A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.     

Cloning, Expression, and Characterization of a Methionyl Aminopeptidase from a Hyperthermophilic Archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1 revealed the presence of an 885-bp open reading frame encoding a protein of 295 amino acids with a calculated molecular mass of 32,981 Da. Analysis of the deduced amino acid sequence showed that amino acid residues important for catalytic activity and the metal binding ligands conserved in all of methionyl aminopeptidases (MetAP) were also conserved and belonged to type IIa MetAP. The protein, designated TNA1_MetAP (Thermococcus sp. NA1 MetAP), was cloned and expressed in Escherichia coli. The recombinant enzyme was a Mn²⁺-, Ni²⁺-, Fe²⁺-, or Co²⁺-dependent metallopeptidase. Optimal MetAP activity against l-methionine p-nitroanilide (Met-pNA) (K _m = 0.68 mM) occurred at pH 7.0 and 80 to 90°C. The MetAP was very unstable compared to Pyrococcus furiosus MetAP, which was completely inactivated by heating at 80°C for 5 min. It seemed likely that the cysteine residue (Cys53) played a critical role in regulating the thermostability of TNA1_MetAP.     

An immune responsive complement factor D/adipsin and kallikrein-like serine protease (PoDAK) from the olive flounder Paralichthys olivaceus

The cDNA encoding of a complement factor D/adipsin and kallikrein-like serine protease, designated PoDAK, was isolated from the olive flounder Paralichthys olivaceus. PoDAK cDNA encodes a polypeptide with 277 amino acids containing conserved catalytic triad residues of serine proteases. The amino acid sequence of PoDAK showed high similarity to the kallikrein-like protein of medaka, mammalian adipsin/complement factor D and tissue kallikrein homolog, KT-14 of trout, complement factor D of zebrafish, and shared 31.6–36.8% homology with complement factor D/adipsin known from other species, including mammals. Phylogenetic analysis revealed that PoDAK clustered with the kallikrein-like protein of medaka and mammalian adipsin/complement factor D and tissue kallikrein homolog KT-14 of trout. The expression of PoDAK mRNA was high in the gills and heart, moderate in muscle, liver, intestine, stomach, kidney, and spleen of healthy flounder, and increased in the kidney, liver, and spleen of flounder challenged by the viral hemorrhagic septicemia virus (VHSV) or Streptococcus iniae. In situ hybridization confirmed that PoDAK mRNA is localized in the kidney and heart of individuals infected with VHSV. Further investigations are needed to clarify the function of PoDAK in vivo and in vitro.     

Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus

We cloned a cDNA and the gene for Japanese flounder TNF. The TNF cDNA consisted of 1217 bp, which encoded 225 amino acid residues. The identities between Japanese flounder TNF and members of the mammalian TNF family were approximately 20-30%. The positions of cysteine residues that are important for disulfide bonds were conserved with respect to those in mammalian TNF-alpha. The Japanese flounder TNF gene has a length of approximately 2 kbp and consists of four exons and three introns. The positions of the exon-intron junction positions of Japanese flounder TNF gene are similar to those of human TNF-alpha. However, the length of the first intron of Japanese flounder is much shorter than that of the human TNF-alpha gene. There are simple CA or AT dinucleotide repeats in the 5'-upstream and 3'-downstream regions of the Japanese flounder TNF gene. Southern blot hybridization indicted that Japanese flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-alpha than mammalian lymphotoxin-alpha, with respect to its gene structure, length of amino acid sequence, number and position of cysteine residues, and regulation of gene expression.     

A novel rat carboxypeptidase, CPA2: characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family

A new member of the carboxypeptidase gene family, carboxypeptidase A2 (CPA2), has been identified from the predicted amino acid sequence of a rat pancreatic cDNA clone. In vivo recombination and in situ hybridization techniques employing the CPA2 cDNA resulted in the isolation of two genomic clones spanning the 25-kilobase pair rat CPA2 gene. Evolutionary trees built from the amino acid sequences of the known pancreatic carboxypeptidases show that CPA2 and carboxypeptidase A1 (CPA1) are the products of genes which duplicated before the mammalian radiation, and that bovine CPA is of the A1 type. The substrate specificities of CPA1 and CPA2 isolated from rat pancreas are similar to bovine CPA in that carboxyl-terminal amino acids with aromatic or branched aliphatic side chains are preferred. However, the substrate preference of rat CPA1 is skewed toward smaller amino acids, while that of rat CPA2 is skewed toward bulkier amino acids as compared to bovine CPA. The differences in the substrate specificities of these three carboxypeptidases are compatible with the nature of the amino acid replacements in their binding pockets for the carboxylterminal amino acid of the substrate.     

