A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Effects of intraspecific larval competition on the development of the African buffalo fly Haematobia thirouxi potans

The effects of larval crowding on development, survival and size of the African buffalo fly, Haematobia thirouxi potans (Bezzi), were examined in small (10 or 20 g) and 1 000 g masses of cattle dung in the laboratory at 25°C. Dung was infested with fly eggs at densities which ranged from 0.05 to 32.0 eggs per g dung; in most cases 90–100% of eggs hatched. The duration of larval developmentin 10 g dung masses was extended by larval crowding at densities above 0.8 eggs per g dung and the development period with 32 eggs per g dung (11 days) was double that observed in uncrowded dung masses. Both size and survival decreased with increasing larval density, but stunting was observed at lower densities than those which reduced survival and so size was the more sensitive index of the adequacy of the larval environment. The effects of larval crowding became evident at lower larval densities in the 1 000 g dung masses than in the 10 g dung masses. There was a minimum size below which third instar larvae failed to pupariate. The probability of survival was related to pupal size and a high proportion of the smaller individuals failed to eclose.

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Effets de la competition intraspécifique sur le développement des larves, la taille des pupes et la survie d'Haematobia thirouxi potans

Résumé Les effets du surpeuplement larvaire sur le développement, la survie et la taille d'Haematobia thirouxi potans, ont été examinés sur de petites quantités de bouse (10 et 20 g) et de plus importantes (1 000 g), au laboratoire à 25°C. Les bouses étaient contaminées avec des oeufs de la mouche à des densités de 0,05 à 32 oeufs par gramme de bouse. Dans la plupart des cas le taux d'éclosion était de 90–100%. La durée du dévelopement larvaire dans 10 g de bouse a été prolongée pour les densités supérieures à 0,8 oeuf per g, et la durée développement pour 32 oeufs per g (11 jours) était le double de celle obtenue grand il n'y avait pas surpeuplement. La taille et la survie diminuent toutes les deux avec la densité larvaire, mais la miniaturisation était observée à des densités où la survie n'était pas modifiée; ainsi la taille est un indice plus sensible de l'adéquation de l'habitat larvaire. Les effets du surpeuplement larvaires deviennent clairs à des densités plus faibles avec 1 000 g de la bouse qu'avec 10 g. Il y a eu une taille minimale endessous de laquelle les larves étaient incapables de former une pupe. La probabilité d'éclosion était liée à la taille de la pupe, et une plus forte proportion de petits individus ne sont pas parvenu à éclore.

     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

The role of HiTI, a serine protease inhibitor from Haematobia irritans irritans (Diptera: Muscidae) in the control of fly and bacterial proteases

Blood-sucking arthropods are vectors responsible for the transmission of several pathogens and parasites to vertebrate animals. The horn fly Haematobia irritans irritans (Diptera: Muscidae) and the tick Boophilus microplus are important hematophagous ectoparasites that cause losses in cattle production. A serine protease inhibitor from a thorax extract of the fly H. irritans irritans (HiTI) was previously isolated, characterized and cloned. In the present study we described the expression, purification, and characterization of the recombinant HiTI (rHiTI) and its possible role in the control of different endogenous and bacterial proteases. rHiTI was successfully expressed using the pPIC9 expression vector with a yield of 4.2 mg/L of active rHiTI. The recombinant HiTI purified by affinity chromatography on trypsin-Sepharose had a molecular mass of 6.53 kDa as determined by LS-ESI mass spectrometry and inhibition constants (Kis) similar to those of native HiTI for bovine trypsin and human neutrophil elastase of 0.4 and 1.0 nM, respectively. Purified rHiTI also showed inhibitory activity against the trypsin-like enzyme of H. i. irritans using its possible natural substrates, fibrinogen and hemoglobin; and also inhibited the OmpT endoprotease of Escherichia coli using fluorogenic substrates. The present results confirm that HiTI may play a role in the control of fly endogenous proteases but also suggest a role in the inhibition of pathogen proteases.     

Purification and characterization of a thermodynamic stable serine protease from Aspergillus fumigatus

A thermostable extracellular serine protease from Aspergillus fumigatus was purified 8.8-fold using a 4-step protocol. The enzyme was produced using a 36 h solid-state culture, had a molecular weight of 88 kDa and exhibited maximal enzyme activity at pH 7 and 60 °C. Structural analysis revealed that the protease is monomeric and non-glycosylated. Thermal inactivation of the pure enzyme followed first-order kinetics. The half-life (t_1/2) of the pure enzyme at 50, 60 and 70 °C was 65, 34 and 14 min, respectively. The denaturation and activation energies were 69 and 62 kJ mol⁻¹, respectively. Thermodynamic parameters (entropy and enthalpy) suggested that the protease was highly thermostable. This is the first report on the thermodynamic parameters of proteases produced by A. fumigatus.     

