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1.
The sequences of the epidermolytic toxins and V8 serine proteinase share about 25% identity, including the catalytic triad at the proteinase active centre. Here we have altered the putative ETA active-site serine-195 to glycine by site-directed mutagenesis. No epidermolytic activity was detected when up to 100-fold greater amounts of the homogeneous mutant ETA were injected subcutaneously into neonatal mice showing that serine-195 is required for toxicogenesis.  相似文献   

2.
The exfoliative toxins (ETs) cause staphylococcal scalded skin syndrome, a disease characterized by specific separation of layers of the skin. Evidence suggests that the toxins act as serine proteases, though the specific substrate and mode of action are not known for certain. The crystal structure of exfoliative toxin A (ETA) was reported earlier and shown to be similar to that of the chymotrypsin-like serine proteases. Here, we report the 2.4 A resolution crystal structure of the other exfoliative toxin, ETB, which is 40% identical to ETA. The overall structures of ETA and ETB are similar including the positions of key residues within the active site. The structure of ETB supports the previous findings that the ETs are serine proteases that cleave substrates after glutamic acid residues. In this study we also discuss a number of structural differences including a large 14 residue loop insertion which may be a key feature involved in the differing biological properties of the ETs, particularly the pyrogenic and lethal activities of ETB not shared by ETA.  相似文献   

3.
Exfoliative toxins produced by certain strains of Staphylococcus hyicus mediate exudative epidermitis in pigs. In this study the genes coding for four different exfoliative toxin from S. hyicus (ExhA, ExhB, ExhC, and ExhD) were cloned and sequenced. The coding sequence of the four toxin genes ranged from 816 to 834 bp. The amino acid sequences of these four toxins were homologous to the earlier described exfoliative toxins SHETB from S. hyicus and ETA, ETB, and ETD from Staphylococcus aureus. The homology between the S. hyicus toxins was at the same level as the homology to the exfoliative toxins from S. aureus. The toxins showed similarity to serine proteases, including preservation of the catalytic tract in ExhA, ExhB, and ExhC. However, in ExhD, Asp in the putative catalytic tract was replaced with Glu. The recombinant toxins could be expressed in Escherichia coli, and three of the four toxins were recognized by monoclonal antibodies raised against native exfoliative toxins.  相似文献   

4.
5.
Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.  相似文献   

6.
G protein-coupled receptors (GPCRs), including endothelin receptor A (ETA) and B (ETB), may form dimers or higher-order oligomers that profoundly influence signaling. Here we examined a PDZ finger motif within the C-terminus of ETA and its role in heterodimerization with ETB, and in homodimerization with itself, when expressed in HEK293 cells. Receptor dimerization was monitored by (i) fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) (FRET donor) and tetracysteine/FlAsH (FRET acceptor) fused to the C-termini of ET receptors, and (ii) coimmunoprecipitation of ET receptors after mild detergent solubilization. Mutations in a PDZ finger motif at threonine403/serine404 eliminated FRET and reduced coimmunoprecipitation of heterodimers and homodimers. Functional consequences were evaluated by measuring mobilization of intracellular Ca2+ and internalization of receptors in response to a 10 nmol/L ET-1 challenge. PDZ mutations converted a sustained Ca2+ signal mediated by ETA:ETB heterodimers into a transient response, similar to that observed for homodimers or monomers. Heterodimers containing PDZ mutations were seen to internalize in a similar time domain (approximately 5 min) to the transient Ca2+ elevation and with similar kinetics to internalization of ETA homodimers or monomers. Without the PDZ mutations, heterodimers did not internalize over 15 min, suggesting the intriguing possibility that sustained Ca2+ signaling was a consequence (at least in part) of delayed internalization. The results are consistent with structural models of ETA-receptor dimerization that place threonine403/serine404 of the PDZ finger motif at the interaction interface between heterodimers and homodimers. Sustained Ca2+ signaling and delayed endocytosis of ETA:ETB heterodimers argues strongly for a unique dimer interface that impacts transmembrane signaling and internalization.  相似文献   

7.
The covalent structures of two, novel, neutrophile, leucocyte-derived, strongly basic proteins of porcine and human origin have been determined by microsequencing in combination with time-of-flight plasma desorption mass spectrometry. The porcine protein primary structure of 219 amino acid residues was shown to contain 6 cysteine residues, 2 putative carbohydrate sites and 14% basic residues. The human protein contained 221 amino acid residues of which 8 were cysteine, 4 putative carbohydrate sites and 12% basic. A 47% direct sequence similarity to human neutrophile elastase was found, but due to mutations of two of the three amino acids in the catalytic triad, proteolytic activity is absent. Modelling and alignment studies unveil a close relationship of both proteins to the serine protease family, the greatest similarity being to those serine proteases present in granules from peripheral blood cells. Both proteins have been shown to be chemotactically active for monocytes and fibroblasts in vitro.  相似文献   

