The complete amino acid sequence of rat submaxillary gland tonin does contain the aspartic acid at the active site: confirmation by protein sequence analysis

The revised amino acid sequence of rat submaxillary gland tonin, a serine protease, does contain the active site Asp residue. The active site of this kallikrein-related enzyme is thus made up of the same catalytic triad (Asp, Ser, and His) found in all known serine proteases. The important Asp residue has now been localized in a 16 amino acid peptide previously reported as missing in the tonin sequence. The complete amino acid sequence thus contains 235 residues corresponding to a molecular weight of 25,658, more in agreement with previously reported molecular weights. Moreover, the revised structure led (a) to the assignment of Arg, Asn, and Val residues instead of His, Asp, and Gly at positions 63, 165, and 169, respectively; (b) to the assignment of residues occupying an overlapping sequence at positions 165-171, and finally (c) to the localization of two N-glycosylation sites at positions 82 and 165. These results further document the close relationship of tonin to the ever expanding kallikrein family.     

黑曲霉脯氨酸蛋白内肽酶基因的克隆及其在酵母中的表达

以A．niger TCCC41013总RNA为模板,通过RT-PCR扩增含自身信号肽的脯氨酸蛋白内肽酶基因pep,将其克隆到pUCm-T载体上进行测序。测序结果表明基因全长1581bp,共编码526个氨基酸,包括22个氨基酸的信号肽序列和504个氨基酸的成熟肽序列。通过生物信息学方法对蛋白质的理化性质进行分析预测,脯氨酸蛋白内肽酶等电点为4．43,具有丝氨酸活性中心,属丝氨酸羧肽酶S28家族。以重组质粒pUCm．T-pep为模板,通过PCR扩增去除自身信号肽的pep,构建毕赤酵母表达载体pPIC9-peP,电转酵母宿主菌GS115。SDS-PAGE分析显示表达产物的分子量为60000Da左右,酶特异性底物法测定酶活力最高可达2275．4mU／mL。     

Characterization of genes encoding rat tonin and a kallikrein-like serine protease

Tissue kallikreins are a group of serine proteases which may function as peptide hormone processing enzymes. Two rat kallikrein genomic clones (RSKG-5 and RSKG-50) were sequenced and characterized. The rat tonin gene and a kallikrein-like gene were found in clones RSKG-5 and RSKG-50, respectively. The tonin gene is 4146 base pairs in length, with both the variant CCAAA and TTTAAA boxes in the 5'-end region and an AATAAA polyadenylation signal at the 3' end of the gene. It has five exons which are separated by four introns. Sequence analysis of 3.7-kb 5' upstream and 7.5-kb 3' downstream of the tonin gene failed to reveal a second kallikrein gene. Sequence comparisons of the RSKG-5 exons with tonin cDNA revealed that only one base in the 3'-noncoding region was different from that in the previously reported rat tonin cDNA. Characteristic TC- and TG-repeated sequences were also found in the first and second introns of the tonin gene. The tonin gene encodes a preprotonin of 259 amino acids (aa). The active enzyme consists of 235 aa and is preceded by a deduced signal peptide of 17 aa and a profragment of 7 aa. Northern blot analysis indicates that RSKG-5 is expressed in a sex-dependent manner in rat submandibular gland, with a higher level expressed in males. The RSKG-50 gene was truncated at an EcoRI site in the second intron, excluding its 5' end. Compared to the coding sequence of pancreatic kallikrein, 12 nucleotides have been deleted in exon 3 of the RSKG-50 gene. The nucleotide sequences of the third, fourth, and fifth exons of the RSKG-50 gene encode a polypeptide of 188 aa residues. The translated peptide is 80% homologous to rat pancreatic kallikrein and 75% homologous to rat tonin in the corresponding regions. Key residues in the RSKG-50 gene product indicate a serine protease with kallikrein-like cleavage specificity at basic amino acids.     

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.     

Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine.

A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.     

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.     