Identification of serine protease,serine protease homolog and prophenoloxidase genes in Spodoptera frugiperda (Lepidoptera: Noctuidae)

In insects, proteolytic cascades medicated by serine proteases (SPs), serine protease homologs (SPHs) and prophenoloxidases (PPOs) control several physiological processes, notably the innate immunity. However, no attempts have been made to identify and characterize these genes in Spodoptera frugiperda, one of the most destructive agricultural pests. In this study, 83 SPs, 26 SPHs and four PPOs were respectively identified in S. frugiperda genome based on homology blast against those of other insects. We then analyzed the domain organization of these proteins and assigned them into different groups by phylogenetic reconstruction. Furthermore, the mRNA levels of clip-domain SPs/SPHs (cSPs/cSPHs) and PPOs were quantified in response to a mixed infection of Micrococcus luteus and Escherichia coli, and obvious accumulations were recorded in immune tissues, including hemocytes and fat body. In the latter study, we profiled the expression patterns of highly expressed cSPs and PPOs in different developmental stages, including egg, larva, pupa, female and male adults. It was shown that most cSPs were abundantly expressed in adults, while PPOs were detected at high levels in both egg and larval stages. These current findings substantially add to our understanding of the roles of S. frugiperda SPs, SPHs and PPOs in immune regulation and further lay a solid foundation for uncovering the interaction mechanisms between insects and pathogens.     

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.     

The serine carboxypeptidase like gene family of rice (Oryza sativa L. ssp. japonica)

Serine carboxypeptidases (SCPs) comprise a large family of protein hydrolyzing enzymes and have roles ranging from protein turnover and C-terminal processing to wound responses and xenobiotic metabolism. The proteins can be classified into three groups, namely carboxypeptidase I, II and III, based on their coding protein sequences and the fact that each family is characterized by a central catalytic domain of unique topology designated as the “α/β hydrolase fold”. The available SCP protein sequences have been utilized as datasets to build a HMM (hidden Markov model) profile, which is used to search the rice (Oryza sativa L. ssp. japonica) proteome. A total of 71 SCP and serine carboxypeptidase-like (SCPL) protein-coding genes exist in rice. The intron-exon structure, chromosome localization, expression and characteristics of encoded protein sequences of the 71 putative genes are reviewed.     

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by nonionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.Abbreviations ISP intracellular serine protease - ISP-A-Bsu A-50 and ISP-B-Bsu A-50 molecular forms A and B of B. subtilis A-50 intracellular serine protease, respectively - SDS sodium dodecyl sulfate - PMSF phenylmethyl sulfonylfluoride - pNA p-nitroanilide - Buffer A 50 mM Tris-(hydroxymethyl)aminomethane-1 mM CaCl₂ adjusted to pH 8.5 with HCl     

A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex

A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60 °C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis.     

丝氨酸蛋白酶抑制剂(SERPIN)家族相关基因在结肠腺癌中预后模型的建立及应用

应用生物信息学方法,构建结肠腺癌(COAD)丝氨酸蛋白酶抑制剂(SERPIN)家族相关基因预后模型。从TCGA数据库和GEO数据库下载结肠腺癌(COAD)转录组和临床数据,根据数据中SERPINs家族基因的表达量对COAD患者进行一致性聚类分析;将数据随机均分为训练集(Train)组和验证集(Test)组,基于两个亚型的差异基因,利用Train组进行COX回归和Lasso回归构建预后模型,根据模型风险评分中位值将样本分为高、低风险两组,绘制高低风险组患者生存曲线;通过ROC曲线评价模型预测能力;利用Test组数据验证模型;构建列线图,评估患者生存率模型预测值与实际值的一致性;并利用利用ESTIMATE算法和CIBERSORT算法评估风险评分和肿瘤微环境(TME)以及免疫浸润的相关性。通过34个SERPIN基因确定了两个亚型,基于2个亚型筛选出了436个预后相关分型差异基因,通过Lasso回归确定出了11个预后相关基因参与风险模型的构建,根据模型评分区分的高低风险组具有明显的生存差异,列线图可以准确预测1、3和5年生存率。肿瘤微环境分析和免疫浸润分析显示高风险评分组患者免疫活性差。SERPIN家族相关基因构建的风险评分模型能够预测COAD的预后,有利于进一步指导临床对COAD的诊治,提高患者生存率。     

A novel serine protease complexed with α2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis)

A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to β-chain of carp α₂-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp α₂-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by α₁-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by α₁-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with α₂-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.     

