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1.
The cDNA encoding BthaTL, a serine peptidase from the venom of the snake Bothrops alternatus, was cloned and sequenced. The deduced primary structure shows over 62% of identity with snake venom thrombin-like enzymes (SVTLEs), molecules with high substrate specificity toward different natural substrates. Indeed, a phylogenetic reconstruction by two different methods clustered this enzyme close to other SVTLEs. These enzymes generally affect the hemostatic system in several ways, and therefore are used as tools in pharmacology and clinical diagnosis. A three-dimensional model of BthaTL was built by homology modeling using TSV-PA (Trimeresurus stejnegeri venom plasminogen activator) crystal structure as template. BthaTL model showed that the typical catalytic triad conformation of serine peptidases was preserved. The calcium coordination ligands were absent or adopt an unfavorable conformation, preventing interactions with metals. On the other hand, the Asp97-Arg174 saline bridge of TSV-PA was not found and its specificity determinant Phe193 is replaced by a Gly in BthaTL. The substitution of essential residues in the neighborhoods of the catalytic site cleft of BthaTL indicates that these two proteins do not share the same enzymatic specificity, what means that BthaTL will probably not activate plasminogen. Such observations may be helpful in the understanding of the molecular mechanism for substrate specificity of these enzymes.  相似文献   

2.

Background

Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders.

Methodology/Principal Findings

In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate.

Conclusions/Significance

A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.  相似文献   

3.
The alpha/beta hydrolase fold.   总被引:21,自引:0,他引:21  
We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.  相似文献   

4.
The amino acid sequence of a protease, protein C activator, from Agkistrodon contortrix contortrix venom was determined. Peptide fragments obtained by chemical or enzymatic cleavage of the S-carboxymethylated protein were purified by gel filtration and reverse-phase high-performance liquid chromatography. The present study demonstrates that protein C activator from A. contortrix contortrix venom is a trypsin-type serine protease that is composed of 231 residues with a molecular weight of 25,095 for the polypeptide portion of the molecule. By analogy to the mammalian serine proteases, the catalytic triad in venom protein C activator consists of His-40, Asp-85, and Ser-177. The protein also contains three N-linked glycosylation sites at Asn-21, Asn-78, and Asn-129. The amino acid sequence of protein C activator exhibits a high degree of sequence identity with other snake venom proteases: 73% with batroxobin, 68% with flavoxobin, and 55% with Russell's viper venom factor V activator.  相似文献   

5.
The complete amino acid sequences of two isoproteins of the factor V-activating enzyme (RVV-V) isolated from Vipera russelli (Russell's viper) venom were determined by sequencing S-pyridylethylated derivatives of the proteins and their peptide fragments generated by either chemical (cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine) or enzymatic (trypsin, alpha-chymotrypsin, and lysyl endopeptidase) cleavages. Both enzymes, designated RVV-V alpha and RVV-V gamma, consist of 236 amino acid residues and have a N-linked oligosaccharide chain at Asn229. The six amino acid substitutions between RVV-V alpha and -V gamma are: Thr22(alpha)-Ala22(gamma), Gly29(alpha)-Ala29(gamma), Gln191(alpha)-Glu191(gamma), Ile192(alpha)-Met192(gamma), Gln193(alpha)-His193(gamma), and Asn224(alpha)-Ser224(gamma). The molecular weights were calculated as 26,182 for RVV-V alpha and 26,167 for RVV-V gamma. The sequences of the RVV-V isoproteins exhibited 62% identity with that of batroxobin, a thrombin-like enzyme present in Bothrops atrox venom, and 33% identity with that of human thrombin B chain. The most interesting difference between the structures of RVV-V and other trypsin-type serine proteases is that the conservative Ser214-Trp215-Gly216 sequence (chymotrypsinogen numbering), considered as the site of antiparallel beta-sheet formation between the protein substrate and most serine proteases, has been replaced by the corresponding sequence Ala-Gly-Gly.  相似文献   

6.
Trypsin and chymotrypsin serine proteases are the main digestive proteases in Diptera midguts and are also involved in many aspects of the vector-parasite relationship. In sand flies, these proteases have been shown to be a potential barrier to Leishmania growth and development within the midgut. Here we describe the sequence and partial characterization of six Phlebotomus papatasi midgut serine proteases: two chymotrypsin-like (Ppchym1 and Ppchym2) and four trypsin-like (Pptryp1-Pptryp4). All six enzymes show structural features typical to each type, including the histidine, aspartic acid, and serine (H/D/S) catalytic triad, six conserved cysteine residues, and other amino acid residues involved in substrate specificity. They also show a high degree of homology (40-60% identical residues) with their counterparts from other insect vectors, such as Anopheles gambiae and Aedes aegypti. The mRNA expression profiles of these six proteases vary considerably: two trypsin-like proteases (Pptryp1 and Pptryp2) are downregulated and one (Pptryp4) upregulated upon blood feeding. The two chymotrypsin-like enzymes display expression behavior similar to that of the early and late trypsins from Ae. aegypti.  相似文献   

