Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils

We have examined the effect of the myeloperoxidase-hydrogen peroxide-halide system and of activated human neutrophils on the ability of serum alpha 1-protease inhibitor (alpha 1-PI) to bind and inhibit porcine pancreatic elastase. Exposure to the isolated myeloperoxidase system resulted in nearly complete inactivation of alpha 1-PI. Inactivation was rapid (10 to 20 s); required active myeloperoxidase, micromolar concentrations of H2O2 (or glucose oxidase as a peroxide generator), and a halide cofactor (Cl- or I-); and was blocked by azide, cyanide, and catalase. Intact neutrophils similarly inactivated alpha 1-PI over the course of 5 to 10 min. Inactivation required the neutrophils, a halide (Cl-), and a phorbol ester to activate secretory and metabolic activity. It was inhibited by azide, cyanide, and catalase, but not by superoxide dismutase. Neutrophils with absent myeloperoxidase or impaired oxidative metabolism (chronic granulomatous disease) failed to inactivate alpha 1-PI, and these defects were specifically corrected by the addition of myeloperoxidase or H2O2, respectively. Thus, stimulated neutrophils secrete myeloperoxidase and H2O2 which combine with a halide to inactivate alpha 1-PI. We suggest that leukocyte-derived oxidants, especially the myeloperoxidase system, may contribute to proteolytic tissue injury, for example in elastase-induced pulmonary emphysema, by oxidative inactivation of protective antiproteases.     

S-nitrosylated human alpha(1)-protease inhibitor

Alpha(1)-protease inhibitor (alpha(1)PI) is an acute phase plasma protein, and possesses a single cysteine residue at position 232. A single cysteinyl sulfhydryl of human alpha(1)PI is found to be readily S-nitrosylated by nitric oxide (NO) in vitro without affecting the inhibitory capacity against bovine trypsin or elastase, a major target protease of alpha(1)PI in vivo. S-nitroso-alpha(1)PI (S-NO-alpha(1)PI) was also formed by the reaction of alpha(1)PI with NO produced excessively by a murine macrophage cell line (RAW264 cells) upon infection with Salmonella typhimurium and in an ex vivo perfusion system of the liver obtained from lipopolysaccharide-treated rats. S-NO-alpha(1)PI (10(-9)-10(-6) M) induces a dose-dependent relaxation of the ring preparation of rabbit aorta. Also, S-NO-alpha(1)PI but not alpha(1)PI shows a potent inhibitory effect on platelet aggregation. Unprecedented observation is that S-NO-alpha(1)PI showed a potent bacteriostatic effect against a wide range of bacteria at the concentration of 1-10 microM, which was 10-1000-fold stronger than that of NO and other S-nitrosylated compounds including S-nitrosylated albumin and S-nitrosylated glutathione. These results suggest that S-NO-alpha(1)PI is produced as an NO sink under inflammatory conditions, where production of both alpha(1)PI and NO is highly up-regulated, and it may function as a soluble factor which consists of an innate defense system through not only the protease inhibitory activity but also its antibacterial activity and facilitating the peripheral blood flow. Therefore, S-nitrosylation of alpha(1)PI occurring under physiological conditions in vivo should diversify the biological functions contributing to cytoprotective effects of alpha(1)PI.     

Structure of the oligosaccharide chains in human alpha 1-protease inhibitor.

Two glycopeptides were obtained from alpha 1-protease inhibitor after extensive pronase digestion and chromatography on Bio-Gel P-10 and concanavalin A-Sepharose. these glycopeptides were characterized by compositional analysis and sequential exoglycosidase digestion followed at each step by methylation analysis. The partially methylated alditol acetates obtained were resolved by gas chromatography and identified by mass spectrometry. The proposes structures of the oligosaccharide moieties of the glycopeptides are given below. (formula: see text) The relative amounts of the two glycopeptides isolated from concanavalin A-Sepharose suggest that each protein molecule contains four carbohydrate chains; one large chain (A) and three small chains (B).     

