Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai

An endopeptidase has been purified from sprouts of bamboo (Pleioblastus hindsii Nakai) to electrophoretic homogeneity by four purification steps. Its Mr was estimated to be 82 kDa by SDS-PAGE. Enzyme activity was inhibited strongly by diisopropyl fluorophosphate, and weakly by p-chloromercuriphenylsulfonic acid, but not at all by EDTA or pepstatin, indicating that it was a serine protease. The preferential cleavage sites for this protease were found to be large hydrophobic and amide residues at the P1 position. The specificity of the bamboo serine protease differed from that of cucumisin [EC 3.4.21.25], which cleaved the charged amino acid residues at the P1 position.     

Covalent structure of two novel neutrophile leucocyte-derived proteins of porcine and human origin. Neutrophile elastase homologues with strong monocyte and fibroblast chemotactic activities

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.     

Active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala / (Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.     

Hemoglobin-binding site on haptoglobin probed by selective proteolysis

Selective proteolysis has been used to delineate the hemoglobin-binding site on haptoglobin heavy chain. Haptoglobin was cleaved specifically by plasmin, trypsin, chymotrypsin, staphylococcal protease, and thermolysin. Haptoglobin-hemoglobin complex was treated with these enzymes to determine which sites were protected from cleavage by the hemoglobin. The modified haptoglobins were tested for changes in their hemoglobin and hemoglobin alpha chain-binding properties. The sites of proteolytic cleavage were identified from the newly generated NH2 termini by automated Edman degradation amino acid-sequencing techniques. The results suggest that residues 128 through 131, 136 and 137, as well as 9 and 10 of the heavy chain may be involved in the binding of hemoglobin. On the other hand, residues 159 and 160, which lie in the 17-residue additional loop that is unique to haptoglobin among its homologous serine protease family, and residues 73 and 74, which lie close to the carbohydrate-binding residues, appear to be remote from the hemoglobin-binding site.     

The molecular evolution of acrosin in placental mammals

Acrosin is thought to fulfill several different roles in fertilization including that of a serine protease and in secondary zona pellucida (ZP) binding. However, acrosin's importance as a fertilization protein has been questioned. Especially since it was discovered that acrosin knockout mice are fertile. In this study, we explored the sites involved in serine protease activity and secondary binding. We also assessed conservation in functional sites across species and examined whether amino acid changes present in the human population have the potential to affect fertility. In addition, since many mammalian reproduction proteins have been found to evolve rapidly, we tested for positive selection. Sequences from 43 mammals from all 19 placental orders, which included a total of 828 nucleotides from acrosin exons 2, 3, 4, and a portion of exon 5, were obtained. We found that all sites of the serine catalytic triad as well as three other sites linked to catalytic activity were completely conserved. Five of six sites proposed to play a role in secondary binding were 100% conserved as basic residues. These results support an evolutionary conserved role for acrosin as a serine protease and secondary binding protein across placental mammals. We found statistically significant support for positive selection within acrosin, but no single amino acid site reached the significance level of P > 0.95 for inclusion within the category omega > 1. Based upon two amino acid mutation scoring systems, three out of seven human residue changing single nucleotide polymorphisms (SNPs) were found to be potentially protein-altering mutations.     

A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1

A collagenolytic protease was purified to homogeneity from thermophilic Bacillus sp. strain MO-1. The protease from strain MO-1 showed high activity toward type I and IV collagens and gelatin. However, peptide substrates (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and 2-furylacryloyl-Leu-Gly-Pro-Ala) for collagenases were inert as substrates. The collagenolytic protease cleaved oxidized insulin B-chain at 11 sites and degraded type I and IV collagens into anonymous small pieces, suggesting that the protease digests collagens at multiple sites. The collagenolytic protease was far more thermostable than a mesophilic Clostridium histolyticum collagenase. The collagenolytic protease possesses two salient features: (1) it has a very large molecular mass, 210 kDa, and consists of two, identical 105-kDa subunits; (2) it belongs to a serine protease group. The high molecular mass is unique among serine proteases but common for collagenases. The features of the enzyme from strain MO-1 suggest that it is a new collagenolytic protease which is distinct from previously reported collagenases and serine proteases.     

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.     

Isolation of two cDNA sequences which encode cytotoxic cell proteases

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.     

Purification and cloning of a novel serine protease, RNK-Met-1, from the granules of a rat natural killer cell leukemia.

We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.     

Knowledge-based modeling of the serine protease triad into non-proteases.

The Asp-His-Ser triad of serine proteases has been regarded, in the present study, as an independent catalytic motif, because in nature it has been incorporated at the active sites of enzymes as diverse as the serine proteases and the lipases. Incorporating this motif into non-protease scaffolds, by rational design and mutagenesis, might lead to the generation of novel catalysts. As an aid to such experiments, a knowledge-based computer modeling procedure has been developed to model the protease Asp-His-Ser triad into non-proteases. Catalytic triads from a set of trypsin family proteases have been analyzed and criteria that characterize the geometry of the triads have been obtained. Using these criteria, the modeling procedure first identifies sites in non-proteases that are suitable for modeling the protease triad. H-bonded Asp-His-Ser triads, that mimic the protease catalytic triad in geometry, are then modeled in at these sites, provided it is stereochemically possible to do so. Thus non-protease sites at which H-bonded Asp-His-Ser triads are successfully modeled in may be considered for mutagenesis experiments that aim at introducing the protease triad into non-proteases. The triad modeling procedure has been used to identify sites for introducing the protease triad in three binding proteins and an immunoglobulin. A scoring function, depending on inter-residue distances, solvent accessibility and the substitution potential of amino acid residues at the modeling sites in the host proteins, has been used to assess the quality of the model triads.     