Molecular cloning of preprocathepsin E cDNA from the stomach of bullfrog Rana catesbeiana

A cDNA library was constructed from a poly(A)⁺ RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn¹³⁹-Leu¹⁴⁰-Thr¹⁴¹) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases.     

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A₂ (PLA₂), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA₂ protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA₂ protein, suggesting the presence of multiple forms in the starfish PLA₂. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA₂. In phylogenetic tree, the starfish PLA₂ should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA₂, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA₂ from porcine pancreas.     

Identification of a novel Japanese flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) CC chemokine gene and an analysis of its function

A cDNA of Japanese flounder (Paralichthys olivaceus) CC chemokine designated as Paol-SCYA104 was cloned and sequenced. The cDNA contains an opening reading frame of 315 nucleotides encoding 104 amino acid residues. The full gene was cloned and sequenced from a BAC library. It has a length of approximately 750 bp from the start codon to the stop codon and is composed of four exons and three introns. Four cysteine residues are conserved in the same positions as those of mammalian and fish CC chemokines. Paol-SCYA104 gene was expressed in several organs, including peripheral blood leukocytes (PBLs), head kidney, trunk kidney, and spleen. The recombinant Paol-SCYA104 was expressed in Escherichia coli and the expressed protein was partially purified. The recombinant Paol-SCYA104 was able to attract Japanese flounder PBLs in a microchemotaxis chamber. On the other hand, a negative control, the fraction of the control cells carrying an expression vector lacking the Paol-SCYA104 cDNA, did not show chemotactic activity. These results indicate that Paol-SCYA104 probably acts as a CC chemokine.     

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H⁺-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe⁵, Met¹⁰, and Gln¹⁴ involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.     

An analysis of nucleotide and amino acid variability in the barcode region of cytochrome c oxidase I (cox1) in fishes

The 655 bp cytochrome c oxidase subunit I barcode region of single specimens of 388 species of fishes (four Holocephali, 61 Elasmobranchii and 323 Actinopterygii) was examined. All but two (Urolophus cruciatus and Urolophus sufflavus) showed different cox1 nucleotide sequences (99.5% species discrimination); the two that could not be resolved are suspected to hybridize. Most of the power of cox1 nucleotide sequence analysis for species identification comes from the degenerate nature of the genetic code and the highly variable nature of the third codon position of amino acids. Variation at the third codon position is bimodally distributed, and the more variable mode is dominated by amino acids with four or six codons, while the less variable mode is dominated by amino acids with two codons. The ratio of nonsynonymous to synomymous changes is much less than one, indicating that this gene is subject to strong purifying selection. Consequently, cox1 amino acid sequence diversity is much less than nucleotide sequence diversity and has very poor species resolution power. Fourteen of the 16 amino acid residues recognized as having important functions in the region of cox1 sequenced were completely conserved over all 388 species (and the bovine cox1 sequence), with one fish species varying at one of these sites, and three fish at another site. No significant differences in amino acid conservation were observed between residues in helices, strands and turns. Patterns of nucleotide and amino acid variability were very similar between elasmobranchs and actinopterygians.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 × 10^{? 9} M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

Amino acids in Tobamovirus coat protein controlling pepper L(1a) gene-mediated resistance

In pepper plants (genus Capsicum), the resistance against Tobamovirus spp. is conferred by L gene alleles. The recently identified L variant L^1a can recognize coat proteins (CPs) of Tobacco mild green mosaic virus Japanese strain (TMGMV‐J) and Paprika mild mottle virus Japanese strain (PaMMV‐J), but not of Pepper mild mottle virus (PMMoV), as the elicitor to induce resistance at 24 °C. Interestingly, L^1a gene‐mediated resistance against TMGMV‐J, but not PaMMV‐J, is retained at 30 °C. This observation led us to speculate that L^1a can discriminate between CPs of TMGMV‐J and PaMMV‐J. In this study, we aimed to determine the region(s) in CP by which L^1a distinguishes TMGMV‐J from PaMMV‐J. By using chimeric CPs consisting of TMGMV‐J and PaMMV‐J, we found that the chimeric TMGMV‐J CP, whose residues in the β‐sheet domain were replaced by those of PaMMV‐J, lost its ability to induce L^1a gene‐mediated resistance at 30 °C. In contrast, the chimeric PaMMV‐J CP with the β‐sheet domain replaced by TMGMV‐J CP was able to induce L^1a gene‐mediated resistance at 30 °C. Furthermore, viral particles were not detected in the leaves inoculated with either chimeric virus. These observations indicated that the amino acids within the β‐sheet domain were involved in both the induction of L^1a gene‐mediated resistance and virion formation. Further analyses using chimeric CPs of TMGMV‐J and PMMoV indicated that amino acids within the β‐sheet domain alone were not sufficient for the induction of L^1a gene‐mediated resistance by TMGMV‐J CP. These results suggest that multiple regions in Tobamovirus CP are implicated in the induction of L^1a gene‐mediated resistance.     

Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Viral Receptors,Neutralizing Antibodies and Lipoproteins

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H³⁸⁶ and R⁴⁰⁸), and the two highly conserved basic residues H⁴⁸⁸ and R⁶⁴⁸ contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.     

Acetylcholinesterase from a Non-membrane Source (Bungarus fasciatus Venom)

Abstract

Acetylcholinesterase from Bungarus fasciatus venom has been purified by a conventional procedure with a specific activity of 230 millimoles acetylcholine hydrolysed mg protein^?1 hr^?1. The enzyme with a molecular weight of 126, 000 has an excess of acidic amino acids over basic amino acids. The N-terminal amino acid analysis gave leucine as the only N-terminal amino acid with a free amino group. There are no common antigenic sites between the B. fasciatus venom acetylcholinesterase and acetylcholinesterases from bovine erythrocytes and electric eel.     

The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components

The effects of serum components and amino acids on the uptake and cytotoxicity of NiCl₂ were examined in cultured Chinese hamster ovary (CHO) cells. CHO cells maintained in a minimal salts/glucose medium accumulated 10-fold more⁶³Ni than did cells maintained in complete medium supplemented with 10% fetal bovine serum. Cell-surface binding of⁶³Ni appeared to account for the majority of this increased accumulation of cell-associated nickel observed in the simple maintenance medium since such increases were reduced 70% by trypsin treatment. The addition of the Ni²⁺-binding amino acids cysteine or histidine to the salts/glucose medium markedly decreased⁶³Ni accumulations, an effect not observed following addition of any of several amino acids that do not bind Ni²⁺. Supplementation of the salts/glucose medium with fetal bovine serum decreased in a concentration dependent fashion both the⁶³Ni²⁺ uptake and cell detachment caused by Ni²⁺, while dialyzed (amino acid-free) serum was 3–5-fold less effective than undialyzed serum at reducing⁶³Ni²⁺ uptake and similarly exhibited only a slight protective effect against nickel-induced cytotoxicity. Supplementation of dialyzed serum with cysteine at levels approximating those in whole serum partially restored its inhibitory activity toward nickel uptake by cells and restored completely its inhibition of nickel's cytotoxicity, indicating the predominant role of specific amino acids over serum proteins in regulating the uptake and subsequent cytotoxicity of Ni²⁺. Addition of cysteine to the salts/glucose medium during a 2 h exposure of cells to either 100 μM HgCl₂ or 1 mM NiCl₂ masked the cytotoxic effects of these metal ions. These results demonstrate the importance of extracellular small molecular weight metal ion chelators in altering the biological effects of metal ions at the level of metal uptake.     

Selective stabilization of a labile mutant form of bovine pancreatic ribonuclease A by antibodies

The region between the amino acids 31-46 was previously identified as being first exposed during thermal unfolding of bovine pancreatic ribonuclease A (RNase). The exchange of one amino acid (Leu35toSer) in this unfolded region of RNase is shown to have a dramatic destabilizing effect (T_m=9 °C). Antibodies raised against a peptide corresponding to the sequence of the labile region, S32-V43, of RNase were effective in stabilizing L35S-RNase against thermal inactivation (65 °C for 2 h) and surpassed the stabilization effect of antiRNase antibodies. An 11% contribution to the stabilizing effect of antiRNase antibodies resulted from antibodies recognizing the unfolding region of the enzyme.     

Cloning,expression, and characterization of fugu CD4, the first ectothermic animal CD4

We have cloned and sequenced the first ectothermic animal CD4 gene from fugu, Takifugu rubripes, using a public database of the third draft sequence of the fugu genome. The fugu CD4 gene encodes a predicted protein of 463 amino acids containing four extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail. Fugu CD4 shares low identity of about 15–20% with avian and mammalian CD4 proteins. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair of the first Ig-like domain, but has a unique possible disulfide bond in the third domain. These differences suggest that fugu CD4 may have a different structure that could affect binding of major histocompatibility complex class II molecules and subsequent T-cell activation. In the putative fugu cytoplasmic region, the protein tyrosine kinase p56^lck binding motif is conserved. The predicted fugu CD4 gene is composed of 12 exons, differing from other CD4 genes, but showing conserved synteny and many conserved sequence motifs in the promoter region. RT-PCR analysis demonstrated that the fugu CD4 gene is expressed predominantly in lymphoid tissues. We also show that fugu CD4 can be expressed on the surface of cells via transfection. Molecular characterization of CD4 in fish provides insights into the evolution of both the CD4 molecule and the immune system.