Schistosoma mansoni: inhibition of cercarial "penetration" proteases by components of mammalian blood.

1. Normal human sera and plasma were fractionated in order to identify inhibitors of the "penetration" proteases of Schistosoma mansoni cercariae. 2. The main inhibitor, accounting for 90% of the total activity of serum, appears to be alpha 1-antitrypsin (alpha 1-AT) as identified by separation on DEAE-cellulose and Sephadex, by immunoelectrophoresis and by anticercarial protease activity of purified alpha 1-AT preparations. 3. The inhibition profiles of purified preparations of the 6 known serum antiproteases suggest that the parasite protease is similar to vertebrate chymotrypsin. 4. On a molar basis, the order of inhibitory activity against the cercarial protease is: alpha 1-AT = alpha 2-macroglobulin; C'-1-inactivator; alpha 1-antichymotrypsin. No inhibition was obtained with inter-alpha-inhibitor or antithrombin III.     

The protease inhibitor system of macaques. The identification of alleles using isoelectric focusing and family analysis

Five alpha-1-antitrypsin (alpha-1-AT) phenotypes have been revealed by isoelectrofocusing (IEF) in sera of 215 crab-eating macaques. Alpha-1-AT was monomorphic in sera of 250 Rhesus monkeys. A new allele of macaque Pi-system, designated as B' was postulated in addition to existing two (B and C) on the basis of IEF data. The above conclusion was supported by family analysis, based on 35 monkey birth cases. Alpha-1-AT phenotype frequencies were in agreement with Hardy-Weinberg equation both in wild and capture born crab-eating macaques. Alpha-1-AT was found to be microheterogeneous: several zones of the protein were revealed by IEF and Western blotting with anti human alpha-1-AT serum. The pregnancy caused a sharp increase of one band. This may lead to false identification of alpha-1-AT phenotypes, particularly when acid starch gel electrophoresis is used for alpha-1-AT identification. Such misinterpretation during alpha-1-AT phenotyping may explain the disagreement with Hardy-Weinberg equation described earlier for crab-eating macaques.     

Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre

Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G. The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity. There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking. We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT. The mutant proteins were produced in E. coli following in vitro mutagenesis of the alpha 1AT cDNA. Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G. Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively. Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G. These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre. In each case, replacement of Met358 led to resistance to oxidative inactivation. Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.     

Augmentation of lung antineutrophil elastase capacity with recombinant human alpha-1-antitrypsin

To evaluate the potential use of recombinant DNA-produced alpha-1-antitrypsin (alpha-1-AT) to augment the lung antineutrophil elastase defenses in alpha-1-AT deficiency, we compared the kinetics of intravenously administered recombinant produced alpha-1-AT (r alpha-1-AT) and purified normal human plasma alpha-1-AT (p alpha-1-AT) in the blood and lung of rhesus monkeys. The r alpha-1-AT was produced in yeast transformed with an expressing plasmid containing a full-length human alpha-1-AT complementary deoxyribonucleic acid and purified to greater than 99% homogeneity. The r alpha-1-AT has a molecular weight of 45,000, no carbohydrates, and is identical in sequence to normal plasma alpha-1-AT except for an additional N-terminal acetylmethionine. Despite its lack of carbohydrates, the r alpha-1-AT inhibited human neutrophil elastase with an association rate constant similar to that of p alpha-1-AT. Rhesus monkeys were infused intravenously with 120 mg/kg of r alpha-1-AT (n = 13) or p alpha-1-AT (n = 12) and the serum, urine, and lung epithelial lining fluid (ELF) concentrations of these molecules quantified at various intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the chemical, physical, and survival properties of normal and Z-variant alpha-1-antitrypsins.

