球孢白僵菌降解昆虫体壁蛋白酶基因 CDEP-1的克隆与序列分析

构建了球孢白僵菌不同诱导时间的混合cDNA文库。根据丝氨酸类蛋白酶的保守区域设计引物,以构建cDNA文库同样的mRNA为模板,采用RT-PCR法得到长度为594bp的片段BbP。序列测定表明BbP是球孢白僵菌类枯草杆菌蛋白酶Prl的一部分,以BbP为探针,从上述cDNA文库筛选得到长度为1557bp的克隆CDEP-1。CDEP-1含有一个1134bp的开放阅读框（ORF）,编码377个氨基酸,分子量为38616、PI=8.302的蛋白酶前体。CEDP-1的核苷酸序列与蛋白酶K、金龟子绿僵菌Prl、球孢白僵菌Prl的同源性分别为：57.9%、54.7%、83.3%。根据cDNA序列扩增到CDEP-1的基因组序列,分析表明其中含有3个内含子。Southern杂交表明CDEP-1在球孢白僵菌是单拷贝。     

Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana

Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album , a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor ( M _r 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an M _r of 26 832.     

Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles.

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.     

Gene structure and expression of the gene from Beauveria bassiana encoding bassiasin I, an insect cuticle-degrading serine protease

Genomic and cDNA encoding Beauveria bassiana bassiasin I, a potential cuticle-degrading serine protease, were isolated and analysed. Bassiasin I gene is comprised of 1137 bp (379 aa) and 3 introns which are 69, 62 and 68 bp long. The comparison of a deduced amino acid sequence with Metarhizium anisopliae Pr1, B. bassiana Pr1, and proteinase K showed high homology. When the cDNA including the intact signal peptide was expressed in E. coli, a clear proteolytic-degraded zone on LB-skimmed milk plates was observed.     

Detection of a serine proteinase gene in Acanthamoeba genotype T6 (Amoebozoa: Lobosea)

Cytopathic proteins are assumed to contribute to the pathogenicity of Acanthamoeba spp. due to their degrading capacity that is required for tissue invasion. In this study, a serine proteinase gene was demonstrated in a highly virulent Acanthamoeba keratitis causing strain with genotype T6. This gene was detected in both, the genomic DNA and the cDNA by PCR and subsequent sequencing. The gene fragment comprises about 500 bp and exhibits high sequence similarity to the serine proteinases of Acanthamoeba strains with genotype T4 and T12. The detection of a serine proteinase in this Acanthamoeba T6 strain is significant, because while T4 is the most common genotype among pathogenic Acanthamoeba strains and also T12 is known to be associated with disease, this is the only virulent Acanthamoeba T6 strain known to date. Obviously, this serine proteinase represents a common tool in pathogenic processes during Acanthamoeba infection.     

Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.     

Cloning of the subtilisin Pr1A gene from a strain of locust specific fungus, <Emphasis Type="Italic">Metarhizium anisopliae,</Emphasis> and functional expression of the protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The subtilisin-like protease Pr1A plays a role in insect cuticle breach and has been used in the development of advanced engineered biopesticides. We have identified and cloned the Pr1A gene from a locust specific Metarhizium anisopliae strain, CQMa102. The cDNA of Pr1A and its deduced protein sequence were deposited in GenBank (accession numbers EF627449 and ABR20899, respectively). Sequence analysis reveals that Pr1A belongs to the subtilisin-like serine protease family. Analysis of homologous species shows that the protein exhibits 99% identity with the subtilisin Pr1A from M. anisopliae var. acridum strain FI-985. The CQMa102 Pr1A protein was expressed in Pichia pastoris to verify its protease activity. Our results show that the Pr1A gene cloned from M. anisopliae strain CQMa102 has cuticle-degrading function and is a potential virulence factor for the development of engineered biopesticides.     

The subtilisins of the invertebrate mycopathogens Verticillium chlamydosporium and Metarhizium anisopliae. are serologically and functionally related

Abstract The major extracellular proteases from the nematophagous fungus Verticillium chlamydosporium and the entomophagous fungus Metarhizium anisopliae , VCP1 and Pr1, respectively, are closely related both functionally and serologically. Antibodies raised against either enzyme cross-reacted with both antigens, suggesting that they have common epitopes. The VCP1 and Prl antisera labelled bovine pancreatic elastase and proteinase K, respectively. Neither antiserum reacted with commercial chymotrypsin. An antiserum to a serine protease from the closely related V. suchlasporium also cross-reacted with VCP1 and Prl. In contrast, a polyclonal antibody to an isoform of Pr1 exclusive to M. anisopliae isolate ME1 failed to recognize Prl from M. anisopliae V245 or VCP1. The N-terminal amino acid sequence of VCP1 revealed similarities with subtilisin-like enzymes from other fungi, but the closest match was with Pr1. The pure enzymes, VCP1 and Prl, failed to hydrolyse mono-aminoacyl-naphthylamide substrates but demonstrated dipeptidyl peptidase activity against Gly-Pro-βNA and Leu-Ala-βNA, respectively. These results are discussed in the context of specificity of invertebrate mycopathogens.     

