Fibrillogenic C-terminal fragment of alpha-1-antitrypsin activates human monocytes via oxidative mechanisms

Production of alpha-1-antitrypsin by human monocytes is an important factor in controlling tissue damage by proteases in the microenvironment of inflammation. Increases of four- to eightfold in levels of native and fragmented forms of alpha-1-antitrypsin have been detected in inflammatory loci in vivo. In this study we have extended our previous observation that the carboxyl-terminal peptide (C-36) of alpha-1-antitrypsin produced by specific proteinase cleavage, when added in its fibrillar form at concentrations of 5 microM or more to monocytes in culture, induces cytotoxic effects. Experiments with synthetic amyloid-forming peptides suggest fibril cytotoxicity to be mediated via a common oxidative stress mechanism. We undertook to determine whether C-36 fibril cytotoxicity also involves this common pathway. Monocytes stimulated with C-36 fibrils for 1 h showed significant elevation in monocyte chemoattractant protein-1 expression, induced reduced nicotinamide-adenine dinucleotide phosphate oxidase activity, increased intracellular lipid peroxidation, altered mitochondrial membrane potential, and increased cytosolic cytochrome c and caspase-3 activity. Treatment of monocytes with C-36 fibrils after 24 h also resulted in increased cytosolic cathepsin D activity, suggesting that lysosomes may also be destabilized over longer periods of time. In contrast, native alpha-1-antitrypsin only showed concentration and time-dependent effects on chemoattractant protein-1 expression, and these appear to be independent of oxidative stress. These results indicate that the cytotoxicity of the fibrillar fragment is mediated via oxidative mechanisms and support important multiple roles for native and also for cleaved forms of alpha-1-antitrypsin in monocyte recruitment and activation during inflammatory processes such as atherosclerosis.     

Affinity chromatography of alpha-1-protease inhibitor using Sepharose-4B-bound anhydrochymotrypsin

Sepharose 4B-bound bovine anhydrochymotrypsin (AnhCT), a catalytically inactive form of chymotrypsin, was shown to be effective for retaining active alpha-1-protease inhibitor (alpha-1-PI, also alpha-1-antitrypsin) from human plasma, while showing no measurable affinity for oxidized or protease complexed alpha-1-PI, or for most other plasma proteins. alpha-1-PI eluted from this resin with 0.1 M chymostatin retained full activity against trypsin, chymotrypsin, and elastase. In addition to alpha-1-PI, AnhCT-Sepharose binds a limited number of other plasma proteins. Using monospecific antisera to plasma protease inhibitors, one of these proteins was identified as inter-alpha-trypsin inhibitor, and it was recoverable in active form. Therefore, an AnhCT-Sepharose 4B resin has been demonstrated to be of value for isolating active forms of alpha-1-PI from solutions, and may also be useful for the isolation of inter-alpha-trypsin inhibitor.     

Detection of inhibitory inactive fraction of alpha-1-antitrypsin in human serum

Pseudomonas aeruginosa elastase is not inhibited by human serum alpha-1-antitrypsin, it damages inhibitory activity of alpha-1-antitrypsin activity instead. A simple method, based on electrophoresis in urea containing polyacrylamide gel, is described for the separation of active part of the inhibitor from that inactivated by the bacterial enzyme.     

Serum concentration of alpha-2-macroglobulin, alpha-1-antitrypsin and alpha-2-antichymotrypsin in patients with lung carcinoma

Serum concentration of alpha-2-macroglobulin, alpha-1-antitrypsin and alpha-2-antichymotrypsin was evaluated in 26 patients with lung carcinoma. We observed an evident decrease in alpha-2-M and alpha-1-antitrypsin level and no differences between tested and control groups in alpha-1-antichymotrypsin concentration. The deficiency of protease inhibitors may be due to the increased level of protease activity in malignant cells. Infiltration of granulocytes near tumor and released enzymes from them may exhaust proteolytic inhibitory capacity, too. Increased protease activity is associated with transformation and uncontrolled proliferation, therefore antiproteases may be accepted as anticancerogenic factors. Further investigations are needed to bring us closer to understanding this question.     

The interaction of alpha-1-antitrypsin with soluble and sepharose-bound elastase.

