A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

竹叶青蛇毒凝血酶样酶氨基酸序列报道

蛇毒凝血酶样酶可作为蛋白酶结构与功能研究的良好模型,并已广泛用于各种血栓疾病的诊断和治疗,因而测定其一级结构具有重要意义．利用逆转录与聚合酶链反应相结合的ＲＴ－ＰＣＲ法,扩增出竹叶青蛇毒凝血酶样酶（ＴＳＶ－ＴＥＬ１）的ｃＤＮＡ;将扩增的ｃＤＮＡ片段克隆入ｐＧＥＭ－Ｔ载体中,经末端终止法测定核苷酸序列,推导出竹叶青蛇毒凝血酶样酶的全序列．竹叶青蛇毒凝血酶样酶由２３４个氨基酸组成并含有１个位于Ａｓｎ２０的Ｎ－型糖基结合位点．竹叶青蛇毒凝血酶样酶序列与其它蛇种来源凝血酶样酶具有较大相似性,其与黄绿烙铁头蛇毒凝血酶样酶序列相似度为８４％,与美洲矛头蝮蛇毒凝血酶样酶序列相似度为６８％,而与牛凝血酶Ｂ链序列相似度仅为２５％．     

Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper

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.     

cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.     

Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme

Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide.     

Molecular cloning and sequence analysis of a cDNA for factor V activating enzyme.

The complete amino acid sequence of a factor V activator (VLFVA) is deduced from the nucleotide sequence of a cDNA encoding the enzyme. The cDNA was isolated by PCR screening a venomous gland cDNA library of Central Asian Vipera lebetina snake. The full-length cDNA clone, derived from two overlapping fragments, comprises 1563 basepairs which encode an open reading frame of 259 amino acids. The amino acid sequence of VLFVA (235 amino acids) shows significant homology with snake venom and mammalian serine proteinases. It contains 12 half-cysteines which form, by analogy with other serine proteinases, 6 disulfide bridges. VLFVA has the catalytic triad His43-Asp88-Ser182. The amino terminal amino acid valine is preceded by 24 amino acids: a putative signal peptide of 18, mainly hydrophobic, amino acids and an activating peptide of 6, mainly hydrophilic amino acid residues. This is the first cloned factor V activating enzyme from snake venom.     

Cloning and localization of C2orf2(ropp120), a previously unknown WD repeat protein

Ropp 120 (restrictedly overexpressed proliferation-associated protein) is a cytoplasmic protein of 120 kDa that is significantly overexpressed in mitotic cells. Protein sequencing of the immunoaffinity purified 120-kDa protein showed it to be an as yet unknown protein. DNA sequencing revealed a cDNA sequence of 3419 bases, which includes the complete coding region of ropp120 of 2943 bases (981 amino acids). Analysis of the deduced amino acid sequence showed that ropp 120 contains four WD repeats and a well-conserved consensus sequence of serine proteases. The gene encoding ropp120 (HGMW-approved gene symbol C2orf2) was assigned to chromosome 2p21-p22 by means of radiation hybrid and fluorescence in situ hybridization mapping.     

Identification and expression analysis of the Steinernema carpocapsae elastase-like serine protease gene during the parasitic stage

A cDNA encoding elastase was isolated from Steinernema carpocapsae by suppression subtractive hybridization and rapid amplification of 5′ cDNA ends. The predicted protein contained a 19-aa signal peptide, a 44-aa N-terminal propeptide, and a 264-aa mature protein with a predicted molecular mass of 28,949 Da and a theoretical pI of 8.88. BLAST analysis showed 27-35% amino acid sequence identity to serine proteases from insects, mammals, fish and other organisms. The Sc-ela gene contains three exons and two introns with at least two copies in the S. carpocapsae genome. Expression analysis indicated that the Sc-ela gene was upregulated during the initial parasitic stage. Sequence comparison and evolutionary marker analysis revealed that Sc-ELA was a member of the elastase serine protease family with potential degradative, developmental and fibrinolytic activities. Homology modeling showed that Sc-ELA adopts a two β-barrel fold typical of trypsin-like serine proteases, and phylogenetic analysis indicates that Sc-ELA branched off early during elastase evolution.     

