Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211bp, consisting of a 5'-terminal untranslated region (UTR) of 72bp, a 3'-terminal UTR of 77bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16h after injury and injection of bacteria.     

Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca(2+)-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a 5'-terminal untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7- and 4.9-fold at 6h after injury and 8h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury healing and the immune response in A. irradians.     

A novel serine protease with clip domain from scallop Chlamys farreri

The serine proteases with clip domain are involved in various innate immune functions in invertebrate such as antimicrobial activity, cell adhesion, pattern recognition and regulation of the prophenoloxidase system. A serine protease with clip-domain cDNA (Cf SP) was obtained by Expressed sequence taggings (ESTs) method and rapid amplification of cDNA ends (RACE). The Cf SP full-length cDNA was of 1,152 bp, including a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 81 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 1,008 bp encoding a polypeptide of 336 amino acids with a putative signal peptide of 19 amino acids. The deduced amino acid sequence of Cf SP contained an amino-terminal clip domain with three disulfide bonds formed six conserved Cys residues, a carboxyl-terminal trypsin-like domain with the conserved His-Asp-Ser catalytic triad, and a low complexity linker sequence. The Cf SP was strongly expressed in hemocytes and the mRNA expression of Cf SP was up-regulated and increased 3.2-fold and 2.6-fold at 16 h after injection of Vibrio anguillarum and Micrococcus luteus. The results suggested that Cf SP gene might be involved in immune response of Gram-negative and Gram-positive microbial infection in scallop.     

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207bp with a 678bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3h post-injection with V. anguillarum and then slightly recovered from 6 to 48h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V. anguillarum infection.     

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3h post-challenge. After a decrease at 6h, the expression level increased again and reached 207.8-fold at 12h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCfKZSPI-12) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (Ki) of rCfKZSPI-12 to trypsin was 173 nmol L(-1). These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response.     

Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraea mylitta

An inhibitor of Aspergillus oryzae fungal protease was purified to homogeneity from the hemolymph of fifth instar larvae of Antheraea mylitta by ammonium sulfate precipitation, anion exchange and gel filtration (FPLC) chromatography, and termed as AmFPI-1. The extent of purification was checked by two-dimensional gel electrophoresis, and the molecular weight of purified inhibitor was determined by SDS-PAGE as 10.4 kDa. Fifteen N-terminal amino acid sequences of this protein were determined, and degenerate oligonucleotides were synthesized on the basis of these sequences. A cDNA library of A. mylitta integument was constructed, and protease inhibitor cDNA was partially amplified by PCR using degenerate oligonucleotides and CDS primers. A full-length inhibitor cDNA clone obtained by screening the library with PCR amplified DNA as probe was sequenced. The cDNA consists of 543 nucleotides with an ORF of 315 bp and encodes a protein of 105 amino acids. The sequence exhibits similarity to several Bombyx mori ESTs, and in particular to N-terminal amino acid sequence of an inducible serine protease inhibitor (ISPI-1) from Galleria mellonella indicating its relatedness to ISPI-1 of G. mellonella. The presence of this protease inhibitor in the hemolymph may play an important role as a natural defense system against invading microorganisms.     

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651bp in length, having a 5' untranslated region (UTR) of 96bp, a 3' UTR of 575bp, and an open reading frame (ORF) of 1980bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8h and lasted to 16h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop.     

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146bp with a 5' UTR of 103bp, an unusually long 3' UTR of 1519bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1521bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12h post-Vibrio infection, then recovered to the original level at 24h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop.     

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops <Emphasis Type="Italic">Argopecten irradians</Emphasis>

Apoptosis is an active process of cell death, which is an integral part of growth and development in multicellular organisms. The defender against cell death 1 (DAD1), the regulatory protein to inhibit the apoptosis process, was first cloned from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA end (RACE). The full-length cDNA of the A. irradians DAD1 was 607 bp, consist of a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 205 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 339 bp. The deduced amino acid sequence of the A. irradians DAD1 showed 75.5% identity to Araneus ventricosus, 74.5% to Drosophila melanogaster, and 73.6% to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. Excluding the Saccharomyces cerevisiae DAD1 homologue, all animal DAD1 including A. irradians DAD1 homologue formed a subgroup and all plant DAD1 proteins formed another subgroup in the phylogenetic analysis. The A. irradians DAD1 was expressed in all examined tissues including adductor muscle, mantle, gills, digestive gland, gonad and hemolymph, suggesting that A. irradians DAD1 is expressed in most body tissues. Furthermore, the mRNA expression levels of A. irradians DAD1 gene of hemolymph were particularly high after injury, suggesting that the gene is responsive to injury stimuli.     

大豆蚜Kazal型丝氨酸蛋白酶抑制剂基因AgKaSPI对蜡蚧刺束梗孢菌侵染的表达响应

[目的]探究Kazal型丝氨酸蛋白酶抑制剂KaSPI在大豆蚜Aphis glycines的生长发育、消化和免疫防御等过程中的作用.[方法]基于大豆蚜转录组数据PCR克隆大豆蚜Kazal型丝氨酸蛋白酶抑制剂基因cDNA序列;qRT-PCR分别检测AgKaSPI在大豆蚜1-4龄若虫和成虫以及蜡蚧刺束梗孢菌Akanthomy...     