8.
Staphylococcus aureus is the major cause of nosocomial infections world-wide, with increasing prevalence of community-acquired diseases. The recent dramatic increase in multi-antibiotic resistance, including resistance to the last-resort drug, vancomycin, together with the lack of an effective vaccine highlight the need for better understanding of S.aureus pathogenicity. Comparative analysis of available bacterial genomes allows for the identification of previously uncharacterized S.aureus genes with potential roles in pathogenicity. A good example is a cluster of six serine protease-like (spl) genes encompassed in one operon, which encode for putative proteases with similarity to staphylococcal glutamylendopeptidase (V8 protease). Here, we describe an efficient expression system for the production of recombinant SplB and SplC proteases in Escherichia coli, together with structural and functional characterization of the purified enzymes. A unique mechanism of cytoplasm protection against activity of misdirected SplB was uncovered. Apparently, the co-translated signal peptide maintains protease latency until it is cleaved by the signal peptidase during protein secretion. Furthermore, the crystal structure of the SplC protease revealed a fold resembling that of the V8 protease and epidermolytic toxins. Arrangement of the active site cleft and substrate-binding pocket of SplC explains the mechanism of enzyme latency and suggests that some Spl proteases possess restricted substrate specificity similar to that of the V8 protease and epidermolytic toxins.  相似文献   

9.
The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na(+) binds to the substrate bound FXa with K(d) approximately 39 mm in the absence and approximately 9.5 mm in the presence of Ca(2+). Sodium-bound FXa (sodium-Xa) has approximately 18-fold increased catalytic efficiency ( approximately 4.5-fold decrease in K(m) and approximately 4-fold increase in k(cat)) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca(2+) further increases this k(cat) approximately 1.4-fold. Ca(2+) binds to the protease domain of substrate bound FXa with K(d) approximately 705 microm in the absence and approximately 175 microm in the presence of Na(+). Ca(2+) binding to the protease domain of FXa (Xa-calcium) has no effect on the K(m) but increases the k(cat) approximately 4-fold in hydrolyzing S-2222, and Na(+) further increases this k(cat) approximately 1.4-fold. In agreement with the K(m) data, sodium-Xa has approximately 5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and approximately 4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca(2+) (+/-Na(+)) has no effect on these interactions. Antithrombin binds to Xa-calcium with a approximately 4-fold faster rate, to sodium-Xa with a approximately 24-fold faster rate and to sodium-Xa-calcium with a approximately 28-fold faster rate. Thus, Ca(2+) and Na(+) together increase the catalytic efficiency of FXa approximately 28-fold. Na(+) enhances Ca(2+) binding, and Ca(2+) enhances Na(+) binding. Further, Na(+) enhances S1 site occupancy, and S1 site occupancy enhances Na(+) binding. Therefore, Na(+) site is thermodynamically linked to the S1 site as well as to the protease domain Ca(2+) site, whereas Ca(2+) site is only linked to the Na(+) site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.  相似文献   

10.
The complete amino acid sequence of Achromobacter lyticus protease I (EC 3.4.21.50), which specifically hydrolyzes lysyl peptide bonds, has been established. This has been achieved by sequence analysis of the reduced and S-carboxymethylated protease and of peptides obtained by enzymatic digestion with Achromobacter protease I itself and Staphylococcus aureus V8 protease and by chemical cleavage with cyanogen bromide. The protease consists of 268 residues with three disulfide bonds, which have been assigned to Cys6-Cys216, Cys12-Cys80, and Cys36-Cys58. Comparison of the amino acid sequence of Achromobacter protease and other serine proteases of bacterial and mammalian origins has revealed that Achromobacter protease I is a mammalian-type serine protease of which the catalytic triad comprises His57, Asp113, and Ser194. It has also been shown that the protease has 9- and 26-residue extensions of the peptide chain at the N and C termini, respectively, and overall sequence homology is as low as 20% with bovine trypsin. The presence of a disulfide bridge between the N-terminal extension Cys6 and Cys216 close to the putative active site in the C-terminal region is thought to be responsible for the generation of maximal proteolytic function in the pH range 8.5-10.7 and enhanced stability to denaturation.  相似文献   