Isolation and sequencing of porcine lipoprotein lipase cDNA and its use in multiallelic restriction fragment length polymorphism detection

Porcine lipoprotein lipase (LPL) cDNA has been cloned and sequenced. The deduced amino acid sequence shows a high degree of identity to LPL from other species, and contains the Ser/His/Asp triade characteristic of serine proteases and esterases. A repetitive element is present in the 3'-untranslated region of the cDNA. A partial cDNA covering the coding region of LPL detects three restriction fragment length polymorphisms with HindIII. This represents the first marker assigned to porcine chromosome 14.     

Amino acid sequence of human D of the alternative complement pathway

The primary structure of human D, the serine protease activating the C3 convertase of the alternative complement pathway, has been deduced by sequencing peptides derived from various chemical (CNBr and o-iodosobenzoic acid) and enzymatic (trypsin, lysine protease, Staphylococcus aureus V8 protease, and chymotrypsin) cleavages. Carboxypeptidase A was also used to confirm the COOH-terminal sequence. The peptides were purified by high-pressure liquid chromatography. The proposed sequence of human D contains 222 amino acids and has a calculated molecular weight of 23 748. It exhibits a high degree of homology with other serine proteases, especially around the NH2-terminus as well as the three residues corresponding to the active-site His-57, Asp-102, and Ser-195 (chymotrypsinogen numbering). This sequence homology is highest (40%) with plasmin, intermediate (35%) with pancreatic serine proteases, such as elastase, trypsin, chymotrypsin, and kallikrein, and least (30%) with the serum enzymes thrombin and factor X. D, however, exhibits only minimal amino acid homology with the other sequenced complement serine proteases, Clr (25%) and Bb (20%). The substitution of a basic lysine for a neutral amino acid three residues NH2-terminal to the active-site serine as well as a small serine residue for a bulky aromatic amino acid at position 215 (chymotrypsinogen numbering) in the binding pocket may be important in determining the exquisite substrate specificity of D. The presence of His-40 which interacts with Asp-194 (chymotrypsinogen numbering) to stabilize other serine protease zymogens [Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., & Xuong, N. H. (1970) Biochemistry 9, 1997] argues in favor of such a D precursor molecule.     

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found.     

Human thrombin: Partial primary structure

Human thrombin, prepared from an extrinsically activated prothrombin, was finally chromatographed on an affinity column of p-chlorobenzylamido-?-aminocaproyl agarose. After reduction and s-pyridylethylation, the A and B chains were separated. The A chain contains 36 residues and no carbohydrate and has a formula weight of 4093. Its sequence corresponds to 14–49 of the bovine A chain sequence with nine replacements. Human B chain contains 264 residues. The carbohydrate content is 3.5% neutral sugar, 2.6% glucosamine, and 1.5% sialic acid. High-speed sedimentation equilibrium in guanidine gave a minimum molecular weight of 33,500. Sequenator analysis yielded the first 50 amino-terminal residues and established the positions of three of the cyanogen bromide fragments. A fourth fragment lacked homoserine and was placed at the carboxyl-terminus. The four remaining fragments, comprising half of the B chain, were tentatively assigned positions by assuming homology with bovine thrombin and with pancreatic serine proteases. Within the B chain, the active site histidine and serine were both in highly conservative regions, and an Arg-Tyr bond has been identified as a biological degradation site. Sixty-nine percent of the primary structure of human thrombin was identified by sequenator analysis. In comparing this sequence with that reported for the bovine molecule, 26 replacements are present.     

Sequencing of bovine enamelin ("tuftelin") a novel acidic enamel protein

Enamelins are a major group of 28-70-kDa acidic proteins rich in aspartic acid, glutamic acid, serine, and glycine found in developing and mature extracellular enamel; a unique and highly mineralized ectodermal tissue covering vertebrate teeth. They have been associated with the mineralization and structural organization of this tissue. In an attempt to elucidate the primary structure of enamelin, a 2674-base pair cDNA isolated from a bovine ameloblast-enriched, lambda Zap 2 expression library, was sequenced. The identity and localization of the deduced protein was confirmed by amino acid composition, enzyme-linked immunosorbent assay, Western blotting, indirect immunohistochemistry, and high resolution protein-A gold immunocytochemistry. The immunological techniques were employed using antibodies directed against synthetic peptides corresponding to the protein sequence deduced from the cloned cDNA sequence. The results reveal the deduced protein to be a novel acidic enamel protein. It contains 389 amino acids and has a calculated molecular weight of 43,814. Its amino acid composition is similar to that of "tuft" proteins (enamel matrix protein fragments remaining in the mature tissue). It contains one potential N-glycosylation site and 5 cysteine residues. Southern hybridization of the cloned cDNA with genomic bovine DNA indicated the existence of a single gene with one or more introns.     

Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans

A novel serine protease, named as scolonase, was purified and characterized from the tissue of the Korean centipede, Scolopendra subspinipes mutilans. Purified scolonase showed an apparent molecular weight of 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and an isoelectric point of 4.8 on isoelectric focusing gel. Scolonase was able to preferentially hydrolyze arginine over lysine at the cleavage site among the several synthetic peptide substrates. Scolonase has also a potent fibrinolytic activity by converting human Glu-plasminogen to activated plasmin due to the specific cleavage of the molecule at the peptide bond Arg(561)-Val(562). The enzyme activity of scolonase was completely inhibited by phenylmethanesulfonyl fluoride and difluorophosphate. The cDNA encoding scolonase was cloned from the cDNA library of the centipede constructed with oligonucleotide probe, which was designed on the basis of the N-terminal amino acid sequence of scolonase. The deduced complete amino acid sequence of scolonase demonstrated that the protein is composed of 277 amino acids including 33 amino acids as a leader sequence, and that it has significant sequence homology with other serine proteases.     

Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

Molecular cloning and sequence analysis of cDNAs coding for a serine proteinase and a Kunitz-type inhibitor in the venom gland of the Vipera nikolskii viper

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.     

Purification and molecular cloning of a serine protease from the mushroom Hypsizigus marmoreus

A protease with a molecular mass of 28 kDa, designated as hmsp, was isolated from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on CM-cellulose. hmsp was thermolabile, and exhibited a temperature optimum at 50 °C and a pH optimum at pH 7.5. The activity of the protease was adversely affected by PMSF, EGTA and aprotinin, indicating that it is a serine protease. Based on the N-terminal sequence, the cDNA of hmsp was cloned by using RACE combined with the TAIL-PCR method. The deduced protease sequence contained a signal peptide with 19 amino acids, a pro-region with 82 amino acids, and a mature protease with 285 amino acids and a molecular mass of 28.07 kDa. It possessed the three active sites characteristic of the subtilisin family (S8A). hmsp demonstrated 63%, 57% and 44% identity in amino acid sequence respectively to Absp1, Absp2, and Gf-spr1, which are serine proteases from Agaricus bisporus and Grifola frondosa.     

Molecular cloning of the Atlantic cod chymotrypsinogen B

The cDNA encoding Atlantic cod (Gadus morhua) chymotrypsinogen B has been isolated and sequenced. Its deduced amino acid sequence consists of a 16-residue signal sequence and a mature polypeptide of 247 residues, being two residues longer than its vertebrate analogs. This mature polypeptide corresponds to a calculated molecular mass of 26.5 kDa and shares 70% sequence identity with cod chymotrypsinogen A. However, the identity between cod chymotrypsinogen B and its other vertebrate analogues is 63-66%. In common with most fish serine proteases, cod chymotrypsinogen B contains a high number of methionine residues. The presence of a threonine instead of a highly conserved serine residue at position 189 is a novel characteristic of this enzyme. Cod chymotrypsin B, as its type B vertebrate analogs, has an alanine at position 226, whereas a glycine is most commonly found at this position in the type A chymotrypsins.     

cDNA cloning of granzyme C, a granule-associated serine protease of cytolytic T lymphocytes

A cDNA clone corresponding to the complete amino acid sequence of a putative protease CCP2 of murine cytotoxic T lymphocytes was isolated and sequenced. The clone encodes a 248-residue long serine esterase. The deduced N-terminal amino acid sequence is identical over 40 residues to that of granzyme C, a protease of unknown function present in granules of cytotoxic lymphocytes. Analysis of the sequence of granzyme C/CCP2 reveals high homology to other granzyme proteases, i.e. granzyme A (40%) and granzyme B (67%) and to rat mast cell protease II (46%). The amino acids lining the specificity pocket are well conserved between granzyme B, C, and rat mast cell protease II, but not granzyme A, suggesting a similar general specificity of these three proteases.