Novel bradykinins and their precursor cDNAs from European yellow-bellied toad (Bombina variegata) skin.

Two novel bradykinin-related peptides (Ala3,Thr6)-bradykinin and (Val1,Thr3,Thr6)-bradykinin, were identified by a systematic sequencing study of peptides in the defensive skin secretion of the yellow-bellied toad, Bombina variegata. These peptides are the first amphibian skin bradykinins to exhibit amino acid substitutions at the Pro3 position of the bradykinin nonapeptide. Previously reported bradykinins from other Bombina species were not detected. Respective precursor cDNAs, designated BVK-1 and BVK-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. BVK-1 contained an open-reading frame of 97 amino acids encoding a single copy of (Ala3,Thr6)-bradykinin and similarly, the open-reading frame of BVK-2 consisted of 96 amino acids encoding a single copy of (Val1,Thr3,Thr6)-bradykinin. Synthetic replicates of each novel bradykinin were found to be active on mammalian arterial and small intestinal smooth muscle preparations. The structural diversity of bradykinins in amphibian defensive skin secretions may be related to defence against specific predators.     

Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine O-sulfation motif

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.     

Bradykinins and their cDNA from piebald odorous frog,Odorrana schmackeri,skin

Bradykinin, (des-Arg(9))-bradykinin and bradykinyl-VAPAS, were identified in the skin secretion of the piebald odorous frog, Odorrana schmackeri. Using 3'- and 5'-RACE reactions, bradykinin precursor cDNA was cloned and found to contain an open-reading frame of 311 amino acid residues. The preprobradykinin was found to consist of a putative signal peptide of approximately 20 amino acid residues, followed by seven tandem repeat coding domains of 43-44 amino acids. Bradykinin and its C-terminal extended molecular form were encoded on this single precursor and could be generated by differential post-translational processing.     

A fish bradykinin (Arg0, Trp5, Leu8-bradykinin) from the defensive skin secretion of the European edible frog, Pelophylax kl. esculentus: structural characterization; molecular cloning of skin kininogen cDNA and pharmacological effects on mammalian smooth muscle

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val¹, Thr⁶)-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes—common predators of these frogs. Here, we report the presence of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg⁰, Trp⁵, Leu⁸)-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg⁰, Trp⁵, Leu⁸)-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.     

Bradykinin-related peptides, including a novel structural variant, (Val1)-bradykinin, from the skin secretion of Guenther's frog, Hylarana guentheri and their molecular precursors

Multiple bradykinin-related peptides including a novel bradykinin structural variant, (Val(1))-bradykinin, have been identified from the defensive skin secretion of Guenther's frog, Hylarana guentheri by a tandem mass spectrometry method. Subsequently, four different preprobradykinin cDNAs, which encoded multiple bradykinin copies and its structural variants, were consistently cloned from a skin derived cDNA library. These preprobradykinin cDNAs showed little structural similarity with mammalian kininogens and the kininogens from the skin of toads, but have regions that are highly conserved in the kininogens from another ranid frog, Odorrana schmackeri. Alignment of these preprobradykinins revealed that preprobradykinin 1, 2 and 3 may derive from a single gene by alternative exon splicing.     

Molecular cloning of mouse and bovine chondromodulin-II cDNAs and the growth-promoting actions of bovine recombinant protein

We previously determined the complete primary sequence of a heparin-binding growth-promoting factor, chondromodulin-II (ChM-II), which stimulated the growth of chondrocytes and osteoblasts in culture. Bovine ChM-II was a 16-kDa basic protein with 133 amino acid residues and exhibited a significant sequence similarity to the repeats of the chicken mim-1 gene product. Here we report the nucleotide sequences of bovine and mouse ChM-II cDNAs. The cDNAs each contained an open-reading frame corresponding to the ChM-II precursor with 151 amino acid residues. The N-terminus of the precursor included a secretory signal sequence of 18 amino acids prior to the mature ChM-II sequence. Unlike MIM-1, there was no repeat structure in the precursor protein, indicating that ChM-II was encoded as a gene product distinct from MIM-1. We then expressed recombinant bovine ChM-II protein which was purified to homogeneity. The recombinant protein stimulated the growth of rabbit growth plate chondrocytes, mouse MC3T3-E1 cells and rat UMR-106 osteoblastic cells in vitro.     

