Substrate and inhibitor studies on proteinase 3.

Various amino acid and peptide thioesters were tested as substrates for human proteinase 3 and the best substrate is Boc-Ala-Ala-Nva-SBzl with a kcat/Km value of 1.0 x 10(6) M-1.s-1. Boc-Ala-Ala-AA-SBzl (AA = Val, Ala, or Met) are also good substrates with kcat/Km values of (1-4) x 10(5) M-1.s-1. Substituted isocoumarins are potent inhibitors of proteinase 3 and the best inhibitors are 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarin and 3,4-dichloroisocoumarin (DCI) with kobs/[I] values of 4700 and 2600 M-1.s-1, respectively. Substituted isocoumarins, peptide phosphonates and chloromethyl ketones inhibited proteinase 3 less potently than human neutrophil elastase (HNE) by 1-2 orders of magnitude.     

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors.

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.     

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.     

Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition

The X-ray crystal structure of the serpin-proteinase complex suggested that the serpin deformed the proteinase thereby inactivating the molecule. Using a variant of alpha(1)-antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition. The tryptophan free variant, alpha(1)-antitrypsin((FF)), is fully active as an inhibitor of thrombin. Thrombin has a fluorescence emission maximum of 340 nm which blue shifts to 346 nm, concomitant with a 40% increase in intensity, upon formation of the serpin-proteinase complex indicative of substantial conformational change within the proteinase. Stopped-flow analysis of the fluorescence changes within the proteinase indicated a two-step mechanism. A fast bimolecular reaction with a rate constant of 2.8x10(6) M(-1) s(-1) is followed by a slow unimolecular process with a rate of 0.26 s(-1) that is independent of concentration. We propose that the first rate is formation of an initial complex which is then followed by a slower process involving the partial unfolding of the proteinase during its translocation to the opposite pole of the serpin.     

Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of 125I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) of 3.5 x 10(-4) s-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.     

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.     

Human skin chymotryptic proteinase. Isolation and relation to cathepsin g and rat mast cell proteinase I

A chymotrypsin-like proteinase was purified 2400-fold from human skin. The procedure involves extraction of the proteinase from skin in 2 M KCl, precipitation with protamine chloride, fractionation by gel filtration chromatography, and fractionation by chromatography using a CH-Sepharose-D-tryptophan methyl ester affinity column. The properties of this proteinase were compared to the rat mast cell proteinase I and human cathepsin G. Differences were observed in the rates at which the proteinases were inhibited by diisopropyl fluorophosphate, the sensitivity of the proteinases to protein proteolytic inhibitors, the relative hydrolytic rates of the proteinases for a series of substrates, and the kinetic constants of the proteinases for synthetic substrates. The human skin proteinase did not react with antiserum to the rat skin proteinase and did not elute in the same position as the rat skin proteinase on gel filtration columns. These data demonstrate that the human skin proteinase is distinct from the other proteinases. Extracts of involved skin from patients with cutaneous mastocytosis had 15-fold higher levels of chymotryptic activity than extracts of uninvolved skin or skin from normal controls. The enzymatic properties of the material extracted from the biopsied skin were similar to those of the proteinase from normal skin, suggesting that the human skin chymotrypsin-like proteinase is a mast cell constituent.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

Affinity purification of serine proteinase from Deinagkistrodon acutus venom

An affinity protocol was developed for the preparation of the main serine proteinase from Deinagkistrodon acutus venom on industrial scales. As affinity ligand, l-arginine was composed to medium and its structure was confirmed by ESI-MS analysis. The purification process consisted of one major affinity chromatography step to remove more than 95% of other proteins, and a polishing step of DEAE ion-exchange chromatography for removal of minor contaminants. The serine proteinase was 100% pure analyzed on HPLC Vydac C4 column, 99.4% on TSK G3000SW column, and 97.7% with SDS-PAGE analysis. The yield of the main serine proteinase was 3.6% of crude venom protein, the recoveries of typical fibrinogen (Fg) clotting activity and arginine esterase activity of serine proteinase were 82.2% and 84%, higher than those of other reported traditional protocols, the proteinase also showed arginine amidase activity. Reducing SDS-PAGE analysis showed that the arginine esterase was a single polypeptide with the mass of approximately 40 kDa, while MALDI-TOF-TOF-MS analysis showed that the purified proteinase should be a approximately 34 kDa glycoprotein. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 9.93 x 10(-5) and 38.1mg/g medium in absorption analysis.     

Protease-specificity of Kunitz inhibitor domain of Alzheimer's disease amyloid protein precursor

The putative inhibitor domain of Alzheimer's disease amyloid protein precursor was purified from E. coli containing a synthetic gene encoding the Kunitz domain. The purified protein (A4 inhibitor) inhibited the activity of trypsin, forming a 1:1 molar complex with the enzyme. It also strongly inhibited plasmin (Ki = 7.5 x 10(-11) M) from human serum and tryptase (Ki = 2.2 x 10(-10) M) from rat mast cells (tryptase M). In addition, it inhibited rat pancreatic trypsin, alpha-chymotrypsin and kallikrein and human serum kallikrein, but did not inhibit rat chymase, pancreatic elastase, alpha-thrombin, urokinase, papain or cathepsin B.     

Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.     