Cell surface alpha 1-protease inhibitor on human peripheral mononuclear cells in culture

We have studied expression of alpha 1-protease inhibitor (alpha 1-PI) by human mononuclear cells. alpha 1-PI was detected on 50% of freshly isolated peripheral mononuclear cells. Unless a proliferative stimulus was provided, alpha 1-PI subsequently disappeared from the cell surfaces. Plant mitogens, periodate, neuraminidase-galactose oxidase, or allogeneic cells all were effective stimuli of alpha 1-PI expression. Concanavalin A stimulated de novo synthesis of alpha 1-PI in cell cultures containing both lymphocytes and mononuclear phagocytes, and alpha 1-PI simultaneously appeared on surfaces of activated lymphocytes. Inhibition of protein synthesis by cycloheximide or monocyte depletion abolished de novo alpha 1-PI synthesis, but only monocyte depletion inhibited alpha 1-PI expression. Lymphocytes, but not monocytes, displayed saturable binding of radioiodinated alpha 1-PI. The data are consistent with the interpretation that human mononuclear phagocytes synthesize and secrete alpha 1-PI. When protein synthesis is inhibited, mitogenic stimuli may provoke release of previously synthesized alpha 1-PI from mononuclear phagocytes. Secreted alpha 1-PI then may bind to specific lymphocyte cell surface receptors. This pattern of alpha 1-PI synthesis, secretion, binding, and expression on lymphoid cell surfaces appears to be a common characteristic of many immunologic reactions in vitro.     

Divergent expression of alpha1-protease inhibitor genes in mouse and human.

The alpha1-protease inhibitor proteins of laboratory mice are homologous in sequence and function to human alpha1-antitrypsin and are encoded by a highly conserved multigene family comprised of five members. In humans, the inhibitor is expressed in liver and in macrophages and decreased expression or inhibitory activity is associated with a deficiency syndrome which can result in emphysema and liver disease in affected individuals. It has been proposed that macrophage expression may be an important component of the function of human alpha1-antitrypsin. Clearly, it is desirable to develop a mouse model of this deficiency syndrome, however, efforts to do this have been largely unsuccessful. In this paper, we report that aside from the issues of potentially redundant gene function, the mouse may not be a suitable animal for such studies, because there is no significant expression of murine alpha1-protease inhibitor in the macrophages of mice. This difference between the species appears to result from an absence of a functional macrophage-specific promoter in mice.     

Role of disulfide exchange in alpha 1-protease inhibitor.

The major endogenous inhibitor of neutrophil elastase in the plasma, alpha 1-protease inhibitor (alpha 1-PI), has a single cysteine residue which has been shown to form mixed disulfides with a number of thiols in vitro. Under normal physiological conditions, the plasma concentrations of reduced and oxidized thiols are such that a major fraction of alpha 1-PI in the circulation in vivo is in the form of mixed disulfides [Laurell, C.-B. (1979) in The Chemistry and Physiology of Human Plasma Proteins (Bing, D. H., Ed.) pp 329-341, Pergamon, New York]. We show here that the mixed disulfide between glutathione or cysteine and alpha 1-PI (alpha 1-PI-SSG or alpha 1-PI-SScys) has an intrinsic fluorescence which distinguishes it from the reduced form of alpha 1-PI. By employing the fluorescence difference, we have measured the ratio of alpha 1-PI-SH to mixed disulfide alpha 1-PI in redox buffers of different ratios of reduced to oxidized glutathione (GSH to GSSG) or reduced to oxidized cysteine (cys to cysSScys) and have calculated an equilibrium constant and redox potential of 0.74 +/- 0.08 and 8 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SSG couple and of 0.32 +/- 0.02 and 29 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SScys couple. We are unable to detect any change in Trp fluorescence in the complex of alpha 1-PI and elastase when the preformed complex is added to the same GSH/GSSG or cys/cysSScys redox buffers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of chymotrypsinogens with alpha 1-protease inhibitor