The products resulting from the interaction of alpha-1-antitrypsin with elastase were examined with polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate, and by affinity chromatography. Five products of the reaction can be identified by polyacrylamide disc gel electrophoresis. Two products are complexes between alpha-1-antitrypsin and elastase (73 800 and 58 300 daltons). Two additional products are identical to fragments of alpha-1-antitrypsin which can be washed from a column of Sepharose-bound elastase immediately after alpha-1-antitrypsin is applied to the column. The larger component about 50 000 daltons, reacts with antiserum to alpha-1-antitrypsin, and does not inhibit enzymes. Together, these two products have an amino acid analysis similar to alpha-1-antitrypsin. These two fragments are probably hydrolytic products of the interaction of elastase with alpha-1-antitrypsin which is biologically inactive. The fifth product is probably a fragment of alpha-1-antitrypsin missing from the low molecular weight complex. The components of the complexes can be separated from each other by a mild nucleophilic attack. Small quantities of alpha-1-antitrypsin can be displaced from the elastase affinity column by phenyl methane sulfonyl fluoride. In conclusion, porcine pancreatic elastase forms two complexes with alpha-1-antitrypsin. One or both complexes can be split by alkali.     

Alpha-1-antitrypsin. Plasma survival studies in the rat of the normal and homozygote deficient forms.

Alpha-1-antitrypsin from normal individuals (Pi type MM) from those with an inherited deficiency of circulatory protein (Pi type ZZ) were labelled with 125I and plasma clearance rates measured in rats either prior to, or following treatment with neuraminidase to remove terminal sialic acid residues. In addition, these proteins and the derivatives were tested for their ability to bind to an hepatic binding protein obtained from rabbit liver membranes that has been shown to be responsible for the clearance of serum asialoglycoproteins. Finally, the two native forms of alpha-1-antitrypsin were treated with galactose oxidase followed by reduction with tritiated potassium borohydride and then analyzed for tritium incorporation in the neutral sugar fraction. The results indicate: (a) clearance from plasma for both forms of alpha-1-antitrypsin is dramatically enhanced upon the loss of terminal sialic acid residues to the liver membrane protein; (b) Z protein does not exhibit terminal galactosyl residues; (c) the low level of Z protein in plasma cannot be accounted for by a faster rate of clearance relative to M protein. The relevance of these findings to the alpha-1-antitrypsin deficiency state are discussed.     

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.     

Cavities of α1-antitrypsin that play structural and functional roles

The native form of inhibitory serine protease inhibitors (serpins) is strained, which is critical for their inhibitory activity. Previous studies on stabilizing mutations of alpha(1)-antitrypsin, a prototype of serpins, indicated that cavities provide a structural basis for the native strain of the molecule. We have systematically mapped the cavities of alpha(1)-antitrypsin that play such structural and functional roles by designing cavity-filling mutations at residues that line the walls of the cavities. Results show that energetically unfavorable cavities are distributed throughout the alpha(1)-antitrypsin molecule, and the cavity-filling mutations stabilized the native conformation at 8 out of 10 target sites. The stabilization effect of the individual cavity-filling mutations of alpha(1)-antitrypsin varied (0.2-1.9 kcal/mol for each additional methylene group) and appeared to depend largely on the structural flexibility of the cavity environment. Cavity-filling mutations that decreased inhibitory activity of alpha(1)-antitrypsin were localized in the loop regions that interact with beta-sheet A distal from the reactive center loop. The results are consistent with the notion that beta-sheet A and the structure around it mobilize when alpha(1)-antitrypsin forms a complex with a target protease.     

Does the PI polymorphism alone control alpha-1-antitrypsin expression?

