Site-directed mutagenesis to identify key residues in structure-function relationship of winged bean chymotrypsin-trypsin inhibitor and 3-D structure prediction

Winged bean chymotrypsin trypsin inhibitor (WbCTI) is a Kunitz type serine protease inhibitor that inhibits both trypsin and chymotrypsin at 1:1 molar ratio. Site-directed mutagenesis study was employed to generate two mutants of WbCTI, with an aim to explore its dual inhibitory properties against the proteases. The mutants were expressed in Escherichia coli and, were purified to homogeneity using a single step immunoaffinity column. The two mutants, each containing a single mutation at the amino acid sequence positions of 63 and 64, were named as L63A and R64A, respectively. Purified L63A-WbCTI exhibited anti-trypsin activity with no anti-chymotrypsin activity whereas R64A-WbCTI could inhibit chymotrypsin but not trypsin. To investigate the binding interactions between the mutated forms of WbCTI with the putative proteases, binding studies were carried out using gel filtration chromatography which further confirmed the formation of enzyme-inhibitor complexes. Finally, 3D model structure of WbCTI was designed using computer simulations which further emphasize the roles of L63 and R64 residues for dual inhibitory characteristics of WbCTI.     

Detection of proteolytic activity by fluorescent zymogram in-gel assays

Proteases are involved in the regulation of many biological functions. This study describes a novel method for detecting protease activity by fluorescent zymogram in-gel protease assays, using SDS polyacrylamide gels copolymerized with a peptide-MCA (4-methyl-coumaryl-7-amide) substrate. This method allows simultaneous determination of protease cleavage specificity and molecular weight. Trypsin was electrophoresed in SDS polyacrylamide gels copolymerized with Boc-Gln-Ala-Arg-MCA, the gel was then incubated in assay buffer, and trypsin cleavage of the peptide-MCA substrate generated fluorescent AMC (7-amino-4-methyl-coumarin), which was subsequently detected under UV transillumination. Chymotrypsin activity was detected in gels copolymerized with Suc-Ala-Ala-Pro-Phe-MCA substrate. Selective detection of these proteases was demonstrated by the absence of trypsin activity in gels containing the chymotrypsin substrate, and the lack of chymotrypsin activity in gels containing the trypsin substrate. Detection of proteolytic activity from secretory vesicles of adrenal medulla (chromaffin granules) was observed with the trypsin substrate, Z-Phe-Arg-MCA, but not with the chymotrypsin substrate. Overall, this sensitive fluorescent zymogram in-gel protease assay method can be used for rapid determination of protease cleavage specificity and enzyme molecular weight in biological samples. This assay should be useful for many research disciplines investigating the role of the many proteases that control cellular functions.     

Detection of proteases in polyacrylamide gels containing covalently bound substrates

Conjugates have been prepared from glutaraldehyde-activated linear polyacrylamide and bovine serum albumin, casein, or gelatin. Incorporation of these conjugates into sodium dodecyl sulfate-polyacrylamide gels has provided a simple and general method for the analysis of proteases following electrophoresis. The conjugates did not migrate during electrophoresis or development, but remained susceptible to proteolytic action following regeneration of enzyme activity. The sensitivity of this procedure was such that 2 pg of trypsin or chymotrypsin, 39 ng of elastase, and 2 ng of thermolysin could be detected. Results obtained with trypsin and chymotrypsin are 5 to 10 times more sensitive than previously reported techniques for protease detection following electrophoresis.     

Evidence that hatching enzyme of the sea urchin Strongylocentrotus purpuratus is a chymotrypsin-like protease

