Subtilisin-like Serine Protease Research Articles

FAF and SufA: Proteins of Finegoldia magna That Modulate the Antibacterial Activity of Histones

Many bacterial pathogens have developed methods to overcome the defences of the host innate immune system. One such defence is the release of antimicrobial peptides (AMPs). Histones have been found to function as AMPs, in addition to their main biological function of packaging and organising DNA into nucleosomes. In this study, the Gram-positive anaerobic coccus Finegoldia magna was found to bind histones by Western blot and immunoprecipitation analysis. F. magna, which is normally a commensal of the skin and mucous membranes, is also known to act as an opportunistic pathogen and has been isolated from various clinical infection sites. It was found to bind to histones extracted from human skin epidermis through its surface and extracellular adhesion protein FAF. Through FAF binding, F. magna was protected from histone bactericidal activity. Furthermore, the histones were found to be degraded by SufA, a subtilisin-like extracellular serine protease of F. magna. Hence, the results of the present study will give more insight into how F. magna persists both as a commensal organism at the basement membrane of the skin and as an opportunistic pathogen during infection.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SpyCEP, a candidate antigen for a vaccine against Streptococcus pyogenes.

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen against which an effective vaccine does not yet exist. The S. pyogenes protein SpyCEP (S. pyogenes cell-envelope proteinase) is a surface-exposed subtilisin-like serine protease of 1647 amino acids. In addition to its auto-protease activity, SpyCEP is capable of cleaving interleukin 8 and related chemokines, contributing to GAS immune-evasion strategies. SpyCEP is immunogenic and confers protection in animal models of GAS infections. In order to structurally characterize this promising vaccine candidate, several SpyCEP protein-expression constructs were designed, cloned, produced in Escherichia coli, purified by affinity chromatography and subjected to crystallization trials. Crystals of a selenomethionyl form of a near-full-length SpyCEP ectodomain were obtained. The crystals diffracted X-rays to 3.3 Å resolution and belonged to space group C2, with unit-cell parameters a=139.2, b=120.4, c=104.3 Å, β=111°.

Molecular cloning of a novel subtilisin-like protease (Pr1A) gene from the biocontrol fungus Isaria farinosa

Fungal proteases, such as subtilisin-like serine protease (Pr1) and trypsin-like serine protease (Pr2), play an important role in penetration through the host cuticle in entomopathogenic fungi, including Isaria farinosa. In the present study, one gene of I. farinosa subtilisin-like protease (Ifa-pr1A), showing high homology to a Beauveria bassiana cuticle-degrading protease, was amplified by rapid amplification of cDNA ends PCR. The resulting full-length cDNA displayed an open reading frame (ORF) of 960 bp, encoding a protein of 319 amino acids. The Ifa-Pr1A protein was expressed in Escherichia coli to verify its protease activity. The recombinant Ifa-Pr1A protein exhibited high enzymatic activity according to the enzyme assay using a synthetic substrate. The expression profiles of Ifa-pr1A and Ifa-pr1H (another subtilisin-like protease gene from I. Farinosa) were analyzed at different induction times by adding cuticle material to the media. Quantitative reverse transcriptase PCR (RT-PCR) analysis revealed that Ifa-pr1A transcripts increased 58,800-fold at 12 h post-induction, whereas Ifa-pr1H transcripts peaked at 12 h with only an 11-fold increase, indicating that Ifa-pr1A may be a major gene of cuticle-degrading proteases in I. farinosa. Furthermore, Ifa-pr1A gene expression under in vivo conditions showed a clear upward trend during the early stages of the infection process. These results suggest that Pr1A cloned from I. farinosa is a potential virulence factor for the development of engineered biopesticides.