Cloning and characterization of the first amphibian bradykinin gene

More than ten bradykinin-related peptides and their cDNAs have been identified from amphibians, but their genes are unknown. In present study, four cDNAs encoding one, two, four and six copies of bradykinin-related peptides were cloned from the frog (Odorrana grahami) skin cDNA library, respectively. Three bradykinin-related peptides (bradykinin, Thr6-bradykinin, Leu5Thr6-bradykinin) were deduced from these four cDNA sequences. Based on the cDNA sequence, the gene sequence encoding an amphibian bradykinin-related peptide from O. grahami was determined. It is composed of 7481 base pairs including two exons and two introns. The first exon codes signal peptide and the second exon codes acidic spacer peptide and Thr6-bradykinin. The promoter region of the bradykinin gene contains several putative recognition sites for nuclear factors, such as SRY, GATA-1, LYF-1, DeltaE, CDXA, NKX-2.5, MIF1 and S8. The current work may facilitate to understand the regulation and possible functions of amphibian skin bradykinin-related peptides.     

Identification and characterization of a putative homolog of a spliceosome component,precursor RNA processing 3 in Drosophila melanogaster

Nuclear pre-mRNA splicing occurs in a large RNA-protein complex that contains four small nuclear ribonucleoprotein particles (snRNPs) as well as many protein factors. The Precursor RNA processing 3 (Prp3) is a U4/U6-associated splicing factor. A putative homologue of Prp3, which showed a 45% identity to the human Prp3 in an amino acid sequence, was identified in Drosophila melanogaster (dPrp3). A full-length cDNA clone was isolated and sequenced from the embryonic cDNA library. This gene consisted of 2 exons and contained an open-reading frame that encoded 550 amino acid residues. A Northern blot analysis showed that dPrp3 is expressed both maternally and zygotically. Immunostaining revealed that dPrp3 was localized to the nuclei of nurse cells and follicle cells in early embryos, which is consistent with its role as a component of spliceosome.     

The substrate recognition site of collagen proline hydroxylase: the hydroxylation of -X-Pro-Gly- sequences in bradykinin analogs and other peptides

A series of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) analogs, in which one or more amino acid residues were substituted or added to the molecule, were tested as substrates of collagen proline hydroxylase. Analogs with substitutions at position 1 (lys¹-bradykinin), at position 2 (ala²-bradykinin), at position 6 (gly⁶-bradykinin) and position 1 and 9 (nitroarg^1,9-bradykinin) were hydroxylated by the enzyme. Analogs in which 1, 2, 3, or 9 amino acid residues had been added at the N-terminus of the molecule were also hydroxylated, and in general were more effective substrates than bradykinin itself. In contrast, modification of bradykinin by introducing substituents at position 3 (d-pro³-bradykinin, val³-bradykinin) or at position 4 (ala⁴-bradykinin, sar⁴-bradykinin, 5-aminoval⁴-bradykinin) yielded analogs which were not hydroxylated. Kinetic data for many of the active analogs are reported. A synthetic bradykinin-potentiating peptide (C) containing one -Pro-Pro-Gly-sequence could also act as a substrate, but the tetrapeptide Gly-Pro-Gly-Gly did not interact with the enzyme. It is concluded that the minimum sequence requirement for proline hydroxylation is an intact -X-Pro-Gly-triplet, and that the affinity of individual prolylcontaining sequences for the enzyme, and the rate at which they are hydroxylated are dependent on the adjacent amino acid residues and the identity of X.     

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog,Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.     

High copy number of highly similar mariner-like transposons in planarian (Platyhelminthe): evidence for a trans-phyla horizontal transfer

Several DNA sequences similar to the mariner element were isolated and characterized in the platyhelminthe Dugesia (Girardia) tigrina. They were 1,288 bp long, flanked by two 32 bp-inverted repeats, and contained a single 339 amino acid open-reading frame (ORF) encoding the transposase. The number of copies of this element is approximately 8,000 per haploid genome, constituting a member of the middle- repetitive DNA of Dugesia tigrina. Sequence analysis of several elements showed a high percentage of conservation between the different copies. Most of them presented an intact ORF and the standard signals of actively expressed genes, which suggests that some of them are or have recently been functional transposons. The high degree of similarity shared with other mariner elements from some arthropods, together with the fact that this element is undetectable in other planarian species, strongly suggests a case of horizontal transfer between these two distant phyla.      

Synthesis, conformation and biological activity of linear and cyclic Thr6-bradykinin analogues containing N-benzylglycine in place of phenylalanine.