The peptide sequences near the bound pyridoxal phosphate are conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli

The blocked amino-terminal residue of rat liver serine dehydratase was shown to be acetylalanine by analysis of an isolated amino-terminal peptide after digestion with acylamino acid-releasing enzyme. Digestion of the borohydride-reduced, carboxymethylated enzyme with lysyl endopeptidase yielded a single epsilon-N-pyridoxyllysine-containing peptide, whose sequence is Met-Asp-Ser-Ser-Gln-Pro-Ser-Gly-Ser-Phe-Lys(Pxy)-Ile-Arg-Gly- His-Leu-Cys(Cm)-Lys. This peptide comprises residues 30-49 of the cDNA-deduced amino acid sequence. The sequence of seven amino acids around the bound pyridoxal phosphate is highly conserved in serine dehydratase from rat liver, and threonine dehydratases from yeast and Escherichia coli.     

Effect in vivo of beta-adrenergic stimulation, angiotensin II, dibutyryl cyclic AMP, and theophylline on tonin concentration in rat saliva and submaxillary gland.

Tonin (an enzyme present in rat submaxillary gland and saliva) has previously been shown to be able, unlike renin and reninlike substances, to release angiotensin II either directly by acting on an appropriate substrate or from angiotensin I. The administration of a beta-adrenergic drug, isoproterenol, produces a rise of tonin concentration in saliva without affecting its concentration in the submaxillary gland. Prior administration of a beta blocker, propranolol, partially prevents this effect. The administration of theophylline increases the tonin concentration in both saliva and the submaxillary gland, whereas dibutyryl cyclic AMP increases tonin concentration in the former. These results suggest that beta-adrenergic stimulation enhances both tonin release into the saliva and tonin synthesis in the submaxillary gland, and that these effects might be mediated by cyclic AMP. Infusion of angiotensin II blocked the stimulatory effect of isoproterenol on salivary tonin. 1Sar-8Ile-angiotensin II is both a weak antagonist of angiotensin II in this respect and a strong agonist in terms of blocking the effect of isoproterenol another role mirrored in other physiological mechanisms of derivatives of angiotensin II.     

Formation of angiotensin II by tonin from partially purified human angiotensinogen

The renin substrate (angiotensinogen) has been purified from outdated human blood bank plasma. A 100-fold purification was achieved by ammonium sulphate protein fractionation and four successive chromatographic procedures. We show that tonin, a serine protease enzyme found in submaxillary glands of the rat, cleaves the human plasma angiotensinogen, devoid of tonin inhibiting factor(s), at a pH optimum of 5--5.5. It generates a pressor substance that was identified as angiotensin (A) II. The rate of cleavage of the human angiotensinogen preparation by 1 nmol of renin or tonin was calculated to be 1320 nmol AI/h for renin and 26 nmol AII/h for tonin.     

Tonin as activator of renin

Tonin, a new serine protease, found in high concentration in rat submaxillary glands, leads to a significant activation of human amniotic fluid renin. The optimum pH on renin activation by tonin was found at pH 6.0. The reaction was time dependent and the initial rate of angiotensin I generation was constant up to 2 h. The two amniontic fluid samples studied showed an increase in renin activity after incubation with tonin to about five times the control level (268 to 1240 pmol x h-1 x mL-1 and 1490 to 7480 pmol x h-1 x mL-1).     

Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.     

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.     

Rat submaxillary gland serine protease, tonin. Structure solution and refinement at 1.8 A resolution

Tonin is a mammalian serine protease that is capable of generating the vasoconstrictive agent, angiotensin II, directly from its precursor protein, angiotensinogen, a process that normally requires two enzymes, renin and angiotensin-converting enzyme. The X-ray crystallographic structure determination and refinement of tonin at 1.8 A resolution and the analysis of the resulting model are reported. The initial phases were obtained by the method of molecular replacement using as the search model the structure of bovine trypsin. The refined model of tonin consists of 227 amino acid residues out of the 235 in the complete molecule, 149 water molecules, and one zinc ion. The R-factor (R = sigma Fo - Fc/sigma Fo) is 0.196 for the 14,997 measured data between 8 and 1.8 A resolution with I greater than or equal to sigma (I). It is estimated that the overall root-mean-square error in the coordinates is about 0.3 A. The structure of tonin that has been determined is not in its active conformation, but one that has been perturbed by the binding of Zn2+ in the active site. Zn2+ was included in the buffer to aid the crystallization. Nevertheless, the structure of tonin that is described is for the most part similar to its native form as indicated by the close tertiary structural homology with kallikrein. The differences in the structures of the two enzymes are concentrated in several loop regions; these structural differences are probably responsible for the differences in their reactivities and specificities.     