The Expression of Genes Encoding Secreted Proteins in Medicago truncatula A17 Inoculated Roots

Subtilisin-like serine protease (MtSBT), serine carboxypeptidase (MtSCP), MtN5, non-specific lipid transfer protein (MtnsLTP), early nodulin2-like protein (MtENOD2-like), FAD-binding domain containing protein (MtFAD-BP1), and rhicadhesin receptor protein (MtRHRE1) were among 34 proteins found in the supernatant of M. truncatula 2HA and sickle cell suspension cultures. This study investigated the expression of genes encoding those proteins in roots and developing nodules. Two methods were used: quantitative real time RT-PCR and gene expression analysis (with promoter: GUS fusion) in roots. Those proteins are predicted as secreted proteins which is indirectly supported by the findings that promoter: GUS fusions of six of the seven genes encoding secreted proteins were strongly expressed in the vascular bundle of transgenic hairy roots. All six genes have expressed in 14-day old nodule. The expression levels of the selected seven genes were quantified in Sinorhizobium- inoculated and control plants using quantitative real time RT-PCR. In conclusion, among seven genes encoding secreted proteins analyzed, the expression level of only one gene, MtN5 , was up-regulated significantly in inoculated root segments compared to controls. The expression of MtSBT1, MtSCP1, MtnsLTP, MtFAD-BP1, MtRHRE1 and MtN5 were higher in root tip than in other tissues examined.

Identification of a Highly Antigenic Region of Subtilisin-Like Serine Protease 1 for Serodiagnosis of Neospora caninum Infection

Neospora caninum is an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that the N. caninum subtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool for Neospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples from N. caninum experimentally infected cattle and mice and cattle from a farm with confirmed cases of Neospora abortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In the N. caninum experimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera from Toxoplasma gondii experimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis of N. caninum.

Purification, characterization and identification of a senescence related serine protease in dark-induced senescent wheat leaves

Senescence-related proteases play important roles in leaf senescence by regulating protein degradation and nutrient recycling. A 98.9kDa senescence-related protease EP3 in wheat leaves was purified by ammonium sulfate precipitation, Q-Sepharose fast flow anion exchange chromatography and gel slicing after gel electrophoresis. Due to its relatively high thermal stability, its protease activity did not decrease after incubation at 40°C for 1-h. EP3 protease was suggested to be a metal-dependent serine protease, because its activity was inhibited by serine protease inhibitors PMSF and AEBSF and metal related protease inhibitor EGTA. It was identified as a subtilisin-like serine protease of the S8A family based on data from both mass spectrometry and the cloned cDNA sequence. Therefore, these data suggest that a serine protease of the S8A subfamily with specific biochemical properties is involved in senescence-associated protein degradation.

In Silico Screening on the Three-dimensional Model of the Plasmodium vivax SUB1 Protease Leads to the Validation of a Novel Anti-parasite Compound

Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. We characterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had Ki values of <50 μM for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1 (compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial.

A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides

Batrachochytrium dendrobatidis (B. dendrobatidis), a chytrid fungus, is one of the major contributors to the global amphibian decline. The fungus infects both tadpoles and adult amphibians. Tadpoles are infected in their keratinized mouthparts, and infected adults exhibit hyperkeratosis and loss of righting reflex. Infections of adults may result in death from cardiac arrest in susceptible species. Thyroid hormone plays a key role in amphibian metamorphosis. The occurrence of B. dendrobatidis in tadpoles during metamorphosis may result in exposure of the fungus to host morphogens including TH. This exposure may induce gene expression in the fungus contributing to invasion and colonization of the host. Here, we demonstrate movement of fungal zoospores toward TH. Additionally, expression of a subtilisin-like serine protease is up-regulated in B. dendrobatidis cells exposed to TH. A gene encoding this protease was cloned from B. dendrobatidis and expressed in Escherichia coli. The protein was partially purified and characterized. The similarity between subtilases of human dermatophytes and the B. dendrobatidis subtilisin-like serine protease suggests the importance of this enzyme in B. dendrobatidis pathogenicity. Cleavage of frog skin antimicrobial peptides (AMPs) by this B. dendrobatidis subtilisin-like serine protease suggests a role for this enzyme in fungal survival and colonization.