Three linear Thr6-bradykinin analogues in which either one or both the two phenylalanine residues in the peptide sequence have been substituted by N-benzylglycine (BzlGly) and their head-to-tail cyclic analogues were synthesized and tested on an isolated rat duodenum preparation. The linear (BzlGly5,Thr6-BK, BzlGly8,Thr6-BK and BzlGly(5,8),Thr6-BK) and the cyclic (cyclo BzlGly5,Thr6-BK, cyclo BzlGly8,Thr6-BK and cyclo BzlGly(5,8),Thr6-BK) peptoid-like analogues were characterized by amino acid analysis, optical rotation, analytical HPLC and MALDI-TOF mass spectroscopy. The conformational features of both the linear and cyclic derivatives were investigated by FT-IR and CD measurements. Preliminary molecular mechanics calculations were also performed on some synthetic peptides. Pharmacological screening using the relaxation of the isolated rat duodenum preparation showed that incorporation of N-benzylglycine at positions 5 and/or 8 in the linear Thr6-BK causes a substantial decrease in potency. Comparable incorporation in cyclo Thr6-BK, at position 8, or 5 and 8, resulted in nearly inactive analogues. However, cyclo BzlGly5,Thr6-BK showed a potency which is of the same order of magnitude as for cyclo-BK and cyclo Thr6-BK.     

Cloning and characterization of cDNAs encoding the GnRH1 and GnRH2 precursors from bullfrog (Rana catesbeiana)

We have isolated the cDNAs encoding the GnRH1 and GnRH2 precursors, respectively, from bullfrog (Rana catesbeiana) brain. The first cDNA consists of 648 bp and contains an open-reading frame of 270 nucleotides, encoding the bullfrog GnRH1 precursor. The second cDNA consists of 1053 bp and contains an open-reading frame of 255 nucleotides, encoding the bullfrog GnRH2 precursor. Both types of bullfrog GnRH precursor have a similar molecular architecture as observed in other GnRH precursors, consisting of a signal peptide, followed by the GnRH decapeptide, a conserved carboxy-terminal amidation and proteolytical processing site, and a GnRH-associated peptide (GAP). In addition, we have identified a third cDNA, containing 24 additional nucleotides in its GAP-coding region. Genomic PCR and sequence analysis confirmed that this cDNA represents an alternative splice variant of the bullfrog GnRH2-precursor pre-mRNA. The bullfrog GnRH1 precursor exhibits 60% and less than 40% amino acid identity to its Xenopus and mammalian counterparts, respectively, whereas the bullfrog GnRH2 precursor displays 50% to 60% amino acid identity to that of its nonmammalian counterparts, but shares only 25% amino acid identity with its mammalian counterparts. Northern blot analysis revealed a single GnRH1-precursor mRNA species of approximately 0.75 kilobases, expressed in bullfrog forebrain, and a single GnRH2-precursor mRNA species of approximately 1.1 kilobases, expressed in bullfrog midbrain/hindbrain. Furthermore, both bullfrog GnRH-precursor mRNAs exhibited a differential spatiotemporal expression pattern. Genomic Southern blot analysis indicated that both bullfrog GnRH genes are present as single copy genes. This is the first report on the molecular cloning of a GnRH2-precursor cDNA from an amphibian species. In addition, we present data showing that alternative splicing is utilized to generate different GnRH2-precursor mRNAs. J. Exp. Zool. 289:190-201, 2001.     

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8--a novel prokineticin from Bombina orientalis

Prokineticins are small (approximately 8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr=7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.     

Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-sensitive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.     

Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the "shotgun" cloning of novel brevinins by means of 3'-RACE, using a "universal" degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.     

PCR-cloning of cadmium-inducible peptides in the barnacle,Megabalanus volcano

A 340 bp DNA fragment was amplified from barnacle (Megabalanus volcano) cDNA by polymerase chain reaction using primers designed based on the amino acid sequences of barnacle cadmium-inducible peptides CdlP1 and CdlP2. The whole sequence was determined by rapid amplification of cDNA ends method. The cDNA contained an open reading frame encoding 71 amino acid residues and the sequences for CdlP1 and CdlP2 were found to be located in the center of this coding region. Although CdlP1 and CdlP2 had been detected only in the cadmium-exposed barnacles, their mRNA was present both in cadmium-exposed barnacles and in unexposed barnacles. These results suggest that posttranslational proteolytic processing may be induced in the presence of cadmium.     

Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state.