Comparison of properties of thiol proteinase inhibitors from rat serum and liver

Thiol proteinase inhibitors in rat serum were purified and their properties were compared with those of rat liver thiol proteinase inhibitor. The inhibitors in rat serum were separated into three forms (S-1, S-2, and S-3) by linear gradient elution from a DE52 column. One inhibitor (S1) was purified to homogeneity by chromatography on ficin-bound Sepharose and Sephadex G-150 columns. The apparent molecular weights of S1, S2, and S3 on Sephadex G-150 columns were 90,000, 95,000, and 160,000, respectively. Serum thiol proteinase inhibitor and liver thiol proteinase differed in the following: 1) all three forms of serum inhibitor had much higher molecular weights than the liver thiol proteinase inhibitor (Mr = 12,500); 2) no cross-reactivity was observed between serum inhibitors and liver inhibitor in tests with either antiserum inhibitor or anti-liver antiserum; 3) both serum inhibitor and liver inhibitor were specific for thiol proteinases, but had different inhibition spectra; 4) the liver inhibitor did not bind to concanavalin A-Sepharose, whereas the serum inhibitor bound and was eluted with alpha-methyl mannoside. A thiol proteinase inhibitor of high molecular weight detected in tissue homogenates inhibited papain markedly but did not inhibit cathepsin H. Its activity was diminished by perfusion of the organ, indicating that it is derived from serum.     

Amino acid sequence of rat mast cell protease I (chymase)

The amino acid sequence has been determined for rat mast cell protease I (RMCP I), a product of peritoneal mast cells. The active enzyme contains 227 residues, including three corresponding to the catalytic triad characteristic of serine protease (His-57, Asp-102, and Ser-195 in chymotrypsin). A computer search for homology indicates 73% and 33% sequence identity of RMCP I with rat mast cell protease II from mucosal mast cells and bovine chymotrypsin A, respectively. When the structure of RMCP I is compared to those of cathepsin G from human neutrophils and two proteases expressed in activated lymphocytes, 48-49% of the sequences are identical in each case. RMCP I has six half-cystine residues at the same positions as in RMCP II, cathepsin G, and the two lymphocyte proteases, suggesting disulfide pairs identical with those reported for RMCP II. A disulfide bond near the active site seryl residue and substrate binding site, present in pancreatic and plasma serine proteases, is not found in RMCP I or in the other cellular proteases. These results indicate that RMCP I and other chymotrypsin-like proteases of granulocyte and lymphocyte origin are more closely related to each other than to the pancreatic or plasma serine proteases.     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

Purification of an acid proteinase from Aspergillus saitoi and determination of peptide bond specificity.

The specificity and mode of action of an acid proteinase (EC 3.4.23.6) from Aspergillus saitoi were investigated with oxidized B-chain of insulin, angiotensin II and bradykinin. Further purification of acid proteinase was performed with N,O-dibenzyloxycarbonyl-tyrosine hexamethylene-diamino-Sepharose 4B affinity chromatography and isoelectric focusing. The purified enzyme was free of any other proteolytic activity demonstrated in Asp. saitoi. Acid proteinase from Asp. saitoi hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu(15)-Tyr(16) bond and the Phe(24)-Phe(25) bond. Additional cleavages of the bonds His(10)-Leu(11), Ala(14)-Leu(15) and Tyr(16)-Leu(17) were also noted. Primary splitting sites at Leu(15)-Tyr(16) and Phe(24-)-Phe(25) with acid proteinase from Asp. saitoi were identical with those reported in the work of cathepsin D (EC 3.4.23.5) from human erythrocyte. Hydrolysis of angiotensin II was observed at the Tyr(4)-Ile(5) bond. In conclusion, peptide bonds which have a hydrophobic amino acid such as phenylalanine, tyrosine, leucine and isoleucine in the P'1 position (as defined by Berger and Schechter, [29]) are preferentially cleaved by the trypsinogenactivating acid proteinase from Asp. saitoi.     

Identification of a chymotrypsin-like proteinase in human mast cells

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.     

Stimulation of proteinase biosynthesis by canavanine in streptococcus faecalis var. liquefaciens

l-Canavanine, an analogue of arginine, was found to stimulate the synthesis of an extracellular proteinase in Streptococcus faecalis var. liquefaciens. Cells grown in a synthetic medium containing 10(-4)m arginine and 10(-4)m canavanine produced almost twice as much proteinase as cells grown in 2 x 10(-3)m arginine alone; total growth was the same in both media. Hydrolyzed proteinase samples were analyzed for arginine and canavanine by means of paper chromatography and electrophoresis. Arginine, but not canavanine, was detected in the purified enzyme sample.     

Guanine nucleotide regulation of B2 kinin receptors. Time-dependent formation of a guanine nucleotide-sensitive receptor state from which [3H]bradykinin dissociates slowly

We have examined the binding of [3H]bradykinin to bovine myometrial membranes and assessed its sensitivity to guanine nucleotides. Total binding displayed a typical B2 kinin receptor specificity. However, saturation binding isotherms were resolved into at least two components with KD values of 8 pM (45%) and 378 pM (55%). Low affinity binding exhibited relatively rapid rates of association (kobs = 1.40 x 10(-2) s-1) and dissociation (k-1 = 3.82 x 10(-3) s-1), while high affinity binding exhibited considerably slower rates (kobs = 9.52 x 10(-4) s-1 and k-1 = 4.43 x 10(-5) s-1). Pre-equilibrium dissociation kinetics revealed that formation of high affinity binding was characterized as a time-dependent accumulation of the slow dissociation rate at the expense of at least one other more rapid dissociation rate. In the presence of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), at least two binding components were resolved with KD values of 37 pM (12%) and 444 pM (88%). Gpp(NH)p apparently specifically perturbed high affinity binding by completely preventing the accumulation of the slow dissociation phase. Instead, two more rapid dissociation rates (k-1 = 8.53 x 10(-3) s-1 and 4.43 x 10(-4) s-1) were observed. These results suggest that [3H]bradykinin interacts with at least two B2 kinin receptor-like binding sites in bovine myometrial membranes. A three-state model for the guanine nucleotide-sensitive agonist interaction with the high affinity binding sites is proposed.