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.     

Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom

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.     

Buried water in homologous serine proteases.

Buried water molecules in the structurally homologous family of eukaryotic serine proteases were examined to determine whether buried waters and their protein environments are conserved in these proteins. We found 16 equivalent water sites conserved in trypsin/ogen, chymotrypsin/ogen, elastase, kallikrein, thrombin, rat tonin and rat mast cell protease, and 5 additional water sites in enzymes which share the primary specificity of trypsin. Based on an alignment of 30 serine protease sequences, it appears that the protein environments of these 21 conserved buried waters are highly conserved. The protein environments of buried waters are comprised primarily of atoms from highly conserved residues or main chain atoms from nonconserved residues. In one instance, the protein environment of a water is conserved even in the presence of an unlikely Pro/Ala substitution. We also note 3 instances in which a histidine side chain substitutes for water, suggesting that the structural role of water at these sites is satisfied by the presence of an alternative hydrogen bonding partner. Buried waters appear to be integral structural components of these proteins and should be incorporated into protein structures predicted on the basis of sequence homology to this family, including the catalytic domains of coagulation proteases.     

Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. A distinct acidic, tetrabasic residue-specific calcium-activated serine protease and a PC2-like enzyme.

Adrenocorticotropin (ACTH) is cleaved at the tetrabasic residue site, in pituitary intermediate lobe secretory vesicles, to yield ACTH1-17 and corticotropin-like intermediate lobe peptide (CLIP). ACTH1-17 is then converted to alpha-melanocyte-stimulating hormone (N-AcACTH1-13NH2) by first removing the Lys15-Lys16-Arg17 residues, followed by amidation of the COOH terminus and acetylation of the NH2 terminus. Bovine intermediate lobe secretory vesicle membranes were screened for proteolytic enzyme activity that will cleave the tetrabasic residues of ACTH. Two activities with pH optima of 5.0-6.0 and 7.5-8.0 were detected. The acidic, ACTH-converting enzyme cleaved ACTH1-39 at the tetrabasic residues between the Arg17-Arg18 bond to yield ACTH1-17 and CLIP, but did not cleave paired basic residues of pro-opiomelanocortin. This enzyme activity was characterized as a Ca(2+)-activated serine protease with unique specificity for the tetrabasic residues of ACTH1-39. The neutral activity preferentially generated ACTH1-17 and to a small extent ACTH1-16 from ACTH1-39 and ACTH1-24. This enzyme activity was Ca(2+)-dependent but was not inhibited by serine or aspartic protease inhibitors. The neutral activity was significantly immunodepleted by antiserum raised against bovine PC2/PC3, and together with specificity studies, suggests that the enzyme is a PC2-like serine protease. The pH optimum, distinct specificity for tetrabasic residues, and subcellular localization of the acidic ACTH-converting enzyme indicate a function of this enzyme in the in vivo conversion of ACTH1-39 to alpha-melanocyte-stimulating hormone in intermediate lobe secretory vesicles which have an acidic internal pH.     

Complete amino acid sequence of streptokinase and its homology with serine proteases

The complete amino acid sequence of streptokinase has been determined by automated Edman degradation of its cyanogen bromide and proteolytic fragments. The protein consists of 415 amino acid residues. Sequence microheterogeneity was found at two positions. The NH2-terminal 245 residues of streptokinase are homologous to the sequences of several serine proteases including bovine trypsin and Streptomyces griseus proteases A and B. The sequence alignment suggests that the active-site histidine-57 has changed to a glycine in streptokinase. The other active-site residues, aspartyl-102 and serine-195, are, however, present at the expected positions. Streptokinase also contains internal sequence homology between the NH2-terminal 173 residues and a COOH-terminal 162-residue region between residues 254 and 415. Moderate homology in predicted secondary structures also exists between these two regions. Although streptokinase is not a protease, these observations suggest that it has evolved from a serine protease by gene duplication and fusion. A COOH-terminal region of about 80 residues is apparently deleted from the second half of the duplicated structures. These observations further suggest that the three-dimensional structure of streptokinase likely contains two independently folded domains, each homologous to serine proteases.     

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii

A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry.     

竹叶青蛇毒丝氨酸蛋白酶的分子克隆和序列比较

利用逆转录酶与聚合酶链反应相结合的RT—PCR法,扩增出5个竹叶青(Trimeresurus stejnegeri)蛇毒丝氨酸蛋白酶的cDNAs;将扩增的cDNA片段克隆入pGEM-T载体中,筛选得到它们的基因,分别命名为TSSP-1、TSSP-2、TSSP-3、TSSP-4和TSSP-5。经末端终止法测定核苷酸序列,推导出5个丝氨酸蛋白酶的全序列;结合纯化的蛋白酶N-末端序列测定结果,推导TSSP-2、-3和-4分别编码凝血酶样酶stejnobin、纤溶酶stejnefibrase 1和2。5个丝氨酸蛋白酶分别含有1～6个N-型糖基结合位点,表明它们的计算分子量与纯化蛋白表观分子量之间的差异是由糖含量的不同造成,而其氨基酸序列相似度在60％～90％。TSSP-1和-2编码的成熟蛋白酶由236个氨基酸残基组成,TSSP-3、-4和-5的则由234个氨基酸残基组成。TSSP-1编码的蛋白酶在组成丝氨酸蛋白酶三联体催化活性中心产生了His^41-Arg^41的天然突变,这与其他自然界已发现的丝氨酸蛋白酶明显不同。