The recombinant serine proteaseXAoz1ofArthrobotrys oligosporaexhibits potent nematicidal activity againstCaenorhabditis elegansandHaemonchus contortus

The nematophagous fungus Arthrobotrys oligospora is a potential biological agent against parasitic gastrointestinal nematodes. Its subtilisin-like serine proteases play an important role in nematode cuticle breach. In this study, the cDNA of the mature serine protease XAoz1 from A. oligospora XJ-XAo1 was expressed in Pichia pastoris to assess the in vitro nematicidal activity of recombinant XAoz1 (reXAoz1) on Caenorhabditis elegans and Haemonchus contortus. The cDNA sequence of the protease XAoz1 was amplified by reverse transcription polymerase chain reaction (RT-PCR) and inserted into the vector pPIC9K for expression in P.pastoris GS115. Our results show that the reXAoz1 had a molecular mass of 50 kDa after 3 days of 1.5%-methanol induction at 28 °C. The highest specific protease activity was achieved at 12 168 U mg(-1) protein. The reXAoz1 had the highest hydrolytic activity at pH 6.5-9.5 with an optimal pH at 8.5. Moreover, the purified reXAoz1 displayed a highly toxic and biological activity to immobilize C. elegans and H. contortus by degrading their cuticles and inducing death.

Enzyme mediated beam house operations of leather industry: a needed step towards greener technology

Abstract Leather industries, due to pollution of water, careless disposal of solid wastes and gaseous emissions, are categorized as a red industry and are under deep pressure to develop environmentally efficient leather-making processes to comply with modern pollution and discharge legislation. In the present study, completely enzyme based beam house operations (dehairing & degreasing) of leather industry has been standardized for skins & hides using Bacillus licheniformis protease and Bacillus subtilis lipase. Owing to its immense importance and demand the productions of these enzymes were optimized and scaled up in a 300 L bioreactor resulting in a maximum yield of 4568 U/mL in 18 h for protease and 34.91 IU/mL of lipase in 36 h. 100% enzymatic dehairing & degreasing of skins and hides could be obtained at pH value of 8.0 and temperature 30–37 °C with the enzyme concentration of 2–5% (w/v) of protease for dehairing and 5–10% (w/v) of lipase for degreasing within 8–12 h. An interesting observation noted for enzyme based leather processing was that natural skin colour was preserved along with a drastic reduction of BOD and COD values of effluent. Crust leather formed using enzymatically processed skins exhibited similar physical and tactile properties as observed for conventional crust leather. On the bases of internally quenched fluorescent substrate specific method, this protease is subtilisin-like serine protease with low collagenase activity. High collagenase activity can damage the hide (leather) grain and the physical-mechanical characteristics of the hides. Successful demonstrations of large size (Industrial trials −100 kg skin/hide at Central Leather Research Institute, Chennai, India) 100% chemical free dehairing and degreasing further supports the authenticity of the work. Thus all this makes the beam house operations of leather tannery enzymatically feasible and environmentally benign.

Truncated Cotton Subtilase Promoter Directs Guard Cell-Specific Expression of Foreign Genes in Tobacco and Arabidopsis

A 993-bp regulatory region upstream of the translation start codon of subtilisin-like serine protease gene was isolated from Gossypium barbadense. This (T/A)AAAG-rich region, GbSLSP, and its 5′- and 3′-truncated versions were transferred into tobacco and Arabidopsis after fusing with GUS or GFP. Histochemical and quantitative GUS analysis and confocal GFP fluorescence scanning in the transgenic plants showed that the GbSLSP-driven GUS and GFP expressed preferentially in guard cells, whereas driven by GbSLSPF2 to GbSLSPF4, the 5′-truncated GbSLSP versions with progressively reduced Dof1 elements, both GUS and GFP expressed exclusively in guard cells, and the expression strength declined with (T/A)AAAG copy decrement. Deletion of 5′-untranslated region from GbSLSP markedly weakened the activity of GUS and GFP, while deletion from the strongest guard cell-specific promoter, GbSLSPF2, not only significantly decreased the expression strength, but also completely abolished the guard cell specificity. These results suggested both guard cell specificity and expression strength of the promoters be coordinately controlled by 5′-untranslated region and a cluster of at least 3 (T/A)AAAG elements within a region of about 100 bp relative to transcription start site. Our guard cell-specific promoters will enrich tools to manipulate gene expression in guard cells for scientific research and crop improvement.

Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensisin detergents and its ability to degrade the abnormal prion protein

BackgroundTk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC).ResultsTk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis.ConclusionsOur results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE).

Structural and Functional Analysis of the CspB Protease Required for Clostridium Spore Germination

Spores are the major transmissive form of the nosocomial pathogen Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission.

Mycoplasma hyopneumoniae in vitro peptidase activities: Identification and cleavage of kallikrein-kinin system-like substrates

Bacterial proteases are important for metabolic processes and pathogenesis in host organisms. The bacterial swine pathogen Mycoplasma hyopneumoniae has 15 putative protease-encoding genes annotated, but none of them have been functionally characterized. To identify and characterize peptidases that could be relevant for infection of swine hosts, we investigated the peptidase activity present in the pathogenic 7448 strain of M. hyopneumoniae. Combinatorial libraries of fluorescence resonance energy transfer peptides, specific inhibitors and pH profiling were used to screen and characterize endopeptidase, aminopeptidase and carboxypeptidase activities in cell lysates. One metalloendopeptidase, one serine endopeptidase, and one aminopeptidase were detected. The detected metalloendopeptidase activity, prominent at neutral and basic pH ranges, was due to a thimet oligopeptidase family member (M3 family), likely an oligoendopeptidase F (PepF), which cleaved the peptide Abz-GFSPFRQ-EDDnp at the F-S bond. A chymotrypsin-like serine endopeptidase activity, possibly a subtilisin-like serine protease, was prominent at higher pH levels, and was characterized by its preference for a Phe residue at the P1 position of the substrate. The aminopeptidase P (APP) activity showed a similar profile to that of human membrane-bound APP. Genes coding for these three peptidases were identified and their transcription was confirmed in the 7448 strain. Furthermore, M. hyopneumoniae cell lysate peptidases showed effects on kallikrein-kinin system-like substrates, such as bradykinin-derived substrates and human high molecular weight kininogen. The M. hyopneumoniae peptidase activities, here characterized for the first time, may be important for bacterial survival strategies and thus represent possible targets for drug development against M. hyopneumoniae swine infections.

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.

ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2

Seed production in angiosperms requires tight coordination of the development of the embryo and the endosperm. The endosperm-specific transcription factor ZHOUPI has previously been shown to play a key role in this process, by regulating both endosperm breakdown and the formation of the embryonic cuticle. To what extent these processes are functionally linked is, however, unclear. In order to address this issue we have concentrated on the subtilisin-like serine protease encoding gene ABNORMAL LEAF-SHAPE1. Expression of ABNORMAL LEAF-SHAPE1 is endosperm specific, and dramatically decreased in zhoupi mutants. We show that, although ABNORMAL LEAF-SHAPE1 is required for normal embryonic cuticle formation, it plays no role in regulating endosperm breakdown. Furthermore, we show that re-introducing ABNORMAL LEAF-SHAPE1 expression in the endosperm of zhoupi mutants partially rescues embryonic cuticle formation without rescuing their persistent endosperm phenotype. Thus, we conclude that ALE1 can normalize cuticle formation in the absence of endosperm breakdown, and that ZHOUPI thus controls two genetically separable developmental processes. Finally, our genetic study shows that ZHOUPI and ABNORMAL LEAF-SHAPE1 promotes formation of embryonic cuticle via a pathway involving embryonically expressed receptor kinases GASSHO1 and GASSHO2. We therefore provide a molecular framework of inter-tissue communication for embryo-specific cuticle formation during embryogenesis.