A procedure has been developed for the purification of Z-type alpha-1-antitrypsin (alpha-1-AT) which is rapid, gentle, and results in good yields. From 4 units (750 ml) of fresh human plasma, obtained from two individuals possessing the Pizz phenotype, 53 mg of pure Z-type alpha-1-AT was obtained. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis, both in the presence and absence of sodium dodecyl sulfate, and by analytical ultracentrifugation. When compared to pure alpha-1-AT from plasma of individuals possessing the normal PiMM phenotype, the two proteins were indistinguishable with respect to amino acid composition, sedimentation coefficient (s20w of 3.33 for both M and Z), molecular weight (51,000 by sodium dodecyl sulfate gel electrophoresis and 47,000 by sedimentation equilibrium for both M and Z), and trypsin-combining ratio (0.91 for Z and 0.99 for M). The only difference which was observed between the variant forms of alpha-1-AT was in the carbohydrate composition. The Z-type alpha1-AT contains between 20 and 25% less carbohydrate than the M-type alpha-1-AT. Specifically, the Z-type alpha-1-AT is deficient in 1 glucosamine residue, 3 neutral sugar residues (1 mannose and 2 galactose), and 2 sialic acid residues. Although the Z-variant is deficient in sialic acid, its survival time in the serum of a rabbit was not significantly different from that of M-type alpha-1-AT.     

Isolation, chemical, and physical properties of alpha-1-antitrypsin.

A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces.     

Inhibitory spectrum of mouse contrapsin and alpha-1-antitrypsin against mouse serine proteases

Contrapsin and alpha-1-antitrypsin have been recently characterized as major protease inhibitors in mouse plasma (Takahara, H. & Sinohara, H. (1982) J. Biol. Chem. 257, 2438-2446). We have studied the effects of the two inhibitors upon various serine proteases prepared from mouse tissues. Trypsin, plasmin and trypsin-like proteases of the submaxillary gland were inhibited by contrapsin but not by alpha-1-antitrypsin. On the other hand, chymotrypsin, elastase, and thrombin were inactivated by alpha-1-antitrypsin but not by contrapsin. Thus, their inhibitory spectra did not overlap each other in spite of their broad specificities. The inhibition of trypsin, chymotrypsin, and elastase was rapid and stoichiometric, whereas the inhibition of the other proteases was relatively slow. Contrapsin accounted for almost the total capacities of mouse plasma to inhibit both trypsin and submaxillary gland trypsin-like proteases, whereas alpha-1-antitrypsin was responsible for nearly all the capacities of plasma to inhibit both chymotrypsin and elastase.     

Isolation and Characterization of Alpha-1-Antitrrpsin from the Cohn Fraction Iv-1 of Human Plasma

Cohn Fraction IV-I from pooled human plasma was used as a starting material in the large-scale purification of alpha-1-antitrypsin (alpha-1-AT). Following ion-exchange, blospecific affinity and gel exclusion chromatographic procedures, material of high biological activity was obtained in 307percnt; overall yield. Homogeneity was demonstrated by acrylamide gel electrophoresis, immunoelectrophoresis, ultracentrifugation, gel filtration and end-group determination. The present preparation should be applicable to large scale industrial processing of alpha-1-AT with the potential for use in protein replacement therapy.     

Active site selective labeling of serine proteases with spectroscopic probes using thioester peptide chloromethyl ketones: demonstration of thrombin labeling using N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl

The feasibility of a new approach to incorporation of spectroscopic probes into the active sites of certain serine proteases has been demonstrated. The method is based on inactivation of a serine protease with a thioester derivative of a peptide chloromethyl ketone. The thiol group generated by reaction of the covalent enzyme-inhibitor complex with NH2OH provides a unique site for subsequent labeling with thiol-reactive probes. To evaluate the method, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl was synthesized by reaction of the thrombin-specific tripeptide chloromethyl ketone with succinimidyl (acetylthio)acetate and purified by sulfopropyl-Sephadex and Sephadex G-10 chromatography. Reverse-phase high-performance liquid chromatography indicated that the product was 90 +/- 3% pure. The compound was quantitated by using 5,5'-dithiobis(2-nitrobenzoic acid) to measure the concentration of thiol produced in the presence of NH2OH. On this basis, titrations of the irreversible loss of human alpha-thrombin activity had end points of 1.1 +/- 0.1 mol of inhibitor/mol of active sites, indicating a 1:1 stoichiometry for inactivation. Incubation of N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-thrombin with 5-(iodoacetamido)fluorescein in the presence of NH2OH resulted in incorporation of 0.96 mol of the fluorescence probe/mol of active sites and the appearance of fluorescein fluorescence associated with the active site containing B-chain on sodium dodecyl sulfate-polyacrylamide gels. Fluorescence labeling of thrombin required reaction of the inhibitor at the active site as well as subsequent generation of the thiol group with NH2OH. It is concluded that active site selective labeling can be achieved by using this approach, which is likely to be applicable to other proteases, peptide chloromethyl ketones, and a wide variety of probes.     