The sea urchin blastula secretes a hatching enzyme (HE) that dissolves the fertilization envelope. HE was collected from the supernatant seawater of cultures of hatched Strongylocentrotus purpuratus blastulae, and concentrated 20 times by ultrafiltration. The proteolytic activity of HE using casein as substrate was inhibited by the chymotrypsin inhibitors, chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone. The activity was not inhibited by inhibitors (antipain, elastatinal, pepstatin, phosphoramidon, soybean trypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone) of other types of proteases. HE did not hydrolyze the synthetic trypsin substrate, alpha-N-benzoyl-L-arginine ethyl ester, but did hydrolyze the synthetic substrate of chymotrypsin, N-benzoyl-L-tyrosine ethyl ester (BTEE). The BTEEase activity of HE was completely inhibited by the chymotrypsin inhibitors chymostatin and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC). Chymostatin inhibited the natural hatching of sea urchin blastulae. Application of HE to freshly fertilized sea urchin eggs, 2 h after insemination, caused premature dispersal of the hardened fertilization envelope. Chymostatin and NCDC inhibited HE-induced lysis of the fertilization envelope, while inhibitors of other types of proteases were ineffective. These data suggest that sea urchin HE is a chymotrypsin-like protease we call "chymotrypsin."     

Conformational change at the active center of serine-proteases upon their binding to alpha 2-macroglobulin

The inhibition rates and spectral characteristics of 2 probes specific for the active-site serine residue of proteases were examined for evidence of conformational change of the proteases upon their binding to alpha 2-macroglobulin (alpha 2M). Elastase, chymotrypsin, trypsin, and plasmin were reacted with (7-nitrobenz-2-oxa-1,3-diazole) aminoethyl- and aminopentyl methylphosphonofluoridate. The inhibition rate constants depend on the chain length of the aminoalkyl moiety of the probe and range from 10(5) to 10(4) M-1 min-1 for elastase and chymotrypsin. They are significantly modified when the proteases are stoichiometrically bound to alpha 2M. The absorption maximum of the chromophore appears in the range of 460-470 nm and 475-480 nm for the aminoethyl- and aminopentyl- conjugates, respectively. The fluorescence emission is maximal around 530 nm with a low quantum yield of about 3%. These spectral characteristics are altered in different ways by the covalent or non-covalent binding mode of the protease to alpha 2M. Finally, the CD spectrum of the NBD aminoethyl and aminopentyl elastase and chymotrypsin conjugates exhibits intense optical activity in the absorbing band of the NBD-moiety. These chiral properties are greatly altered upon binding of the protease to alpha 2M. All these results strongly suggest a conformational change in the protease at its active center upon its binding to alpha 2M; this conformational change could be taken into account to explain the alteration of the catalytic properties of the alpha 2M-bound proteases.     

Conformation and protease binding activity of binary and ternary human alpha 2-macroglobulin-protease complexes

Human alpha 2-macroglobulin (alpha 2M) undergoes a conformational change after reaction with proteases. In this report, it is shown that although two trypsin molecules may bind simultaneously to each alpha 2M, only one trypsin is necessary to induce alpha 2M conformational change. Ternary complexes of alpha 2M and either two radioiodinated trypsins or two nonradioiodinated trypsins were purified by gel filtration chromatography. The nonradioactive complex did not bind 125I-trypsin, even after incubation for 24 h with the free protease present at a large molar excess. Under comparable conditions, a large molar excess of nonradioactive trypsin did not cause significant dissociation of the complex prepared with radioiodinated protease. Equations are presented that distinguish between two separate models of protease binding and demonstrate that binary alpha 2M-trypsin complex retains no significant trypsin binding activity despite the presence of a vacant protease binding site. Purified alpha 2M-plasmin complex, with 1.10 mol of plasmin/mol of inhibitor, also retained no trypsin binding activity as assessed with radioiodinated protein binding experiments. These studies suggest that reactions of alpha 2M with proteases are accurately described by the "trap hypothesis" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724) independent of protease size or binding stoichiometry.     

Midgut proteases of the cardamom shoot and capsule borer Conogethes punctiferalis (Lepidoptera: Pyralidae) and their interaction with aprotinin

