Major Serine Protease Inhibitor Research Articles

Quantification of circulating alpha-1-antitrypsin polymers associated with different SERPINA1 genotypes.

Alpha-1-antitrypsin deficiency is a genetic disorder caused by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin (AAT), the major serine protease inhibitor in plasma. Reduced AAT levels are associated with elevated risk of developing emphysema mainly due to uncontrolled activity of neutrophil elastase in the lungs. The prevalent Z-AAT mutant and many rare pathogenic AAT variants also predispose to liver disease due to their accumulation as polymeric chains in hepatocytes. Part of these polymers are secreted into the bloodstream and could represent biomarkers of intra-hepatic accumulation. Moreover, being inactive, they further lower lung protection against proteases. Aim of our study is to accurately quantify the percentage of circulating polymers (CP) in a cohort of subjects with different SERPINA1 genotypes. CP concentration was measured in plasma or Dried Blood Spot (DBS) by a sensitive sandwich ELISA based on capture by the polymer-specific 2C1 monoclonal antibody. CP were significantly elevated in patients with the prevalent PI*SZ and PI*ZZ genotypes, with considerable intra-genotype variability. Notably, higher percentage of polymers was observed in association with elevated C-reactive protein. CP levels were also increased in carriers of the Mmalton variant, and of Mprocida, I, Plowell and Mherleen in heterozygosity with Z-AAT. These findings highlight the importance of implementing CP quantification in a clinical laboratory. Indeed, the variable amount of CP in patients with the same genotype may correlate with the variable severity of the associated lung and liver diseases. Moreover, CP can reveal the polymerogenic potential of newly discovered ultrarare AAT variants.

Therapeutic benefits of recombinant alpha1-antitrypsin IgG1 Fc-fusion protein in experimental emphysema

BackgroundAlpha-1 antitrypsin (AAT) is a major serine protease inhibitor. AAT deficiency (AATD) is a genetic disorder characterized by early-onset severe emphysema. In well-selected AATD patients, therapy with plasma-derived AAT (pAAT), “augmentation therapy”, provides modest clinical improvement but is perceived as cumbersome with weekly intravenous infusions. Using mouse models of emphysema, we compared the effects of a recombinant AAT-IgG1 Fc-fusion protein (AAT-Fc), which is expected to have a longer half-life following infusion, to those of pAAT.MethodsIn an elastase model of emphysema, mice received a single intratracheal instillation of porcine pancreatic elastase (PPE) or human leucocyte elastase (hLE). AAT-Fc, pAAT, or vehicle was administered intraperitoneally 1 day prior to or 3 weeks following elastase instillation. Lung function and histology assessments were performed at 7 and 32 days after elastase instillation. In a cigarette smoke (CS) model of emphysema, mice were exposed to CS daily, 5 days a week, for 6 months and AAT-Fc, pAAT, or vehicle were administered every 10 days during the last 3 months of CS exposure. Assessments were performed 3 days after the last CS exposure. Immune responses to lung elastin peptide (EP) and the effects of AAT-Fc or pAAT treatment on dendritic cell (DC) function were determined ex vivo.ResultsBoth elastase instillation and CS exposure triggered emphysema-like alveolar enlargement, increased lung compliance, and increased markers of inflammation compared to controls. Administration of AAT-Fc either prior to or following elastase instillation or during CS exposure provided greater protection than pAAT against alveolar enlargement, lung dysfunction, and airway inflammation. When challenged ex vivo with EP, spleen mononuclear cells from elastase-exposed mice exhibited dose-dependent production of IFNγ and IL-17, suggesting immune reactivity. In co-culture experiments with splenic CD4+ T cells isolated from elastase-exposed mice, AAT-Fc treatment prior to EP-priming of bone marrow-derived dendritic cells inhibited the production of IFNγ and IL-17.ConclusionsCompared to pAAT, AAT-Fc more effectively prevented or attenuated elastase- and CS-induced models of emphysema. These effects were associated with immunomodulatory effects on DC activity. AAT-Fc may provide a therapeutic option to individuals with AATD- and CS-induced emphysema.