In a previous report [Largman, C., Brodrick, J.W., Geokas, M.C., Sischo, W.M., & Johnson, J.H. (1979) J. Biol. Chem. 254, 8516-8523] it was demonstrated that human proelastase 2 and alpha 1-protease inhibitor react slowly to form a complex that is stable to denaturation with sodium dodecyl sulfate and beta-mercaptoethanol and that the zymogen can be recovered from the isolated complex following dissociation by hydroxylamine. The present report demonstrates that bovine chymotrypsinogen A reacts with human alpha 1-protease inhibitor in a very similar manner. The rate of complex formation was measured by two methods. In the first, the reaction was followed by determining the loss of the inhibitory activity of alpha 1-protease inhibitor as a function of time. A second-order rate constant for complex formation formation (pH 7.6, 36 degrees C) of 12.9 +/- 2.4 M-1s-1 was obtained. In the second procedure, the reaction of fluorescein isothiocyanate labeled chymotrypsinogen A with alpha 1-protease inhibitor was measured by fluorescence polarization. A second-order rate constant (pH 7.6, 37 degrees C) of 13.9 +/- 2.1 M-1s-1 was obtained. The rate of complex formation is approximately 10(-5) of that measured for the reaction of bovine chymotrypsin with alpha 1-protease inhibitor. Dissociation of the complex was not observed after dilution or the addition of excess bovine alpha-chymotrypsin. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments, human chymotrypsinogens I and II react with alpha 1-protease inhibitor at rates that are approximatley equivalent to that determined for bovine chymotrypsinogen A. In contrast, bovine trypsinogen reacts very slowly with alpha 1-protease inhibitor, at a rate that is at most 10(-2) of that of bovine chymotrypsinogen A. These results suggest that zymogens react with alpha 1-protease inhibitor by virtue of partially formed active sites and that the potential active-site specificity of the zymogen in part determines the rate of complex formation.     

Growth-associated glycosylation of transferrin secreted by HepG2 cells.

Growth-associated alterations in the oligosaccharide structures of transferrin secreted by HepG2 cells were examined by concanavalin A-crossed affinoimmuno-electrophoresis. Slowly dividing, confluent cultures produced transferrin with a approximately 4.5-fold greater proportion of biantennary complex-type oligosaccharides than rapidly dividing, subconfluent cultures. The activity of N-acetylglucosaminyltransferase V (GlcNAc-T V) and galactosyltransferase were measured in cell extracts from subconfluent and confluent cultures. While the activity of galactosyltransferase remained relatively constant, the activity of GlcNAc-T V was approximately 3.2-fold lower in confluent cultures than in subconfluent cultures. These results suggest that the growth-associated alteration in the oligosaccharides of transferrin is due, at least in part, to the regulation of GlcNAc-T V activity.     

Inhibition of neutrophil elastase by alpha1-protease inhibitor at the surface of human polymorphonuclear neutrophils

The uncontrolled proteolytic activity in lung secretions during lung inflammatory diseases might be due to the resistance of membrane-bound proteases to inhibition. We have used a new fluorogenic neutrophil elastase substrate to measure the activity of free and membrane-bound human neutrophil elastase (HNE) in the presence of alpha1-protease inhibitor (alpha1-Pi), the main physiological inhibitor of neutrophil serine proteases in lung secretions. Fixed and unfixed neutrophils bore the same amounts of active HNE at their surface. However, the HNE bound to the surface of unfixed neutrophils was fully inhibited by stoichiometric amounts of alpha1-Pi, unlike that of fixed neutrophils. The rate of inhibition of HNE bound to the surface of unfixed neutrophils was the same as that of free HNE. In the presence of alpha1-Pi, membrane-bound elastase is almost entirely removed from the unfixed neutrophil membrane to form soluble irreversible complexes. This was confirmed by flow cytometry using an anti-HNE mAb. HNE activity rapidly reappeared at the surface of HNE-depleted cells when they were triggered with the calcium ionophore A23187, and this activity was fully inhibited by stoichiometric amounts of alpha1-Pi. HNE was not released from the cell surface by oxidized, inactive alpha1-Pi, showing that active inhibitor is required to interact with active protease from the cell surface. We conclude that HNE activity at the surface of human neutrophils is fully controlled by alpha1-Pi when the cells are in suspension. Pericellular proteolysis could be limited to zones of contact between neutrophils and subjacent protease substrates where natural inhibitors cannot penetrate.     

Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in human hepatoma cells (HepG2). Up-regulation by interleukin-6 and transforming growth factor beta 1.

Metalloproteinases and their specific inhibitors, believed to play a role in extracellular matrix metabolism, are regulated by inflammatory cytokines. Here we have addressed the question of whether liver, the major site of synthesis of plasma proteinase inhibitors, is also capable of synthesizing the tissue inhibitor of metalloproteinase-1 (TIMP-1). We show at mRNA and protein levels that TIMP-1 is expressed in differentiated human hepatoma cells (HepG2) and that its synthesis is up-regulated by interleukin-6 (IL-6), transforming growth factor beta 1 and phorbol 12-myristate 13-acetate. The physiological role of this phenomenon is underlined by the fact that lipopolysaccharide administration into rats in vivo, as well as IL-6-stimulation of rat hepatocytes in primary culture, also leads to an increase of TIMP-1 mRNA in liver cells.     

A new method for the determination of alpha1-protease inhibitor (alpha1-antitrypsin) phenotypes based on the formation of alpha1-protease inhibitor allele product-elastase complexes.

Up until now it has been assumed that the protease-binding property of alpha1-protease inhibitor (alpha1PI) was destroyed by acid starch gel electrophoresis (pH 4.9). Analyses on acid starch gel blocks for pH and conductivity changes during and following a typical electrophoretic run showed that it was unlikely that the separating alpha1PI would be exposed to pH values lower than 6.2, and that the allele products, following the passage of the buffer front, were in an environment of constant pH(6.3), extremely low conductivity and high field strength. These results strongly suggested the likelihood that alpha1-PI would be chemically and physically unchanged as a result of exposure to acid starch gel electrophoresis. In order to test this likelihood, human serum was electrophoretically separated in acid starch gel and following electrophoresis, was immersed in 0.1 M diethylbarbiturate buffer, pH 8.6, containing 20 mug/ml of pancreatic elastase. The pH-adjusted (8.15) and elastase-impregnated starch gel layer was superimposed on hemoglobin-agar for 2.5 h at 37 degrees C followed by immersion of the hemoglobin-agar layer in 1% NaCl overnight, distilled water for 2 h, drying under filter paper and staining. The results showed zones of undigested hemoglobin indicating, unequivocally, that the separated alpha1PI allele products are capable of forming complexes with proteases and that alpha1PI is not inactivated following exposure to acid starch gel electrophoresis. Densitometric analysis of the transparent stained zones on a clear agar gel background offers an alternative to analysis of the acid starch gel-separated zones by antigen-antibody crossed electrophoresis and as such is suitable for identification of alpha1-protease inhibitor phenotypes. Further, the method is specific for alpha1PI and a densitometric scan provides direct information relative to the protease-binding capacity of the sample as well as the contribution of each alpha1PI allele product to that capacity.     

Linear structure of the oligosaccharide chains in alpha1-protease inhibitor isolated from human plasma.

Two glycopeptides present in equal amounts were isolated from a pronase digest of alpha1-protease inhibitor of human plasma by gel filtration on Sephadex G-50 and chromatography on DEAE-cellulose. The carbohydrate side chains in both glycopeptides are linked through asparaginyl residues. The glycopeptides were digested sequentially with specific glycosidases; and after each step, the released sugars as well as the composition of the residual peptides were determined. The linear structures of these glycopeptides deduced from these data are shown below. Based on the total carbohydrate content of the intact protein and with these structural data, it is postulated that 4 oligosaccharide units are attached to 1 molecule of the protein; 2 of these were represented as in Equation 1, the other 2 as in Equation 2.     