Immunohistochemical localization of tonin and its relation to kallikrein in rat salivary glands

Tonin and kallikrein are serine proteases present in high concentrations in the submandibular gland of the rat. These enzymes release the vasoactive peptides angiotensin II and lysyl-bradykinin from the precursors angiotensinogen and kininogen, respectively. Tonin and kallikrein were purified from homogenates of rat submandibular gland, and antisera against each protein were raised in rabbits. The anti-kallikrein antibody also reacted with tonin, showing partial cross-reactivity between kallikrein and tonin when tested by double immunodiffusion and by immunoelectrophoresis. The anti-tonin antibody did not appear to react with kallikrein in immunodiffusion systems. The cellular localization of tonin was investigated by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. In the granular tubular cells tonin-specific staining was abundantly present with a granular distribution; in the striated duct cells tonin-specific staining was observed as a thin luminal rim. Tonin was not detected in any other structures of the gland. When the localization of tonin was compared with that of kallikrein, both enzymes were found within the same granular tubular cells. However, more kallikrein than tonin was detected in the striated duct cells. Furthermore, kallikrein but not tonin was found in the ductal cells of the parotid and sublingual glands.     

脯氨酸内肽酶研究进展

脯氨酸内肽酶是一类能够特异性水解多肽链中脯氨酸残基羧基端的内切酶,是丝氨酸蛋白酶家族成员之一,其能有效降解小于30个含有脯氨酸残基的多肽链,在对PEP的研究中发现它能特异性地水解许多含脯氨酸的多肽类神经递质和激素,如促甲状腺激素释放激素、P物质(Substance P)等,因此研究认为PEP可能参与一些神经类疾病的发生,如老年性记忆障碍等。本研究经过阅读相关期刊文献,对脯氨酸内肽酶的分布情况、基本理化和酶学性质、分子结构、作用机理以及在食品、医药领域的最新研究和应用进行了综合性评述,以期能对脯氨酸内肽酶相关性质、机理的后续研究以及在食品、医药领域更广泛的应用提供一定的参考。     

The effects of intracerebroventricular administration of tonin on water drinking and blood pressure in rats

Intraventricular administration of tonin, an enzyme which forms angiotensin II directly from a natural protein substrate (angiotensinogen), stimulated water drinking and increased systemic blood pressure in rats. These responses were abolished by the simultaneous administration of an angiotensin II analogue, but unaffected by an angiotensin I- converting enzyme inhibitor. We confirmed, in vitro, that angiotensin II was generated when the brain homogenate was incubated with tonin in the presence of DFP and EDTA. These results indicate that the central effects of tonin on water drinking and systemic blood pressure are mediated via the direct formation of angiotensin II in the brain, and suggest the possibility that the tonin-angiotensin II system in the central nervous system might be important in controlling water balance and blood pressure.     

A structural model for the prostate disease marker, human prostate-specific antigen.