Protease inhibitors cause mortality in a range of insects, and transgenic plants expressing protease inhibitors have been protected against pest attack, particularly internal feeders that are not amenable to control by conventional means. A study of luminal proteases in Conogethes punctiferalis Guenée was performed to identify potential targets for proteinaceous biopesticides, such as protease inhibitors. The midgut protease profile of the gut lumen from C. punctiferalis was studied to determine the conditions for optimal protein hydrolysis. Optimum conditions for peptidase activity were found to be in 50 mm Tris-HCl, pH 10 containing 20 mm CaCl2; incubation for 30 min at 40 degrees C. Four synthetic substrates, i.e. benzoyl-arg-p-nitroanilide, benzoyl-tyr-p-nitroanilide, succinyl-ala-ala-pro-leu-p-nitroanilide (SAAPLpNA) and leu-p-nitroanilide were hydrolysed by C. punctiferalis gut proteases in Tris-HCl buffer pH 10. Trypsin and elastase-like chymotrypsin were the prominent digestive proteases, and age-related modulation of midgut proteases existed for trypsin, chymotrypsin, elastase-like chymotrypsin and leucine aminopeptidase. Serine protease inhibitors such as aprotinin, soybean trypsin inhibitor and phenylmethanesulfonyl fluoride inhibited peptidase activity. Some metal ions such as Ca(2+), Mg(2+), Pb(2+) and Co(2+) enhanced BApNA-ase activity whereas others like Mn(2+), Zn(2+), Cu(2+), Fe(2+) and Hg(2+) were inhibitory at 6 mm concentration. Trypsin and elastase-like chymotrypsin were significantly inhibited by 94% and 29%, respectively, by aprotinin (150 nm) under in vitro conditions. A possible incorporation of protease inhibitors into transgenic plants is discussed.     

Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M _r) of 28.7 kDa, whereas protease B, with a M _r of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K _m values of these two proteases on SAAPF-pNa were higher than that for α-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH₂-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family. Journal of Industrial Microbiology & Biotechnology (2001) 26, 387–393. Received 05 November 2000/ Accepted in revised form 23 April 2001     

Removal of proteases from DNase I by chromatography over agarose with covalently attached lima bean protease inhibitor

Lima bean protease inhibitor (LBI) can be convalently attached to agarose using the cyanogen bromide activation procedure. The LBI-agarose complex retains full capacity to inhibit chymotrypsin and 30% capacity to inhibit trypsin; the dissociation constant for chymotrypsin bound to LBI-agarose is 3.7 × 10^?6m. Bovine pancreatic deoxyribonuclease is contaminated by 3% by weight of chymotrypsin plus chymotrypsinogen. The contaminating proteases may be removed from DNase by sequential reaction with LBI-agarose followed by filtration. The most effective method for the removal of proteases from DNase is chromatography over LBI-agarose. DNase which has been chromatographed over LBI-agarose is at least ten times more stable than DNase prepared by earlier procedures.     

Proteolytic activities in body and faecal extracts of the storage mite, Acarus farris

Trypsin, chymotrypsin, cathepsins B and D, aminopeptidase and carboxypeptidases A and B were detected in body extracts of the storage mite Acarus farris (Oudemans) (Astigmata: Acaridae). Faeces-enriched medium exhibited higher (10-50-fold) specific protease activity rates than those measured with mite body extracts for trypsin, chymotrypsin and carboxypeptidases A and B, suggesting that they are involved in mite digestion. However, the activity of cathepsin B was only three-fold higher in faecal than in body extracts, indicating that its presence in the lumen of the digestive tract is low compared to that of serine proteases. The activity of aminopeptidases was higher in mite bodies, indicating that they might be membrane bound. Cathepsin D activity was only detected in body extracts, indicating that this enzyme is not a digestive protease in this species. Zymograms resolved three major bands of gelatinolytic activity, but at least one protease form was only present in body extracts. Protease inhibitors of different specificity were tested in vivo to establish their potential as control agents. The development of A. farris was significantly retarded when the immature stages were fed on artificial diet containing inhibitors of serine and cysteine proteases and aminopeptidases, whereas no such effect was found with inhibitors of aspartyl proteases and carboxypeptidases. Interestingly, the most significant effects on A. farris occurred when a combination of inhibitors targeting different enzyme classes was supplied mixed in the diet, suggesting a synergistic toxicity. Several plant lectins were also tested, but only wheat germ agglutinin and concanavalin-A affected development.     