The Utilization of Alpha-1 Anti-trypsin (A1AT) in Infectious Disease Monitoring and Treatment

Alpha one anti-trypsin (A1AT) is a major serine protease inhibitor found circulating in human blood. A1AT related studies mainly focus on A1AT potential biomarker as well as therapeutic target in non-infectious diseases. Their findings indicate A1AT beneficial features may also be applied for monitoring and treating infectious disease. However, only a few studies have reviewed A1AT’s useful properties as a biomarker and therapeutic agent for infectious diseases. This narrative review aims to summarize growing evidences that support the idea of utilizing A1AT as a tool for monitoring and therapy for infectious diseases. A1AT showed potential as a biomarker for a wide spectrum of infectious disease, from virus, bacteria, to parasite. Its level and functionality were proposed to predict risk for disease susceptibility or progression and to indicate response therapy. As promising therapeutic agent in various infectious diseases, the administration of A1AT has shown antimicrobial activity, immunomodulatory and anti-apoptotic effect, in addition to more familiar function, suppressing excessive proteolysis. The broad utilization of A1AT, both as biomarker and therapeutic agent, in studies on infectious diseases seems promising. However, there are issues need to be investigated further before establishing its feasibility as a monitoring and therapy tool against infection diseases . J Microbiol Infect Dis 2019; 9(1): 51-58.

Isolation of recombinant human antithrombin isoforms by Cellufine Sulfate affinity chromatography

Human antithrombin (hAT) is a major serine protease inhibitor that regulates blood coagulation in human plasma, and it has been applied for the treatment of antithrombin (AT) deficiency and disseminated intravascular coagulation (DIC). In the past, hAT for therapeutic use has been obtained from human plasma; however, hAT can now be sourced from transgenic animals and Chinese hamster ovary (CHO) cells by recombinant technology. The dominant form of hAT in plasma is the α form, which is glycosylated with four oligosaccharides and sialylated at its terminals. However, it would be preferable to remove the poorly sialylated α form of hAT, the minor β form with unoccupied glycosylation sites, and the inactive forms. Cellufine Sulfate, a heparin-mimic affinity resin made of cellulose and modified with sulfate groups, has an affinity for heparin-binding proteins and can be used in cation exchange chromatography. Based on these properties, the α and β forms of hAT could be separated and purified depending on the rate of sialylation. Consequently, Cellufine Sulfate was used to enrich the highly sialylated α form with a considerably higher step yield than that obtained using conventional heparin-immobilized resins. Furthermore, subsequent hydrophobic interaction chromatography could eliminate the inactive forms. These results suggested that Cellufine Sulfate was more effective than heparin-immobilized resins in purifying the highly sialylated α form of hAT for therapeutic applications.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2′ site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2′ sites on ShPI-1 specificity.

Elastase Inhibitory Activity of Airway α1-Antitrypsin Is Protected by Treatment With a Catalytic Antioxidant in a Baboon Model of Severe Bronchopulmonary Dysplasia

Recent studies in animal models of bronchopulmonary dysplasia (BPD) suggest that antioxidant treatments may be beneficial for the disease. However, the mechanisms by which these drugs improve the course of BPD are not completely known. Alpha1-antitrypsin (α1-AT) is one of the major serine protease inhibitors in human plasma that has antielastase and antiapoptotic activities. Both activities of α1-AT are dependent on its reactive site loop (RSL), which is highly susceptible to oxidative inactivation. In this study, we investigated the elastase inhibitory activity of α1-AT in two different baboon models of BPD, the "new BPD" and the "severe BPD" models, and determined the effect of treatment with a catalytic antioxidant, Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP), on the elastase inhibitory activity of α1-AT in the severe BPD model. Our results demonstrate the presence of sufficient elastase inhibitory activity of the airway α1-AT in the new but not in the severe BPD model. Treatment of severe BPD group baboons with the catalytic antioxidant MnTE-2-PyP resulted in augmentation of the elastase inhibitory activity of α1-AT. These findings suggest that prevention of the oxidative inactivation of α1-AT may be one of the mechanisms by which antioxidant therapy improves the pulmonary outcomes in animal models of severe BPD.