Overexpression and characterization of thermostable serine protease in Escherichia coli encoded by the ORF TTE0824 from Thermoanaerobacter tengcongensis

A novel extracellular serine protease derived from Thermoanaerobacter tengcongensis, designated tengconlysin, was successfully overexpressed in Escherichia coli as a soluble protein by recombination of an N-terminal Pel B leader sequence instead of the original presequence and C-terminal 6× histidine tags. The purified protein was activated by 0.1% sodium dodecyl sulfate (SDS) treatment but not by thermal treatment. The molecular weight of tengconlysin estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration chromatography was 37.9 and 36.2 kDa, respectively, suggesting that the enzyme is monomeric. The N-terminal sequence of mature tengconlysin was LDTAT, suggesting that it is a preproprotein containing a 29 amino acid presequence (predicted from the SigP program) and a 117 amino acid prosequence in the N-terminus. The C-terminal putative propeptide (position 469–540 in the preproprotein) did not inhibit the protease activity. The optimum temperature for tengconlysin activity was 90°C in the presence of 1 mM calcium ions and the optimum pH ranged from 6.5 to 7.0. Activity inhibition studies suggest that the protease is a serine protease. The protease was stable in 0.1% SDS and 1–4 M urea at 70°C in the presence of calcium ions and was activated by the denaturing agents.     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

A novel fibrinolytic serine protease from the polychaete Nereis (Neanthes) virens (Sars): purification and characterization

A novel fibrinolytic serine protease has been identified and purified to homogeneity from the coelomic fluid of polychaete Nereis (Neanthes) virens (Sars), and named N-V protease. N-V protease is a 29kDa single chain protein with an isoelectric point of pH 4.5. It hydrolyzes Aalpha-chain of fibrinogen with a high efficiency, and the Bbeta- and gamma-chains (Aalpha>Bbeta>gamma) with a lower efficiency. The proteolytic activity peaks at pH 7.8 is 45 degrees C. The activity is completely inhibited by serine protease inhibitors DFP (I(50)=5.8 x 10(-4)M) and PMSF (I(50)=5.5 x 10(-2)M), and almost completely by TLCK (I(50)=7.7 x 10(-1) M). But aprotinin, elastinal, SBTI, benzamidine, PCMB, EDTA, EGTA, iodoacetate, E64, and beta-mercaptoethanol have no effect on the protease activity. Therefore, N-V protease is identified as a serine protease. The primary amino acid sequence of N-V protease was determined by mass spectrometry (N-V protease, No. P83433). According to the MALDI-TOF MS analysis, there is no existing protein in the NCBI Non-redundant Protein Sequence Database that matches the N-V protease sequence. Therefore, N-V protease is a novel and special protein in N. virens. Furthermore, we have successfully established an expression cDNA library from the whole body of N. virens (data not shown).     

Transport and kinetics of diflubenzuron and pyriproxyfen in the beet armywormSpodoptera exigua and its predatorPodisus maculiventris

Transport and retention of the insect growth regulators (IGRs) diflubenzuron and pyriproxyfen in larvae of the beet armywormSpodoptera exigua (Hübner) and in nymphs of the predatory bugPodisus maculiventris (Say) were investigated. In a first experiment, the retention of orally administered [¹⁴C]radiolabeled isotopes of both compounds in fifth-instar larvae of the beet armyworm was studied. Rate of excretion of both IGRs inS. exigua caterpillars was high, with a 50% excretion time of approximately 6 h after intake. In a second experiment, the transport of the compounds from prey to predator and their retention inside the predator were studied. Fifthinstar nymphs ofP. maculiventris were allowed to feed on caterpillars that had been given contaminated food. For both diflubenzuron and pyriproxyfen, more than 80% of the amount of radiolabel applied was recovered in consumed prey. Low levels of radioactivity (c. 3% of the applied amount of radiolabel) were also found in the fluid regurgitated by the prey larvae when attacked by the predatory bugs. Relatively small amounts of radiolabel (c. 8 and 15% of the amount orally applied to the prey for diflubenzuron and pyriproxyfen, respectively) were ingested byP. maculiventris nymphs when feeding on beet armyworm caterpillars. The data suggest that the predators did not use gut content as food. The pattern of excretion in nymphs ofP. maculiventris differed between compounds. For diflubenzuron, there was a drastic decrease of radioactivity inside the predator body of around 40% within the first 6 h and then the level of retained radiolabel remained stable at 3–4% up to 72 h. For pyriproxyfen, a slow decrease of radioactivity inside the body was observed and at 72 h only 2% of the applied quantity was detected. Results of this study are discussed in relation to the findings from previous studies on the toxicity of both IGRs toP. maculiventris.     

A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes

A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.