7.
Serine proteinases and Kunitz-type inhibitors are widely represented in the venoms of snakes belonging to different genera. During the studies of the venoms of snakes inhabiting Russia, we have cloned cDNAs coding for novel proteins of these families. A novel serine proteinase that we named nikobin was identified in the venom gland of the Nikolsky viper. The amino acid sequence of nikobin deduced from the cDNA sequence slightly differs from those of the serine proteinases found in other snakes, displaying 15 unique amino acid substitutions. This is the first serine proteinase from a viper of the Vipera genus for which the complete amino acid sequence has been determined. A cDNA coding for a Kunitz-type inhibitor has also been cloned. The deduced amino acid sequence of the inhibitor displays overall homology to the already known sequences of analogous proteins from vipers of the Vipera genus. However, several unusual amino acid substitutions that can cause a change of the inhibitor activity have been detected.  相似文献   

8.
The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50–64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes.  相似文献   

9.
10.
Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.  相似文献   

11.
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   

12.
Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin-like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of como-, nepo- and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re-identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C-terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N-terminal, and Cys in the C-terminal beta-barrel domain. These residues are suggested to form a charge-transfer system similar to that formed by the catalytic triad of chymotrypsin-like proteases. Based on the structural analogy with chymotrypsin-like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate-binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.  相似文献   

13.
To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between various members of the large gene family of trypsin-related serine proteases, over two highly conserved regions, those of the histidine and the serine of the catalytic triad. The partial cDNA clones were used to isolate full-length or almost full-length cDNA clones for three of these proteases from a platypus spleen cDNA library. By phylogenetic analysis, these three clones were identified as being the platypus homologues of human coagulation factor X, neutrophil elastase, and a protease distantly related to the T-cell granzymes. The remaining partial clone was found to represent a close homologue of human complement factor D (adipsin). The isolation of these four clones shows that several of the major subfamilies of serine proteases had evolved as separate subfamilies long before the radiation of the major mammalian lineages of today, the monotremes, the marsupials, and the placental mammals. Upon comparison of the corresponding proteases of monotremes and eutherian mammals, the coagulation and complement proteases were shown to display a higher degree of conservation compared to the hematopoietic proteases N-elastase and the T-cell granzymes. This latter finding indicates a higher evolutionary pressure to maintain specific functions in the complement and coagulation enzymes compared to many of the hematopoietic serine proteases.  相似文献   

14.
Dipeptidyl peptidase activity was investigated in snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis and Crotalus atrox. The strongest dipeptidyl peptidase IV (DPP IV) activity was found in venom from G. blomhoffi brevicaudus. The substrate specificity, susceptibility to inhibitors, and pH optimum of the partially purified enzyme were similar to those of known DPP IVs from bacteria and eukaryotes. The G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on amino acid sequences highly conserved in known DPP IVs. Two cDNA species encoding DPP IV were obtained, and designated as DPP IVa and DPP IVb. This is the first study to report the primary structure of DPP IV from a reptile. The deduced amino acid sequences for DPP IVa and DPP IVb both consist of 751amino acid residues and are highly homologous to each other. A putative catalytic triad for serine proteases, Ser-616, Asp-694, and His-726, is present. It is of particular interest that the deduced NH(2)-terminal sequence associated with the characteristic signal peptide is identical to that determined from the purified DPP IV. This indicates that the signal peptide of snake venom DPP IV is not cleaved off during biosynthesis, unlike those of other snake venom proteins.  相似文献   

15.
Isolation of two cDNA sequences which encode cytotoxic cell proteases   总被引:4,自引:0,他引:4  
Two cDNAs which cross-hybridized with cytotoxic cell protease genes were identified in a library generated from a cytotoxic T cell line. Sequence analysis revealed that the two new members of the family contained the three catalytic triad residues which characterize the active sites of serine proteases. A comparison of the protein sequences revealed not only a high degree of homology but also the conservation of some unusual structural features. These include the lack of a disulphide bond which spans the active site serine, the presence of a signal sequence and the inference of a dipeptide activation sequence.  相似文献   

16.
本文报道烙铁头(Trimeresurusmucrosquamatus)蛇毒纤维蛋白原溶酶(TMVFg),眼镜王蛇(Ophiophagushannah)蛇毒纤维蛋白原溶酶(ohS1),竹叶青(Trimeresurusstejnegeri)蛇毒专一纤溶酶原激活剂(sv-pA)对5种小分子多肽底物的底物专一性,及这些蛇毒丝氨酸蛋白酶对各种凝血因子(第X因子、凝血酶原、纤溶酶原、蛋白C)的作用,并和其它蛇毒丝氨酸蛋白酶如矛头蝮(Bothropsatrox)蛇毒凝血酶样酶(Batroxobin)、铜头蝮(Agkistrodoncontortrixcontortrix)蛇毒蛋白C激活剂ACC-C、蝰蛇(Viperarusselli)毒第Ⅴ因子激活剂RVV-V进行比较研究。通过酶标偶联免疫反应研究了抗sv-PA抗体与各种丝氨酸蛋白酶的免疫交叉反应,并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。  相似文献   

