determined and it was found to encompass the complete protein coding region of KTI excet for three C-terminal residues. Trypsin inhibitor is synthesized with a 25 amino acid hydrophobic N-terminal sequence presumed to be a signal peptide. The mature polypeptide encoded by pSTI 9-2 agrees with the published amino acid composition of KTI, but contains two discrepancies at the peptide sequence level.     

Purification and cloning of a novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin-B was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY by Edman degradation. The cDNA encoding ixosin-B was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 89 amino acids including mature ixosin-B. Purified ixosin-B exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide. It is also the fourth family of antimicrobial peptide from hard ticks.     

Molecular cloning and nucleotide sequence of full-length cDNA for ascorbate oxidase from cultured pumpkin cells

A lambda gt11 cDNA expression library was constructed from size-fractionated poly(A)-rich RNA of cultured pumpkin cells. A full-length cDNA clone for ascorbate oxidase mRNA was selected from the library by screening with synthetic oligonucleotides designed from the amino-terminal sequence of ascorbate oxidase protein. The identity of the clone was confirmed by comparing the amino acid sequence deduced by nucleotide sequence analysis with that determined for the amino-terminal sequence of pumpkin ascorbate oxidase. The nucleotide sequence of the cDNA insert was found to contain an open reading frame of 579 codons corresponding to a signal peptide of 30 amino acids and the mature 549-residue ascorbate oxidase. Furthermore, it was found that the amino acid sequence deduced from the nucleotide sequence of the cDNA insert contained four potential N-glycosylation sites and copper-binding amino acid residues located in four regions where the sequence was identical or nearly identical to those of the other known blue multicopper oxidases Neurospora crassa laccase and human ceruloplasmin.     

Amino acid and cDNA sequences of a neutral phospholipase A2 from the long-nosed viper (Vipera ammodytes ammodytes) venom.

The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and 100%, respectively.     

Identification and characterization of novel reptile cathelicidins from elapid snakes

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576bp and coded for 191 amino acid residue protein precursors. Each of the deduced elapid cathelicidin has a 22 amino acid residue signal peptide, a conserved cathelin domain of 135 amino acid residues and a mature antimicrobial peptide of 34 amino acid residues. Unlike the highly divergent cathelicidins in mammals, the nucleotide and deduced protein sequences of the three cloned elapid cathelicidins were remarkably conserved. All the elapid mature cathelicidins were predicted to be cleaved at Valine157 by elastase. OH-CATH, the deduced mature cathelicidin from king cobra, was chemically synthesized and it showed strong antibacterial activity against various bacteria with minimal inhibitory concentration of 1-20microg/ml in the presence of 1% NaCl. Meanwhile, the synthetic peptide showed no haemolytic activity toward human red blood cells even at a high dose of 200microg/ml. Phylogenetic analysis of cathelicidins from vertebrate suggested that elapid and viperid cathelicidins were grouped together in the tree. Snake cathelicidins were evolutionary closely related to the neutrophilic granule proteins (NGPs) from mouse, rat and rabbit. Snake cathelicidins also showed a close relationship with avian fowlicidins (1-3) and chicken myeloid antimicrobial peptide 27. Elapid cathelicidins might be used as models for the development of novel therapeutic drugs.     

Molecular cloning and characterization of an endo-1,3-beta-D-glucanase from the mollusk Spisula sachalinensis

cDNA encoding the endo-1,3-beta-d-glucanase from Spisula sachalinensis (LIV) was amplified by PCR using oligonucleotides deduced from the N-terminal end peptide sequence. Predicted enzyme structure consists of 444 amino acids with a signal sequence. The mature enzyme has 316 amino acids and its deduced amino acid sequence coincides completely with the N-terminal end (38 amino acids) of the beta-1,3-glucanase (LIV) isolated from the mollusk. The enzyme sequence from Val 121 to Met 441 reveals closest homology with Pacifastacus leniusculus lipopolysaccharide- and beta-1,3-glucan-binding protein and with coelomic cytolytic factors from Lumbricus terrestris. The mollusk glucanase also shows 36% identity and 56% similarity with beta-1,3-glucanase of the sea urchin Strongylocentrotus purpuratus. It is generally considered that invertebrate glucanase-like proteins containing the bacterial glucanase motif have evolved from an ancient beta-1,3-glucanase gene, but most of them lost their glucanase activity in the course of evolution and retained only the glucan-binding activity. A more detailed evaluation of the protein folding elicited very interesting relationships between the active site of LIV and other enzymes, which hydrolyze native glucans.