A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis.

A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion-exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N-terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4-Cys37, Cys8-Cys30 and Cys16-Cys51. The structure of the inhibitor is homologous to Kazal-type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 nM whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C-terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C-terminal sequence is unusual for Kazal-type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal-type inhibitors.     

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.     

苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究

【目的】丝氨酸蛋白酶(Serine protease,SP)是以丝氨酸为活性中心的重要的蛋白水解酶。在昆虫中,丝氨酸蛋白酶参与消化、发育、先天免疫反应和组织重建等重要的生理过程。本试验以苜蓿夜蛾Heliothis viriplaca为材料,克隆其丝氨酸蛋白酶基因的cDNA序列,再对该基因进行原核表达并对表达产物进行活性测定研究。【方法】从苜蓿夜蛾中肠中提取总RNA,通过RT-PCR和RACE技术,扩增获得丝氨酸蛋白酶基因cDNA全长序列,用大肠杆菌E.coli表达系统进行表达;再对表达的重组蛋白进行变性、纯化与复性,并以BTEE为底物进行活性测定。【结果】克隆得到的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为Hv SP,该基因已登录Gen Bank,登录号为KT907053。该基因全长1 017 bp,开放阅读框为886 bp,编码295个氨基酸,分子量约为30.8 ku,等电点为8.27,推导的氨基酸序列与其他昆虫丝氨酸蛋白酶氨基酸序列相似性在46%~92%之间。在Tris-HCl缓冲液中,p H为8.5时,复性的重组蛋白活性最高,为28.7 U/m L。荧光定量PCR结果表明,Hv SP基因的m RNA在苜蓿夜蛾的多个组织中特异性表达,且在中肠中表达量最高,但在唾腺中未检测到Hv SP的m RNA表达。【结论】该研究克隆了一个新的苜蓿夜蛾丝氨酸蛋白酶基因的cDNA序列,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性,为进一步探索丝氨酸蛋白酶在昆虫体内的生理生化功能奠定了基础。     

Molecular cloning, mRNA expression, and characterization of heat shock protein 10 gene from humphead snapper Lutjanus sanguineus

Heat shock protein 10 (HSP10) gene of humphead snapper (Lutjanus sanguineus), designated as ByHSP10, was cloned by rapid amplification of cDNA ends (RACE) techniques with the primers designed from the known EST sequence identified from the subtracted cDNA library of the head kidney of humphead snapper. Sequence analysis showed the full length cDNA of ByHSP10 was 529 bp, containing a 5′ terminal untranslated region (UTR) of 51 bp, a 3′ terminal UTR of 181 bp, and an open reading frame (ORF) of 297 bp encoding a polypeptide of 99 amino acids. Based on the deduced amino acid sequence, the theoretical molecular mass of ByHSP10 was calculated to be 10.92 kDa with an isoelectric point of 9.46. Moreover, chaperonins hsp10/cpn10 signature was found in the amino acids sequence of ByHSP10 by PredictProtein. BLAST analysis revealed that the amino acids of ByHSP10 had the highest homology of 88% compared with other HSP10s. Fluorescent real-time quantitative RT-PCR was used to examine the expression of ByHSP10 gene in eight kinds of tissues of humphead snapper after the challenge with Vibrio harveyi. There was a clear time-dependent expression pattern of ByHSP10 in head kidney, spleen and thymus after bacteria challenge. The expression of mRNA reached the maximum level at the time point of 9 h, 6 h and 24 h, respectively and then returned to control level in 36 h. The up-regulated mRNA expression of ByHSP10 in humphead snapper after bacteria challenge indicated that the HSP10 gene was inducible and might be involved in immune response. A phylogenetic tree was constructed based on the ORF nucleotide sequences of HSP10 for 30 species. The relatonships among them were generally in agreement with the traditional taxonomy which suggested that HSP10 genes could aid in the system classification research.     

cDNA sequence coding for a rat glia-derived nexin and its homology to members of the serpin superfamily

Rat glial cells release a neurite-promoting factor with serine protease inhibitory activity. By using a rat glioma cDNA clone as a probe, it was possible to isolate rat cDNAs containing the entire sequence coding for this neurite-promoting factor. The largest rat cDNA (approximately 2100 bp) was characterized by DNA sequencing. It contained the entire coding region, 135 bp of the 5' nontranslated region, and about 750 bp of the 3' nontranslated region. The open reading frame coded for 397 amino acids including a putative signal peptide of 19 amino acids. The correct identity of the coding sequence was substantiated by the fact that the sequence of tryptic peptides, derived from the purified rat factor, matched exactly with the deduced amino acid sequence. The rat protein sequence had 84% homology with the corresponding protein from human glioma cells. Both amino acid sequences indicated that the proteins belong to the protease nexins [Baker, B.J., Low, D. A., Simmer, R. L., & Cunningham, D.D. (1980) Cell (Cambridge, Mass.) 21, 37-45] and therefore can be defined as glia-derived nexins (GDNs). Further analysis showed that both rat and human GDN belong to the serpin superfamily and share 41%, 32%, and 25% homology with human endothelial-cell-type plasminogen activator inhibitor, antithrombin III, and alpha-1 proteinase inhibitor, respectively.