Haplotypes of the alpha-1 antitrypsin gene in healthy controls and Z deficiency patients

Alpha-1 antitrypsin (AAT; HUGO symbol, SERPINA1) is one of the major serine protease inhibitors (serpins) in human plasma. Deficiency of AAT is a recognised risk factor for chronic obstructive pulmonary disease (COPD), attributed to uninhibited neutrophil elastase released into the lung tissue during inflammatory states. In this study we used sequencing to screen the exonic regions, 5' and 3' flanking sequence of the AAT gene in order to generate a high density map of single nucleotide polymorphisms (SNPs). 16 SNPs were identified throughout AAT. Haplotypes based on SNPs with a minor allele frequency of > or =5% were estimated using genotypic information from 225 healthy control individuals and 41 AAT deficient Pi-ZZ individuals. AAT shows a large amount of variation in the control population, with 17 haplotypes accounting for 88% of the observed variation. The haplotype distribution of the common deficiency Pi-Z variant of AAT was significantly different when compared to the normal variants. In addition to the haplotype information, we present evidence for a functional effect of a SNP in intron 1.

Cathepsin B, L, and S Cleave and Inactivate Secretory Leucoprotease Inhibitor

A number of serine proteases, matrix metalloproteases, and cysteine proteases were evaluated for their ability to cleave and inactivate the antiprotease, secretory leucoprotease inhibitor (SLPI). None of the serine proteases or the matrix metalloproteases examined cleaved the SLPI protein. However, incubation with cathepsins B, L, and S resulted in the cleavage and inactivation of SLPI. All three cathepsins initially cleaved SLPI between residues Thr(67) and Tyr(68). The proteolytic cleavage of SLPI by all three cathepsins resulted in the loss of the active site of SLPI and the inactivation of SLPI anti-neutrophil elastase capacity. Cleavage and inactivation were catalytic with respect to the cathepsins, so that the majority of a 400-fold excess of SLPI was inactivated within 15 min by cathepsins L and S. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of cleaved SLPI in these samples whereas only intact SLPI was observed in control epithelial lining fluid samples. Active cathepsin L was shown to be present in emphysema epithelial lining fluid and inhibition of this protease prevented the cleavage of recombinant SLPI added to emphysema epithelial lining fluid. Taken together with previous data that demonstrates that cathepsin L inactivates alpha(1)-antitrypsin, these findings indicate the involvement of cathepsins in the diminution of the lung antiprotease screen possibly leading to lung destruction in emphysema.

Glycosylation of human alpha 1-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts.

Human alpha 1-antitrypsin (alpha 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human alpha 1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant alpha 1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant alpha 1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted alpha 1-AT. The human alpha 1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of alpha 1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of alpha 1-AT in S. cerevisiae. The alpha 1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of alpha 1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.

Serine protease inhibitors in patients with chronic viral hepatitis

Background/Aims: This study aimed to determine whether deficiency of the major serine protease inhibitors (α1-antitrypsin (AAT) or α1-antichymotrypsin (ACT) is associated with increased risk for chronic hepatitis B or C virus (HBV or HCV) infection.Methods: We studied 709 adults with chronic liver disease who had undergone liver biopsy during the 14-year period 1978–92. Anti-HCV testing was carried out with second-generation ELISA and immunoblot assays (RIBA 2). HBV markers were tested with commercially available radioimmunoassays. ACT and AAT concentrations in plasma were measured with electroimmunoassay and immune nephelometry. Plasma samples were screened for the AAT PiZ deficiency' with ELISA technique and phenotyped by isoelectric focusing. The 229Pro→Ala mutation for ACT deficiency was identified by PCR techniques.Results: Of the 709 patients, 132 (18.6%) were positive for anti-HCV according to RIBA 2. PiZ AAT deficiency was found in 44 (6.2%) of patients (one PiZZ, 38 PiMZ, and PiSZ), while subnormal ACT levels were found in 33 (4.6%) patients, frequencies that were higher than expected in the general population (p=0.0375 and p<0.0001, respectively). Of the PiZ-carriers, 8/44 (18%) were found to be anti-HCV positive according to RIBA 2, as compared to 123/662 (19%) non-PiZ-carriers (p> 0.05). One of these patients had cirrhosis, four chronic active hepatitis and three chronic persistent hepatitis. In contrast, 17/33 (51.5%) of the patients with subnormal ACT were anti-HCV positive (OR=5.2, CI=2.6–10.6; p<0.0001). No relationship was found between HBV infection and AAT deficiency or subnormal ACT levels. Only one patient with subnormal ACT levels was heterozygous for the 229Pro→Ala mutation of ACT deficiency. There was not significant difference in the histological findings when the patients with subnormal ACT levels or PiZ allele were subgrouped according to HCV status.Conclusions: There is no overrepresentation of chronic HBV or HCV in heterozygous AAT deficiency, although an association with more severe liver disease in such patients cannot be excluded. In contrast, low plasma levels of ACT that may be acquired or hereditary, due to mutations other than 229Pro→Ala, are frequent in HCV infection.