Genetic Heterogeneity of Rabbit Alpha-1-Antitrypsin

Sixteen inbred or partially inbred strains of rabbits were investigated for electrophoretic and quantitative variations of alpha-1-antitrypsin (A-1-AT). We found interindividual differences in the electrophoretic A-1-AT patterns as well as quantitative differences in the concentrations of A-1-AT and the serum trypsin-inhibiting activity.

Three electrophoretic phenotypes were distinguished: M, P and MP. M was characterized by a predominant anodal A-1-AT band, and P had a major cathodal component. The MP pattern can be explained by the occurrence of the M and P components in the same serum due to heterozygosity.

The P pattern was associated with an A-1-AT concentration of approximately 56% of that in sera with the M phenotype. The levels of A-1-AT in sera with the MP phenotype were intermediate between those in M and P types.

In addition to the type-specific quantitative variation, we found a quantitative sexual dimorphism of a moderate degree: Female rabbits had A-1-AT concentrations 16% less than males.

     

Analysis of the Alpha-1-Antitrypsin Deficient Alleles M3S, MZ, and ZZ by Biochemical and Molecular Methods: A Family Study

Deficiency of alpha-1-antitrypsin (α₁-AT, a major protease inhibitor controlling tissue degradation) is a genetic disorder transmitted in a codominant autosomal form. It has more than 100 genetically determined variants. This study attempted to determine the degree of association between serum α₁-AT levels and phenotypes and to provide a strategy for reliable laboratory evaluation of deficiencies. The study group consisted of a 38-year-old male proband with clinical features of emphysema, his first-degree relatives, and healthy controls. Family history revealed a four-generation pedigree. Genomic DNA was isolated from peripheral blood leukocytes. Alpha-1-AT levels were determined from human serum by immunonephelometry. Phenotypes were determined by isoelectric focusing of blood samples. DNA sequences of coding exons were analyzed by the amplification DNA technique and direct sequencing. Inheritance and plasma levels of the ZZ, MM, M3S, and MZ phenotypes were confirmed by the family study. In the family members with deficiencies, plasma concentrations were 22.55% ± 5.15 (ZZ), 84.18% ± 5.18 (M3S), and 61.06% ± 7.15 (MZ) of the normal MM level. We found a close association between α₁-AT level and genotype. A combination of genotyping, quantification, and phenotyping is the optimal strategy for the laboratory evaluation of α₁-AT deficiency.     

Interactions of serine proteases with cultured fibroblasts

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.     

Molecular cloning and biochemical characterisation of proteases from Staphylococcus epidermidis.

We report the complete coding sequence and the partial amino acid sequence (determined by chemical sequencing) of Staphylococcus epidermidis extracellular cysteine (Ecp) and serine (Esp) proteases. The first enzyme shows an extended sequence similarity to Staphylococcus aureus cysteine protease (staphopain) and the second one resembles the serine protease produced by that species. The region directly upstream of the sequence coding for the mature protein in both enzymes displays significant homology to the profragments encoded by sspB and sspA, respectively, thus suggesting that the characterised enzymes may also be produced as proproteins. Furthermore, we report some biological properties of the cysteine protease, contributing to a better understanding of its role as a possible virulence factor. The proteolytic activity of this enzyme was rapidly and efficiently inhibited by human alpha-2-macroglobulin; however, human kininogen as well as cystatins (A, C and D) were not inhibitory. Moreover, the protease was capable of inactivating, by limited proteolysis, both alpha-1-antitrypsin and HMW-kininogen, but neither alpha-1-antichymotrypsin nor antithrombin III.     

Inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin A plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.     

Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes

By studying the inactivation of malaria parasite culture by cysteine protease inhibition using confocal microscopy of living cells and electron microscopy of high-pressure frozen and freeze-substituted cells, we report the precise step in the release of malaria parasites from erythrocytes that is likely regulated by cysteine proteases: the opening of the erythrocyte membrane, liberating parasites for the next round of infection. Inhibition of cysteine proteases within the last few minutes of cycle does not affect rupture of the parasitophorus vacuole but irreversibly blocks the subsequent rupture of the host cell membrane, locking in resident parasites, which die within a few hours of captivity. This irreversible inactivation of mature parasites inside host cells makes plasmodial cysteine proteases attractive targets for antimalarials, as parasite-specific cysteine protease inhibitors may significantly augment multi-target drug cocktails.     

Conformational properties of the disease-causing Z variant of α1-antitrypsin revealed by theory and experiment

The human serine protease inhibitor (serpin) α-1 antitrypsin (α1-AT) protects tissues from proteases of inflammatory cells. The most common disease-causing mutation in α1-AT is the Z-mutation (E342K) that results in an increased propensity of α1-AT to polymerize in the ER of hepatocytes, leading to a lack of secretion into the circulation. The structural consequences of this mutation, however, remain elusive. We report a comparative molecular dynamics investigation of the native states of wild-type and Z α1-AT, revealing a striking contrast between their structures and dynamics in the breach region at the top of β-sheet A, which is closed in the wild-type simulations but open in the Z form. Our findings are consistent with experimental observations, notably the increased solvent exposure of buried residues in the breach region in Z, as well as polymerization via domain swapping, whereby the reactive center loop is rapidly inserted into an open A-sheet before proper folding of the C-terminal β-strands, allowing C-terminal domain swapping with a neighboring molecule. Taken together, our experimental and simulation data imply that mutations at residue 342 that either stabilize an open form of the top of β-sheet A or increase the local flexibility in this region, may favor polymerization and hence aggregation.     

Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses.

A computer-assisted comparative analysis of the amino acid sequences of (putative) thiol proteases encoded by the genomes of several diverse groups of positive-stranded RNA viruses and distantly related to the family of cellular papain-like proteases is presented. A high level of similarity was detected between the leader protease of foot-and-mouth-disease virus and the protease of murine hepatitis coronavirus which cleaves the N-terminal p28 protein from the polyprotein. Statistically significant alignment of a portion of the rubella virus polyprotein with cellular papain-like proteases was obtained, leading to tentative identification of the papain-like protease as the enzyme mediating processing of the non-structural proteins of this virus. Specific grouping between the sequences of the proteases of alpha-viruses, and poty- and bymoviruses was revealed. It was noted that papain-like proteases of positive-stranded RNA viruses are much more variable both in their sequences and in genomic locations than chymotrypsin-related proteases found in the same virus class. A novel conserved domain of unknown function has also been identified which flanks the papain-like proteases of alpha-, rubi- and coronaviruses.     

Multiple forms of Ca-activated protease from rat brain and muscle

Three Ca-dependent proteases have been identified in rat brain and skeletal muscle using ion exchange, gel filtration, and substrate affinity chromatography. A high degree of homology exists among three enzymes from different sources. Both the high molecular weight protease (154,000) and lower molecular weight protease (96,000) show high affinity for calcium while the third protease (76,000) had low affinity for calcium. Transformation among the three enzymes was calcium-induced and the process was unidirectional, generating a lower molecular weight form with decreased affinity for calcium. The protease with low affinity for calcium was susceptible to calcium-induced inactivation by autocatalysis. Immunologically the three proteases were equivalent, if not identical, and the brain and muscle proteases cross-react. All three proteases degraded neurofilament proteins; however, the protease with low affinity for calcium had 3 to 6 times higher specific activity. It is suggested that the high molecular weight enzyme (154,000) may be the native form of the Ca-dependent protease present in vivo.     

Disruption of the murine alpha1-antitrypsin/PI2 gene.

Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.