11.
Exfoliative toxins (ETs) from Staphylococcus aureus blister the superficial epidermis by hydrolyzing a single peptide bond, Glu381-Gly382, located between extracellular domains 3 and 4 of desmoglein 1 (Dsg1). Enzyme activity is dependent on the calcium-stabilized structure of Dsg1. Here we further define the characteristics of this cleavage. Kinetic studies monitoring the cleavage of Dsg1 by ETA, ETB, and ETD demonstrated kcat/Km values of 2-6 x 10(4) m(-1) s(-1), suggesting very efficient proteolysis. Proteolysis by ETA was not efficiently inhibited by broad spectrum serine protease inhibitors, suggesting that the enzyme cleavage site may be inactive or inaccessible before specific binding to its substrate. Using truncated mutants of human Dsg1 and chimeric molecules between human Dsg1 and either human Dsg3 or canine Dsg1, we show that for cleavage, human-specific amino acids from Dsg1 are necessary in extracellular domain 3 upstream of the scissile bond. If these residues are canine rather than human, ETA binds, but does not cleave, canine Dsg1. These data suggest that the exquisite specificity and efficiency of ETA may depend on the enzyme's binding upstream of the cleavage site with a very specific fit, like a key in a lock.  相似文献   

12.
The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (phiETA) that encodes ETA and determined the complete nucleotide sequence of the phiETA genome. phiETA has a head with a hexagonal outline and a non-contractile and flexible tail. The genome of phiETA is a circularly permuted linear double-stranded DNA, and the genome size is 43 081 bp. Sixty-six open reading frames (ORFs) were identified on the phiETA genome, including eta, which was found to be located very close to a putative attachment site (attP). phiETA converted ETA non-producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that phiETA or related phages are responsible for the acquisition of eta genes in S. aureus.  相似文献   

13.
Rhomboid is an intramembrane serine protease responsible for the proteolytic activation of Drosophila epidermal growth factor receptor (EGFR) ligands. Although nothing is known about the function of the approximately 100 currently known rhomboid genes conserved throughout evolution, a recent analysis suggests that a Rhomboid from the pathogenic bacterium Providencia stuartii is involved in the production of a quorum-sensing factor. This suggests that an intercellular signaling mechanism may have been conserved between prokaryotes and metazoans. However, the function of prokaryotic Rhomboids is unknown. We have examined the ability of eight prokaryotic Rhomboids to cleave the three Drosophila EGFR ligands. Despite their striking sequence divergence, Rhomboids from one Gram-positive and four Gram-negative species, including Providencia, specifically cleaved Drosophila substrates, but not similar proteins such as Transforming Growth Factor alpha (TGFalpha) and Delta. Although the sequence similarity between these divergent Rhomboids is very limited, all contain the putative serine catalytic triad residues, and their specific mutation abolished protease activity. Therefore, despite low overall homology, the Rhomboids are a family of ancient, functionally conserved intramembrane serine proteases, some of which also have conserved substrate specificity. Moreover, a function for Rhomboids in activating intercellular signaling appears to have evolved early.  相似文献   

14.
Trypsin-like serine proteases are involved in diverse biological processes such as complement activation, tissue remodeling, cellular migration, tumor invasion, and metastasis. Here we report a novel human C1r-like serine protease analog, CLSPa, derived from dendritic cells (DC). The 487-residue CLSPa protein contains a CUB domain and a serine protease domain, possessing characteristic catalytic triad but lacking typical activation/cleavage sequence. It shares great homology with complement C1r/C1s and mannose-associated serine proteases. CLSPa mRNA is widely expressed, especially abundant in placenta, liver, kidney, pancreas, and myeloid cells, which are a major resources of serine proteases. Upon stimulation by agonistic anti-CD40 Ab, TNF-alpha, or LPS, CLSPa mRNA expression was significantly up-regulated in monocytic cells and monocyte-derived immature DC. When overexpressed in 293T cells, CLSPa protein was synthesized into the culture supernatants as a secretory protein, which had an inhibitory effect on complement-mediated cytotoxicity to antibody-sensitized erythrocytes. However, CLSPa itself possesses little protease activity, but it plays an inhibitory role in other active protease catalytic processes. The identification of human CLSPa as a novel Clr-like protein might facilitate future investigation of the regulatory mechanism of CLSPa in complement pathways during inflammation.  相似文献   

15.
Analysis of amino acid sequences of barley stripe mosaic virus (BSMV) proteins revealed the pentapeptide GDSGG, the sequence unique for catalytic centers of serine chymotrypsin-like proteases, in protein p14 encoded by open reading frame 4 of RNA beta. Computer-assisted comparisons revealed a statistically significant similarity between amino acid sequences of p14 and chymotrypsin-like proteases. The catalytic His and Asp residues tentatively identified in p14 together with the Ser residue of the GDSGG sequence, presumably, constitute the "catalytic triad" characteristic of chymotrypsin-like proteases. Based on these observations and on the presence of a potential N-proximal transmembrane domain in p14, this protein may be suggested to be a serine protease involved in processing of the replicase precursor within a membrane-bound replication complex of BSMV.  相似文献   