Molecular basis of proteolytic activation of Sendai virus infection and the defensive compounds for infection.

It has been proposed that the pathogenicity of Sendai virus is primarily determined by a host cellular protease(s) that activates viral infectivity by proteolytic cleavage of envelope fusion glycoproteins. We isolated a trypsin-like serine protease, tryptase Clara, localized in and secreted from Clara cells of the bronchial epithelium of rats. The enzyme specifically cleaved the precursor of fusion glycoprotein F0 of Sendai virus at residue Arg116 in the consensus cleavage motif, Gln(Glu)-X-Arg, resulting in the presentation of the membrane fusion domain in the amino-terminus of the F1 subunit. Administration of an antibody against tryptase Clara in the airway significantly inhibited the activation of progeny virus and multiple cycles of viral replication, thus reducing the mortality rate. These findings indicate that tryptase Clara in the airway is a primary determinant of Sendai virus infection and that proteolytic activation occurs extracellularly. We identified two cellular inhibitory compounds against tryptase Clara in bronchial lavage. One was a mucus protease inhibitor, a major serine protease inhibitor of granulocyte elastase in the lining fluids of the human respiratory tract, and the other was a pulmonary surfactant which may adsorb the enzyme, resulting in its inactivation. These compounds inhibited virus activation by tryptase Clara in vitro and in vivo, but did not themselves affect the hemagglutination and the infectivity of the virus. The functional domain of the mucus protease inhibitor against the enzyme, which is organized in two homologous N- and C-terminal domains, is located in the C-terminal. Administration of these compounds in the airway may be useful for preventing infection with Sendai virus.

SV40 Large T Antigen Directed by Regulatory Elements of the Human Alpha-1-Antitrypsin Gene

SV40 large T antigen (T-ag) is a potent oncogene able to transform many cell types. The human alpha-1-antitrypsin (AAT) gene encodes the major serine protease inhibitor in plasma. The 5' flanking sequence of the AAT gene contains cis-acting elements that specify the expression of reporter genes in transgenic mice. Although the AAT gene is expressed primarily in the liver, it also is expressed in epithelial cells of the stomach, kidney, intestine and pancreas. A fusion gene with the 5' flanking region of AAT driving the SV40 large T-ag gene was used to generate transgenic mice. Between 2 and 6 months of age, 6 of 7 founder mice developed hepatic neoplasms. Kidney hyperplasia (3/7) and carcinomas of the stomach (4/7) and pancreas (1/7) also were observed. Tumor cells in the different sites expressed T-ag that was detectable by immunohistochemical staining. A transgenic mouse line (1812) was established; liver tumors develop reproducibly in all offspring. Cellular changes progressing to liver tumor formation occur in discrete stages with predictable kinetics in animals of the 1812 line: (i) embryonal/fetal stage, no major histological changes; (ii) newborn to 2 weeks of age, hyperplastic livers with minor cellular changes; (iii) mice between 3 and 8 weeks of age, diffuse liver cell dysplasia without observable tumor nodules; (iv) mice 8 weeks of age and older, hepatocellular carcinomas and hepatoblastomas in a background of liver dysplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and characterization of a soluble dextran α1 proteinase inhibitor complex

Purified human alpha(1) proteinase inhibitor, a plasma glycoprotein with a molecular weight of 5.3 x 10(4) daltons and a major serine protease inhibitor has been covalently coupled to dextrans with molecular weights of 1.77 x 10(4) and 1.03 x 10(4) daltons. The coupled conjugates were soluble in aqueous medium and stable up to 6 months at +5 degrees C. Increased moles of dextran/mole protein ratio during coupling resulted in progressive decreases of inhibitory capacity, immunogenicity, and the association constant (k(assoc)) between the enzyme and the inhibitor. Compared to the native protein, the soluble conjugates showed improved stability at pH 3.0 and heat stability at 60 degrees C. At 60 degrees C, no loss of inhibitory capacity has been seen up to 60 min for the conjugates during which time the native protein lost greater than 90% of its inhibitory capacity. The presence of antioxidant catalase was needed to prevent oxidative degradation by hydrogen peroxide.

