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.     

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.     

Immunofluorescent evaluation of platelet alpha-1-antitrypsin and alpha-2-macroglobulin.

Rabbit raised anti-alpha-1-antitrypsin or anti alpha-2-macroglobulin antisera at dilution of less than 1:80 yielded non-specific staining on human platelets by indirect immunofluorescent technique. A similar pattern was in fact obtained by using normal rabbit sera at the same dilution and was due to the presence of smooth muscle autoantibodies. This indicates that human platelets do not contain significant quantities of these antigens. In agreement with the above, only microamounts of alpha-1-antitrypsin and alpha-2-macroglobulin were found to be present in human platelets by means of the electroimmunoassay.     

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.     

Granular cell tumors: evidence for heterogeneous tumor cell differentiation

Eighteen granular cell tumors from various sites were examined with antisera directed against protein S-100, neuron specific enolase (NSE), alpha-1-antichymotrypsin, and alpha-1-antitrypsin, glial fibrillary acidic protein (GFAP), lysozyme, factor VIII-related antigen, myoglobin and vimentin, as well as with a monoclonal antibody (lu-5) directed against a panepithelial marker. The immunocytochemical reaction pattern of the tumors was heterogeneous. The brain and pituitary tumors and one thyroid tumor reacted for alpha-1-antichymotrypsin and alpha-1-antitrypsin, but not for S-100 protein and NSE. However, tumors from other sites showed immunoreactions for S-100 protein and NSE and some also for vimentin. Reactions for alpha-1-antichymotrypsin and alpha-1-antitrypsin were not observed. All other reactions were similarly negative. We conclude that the morphologically homogeneous group of granular cell tumors is biologically heterogeneous.     

Low pH stability of alpha-1-antititrypsin.

According to the previous findings of others, the trypsin inhibitory capacity of alpha-1-antitrypsin is irreversibly lost in acidic solutions below pH 5.0. In contrast, experiments reported herein show that considerable inhibitory activity can be regenerated as a time-dependent phenomena following titration to basic media. The rate of recovery of activity is accompanied by a decreasing amplitude in the fluorescent emission spectrum at 335 nm of acidified alpha-1-antitrypsin solutions following adjustment to pH 8.0. Acidic media also results in the slow, progressive formation of protein aggregates as measured using Sephadex gel filtration. This latter process is more prominent at pH 4.0, near the isoelectric point of alpha-1-antitrypsin than at pH 3 or 2. Both monomer and polymeric forms of alpha-1-antititrypsin were isolated before or after adjustment to basic media. Isolated monomeric material shows a high recovery of biological and immunological activity; aggregate forms, however, are immunologically cross-reactive but show little enzyme inhibitory activity.     

The biological activity of alpha-1-antichymotrypsin: the change of chymotrypsin-inhibitory and immunoenhancing activities by heat treatment

The relationship between chymotrypsin-inhibitory and immunoenhancing activity of alpha-1-antichymotrypsin was studied. alpha-1-Antichymotrypsin was treated at 50 degrees C, 55 degrees C or 60 degrees C for 15 min. It was found that antichymotryptic activity was reduced by half when alpha-1-antichymotrypsin was heated at 55 degrees C and was not detected at all when heating was carried out at 60 degrees C. alpha-1-Antichymotrypsin which was heated at 60 degrees C did not form a complex with chymotrypsin, but became a substrate for chymotrypsin. The effect of native and heated alpha-1-antichymotrypsin on antibody response was studied in mice. alpha-1-Antichymotrypsin increased the number of anti-sheep erythrocytes antibody producing cells even when it was heated at 60 degrees C. Circular dichroism and single radial immunodiffusion were used to detect conformational changes. Circular dichroism in the region of side chain absorption showed that the intensities of the spectra at 296, 284, and 265 nm decreased with a rise in temperature from 50 to 60 degrees C. In single radial immunodiffusion analysis, alpha-1-antichymotrypsin did not form a halo after being heated at 60 degrees C. In conclusion, when alpha-1-antichymotrypsin was heated at 60 degrees C, the immunoenhancing activity remained intact while the antichymotryptic activity was lost with the conformational change.     

Inactivation of human alpha-1-antitrypsin by a tissue-destructive protease of Legionella pneumophila

Three extracellular proteases produced by Legionella pneumophila during growth in liquid medium were examined for their effects on human alpha-1-antitrypsin (alpha-1-AT). One of these proteases, tissue-destructive protease (TDP) destroyed completely the trypsin-inhibitory capacity of alpha-1-AT at protease: inhibitor molar ratios down to 0.002:1. After inactivation by TDP, the Mr of alpha-1-AT was reduced by 5000 in SDS-PAGE. This suggested that inactivation entailed only limited cleavage.     

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.     

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.     

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.     

Antiproteinase activity of alpha 2-macroglobulin

Blood serum separation by the method of gel filtration on Sephadex G-200 with the subsequent immunochemical determination of the quantitative content of basic proteolysis inhibitors permitted isolating the alpha 2-macroglobulin fraction while alpha 1-antitrypsin and alpha 1-antichymotrypsin separation was a failure. The immunochemical analysis of the antienzymic activity of the isolated inhibitors showed that 32.3 +/- 3.5% of the introduced kallikrein, 18.7 +/- 0.6% of trypsin and 14.4 +/- 4.1% of chymotrypsin were bound in the zone of alpha 2-macroglobulin. The rest of antienzymic activity was localized in the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin. After a preliminary saturation of blood serum with trypsin in the amount equivalent to its antitryptic capacity (200 micrograms/ml) the ability of alpha 2-macroglobulin to bind kallikrein and chymotrypsin lowers considerably (by 69 and 72%, respectively). In the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin a decrease in the ability to bind kallikrein and chymotrypsin amounted to 44 and 12% respectively. Thus, alpha 2-macroglobulin being bound with trypsin looses considerably its ability to bind other enzymes.     

A review and comparison of the murine alpha1-antitrypsin and alpha1-antichymotrypsin multigene clusters with the human clade A serpins

The major human plasma protease inhibitors, alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, are each encoded by a single gene, whereas in the mouse they are represented by clusters of 5 and 14 genes, respectively. Although there is a high degree of overall sequence similarity within these groupings, the reactive-center loop (RCL) domain, which determines target protease specificity, is markedly divergent. The literature dealing with members of these mouse serine protease inhibitor (serpin) clusters has been complicated by inconsistent nomenclature. Furthermore, some investigators, unaware of the complexity of the family, have failed to distinguish between closely related genes when measuring expression levels or functional activity. We have reviewed the literature dealing with the mouse equivalents of human alpha(1)-antitrypsin and alpha(1)-antichymotrypsin and made use of the recently completed mouse genome sequence to propose a systematic nomenclature. We have also examined the extended mouse clade "a" serpin cluster at chromosome 12F1 and compared it with the syntenic region at human chromosome 14q32. In summarizing the literature and suggesting a standardized nomenclature, we aim to provide a logical structure on which future research may be based.     

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.     

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 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.     

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.     

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.