Identification of major cellular proteins synthesized in response to interleukin-1 and interleukin-6 in human hepatoma HepG2 cells

Interleukin-1 (IL-1) and interleukin-6 (IL-6) are principal proinflammatory cytokines inducing the acute phase response of various tissues, including liver. Cultured human hepatoma HepG2 cells were stimulated with IL-1 (10 ng/ml) and IL-6 (10 ng/ml). After 24 h the cells were collected and disrupted by sonication in a lysis buffer containing 8M urea. The extracted cellular proteins were separated by 2D polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 and the protein spots showing different intensities in comparison to control (unstimulated) cells were excised and subjected to analysis by LC-MS/MS. Alternatively, proteins were stained with SYPRO Ruby. These differentially expressed proteins include seven up-regulated and two down-regulated intracellular proteins of various functions. The identification of three cytokine-responsive proteins was confirmed by biosynthetic labeling with [35S]methionine after incubation of HepG2 cells, and by western blot with specific antisera.     

Partial preparative separation and properties of the isoinhibitors of human alpha 1-antitrypsin (alpha 1-protease inhibitor).

Human lapha 1-antitrypsin (alpha 1-protease inhibitor) was chromatographed on a DEAE-cellulose column at pH 6.4. After elution with a linearly increasing concentration of NaCl, five pools (pools I-V) were formed from the eluate, pool I corresponding to the lowest and pool V to the highest concentration of salt. As demonstrated by analytical isoelectric focusing, with increasing concentrations of NaCl the concentration of the cathodal isoinhibitors gradually decreased and the concentration of the anodal ones increased in the pools. Pool I contained only three cathodal and pool V only three anodal isoinhibitors with a limited overlap between the pools. In contrast with the isoinhibitor composition, the sialic acid contents of the pools did not vary with the elution conditions. In line with the chemical evidence, desialylation of the fractions did not affect their electrofocusing positions relative to one another and did not abolish the microheterogeneity of the protein.     

Preparation of neuraminidase-resistant human alpha 1-protease inhibitor and its clearance in rat blood circulation

The sialic acid residues of human alpha 1-protease inhibitor were modified by periodate oxidation and subsequent reductive amination with ethanolamine and sodium cyanoborohydride. The modified inhibitor retained its original trypsin inhibitory activity and was not digested by neuraminidase from Clostridium perfringens. The modified inhibitor disappeared from rat blood circulation at the same rate as the native inhibitor.     

Carbohydrate composition of normal and variant human alpha 1-protease inhibitors.

Carbohydrate composition of normal human alpha 1-protease inhibitor (PiM₁) and several variant inhibitors (PiM₂, PiM₃, PiA, PiS, and PiZ) was determined by methanolysis of the samples followed by quantitative analysis of both neutral and amino sugars using gas-liquid chromatography. All normal and variant inhibitors contained nine mannose, seven galactose, ten N-acetylglucosamine, and eight N-acetylneuraminic acid residues per molecule, and no significant difference was found in their carbohydrate compositions. PiA is a variant with the fastest anodal electrophoretic mobility, and PiZ is a variant with the slowest mobility thus far reported. The differences in electrophoretic mobility of these Pi variants are entirely due to their amino acid substitutions determined previously. These amino acid substitutions have no effect on the carbohydrate structure of the protease inhibitor.     

Primary structure of human alpha1-protease inhibitor. The complete amino acid sequence of cyanogen bromide fragment II.

The complete amino acid sequence of a CNBr-fragment from human alpha1-protease inhibitor has been determined and is shown below. The peptide consists of 109 amino acid residues with 1 oligosaccharide unit. The 2 glutamic acid residues which have previously been shown to be substituted by lysine in the Z and by valine in the S mutant proteins are both located in this CNBr fragment. (formula: see text).