Peptide-induced fluorescence quenching of conjugated polyelectrolyte for label-free, ultrasensitive and selective assay of protease activity

We report here a label-free method for ultrasensitive and selective assay of protease activity based on the peptide-induced fluorescence quenching of conjugated polyelectrolyte (PPESO(3)). It is very interesting to find that there is a critical length of oligo-polyarginine (i.e., Arg(5)) below which 1) the quenching efficiency of PPESO(3) is sharply decreased, and more importantly, 2) the trypsin-catalyzed hydrolysis is greatly slowed down. This opens good opportunities for not only the ultrasensitive assay of trypsin, but the specific detection of other proteases if carefully designing an appropriate peptide length and the cleavage site. Herein, the enzyme selected as a proof of concept is chymotrypsin. Due to the essence that any cleavage of the designed peptide probes will result in a notable decrease or even a complete loss of their capability to quench the emission of PPESO(3), the limits of detection for trypsin and chymotrypsin have been found as low as 0.25 ng/mL (11 pM) and 0.15 ng/mL (6 pM), respectively. Both are superior to those of most previous methods by 1-2 orders or higher.     

Chromatography of serine proteases on chitin and its derivatives

Chitin containing sorbents have been obtained for isolation and purification of serine proteases. Serine proteases from Bacillus subtilis have been purified 4-5 times and commercial preparations of trypsin and chymotrypsin 1.5-2 times by chromatography on nondeproteinized chitin. On the benzylated derivative of nondeproteinized chitin complete separation of trypsin and chymotrypsin has been achieved by chromatography of crude pancreatin. It has been shown that the protein moiety of chitin is important for preferential sorption of serine type proteases.     

Contribution of conserved Asn residues to the inhibitory activities of Kunitz-type protease inhibitors from plants

Plant Kunitz-type protease inhibitors contain a conserved Asn residue in the N-terminal region. To investigate the role of Asn residue in protease inhibitory activities, Erythrina variegata trypsin inhibitor a (ETIa), E. variegata chymotrypsin inhibitor (ECI), and their mutants, ETIa-N12A and ECI-N13A, were used. Both mutants exhibit weaker inhibitory activities toward their cognate proteases than the wild-type proteins and were readily cleaved at reactive sites. Furthermore, kinetic analysis of the interactions of the mutated proteins with their cognate proteases by surface plasmon resonance (SPR) measurement indicated that replacements of the Asn residue mainly affected dissociation rate constants. The conserved Asn residues of Kunitz-type inhibitors play an important role in exhibiting effective inhibitory activity by stabilizing the structures of the primary binding loop and protease-inhibitor complex.     

Thioltrypsin. Chemical transformation of the active-site serine residue of Streptomyces griseus trypsin to a cysteine residue.

The active-site serine residue of Streptomyces griseus trypsin was converted to a cysteine residue, and the product, thioltrypsin, was purified through two chromatographic steps with organomercurial-Sepharose and soybean trypsin inhibitor-Sepharose as specific adsorbents. The purified preparation of thioltrypsin was found to contain a single residue of cysteine and to react with almost equimolar amounts of normality titrants. It exhibited only traces of catalytic activity toward typical trypsin substrates such as Nalpha-tosyl-L-arginine methyl ester, whereas it retained some activity toward "active ester" substrates such as Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester. The activity was inhibited by sulfhydryl-blocking reagents, but no inhibition was observed by reagents reactive with the active hydroxyl group of serine proteases. Leupeptin, a natural trypsin inhibitor of peptidyl nature, also inhibited thioltrypsin. Some difference in the mode of leupeptin inhibition, however, was detected between trypsin and thioltrypsin. The bindings of small synthetic ligands and soybean trypsin inhibitor to thioltrypsin were compared with those to trypsin.     

Further evidence for a partially folded intermediate in penicillinase secretion by Bacillus licheniformis.

