Identification of the substrate-binding exosites of two snake venom serine proteinases: molecular basis for the partition of two essential functions of thrombin

The venom of the South American snake Bothrops jararaca contains two serine proteinases, bothrombin and the platelet-aggregating enzyme PA-BJ, which share 66% sequence identity. Each of these proteinases possesses one of the two essential procoagulant functions of thrombin-the clotting of fibrinogen and platelet aggregation. Thus, bothrombin clots fibrinogen but has no direct effect on platelets, unless in the presence of exogenous fibrinogen. PA-BJ induces platelet aggregation by interacting with the protease-activated platelet receptor without clotting fibrinogen. On the other hand, thrombin possesses two extended surfaces. One is composed of basic and hydrophobic residues (exosite I) and the other one of basic residues only (exosite II). These exosites are involved in the recognition of physiological macromolecular substrates. In order to identify the corresponding exosites in bothrombin and PA-BJ and understand the molecular basis of the partition of the two procoagulant functions of thrombin among the two snake venom enzymes, we used molecular modeling to obtain models of their complexes with their natural substrates fibrinogen and a fragment of the protease-activated platelet receptor, respectively. In analogy to thrombin, each of the enzymes presents two exosites. Nonetheless, the exosites contain a smaller proportion of basic residues than thrombin does (45-72%), reducing thus the functional diversity of the enzymes. In addition, the composition of exosite I is different in both enzymes. We identify those residues in exosite I that could contribute to the differences in specificity. Finally, allostery does not seem to mediate macromolecular substrate recognition by these enzymes.     

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.     

Inactivation of human plasma serine proteinase inhibitors (serpins) by limited proteolysis of the reactive site loop with snake venom and bacterial metalloproteinases

Human plasma serine proteinase inhibitors (serpins) gradually lost activity when incubated with catalytic amounts of snake venom or bacterial metalloproteinases. Electrophoretic analyses indicated that antithrombin III, C1-inhibitor, and alpha 2-antiplasmin had been converted by limited proteolysis into modified species which retained inhibitory activity. Further proteolytic attack resulted in the formation of inactivated inhibitors; alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 1-antichymotrypsin were also enzymatically inactivated, but active intermediates were not detected. Sequence analyses indicated that the initial, noninactivating cleavage occurred in the amino-terminal region of the inhibitors. Inactivation resulted in all cases from the limited proteolysis of a single bond near, but not at, the reactive site bond in the carboxy-terminal region of the inhibitors. The results indicate that the serpins have two regions which are susceptible to limited proteolysis--one near the amino-terminal end and another in the exposed reactive site loop of the inhibitor.     

Surface interaction of human granulocytes and lymphocytes with staphylococcal extracellular serine proteinase

Enzymatic activity of purified staphylococcal extracellular serine proteinase decreases as a result of incubation with granulocytes as well as with lymphocytes taken from peripheral blood of healthy donors. However, specific proteinase binding was observed only in the case of granulocytes but not in peripheral lymphocytes.     

Preliminary estimation of chemoattractant activity of staphylococcal serine proteinase in vitro

Human polymorphonuclear leukocytes isolated from peripheral blood of healthy donors migrated toward the staphylococcal serine proteinase.     

N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - BSA bovine serum albumin - Bz benzoyl - EACA 6()-aminocaproic acid - HEPES N-2-hydroxyethylpiperazine-N'-propanesulfonic acid - HPLC high-performance liquid chromatography - PEG polyethylene glycol-3350 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids     

Mechanism of staphylococcal serine proteinase inactivation by lymphocytes and granulocytes

Stricking differences were observed in the mechanism of interaction between staphylococcal serine proteinase and surface of human granulocytes or lymphocytes despite the fact that incubation of this enzyme with both types of cells leads to analogical decrease of proteinase activity. Interaction of proteinase with lymphocytes releases peptides smaller than these released spontaneously by non-treated lymphocytes or lymphocytes treated with DFP-proteinase. However, in supernatants of lymphocytes neither complex of proteinase with cell derived molecules nor changes of electrophoretic mobility of proteinase was found. Products of proteinase—lymphocyte reaction have a proliferative effect on intact lymphocytes, which is greater that the one of active proteinase. On the other hand granulocytes are resistant to proteinase and bind active proteinase as well as the DFP-proteinase in the receptor mediated way, followed by endocytosis with the affinity similar to the one in monocytes.     

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.     

Sequence of the gene encoding an alkaline serine proteinase of Bacillus pumilus TYO-67

The complete nucleotide sequence of the gene encoding an alkaline serine proteinase (aprP) of Bacillus pumilus TYO-67 was determined. The sequence analysis showed an open reading frame of 1,149 bp (383 amino acids) that encoded a signal peptide consisting of 29 residues and a propeptide of 79 residues. The deduced 3 amino acid residues, D32, H64, and S221, were identical with 3 essential amino acids in the catalytic center of subtilases. The sequence around these residues revealed that APRP was a new member of the true subtilisin subgroup of the subtilisin family. The highest homology was found in subtilisin NAT at 64.4% in the DNA sequence. The residue S189 of APRP was different from those of other subtilases.     