Whether genetic factors other than the protease-inhibitor (PI) polymorphism itself contribute to variation in alpha-1-antitrypsin is of both theoretical and practical interest. We have measured the quantity of alpha-1-antitrypsin (by an immunoturbidometric assay) and its activity (by assaying elastase inhibitory capacity [EIC]) in 583 individuals from 114 twin kinships who were also typed for PI by isoelectric focusing. Models of variation were fitted directly to the raw observations by a maximum-likelihood method. Specification of phenotypic means led to highly significant improvements in fit over models including only individual environment variance and additive genetic variance. The 29 phenotype means could also be described as the appropriate additive combinations of the 12 allelic effects. Only small improvements in fit could then be obtained by addition of polygenic components of variance. We conclude that nearly all genetic variation in alpha-1-antitrypsin quantity and activity can be explained by detectable variation at the PI locus and that this variance is largely additive. Bivariate analysis of alpha-1-antitrypsin and EIC revealed marginal evidence for differences in specific activities of molecules coded by different PI alleles. The correlation between environmental deviations for the two measures was only .63, which may reflect, in part, the rather low reliability of the assays and account for the modest heritabilities (less than .5) of the two measures. An intriguing finding was the presence of significant differences in E1 variance for different PI types, suggesting that different phenotypes have differing capacities to react to environmental challenges.     

Concerted regulation of inhibitory activity of alpha 1-antitrypsin by the native strain distributed throughout the molecule

The native forms of common globular proteins are in their most stable state but the native forms of plasma serpins (serine protease inhibitors) show high energy state interactions. The high energy state strain of alpha(1)-antitrypsin, a prototype serpin, is distributed throughout the whole molecule, but the strain that regulates the function directly appears to be localized in the region where the reactive site loop is inserted during complex formation with a target protease. To examine the functional role of the strain at other regions of alpha(1)-antitrypsin, we increased the stability of the molecule greatly via combining various stabilizing single amino acid substitutions that did not affect the activity individually. The results showed that a substantial increase of stability, over 13 kcal mol(-1), affected the inhibitory activity with a correlation of 11% activity loss per kcal mol(-1). Addition of an activity affecting single residue substitution in the loop insertion region to these very stable substitutions caused a further activity decrease. The results suggest that the native strain of alpha(1)-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.     

Cytidine-5'-monophosphate-N-acetylneuraminic acid. Asialoglycoprotein sialic acid transferase activity in liver and serum of patients with juvenile hepatic cirrhosis and alpha-1-antitrypsin deficiency.

The molecular basis for the accumulation of a substance which displays the immunological reactivity of alpha-1-antitrypsin within vesicles of liver parenchymal cells of individuals with hepatic cirrhosis and serum alpha-1-antitrypsin deficiency remains unclear. We recently reported that serum from a patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis was substantially deficient in sialyltransferease (EC 2.4.99.1) an enzyme which transfers sialic acid from cytidine 5'-monophosphate-N-acetylneuraminic acid to a variety of asialoglycoprotein acceptors. In the present report we have extended these studies to include serum from five additional patients with alpha-1-antitrypsin deficiency and juvenile hepatic cirrhosis as well as a liver specimen obtained at autopsy of one of these patients. We find the sialytransferase activity in serum from six patients with alpha-1-antitrypsin deficiency and hepatic cirrhosis to be 50% of healthy pediatric control values and 30% of pediatric patients with liver disease. However, serum from family members homozygous for alpha-1-antitrypsin deficiency but without hepatic cirrhosis, and serum from patients with a variety of other kinds of liver disease, failed to exhibit the marked sialytransferase deficiency. Similar assays carried out on a homogenate of a liver sample from one patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis indicated that the deficiency of sialyltransferase activity was not demonstrable in liver. Furthermore, a comparative kinetic analysis of serum and liver sialytransferase in normal and afflicted individuals failed to detect differences in substrate affinities which might account for a decrease in functional sialyltransferase capacity in individuals with alpha-1-antitrypsin deficiency and hepatic cirrhosis. These observations suggest that the serum sialyltransferase deficiency in such patients probably arises after chronic and extensive liver disease involving hepatic accumulation of alpha-1-antitrypsin rather than the enzyme deficiency being the primary cause of the hepatic cirrhosis and alpha-1-antitrypsin deficiency.     

Tissue alpha-globulins in keloid formation.