17.
The diversity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an example of concerted evolution of a trypsin gene in Chrysomya bezziana.  相似文献   

18.
Endopeptidase classification based on catalytic mechanism and evolutionary history has proven to be invaluable to the study of proteolytic enzymes. Such general mechanistic- and evolutionary- based groupings have launched experimental investigations, because knowledge gained for one family member tends to apply to the other closely related enzymes. The serine endopeptidases represent one of the most abundant and diverse groups, with their apparently successful proteolytic mechanism having arisen independently many times throughout evolution, giving rise to the well-studied soluble chemotrypsins and subtilisins, among many others. A large and diverse family of polytopic transmembrane proteins known as rhomboids has also evolved the serine protease mechanism. While the spatial structure, mechanism, and biochemical function of this family as intramembrane proteases has been established, the cellular roles of these enzymes as well as their natural substrates remain largely undetermined. While the evolutionary history of rhomboid proteases has been debated, sorting out the relationships among current day representatives should provide a solid basis for narrowing the knowledge gap between their biochemical and cellular functions. Indeed, some functional characteristics of rhomboid proteases can be gleaned from their evolutionary relationships. Finally, a specific case where phylogenetic profile analysis has identified proteins that contain a C-terminal processing motif (GlyGly-Cterm) as co-occurring with a set of bacterial rhomboid proteases provides an example of potential target identification through bioinformatics. This article is part of a Special Issue entitled: Intramembrane Proteases.  相似文献   

19.
We have cloned a human cDNA encoding a new serine protease that has been called polyserase-2 (polyserine protease-2) because it is the second identified human enzyme with several tandem serine protease domains in its amino acid sequence. The first serine protease domain contains all characteristic features of these enzymes, whereas the second and third domains lack one residue of the catalytic triad of serine proteases and are predicted to be catalytically inactive. This complex domain organization is also present in the sequences of mouse and rat polyserase-2 and resembles that of polyserase-1, which also contains three serine protease domains in its amino acid sequence. However, polyserase-2 lacks additional domains present in polyserase-1, including a type II transmembrane motif and a low-density lipoprotein receptor A module. Enzymatic analysis demonstrated that both full-length polyserase-2 and its first serine protease domain hydrolyzed synthetic peptides used for assaying serine proteases. Nevertheless, the activity of the isolated domain was greater than that of the entire protein, suggesting that the two catalytically inactive serine protease domains of polyserase-2 may modulate the activity of the first domain. Northern blot analysis showed that polyserase-2 is expressed in fetal kidney; adult skeletal muscle, liver, placenta, prostate, and heart; and tumor cell lines derived from lung and colon adenocarcinomas. Finally, analysis of post-translational processing mechanisms of polyserase-2 revealed that, contrary to those affecting to the membrane-bound polyserase-1, this novel polyprotein is a secreted enzyme whose three protease domains remain as an integral part of a single polypeptide chain.  相似文献   

20.
The sequence of all presently known trypsin-related serine proteases and their zymogens of animal and bacterial origin were optimally aligned on the basis of three different scoring schemes for amino acid comparisons. Sequence homology was found to extend into the activation peptides. The gaps resulting from the alignment of the sequences of the active enzymes formed the basis for a new procedure based on position and number of gaps, which allowed the correct topology of the evolutionary relationship of thrombin and the pancreatic enzymes trypsin, chymotrypsin and elastase to be determined. The procedure was applied in an analogous manner to changes in disulfide bridges as well as to a selected set of amino acid positions.Evolutionary distances between proteins were estimated by minimum, base differences as well as according to the stochastic model of evolution. These distances were used successfully to find the best topology of evolutionary relationships. The fact that the branch lengths in evolutionary trees were less affected by the number of sequences considered when evolutionary distances between contemporary sequences were measured in minimum base differences than when measured according to the stochastic model of evolution, suggested in our specific case, that minimum base differences yielded estimates of evolutionary distance closer to reality than the stochastic model of evolution.All these techniques combined yielded the following picture for the evolution of the four protease families. Prothrombin and the zymogens of the pancreatic serine proteases had a common ancestor with tryptic specificity. After the initial divergence, the gene for trypsinogen duplicated. Evidence was found that the duplicated gene underwent drastic changes for a short period of time to become eventually the common ancestor of chymotrypsin and elastase. The phylogenetic tree elaborated for these enzyme families and the methods introduced to determine its topology, should readily allow determination of the attachment site of branches leading to newly sequenced serine proteases, provided their amino acid sequence can be aligned fairly unambiguously. In addition, the consequences of the alignment of the different serine proteases for the relationship of zymogen to enzyme are discussed.  相似文献   

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