Characterization, cloning, and heterologous expression of a subtilisin-like serine protease gene VlPr1 from Verticillium lecanii

The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg(2+) and Ca(2+) concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

The Discovery of New Cyanobactins from Cyanothece PCC 7425 Defines a New Signature for Processing of Patellamides

Cyanobactins, including patellamides, are a group of cyanobacterial post-translationally modified ribosomal cyclic peptides. The final product should in theory be predictable from the sequence of the precursor peptide and the associated tailoring enzymes. Understanding the mechanism and recognition requirements of these enzymes will allow their rational engineering. We have identified three new cyanobactins from a Cyanothece PCC 7425 culture subjected to a heat shock. One of these compounds revealed a novel signature signal for ThcA, the subtilisin-like serine protease that is homologous to the patellamide protease PatA. The crystal structure of the latter and modelling studies allow rationalisation of the recognition determinants for both enzymes, consistent with the ribosomal biosynthetic origin of the new compounds.

Characterization of a subtilisin-like protease with apical localization from microsporidian Nosema bombycis

The microsporidian Nosema bombycis is the pathogen causing pébrine leading to heavy economic loss in sericulture. Little is known of the proteases of microsporidia that are important for both parasite development and pathogenesis. Here we identified a subtilisin-like serine protease NbSLP1 which contains an inhibitor_I9 and a peptidase_S8 domain. Three dimensional modeling of the catalytic domain of the NbSLP1 exhibited a typical 3-layer sandwich structure with S1 pocket substituted by Y359. Phylogenetic analysis confirms that subtilisin-like serine proteases of microsporidia fall into two clades: SLP1 and SLP2, suggesting the initial subtilisin gene duplication events preceded microsporidia speciation. Furthermore, transcripts of Nbslp1 were detected in the midgut of Bombyx mori infection by N. bombycis by RT-PCR. Antibodies against NbSLP1 recognized both the precursor and mature enzyme by 2D Western blotting. Besides, indirect immunofluorescence assay revealed that the NbSLP1 is mainly localized at the two poles of spore which make the spore look like “safety pins”. Remarkably, the mature protease is only detected in the apical region of the spore after germination. These studies demonstrate that NbSLP1 is a conserved subtilisin protease in microsporidia and suggest that NbSLP1 play a significant role in polar tube extrusion process.

Slow Unfolding Pathway of Hyperthermophilic Tk-RNase H2 Examined by Pulse Proteolysis Using the Stable Protease Tk-Subtilisin

The unfolding speed of some hyperthermophilic proteins is significantly slower than those of their mesostable homologues. Ribonuclease H2 from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-RNase H2) is stabilized by its remarkably slow unfolding rate. In this work, we examined the slow unfolding pathway of Tk-RNase H2 by pulse proteolysis using a superstable subtilisin-like serine protease from T. kodakarensis (Tk-subtilisin). Tk-subtilisin has enzymatic activity in highly concentrated guanidine hydrochloride (GdnHCl), in which Tk-RNase H2 unfolds slowly. The native state of Tk-RNase H2 was completely resistant to Tk-subtilisin, whereas the unfolded state (induced by 4 M GdnHCl) was degraded by Tk-subtilisin. Degradation products of Tk-RNase H2 created from pulse proteolysis during its unfolding were detected by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We identified the cleavage sites in Tk-RNase H2 by N-terminal sequencing and mass spectrometry and constructed mimics of the unfolding intermediate of Tk-RNase H2 by protein engineering. The mimics were biophysically characterized. We found that the native state of Tk-RNase H2 (N-state) changed to the I(A)-state that was digested by Tk-subtilisin in the early stage of unfolding. In the slow unfolding pathway, the I(A)-state shifted to two intermediate forms, I(B)-state and I(C)-state. The I(B)-state was digested by Tk-subtilisin in the C-terminal region, but the I(C)-state was a Tk-subtilisin resistant form. These states gradually unfolded through the I(D)-state, in which the N-terminal region was digested. The results indicate that pulse proteolysis, by a superstable protease, was a suitable strategy and an effective tool for analyzing intermediate structures of proteins with slow unfolding properties. We also showed that the N-terminal region contributes to the slow unfolding of Tk-RNase H2, and the C-terminal region is important for folding and stability.