Acquisition of alpha 1-Antitrypsin by a pathogenic strain of Trichomonas vaginalis.

The interaction of alpha 1-Antitrypsin, the major serine protease inhibitor in plasma, with pathogenic Trichomonas vaginalis and the acquisition by trichomonads of this host protein from normal human plasma were investigated. alpha 1-Antitrypsin acquired by intact parasites could not be removed by repeated washings in phosphate-buffered saline. Saturation kinetics were observed after incubation of glutaraldehyde-fixed organisms with 125I-labeled alpha 1-antitrypsin. Evidence suggesting that specific parasite membrane sites were responsible for trichomonal acquisition of alpha 1-antitrypsin was obtained through competitive binding experiments using purified preparations of homologous versus heterologous plasma proteins. No evidence of degradation of bound antitrypsin by live parasites was observed. The avid binding of alpha 1-antitrypsin by pathogenic T. vaginalis after incubation in normal human plasma was demonstrated by using sensitive electrophoretic and immunodetection techniques. Radioimmunoprecipitation of intrinsically labeled, detergent-solubilized extracts of T. vaginalis incubated with monospecific antisera against alpha 1-antitrypsin and other human plasma proteins revealed the inability of parasites to biosynthesize any substance cross-reactive with host plasma proteins. Finally, T. vaginalis organisms pretreated with alpha 1-antitrypsin inhibited trypsin caseinase activity in an in vitro assay. The implications of these observations are discussed.

Kinetics of thermal denaturation of antithrombin III

Purified antithrombin III (AT III), a single-chain human plasma glycoprotein, molecular weight 58,000 daltons, and one of the major serine protease inhibitors, was heated in the 60-70 degrees C range for inactivating possible contaminations by hepatitis B virus (HBV). Loss of inhibitory activity, unfolding of tertiary structure, and the rate of aggregate formation of AT III were monitored experimentally during heatig. Sucrose and sodium citrate were demonstrated to stabilize the protein. From the rate data the calculated activation energies (E) showed E(tert. struct.) < E(biol. act.) < E(aggreg.) indicating the order (lower activation energy process first) in which heat causes these changes in the protein molecule. The activation energy corresponding to denaturation of HBV was estimated to be at least fourfold lower than that associated with the unfolding of the tertiary structure of the protein. Purified AT III, thus stabilized and pasteurized, should be therapeutically effective, and the risk for transmission of hepatitis B should be decreased significantly.

Cloning and sequence of cDNA coding for alpha 1-antitrypsin.

Recombinant plasmids containing human and baboon cDNA have been screened for alpha 1-antitrypsin, a major serine protease inhibitor present in blood. One plasmid, designated pBa alpha 1a2, was found to contain a cDNA insert of 1352 base pairs coding for the baboon inhibitor. It included 45 nucleotides that code for 15 amino acids present in the amino-terminal signal sequence of the protein, 1182 nucleotides that code for 394 amino acids in the mature protein, a stop codon, and a noncoding region of 76 nucleotides. Comparison of the amino acid sequences of baboon alpha 1-antitrypsin, human antithrombin III, and chicken ovalbumin indicated that these three proteins are about 230% homologous. A second plasmid, designated pH alpha 1a1, was found to contain a human cDNA insert of 306 base pairs. This plasmid coded for 69 amino acids present in the carboxyl-terminal region of human alpha 1-antitrypsin. The human and baboon cDNAs and their amino acid sequences are greater than 96% homologous.

Isolation of the major serine protease inhibitor from the 5-day serum-free conditioned medium of human embryonic lung cells and demonstration that it is fetuin.

Human embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin. Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets. It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction. This paper reports the isolation and purification of the major serine protease inhibitor in 5-day serum-free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS). Both the CM-derived inhibitor and the FBS-derived inhibitor are identical with fetuin, the major glycoprotein of FBS. The CM-derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum-free CM collection periods. It is not labeled metabolically with 3H-leucine. Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS-PAGE, non-SDS basic pH,PAGE, and isoelectric focusing. The CM-derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator. They are immunologically indistinguishable. The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum. These molecules may serve to protect HuEL cells against proteases they generate.