Protoplasis of Bacillus licheniformis 749/C (a mutant constitutive for penicillinase production) continued to synthesize and release penicillinase in hypertonic growth medium in the presence of trypsin and chymotrypsin at 25 mug each per ml. When the protoplasts were stripped of about half of their membrane-bound penicillinase by pretreatment at pH 9.5 or with a higher level of trypsin, penicillinase activity no longer increased in the presence of the proteases. This effect was immediately eliminated after addition of soybean trypsin inhibitor. These proteases do not significantly inhibit general protein synthesis. Stripped protoplasts of strain 749/C and of uninduced strain 749 (unable to synthesize penicillinase) were incubated with 50 mug of chymotrypsin per ml, and the supernatent fluids were examined immunochemically for peptides derived from the penicillinase chain. Such fargments were found only with the protoplasts capable of synthesizing penicillinase (strain 749/C). The direct detection of the products of protease degradation of a susceptible form of penicillinase provides strong evidence that, in stripped protoplasts of B. licheniformis 749/C, penicillinase synthesis continues in the presence of trypsin or chymotrypsin and that, in these modified membranes, the protease is able to act on an early form of the enzyme that has not yet attained the protease-resistant conformation characteristic of the membrane-bound and exopenicillinases. This finding is discussed in terms of the current models of penicillinase secretion.     

Ecotin: lessons on survival in a protease-filled world.

Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has been shown to be a potent inhibitor of a group of homologous serine proteases with widely differing substrate recognition. It is highly effective against a number of enzymes, including both pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. Recent structural and functional studies on ecotin and its interactions with different serine proteases have clarified these initial observations and revealed the remarkable features of this protein in inhibiting a strikingly large subset of the chymotrypsin family of serine proteases. The structures of the ecotin:serine protease complexes provide the first examples of protein-protein recognition where the concept of specificity of interactions needs to be reexamined. The binding sites show a fluidity of protein contacts derived from ecotin's innate flexibility in fitting itself to proteases while strongly interfering with their function.     

Specific fluorescent labeling of alpha 2-macroglobulin-bound proteases: accessibility and localization of their active site within the complex

Pyrenebutylmethylphosphonofluoridate reacts with trypsin and elastase to yield a conjugate with a stoichiometry of one fluorescent label per enzyme molecule as already observed with chymotrypsin. The kinetics of inactivation indicate that the serine active center of the proteases is involved in the labeling reaction. The binding of the proteases to alpha 2-macroglobulin does not modify the specificity of the reaction but drastically diminishes the labeling rate which also depends upon alpha 2-macroglobulin protease binding ratio. Dynamic quenching of the conjugated pyrene moiety by acrylamide, and iodide ions is markedly reduced upon reaction of the protease with alpha 2-macroglobulin, indicating a reduced accessibility of the protease active center in the complex. Singlet--singlet energy transfer measurements from the donor pyrene labeled active center of the proteases to the alpha 2-macroglobulin acceptor labeled thiol groups which are liberated upon protease fixation, gave a rough estimate of the distance (about 25 A) between the active center of the two alpha 2-macroglobulin bound protease molecules.     

Characterization of proteases from a stored product mite, Tyrophagus putrescentiae

Extracts of Tyrophagus putrescentiae feces exhibited higher (>50-fold) specific protease activity rates than those measured using mite body extracts for the substrates azocasein, BApNa, SA(2)PPpNa, HA, and HPA. This suggests that trypsin, chymotrypsin, and carboxypeptidases A and B are involved in mite digestion. Hydrolysis of the substrates ZAA(2)MNA and LpNa was only 3 times higher in fecal extracts, suggesting that levels of cathepsin B and aminopeptidases in the lumen of the digestive tract are low compared to the other enzymes. The hydrolysis of hemoglobin was only detected in body extracts indicating that cathepsin D is not a digestive protease in this species. Protease inhibitors of different specificity were tested invivo to establish their potential as control agents. We found that development from larvae to adult was significantly retarded in larvae fed on brewers' yeast containing inhibitors of serine proteases, whereas no such effect was found with inhibitors of cysteine and aspartyl proteases. Interestingly, when dietary mixtures of serine protease, aminopeptidase and carboxypeptidase inhibitors were fed to T.putrescentiae, a synergistic effect was observed that retarded development. Several plant lectins were also tested, but none affected development.     

Phospholipid protection against proteolysis of D-beta-hydroxybutyrate dehydrogenase, a lecithin-requiring enzyme

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly inactivates D-beta-hydroxybutyrate dehydrogenase; the addition of NAD+ or NADH, together with dithiothreitol and increased salt (to 50 mM), decreases the rate of inactivation, and with trypsin, virtually eliminates inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)