The deposition of alpha-1-antitrypsin and alpha-2-macroglobulin, both known to be inhibitors of human skin collagenase, is significantly increased in keloids and in hypertrophic scars (as compared to normal skin). However, following intralesional triamcinolone treatment, a marked resorption of these abnormal scars occurs along with a significant reduction of the alpha-1-antitrypsin deposits. These findings suggest that alpha-globulins are involved in abnormal scar formation, and that triamcinolone may remove collagenase and/or protease inhibitors--thereby allowing activation of the collagenase with subsequent breakdown and resorption of the excessive collagen.     

Modifications of serum glycoproteins the days following a prolonged physical exercise and the influence of physical training.

Eight male subjects (mean age 24.1 +/- 2.6 years) performed at intervals of 2 weeks successively a 3 h and two 2 h runs of different running speed. The days following the running there were moderate elevations of C-reactive protein, haptoglobin, alpha-1-acid glycoprotein, coeruloplasmin, transferrin, alpha-1-antitrypsin and plasminogen. There were small or no changes of albumin, alpha-2-macroglobulin and hemopexin. The elevations of the "acute phase reactants" were examined in three male subjects following a 2 h run before and after an endurance training period of 9 weeks. This demonstrated a decreased acute phase response after training as illustrated by the changes of C-reactive protein, haptoglobin and alpha-1-acid glycoprotein in spite of higher posttraining running speeds. Well-trained athletes have elevated levels of the serum protease inhibitors alpha-1-antitrypsin, alpha-2-macroglobulin and C1-inhibitor. These antiproteolytic glycoproteins might limit exercise-induced inflammatory reactions.     

Activity of selected proteinase inhibitors in patients with disorders of lipid metabolism]

The study was aimed at verification of previously found, in animals with experimentally induced atherosclerosis, the disturbances of serum proteinases inhibitors: alpha-2-antiplasmin, alpha-1-antitrypsin and alpha-2-macroglobulin. In humans with hypercholesterolemia the decrease of serum activity of alpha-2-antiplasmin was observed. In humans with hypercholesterolemia and increased serum concentration of triacylglycerols no significant changes in activity of alpha-1-antitrypsin and alpha-2-macroglobulin were found--in comparison with control subjects.     

Isolation and characterization of alpha-1-antitrypsin from rhesus-monkey serum.

A simple, relatively gentle, procedure for isolation of rhesus-monkey alpha-1-antitrypsis from serum is described. The method consists of chromatographic separation of the fraction precipitated by 50-75%-satd. (NH4)2SO4 from pooled monkey serum on DEAE-cellulose followed by affinity chromatography on Sepharose-bound concanavalin A. Approx. 30% of the trypsin-inhibitory activity present in the original serum was recovered when alpha-1-antitrypsin was reconstituted with physiological saline (0.85% NaCl). Pure alpha-1-antitrypsin exhibitied a single band on sodium docecyl sulphate/polyacrylamide-gel electrophoresis, with an estimated mol.wt. of 60000 and four bands in acid/starch-gel electrophoresis. The acid/starch-gel-electrophoretic pattern and mobility of isolated material were identical with those of the alpha-1-antitrypsin bands in the original serum sample. The most rapdily migrating bands resembled the pattern and mobility for the normal human phenotype PiM in 28 monkeys. A starch strip from the acid/starch-gel-electrophoresis as the origin for antigen-antibody electrophoresis was used to examine alpha-1-antitrypsin for microheterogeneity; no evidence for microheterogeneity was observed in samples from 18 monkeys. In addition, isolated alpha-1-antitrypsin exhibited a single arc when subjected to immunoelectrophoresis. Amino acid and carbohydrate compositions of isolated monkey alpha-1-antitrypsin were similar to those of human alpha-1-antitrypsin.     

Serum trypsin inhibitory activity and alpha-1-antitrypsin level in liver diseases

Alpha-1-antitrypsin level and serum trypsin inhibitory activity were measured in patients with viral hepatitis, chronic hepatitis and liver cirrhosis. The most pronounced discrepancy between these two parameters were observed in patients with liver cirrhosis: the increase of alpha-1-At level was not accompanied by adequate increase of trypsin inhibitory activity. Some mechanisms potentially responsible for this discrepancy are discussed.     