Structure and expression of mRNA for a pupal cuticle protein of the silkworm,Bombyx mori

Electrophoretic and immunoblot analyses of proteins extracted from the salt-washed integuments of the silkworm Bombyx mori demonstrated that the pupal cuticle contains structural proteins distinct from those present in the larval cuticle. The cDNA clone encoding a pupal cuticle protein was isolated from the cDNA library constructed from epidermal mRNA of pharate pupae. Northern blot hybridization by use of a cDNA probe provided evidence that mRNA for the pupal cuticle protein accumulate in integument during larval-pupal transformation, though temporal rise of the mRNA level was also noticed at the stages of larval molting. Primary structure of the pupal cuticle protein was deduced from the nucleotide sequence of cDNA. The cloned mRNA sequence encodes a 27 kDa protein rich in alanine and proline, containing characteristic repeats of Ala-Pro-Ala-His-Gln-(Asp/Ser)-Trp-Asn sequence in the carboxyl-proximal domain. The sequence (Ile/Val)-(Leu/Ala)-(Asp/Glu)-Thr-Pro-Glu-Val-Ala-(Gln/Ala)-Ala-Arg-Ala-Ala-His-(Leu/Ile)-(Ala/Ser)-Ala-(Leu/His) occurs in three hydrophobic domains of the molecule.     

Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 micromol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.     

Metarhizium anisopliae var. Anisopliae Pr1A基因的克隆表达及抗血清研究

采用RT-PCR方法从本实验室分离筛选到的金龟子绿僵小孢变种Metarhizium anisopliae vat．anisopliae中,扩增得到PrlA基因全长,此基因全长为1242bp,经Blastn分析此基因序列与M．anopliae的PrlA基因(M73795)同源率为98%。以pET- 22b( )为基础载体,构建pET-PrlA重组表达载体,在大肠杆菌(Escherichia．coli)BL 21(DE3)中进行表达。经SDS—PAGE分析,获得了约42kDa大小的重组目的蛋白,目的蛋白占表达总蛋白含量的63．2%。将表达的PrlA蛋白切胶回收后制备成抗原,免疫家兔4次后,采血收集抗血清,用ELISA测定效价为1/10000。结果表明,获得的抗体可用于更进一步的研究,将有利于我们进一步了解M．anisopliaeis的侵染机理,弄清楚各Pr蛋白酶的作用方式和对寄主的选择优势,提高生防控制的有效性。     

Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae

Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.     

Cuticular proteins from the horseshoe crab,Limulus polyphemus

Proteins were purified from the carapace cuticle of a juvenile horseshoe crab, Limulus polyphemus, and several of them were characterized by amino acid sequence determination. The proteins are small (7-16 kDa) and their isoelectric points range from 6.5 to 9.2. They have high contents of tyrosine, ranging from 13.5 to 35.4%. Some of the proteins show sequence similarity to cuticular proteins from other arthropod groups, with the most pronounced similarity to proteins from the cuticle of the spider Araneus diadematus. Two proteins show sequence similarity to a hexamerin storage protein from Blaberus discoidalis.     

Molecular cloning of cDNAs for two subunits of rat multicatalytic proteinase. Existence of N-terminal conserved and C-terminal diverged sequences among subunits

cDNA clones for two subunits (designated subunits K and L) of rat liver multicatalytic proteinase (MCP) were isolated using oligonucleotide probes synthesized according to their partial amino acid sequences. The encoded polypeptides of subunits K and L consisted of 255 and 261 amino acid residues with calculated molecular mass of 28.3 kDa and 29.5 kDa, respectively. Northern blot analysis revealed that subunits K and L were expressed in all tissues examined and their expression patterns were almost identical. The deduced amino acid sequences showed no similarities to known protein sequences other than the recently reported sequences of rat and Drosophila MCP subunits. Sequence comparison of MCP subunits of rat and Drosophila revealed that the N-terminal two-thirds of the sequence (especially the N-terminal approximately 20 residues) is conserved, but the C-terminal third of the sequence shows no similarity, suggesting functional and structural roles for both regions. Implications for the structural and functional aspects of MCP subunits are discussed based on the sequence similarity.     

Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella.

Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.     

Biochemical characterization and molecular cloning of a plasminogen activator proteinase (LV-PA) from bushmaster snake venom

The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for approximately 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver-Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.     

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.     

Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin.

The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is a secreted thiol proteinase. Sequencing of the MEP cDNA shows the coding region for the protein to be identical with the sequence for a mouse cysteine proteinase isolated from macrophages, but the MEP cDNA is polyadenylated at a different site in the 3' non-coding region. Strong homology of MEP with human cathepsin L suggests that MEP is the mouse analogue of cathepsin L. Amino acid sequencing of the N-terminus of the secreted form of MEP indicates that, during secretion, the polypeptide is cleaved between amino acids 17 and 18. We have placed the MEP cDNA in a eukaryotic expression vector and demonstrated the production of the 39 kDa polypeptide form of mouse MEP in monkey CV-1 cells.