Prostate-specific antigen (PSA) provides an excellent serum marker for prostate cancer, the most frequent form of cancer in American males. PSA is a 237-residue protease based on sequence homology to kallikrein-like enzymes. To predict the 3-dimensional structure of PSA, homology modeling studies were performed based on sequence and structural alignments with tonin, pancreatic kallikrein, chymotrypsin, and trypsin. The structurally conserved regions of the 4 reference X-ray proteins provided the core structure of PSA, whereas the loop structures were modeled on the loops of tonin and kallikrein. The unique "kallikrein loop" insert, between Ser 95b and Pro 95k of kallikrein, was constructed using molecular mechanics, dynamics, and electrostatics calculations. In the resulting PSA structure, the catalytic triad, involving residues His 57, Asp 102, and Ser 195, and hydrophobic and electrostatic interactions typical of serine proteases were extremely well conserved. Similarly, the 5-disulfide bonds of kallikrein were also conserved in PSA. These results, together with the fact that no major steric clashes arose during the modeling process, provide strong evidence for the validity of the PSA model. Calculation of the electrostatic potential contours of kallikrein and PSA was carried out using the finite difference Poisson-Boltzmann method. The calculations revealed matching areas of negative potential near the catalytic triad, but differences in the positive potential surrounding the active site. The PSA glycosylation site, Asn 61, is fully accessible to the solvent and is enclosed in a positive region of the isopotential map. The bottom of the substrate specificity pocket, residue S1, is a serine (Ser 189) as in chymotrypsin, rather than aspartate (Asp 189) as in tonin, kallikrein, and trypsin. This fact, plus other features of the S1 binding-pocket region, suggest that PSA would prefer substrates with hydrophobic residues at the P1 position. The location of a potential zinc ion binding site involving the side chain of histidines 91, 101, and 233 is also suggested. This PSA model should facilitate the understanding and prediction of structural and functional properties of this important cancer marker.     

Ala657 and conserved active site residues promote fibroblast activation protein endopeptidase activity via distinct mechanisms of transition state stabilization

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.     

A novel serine protease with vasoconstrictor activity coded by the kallikrein gene S3

A fraction separated from rat submandibular gland homogenates was found to contain a potent vasoconstrictor when tested on isolated rabbit aortic rings. The vasoconstrictor was purified by a series of chromatographic steps. The purified compound (2.77 x 10(-9) M) induced 40% of the maximum contractile response to 60 mM KCl. Constriction was slow in onset, long-lasting, rinse-resistant, and unchanged by de-endothelialization; in addition, it was dose-related and inhibited by both EGTA and verapamil, but it was not affected by DUP753, an angiotensin II receptor antagonist. The compound was found to be a protein having a pI of 7.36 and a molecular weight of approximately 29,000 and exhibiting partial immunologic identity to rat glandular kallikrein and rat tonin. After 2-mercaptoethanol treatment, it separated into heavy (approximately 19,900) and light (approximately 10,700) chains having amino-terminal sequences of AY(X)HNNDLMLL and VVGGYN(X)ETNSQ, respectively. We found that they correspond to the amino-terminal and internal sequence of a previously unidentified kallikrein-like serine protease whose mRNA, named S3, has been found in the rat submandibular gland and prostate. The vasoconstrictor is able to hydrolyze t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide (a thrombin substrate), although its Kcat/Km was only 0.02% that of rat thrombin. Both vasoconstrictor and enzymatic activity on t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide were completely suppressed by amidinophenylmethylsulfonyl fluoride and soybean trypsin inhibitor; however, they were unaffected by hirudin, a thrombin inhibitor. At pH 6.5, it released angiotensin II when incubated with sheep angiotensinogen, although it had approximately one-tenth the activity of tonin. The submandibular enzymatic vasoconstrictor is a kallikrein-like enzyme, having some properties of both tonin and thrombin. It directly contracts vascular smooth muscle, acting via a mechanism that requires intact enzymatic activity.     

Specific cleavage of beta-LPH and ACTH by tonin: release of an opiate-like peptide beta-LPH (61-78).

Tonin, a rat enzyme capable of cleaving angiotensinogen, the tetradecapeptide renin substrate and angiotensin I directly to antiotensin II is also shown to cleave beta-lipotropin into beta-LPH 1–50, 1–51, 51–60, 52–60, 61–78 and 79–91, thereby selectively releasing the opiate-like segment beta-LPH 61–78. Its action on ACTH was similar, releasing ACTH 1–8, 1–7, 3–8, 3–7 and 9–39. In both situations the cleavages are of a selective tryptic-chymotryptic type at specific arginine, phenylalanine residues. Comparison of the tonin cleavage with those of trypsin, trypsin in combination with citraconylation of the lysine residues of beta-LPH is made. The data presented show that tonin does not cleave Met-enkephalin and can be used as an enzyme to study the presence of endorphin-like sequences in polypeptides.