16.
Rezaie AR  He X 《Biochemistry》2000,39(7):1817-1825
The nature of residue 225 on a consensus loop in serine proteases determines whether a protease can bind Na(+). Serine proteases with a Pro at this position are unable to bind Na(+), but those with a Tyr or Phe can bind Na(+). Factor Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na(+) is also known to stimulate the amidolytic activity of FXa toward cleavage of small synthetic substrates, but the role of Na(+) in the prothrombinase complex has not been investigated. In this study, we engineered a Gla-domainless form of FX (GDFX) in which residue Tyr(225) was replaced with a Pro. We found that Na(+) stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa approximately 8-24-fold with apparent dissociation constants [K(d(app))] of 37 and 182 mM in the presence and absence of Ca(2+), respectively. In contrast, Na(+) minimally affected the cleavage rate of these substrates by the mutant, and no K(d(app)) for Na(+) binding to the mutant could be estimated. Unlike the wild-type enzyme, the reactivity of the mutant with antithrombin was independent of Na(+) and impaired approximately 32-fold. Ca(2+) improved the reactivity of the mutant with antithrombin approximately 5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further studies with the wild-type prothrombinase complex revealed that FXa binds to factor Va with a similar K(d(app)) of 1. 1-1.8 nM in the presence of Na(+), K(+), Li(+), Ch(+), and Tris(+) and that the catalytic efficiency of prothrombinase is enhanced less than 1.5-fold by the specific effect of Na(+) in the reaction buffer. These results suggest that (1) the loop including residue 225 (225-loop) is a Na(+) binding site in FXa, (2) the Na(+)- and Ca(2+)-binding loops of FXa are allosterically linked, and (3) the Tyr conformer of the 225-loop is critical for factor Xa function; however, both Na(+)-bound and Na(+)-free forms of factor Xa in the prothrombinase complex can efficiently activate prothrombin.  相似文献   

17.
Abstract The quantum yields of intrinsic fluorescence of staphylococcal exfoliative toxins ETA and ETB were determined after excitation at 295 nm and 275 nm respectively. The variations in the intrinsic fluorescence and degree of fluorescence polarization showed conformational modifications of ETA around 57–59°C and of ETB around 52–54°C. Above these transition temperatures, ETB precipitated out and its biological activity was lost, whereas ETA showed almost no precipitation and no change in its biological activity, despite the structural changes in the molecule.  相似文献   

18.
A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent K(m) and V(max) values of Bafibrinase towards fibrin were determined as 0.24 μM and 2.8 μmol/min, respectively. It showed a K(m) value of 0.139 mM towards a chromogenic substrate for plasmin (D-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,β-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.  相似文献   

19.
A linear peptide analog of endothelin (ET)-1, [Ala1,3,11,15]ET-1 (4AlaET-1), and its truncated peptide analogs were synthesized to study the structural requirements of ET-1 for the recognition of ETs-nonselective ETB receptors. ET-1 exhibited sub-nanomolar binding to two distinct ET receptor subtypes (ETA and ETB), but 4AlaET-1 bound to ETB with an affinity 1,700 times higher than that seen during binding to ETA. The truncated linear peptides 4AlaET-1(6-21), 4AlaET-1(8-21) and N-acetyl-4AlaET-1(10-21) still had high affinity for ETB, whereas 4AlaET-1(6-20) and 4AlaET-1(11-21) displayed remarkably reduced affinity for ETB. Therefore, ET-1 requires the Glu10-Trp21 sequence for ETB binding, but not the disulfide bridges. These ETB-binding peptides elicit endothelium-dependent vasorelaxation of porcine pulmonary arteries in parallel with the binding affinity for ETB, suggesting that they are ETB agonists.  相似文献   

20.
A family of hypothetical proteins, identified predominantly from archaeal genomes, has been analyzed in order to understand its functional characteristics. Using extensive sequence similarity searches it is inferred that this family is remotely related (best sequence identity is 19%) to ClpP proteinases that belongs to serine proteinase class. This family of hypothetical proteins is referred to as SDH proteinase family based on conserved sequential order of Ser, Asp and His residues and predicted serine proteinase activity. Results of fold recognition of SDH family sequences confirmed the remote relationship between SDH proteinases and Clp proteinases and revealed similar tertiary location of putative catalytic triad residues critical for serine proteinase function. However, the best sequence alignment we could obtain suggests that while catalytic Ser is conserved across Clp and SDH proteinases the location of the other catalytic triad residues, namely, His and Asp are swapped in their amino acid alignment positions and hence in 3-D structure. The evidence of conserved catalytic triad suggests that SDH could be a new family of serine proteinases with the fold of Clp proteinase, however sharing the catalytic triad order of carboxypeptidase clan. Signal peptide sequence identified at the N-terminus of some of the homologues suggests that these might be secretory serine proteinases involved in cleavage of extracellular proteins while the remote homologues, ClpP proteinases, are known to work in intracellular environment.  相似文献   

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