Purification and properties of Streptococcus pneumoniae neuraminidase

Considerable amounts (200 units/ml) of neuraminidase activity were detected in middle ear effusion of children (age 1 month-10 years) and its presence was highly correlated with the presence of Streptococcus pneumoniae. When isolates of this organism are cultured, neuraminidase activity appears in the growth medium during the exponential phase of growth. In order to study the role of this enzyme in the pathology of otitis media we have developed a method for its purification. The enzyme was purified over 5,800-fold by removing the organism and passing the culture broth through a series of affinity and ion-exchange columns. The overall yield was 2 mg enzyme protein and the final specific activity was 1.8 X 10(6) units/mg protein. A molecular weight of 65,000 was estimated by SDS-PAGE and gel filtration chromatography. The Stokes radius of neuraminidase was calculated to be 32 A, its isoelectric point was 7.2, and its pH optimum was 6.0. In terms of specificity, the enzyme catalyzed the hydrolysis of sialic acid linkages in mucin, glycoproteins, and gangliosides: bovine submaxillary mucin supported the highest catalytic efficiency, and alpha-1-antitrypsin the lowest. Neuraminidase acted on at least three linkage classes of substrates, alpha-2,6 and alpha-2,3 linkages of N-acetylneuraminic acid to galactose, and alpha-2,6 linkages to N-acetyl-galactosamine.     

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.     

A positively charged loop on the surface of kallistatin functions to enhance tissue kallikrein inhibition by acting as a secondary binding site for kallikrein

Kallistatin is a serine proteinase inhibitor (serpin) that specifically inhibits tissue kallikrein. The inhibitory activity of kallistatin is abolished upon heparin binding. The loop between the H helix and C2 sheet of kallistatin containing clusters of basic amino acid residues has been identified as a heparin-binding site. In this study, we investigated the role of the basic residues in this region in tissue kallikrein inhibition. Kallistatin mutants containing double Ala substitutions for these basic residues displayed a 70-80% reduction of association rate constants, indicating the importance of these basic residues in tissue kallikrein inhibition. A synthetic peptide derived from the sequence between the H helix and C2 sheet of kallistatin was shown to suppress the kallistatin-kallikrein interaction through competition for tissue kallikrein binding. To further evaluate the function of this loop, we used alpha1-antitrypsin, a non-heparin-binding serpin and slow tissue kallikrein inhibitor as a scaffold to engineer kallikrein inhibitors. An alpha1-antitrypsin chimera harboring the P3-P2' residues and a sequence homologous to the positively charged region between the H helix and C2 sheet of kallistatin acquired heparin-suppressed inhibitory activity toward tissue kallikrein and exhibited an inhibitory activity 20-fold higher than that of the other chimera, which contained only kallistatin's P3-P2' sequence, and 2300-fold higher than that of wild-type alpha1-antitrypsin. The alpha1-antitrypsin chimera with inhibitory characteristics similar to those of kallistatin demonstrates that the loop between the H helix and C2 sheet of kallistatin is crucial in tissue kallikrein inhibition, and this functional loop can be used as a module to enhance the inhibitory activity of a serpin toward tissue kallikrein. In conclusion, our results indicate that a positively charged loop between the H helix and C2 sheet of a serpin can accelerate the association of a serpin with tissue kallikrein by acting as a secondary binding site.     

Comparison of the carbohydrate and amino acid composition of normal and S-variant alpha-1-antitrypsin.

alpha-1-Antitrypsin has been isolated and purified from the serum of an individual with the Pi S phenotype whose serum contains only 50--60% as much alpha-1-antitrypsin as normal M-type serum. The preparation was homogeneous by the criteria of sodium dodecyl sulfate polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrigufation. When analyzed in the ultracentrifuge, the S-type alpha-1-antitrypsin exhibited a molecular weight of 47,500 which was essentially the same as that of the M-type (47,300) and the Z-type (47,500) alpha-1-antitrypsin. The S-type alpha-1-antitrypsin contains 15.2% carbohydrate consisting of 16.4 residues/mol of N-acetylglucosamine, 7.8 residues/mol of mannose. 6.7 residues/mol of galactose and 7.1 residues/mol of sialic acid which is essentially the same as the carbohydrate composition of the M-type alpha-1-antitrypsin. In addition, M- and S-type alpha-1-antitrypsin have very similar amino acid compositions.