Effects of proteinase inhibitors on western corn rootworm life parameters

Abstract: Plants have developed defensive mechanisms to minimize predation by insect pests. Proteinase inhibitors are an example of plant compounds synthesized as a mechanism for defence. The objective of this study was to determine the impact of trans‐epoxysuccinyl‐l ‐leucylamido (4‐guanidino) butane (E‐64), phenylmethylsulfonyl fluoride (PMSF‐serine protenase inhibitor) and Kunitz trypsin inhibitors on the pre‐ovipositional and ovipositional periods, the mean number of eggs laid per female, and the longevity of western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, adults. This study provides information on the effectiveness of proteinase inhibitors as a host‐plant resistance tool for managing WCR beetles. The study was conducted in 1997, 1998, and 2000. In 1997, E‐64 was added to an artificial diet at the concentrations of 0.05, 0.025 and 0.0125% (w/w), corresponding to 500, 250, and 125 ppm respectively. In 1998, PMSF was added to the artificial diet at the same concentrations. In 2000, Kunitz trypsin inhibitor was added to the artificial diet at concentrations of 0.2, 0.1 and 0.05% (w/w), corresponding to 2000, 1000, and 500 ppm respectively. The mean fecundity of beetles fed the untreated diet was between 67 and 111 eggs per female. The fecundity of beetles fed E‐64 and PMSF at different concentrations, ranged between 162 and 246 eggs per female for E‐64 and 61 and 80.5 eggs per female for PMSF. The fecundity of the beetles fed Kunitz trypsin inhibitor was between 155 and 225 eggs per female. When beetles fed on the diet which consisted of the lowest dosage of Kunitz trypsin inhibitor (500 ppm), fecundity was higher than that on untreated control. The proteinase inhibitors investigated did not show a negative impact on WCR adults. Beetle fecundity, the length of the pre‐ovipositional and ovipositional periods and the longevity of the beetles fed with proteinase inhibitors were not lower than that of the beetles fed only the artificial diet. This study does not support the use of investigated proteinase inhibitors at applied concentrations as effective host‐plant resistance tools for managing WCR beetles.     

Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono-and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1–1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25–30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca²⁺, Mg²⁺, and Mn²⁺ stimulated biosynthesis of proteinase in the late stationary phase (by 20–60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.     

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

Abstract

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10?kDa, respectively, and under non-reducing conditions, 26?kDa was observed. The inhibitor was shown to inhibit bovine trypsin (K_i of 3.45?nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.     

蛋白酶抑制剂在抗虫基因工程中的应用

简要介绍了蛋白酶抑制剂的种类及其在抗虫基因工程中的应用,并根据当前的研究情况,对蛋白酶抑制剂的研究前景和方向做了一些探讨。     

Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.     

The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.     

Structural models of the snake venom factor V activators from Daboia russelli and Daboia lebetina

Blood coagulation factor V (FV) is a multifunctional protein that circulates in human plasma as a precursor molecule which can be activated by thrombin or activated factor X (FXa) in order to express its cofactor activity in prothrombin activation. FV activation is achieved by limited proteolysis after Arg709, Arg1018, and Arg1545 in the FV molecule. The venoms of Daboia russelli and Daboia lebetina contain a serine protease that specifically activates FV by a single cleavage at Arg1545. We have predicted the three-dimensional structure of these enzymes using comparative protein modeling techniques. The plasminogen activator from Agkistrodon acutus, which shows a high degree of homology with the venom FV activators and for which a high-quality crystallographic structure is available, was used as the molecular template. The RVV-V and LVV-V models provide for the first time a detailed and accurate structure of a snake venom FV activator and explain the observed sensitivity or resistance toward a number of serine protease inhibitors. Finally, electrostatic potential calculations show that two positively charged surface patches are present on opposite sides of the active site. We propose that both FV activators achieve their exquisite substrate specificity for the Arg1545 site via interactions between these exosites and FV.     

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.     

Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice

Low molecular weight, acid-stable proteinase inhibitors from epididymal and seminal vesicle homogenates were isolated and characterized. The isolation procedure consisted of gel filtration, trypsin affinity, and ion exchange chromatography. The inhibitor from seminal vesicle homogenates has a molecular weight of approximately 6,200, and that of the epididymal inhibitor was estimated at 4,000. Antiserum directed against the seminal vesicle inhibitor did not react with epididymal components. The epididymal inhibitor shows competitive, whereas the seminal vesicle inhibitor shows noncompetitive inhibition against trypsin on double reciprocal plots. Both inhibitors are effective against trypsin and acrosin but not against chymotrypsin, kallikrein, thrombin, or plasmin. To verify site of origin and to investigate androgen dependency of the epididymal inhibitor, mice were efferentiectomized, orchiectomized, or orchiectomized with androgen supplementation. Gel filtration profiles of acid-treated epididymal homogenates from normal and efferentiectomized animals show inhibitor peaks in the same regions. The concentration of acid-stable inhibitor from epididymal homogenates decreased with orchiectomy but returned to normal values when exogenous androgen was supplied. These observations suggest that the low molecular weight inhibitor in the epididymal homogenates is distinct from that in the seminal vesicles. Furthermore, the inhibitor associated with epididymal homogenates is androgen-dependent, and the epididymis is the site of origin of this inhibitor.