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.     

Purification, characterization, and acute phase response of plasma alpha-1-antiproteinase in the hamster, Mesacricetus auratus.

1. alpha-1-Antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) with a molecular mass of 60 kDa was purified to apparent homogeneity from hamster plasma. 2. It inhibited elastase, chymotrypsin and trypsin, but did not significantly affect pancreatic kallikrein, plasma kallikrein or plasmin. 3. It has the same N-terminal heptapeptide sequence as that of rat alpha-1-antiproteinase. 4. Its plasma level decreased after injection of bacterial lipopolysaccharide.     

Guinea pig plasma trypsin inhibitors. Purification and characterization of two functionally distinct proteinase inhibitors.

Two trypsin inhibitors (TI-1, TI-2) were isolated from guinea pig plasma and purified to homogeneity. In amino-acid composition as well as molecular masses, TI-1 (Mr 58,000) and TI-2 (Mr 57,000) are similar to each other and to human and mouse alpha 1-proteinase inhibitors, and mouse con-trapsin. The two inhibitors form equimolar complexes with proteinases. The effectiveness of the inhibitors was characterized by association rate constants under second-order rate conditions. The inhibitory action of TI-1 was rapid for bovine trypsin, porcine pancreatic elastase and guinea pig plasma kallikrein, but slow for bovine thrombin and guinea pig plasmin and not detectable for bovine chymotrypsin and porcine pancreatic kallikrein. The inhibitory action of TI-2 was rapid for trypsin and chymotrypsin, but slow for guinea pig plasma kallikrein and not detectable for other proteinases. These results show that TI-1 and TI-2 are physicochemically similar but functionally distinct from each other and from human alpha 1-proteinase inhibitor that inhibits trypsin, chymotrypsin and elastase.     

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.     

Plasma proteinase inhibitors in Morris hepatoma-bearing rats: changes in the blood level and synthesis in tissue slices

The antiproteinase activities against trypsin, chymotrypsin, elastase, papain and rat leucocyte proteinases were determined in plasma from control and Morris hepatoma-bearing rats. Bovine trypsin and chymotrypsin were similarly inhibited by the two types of plasma whereas porcine pancreatic elastase, papain and rat leucocyte neutral proteinases were more efficiently inhibited by plasma from tumour-bearing rats. The increased plasma concentrations of some proteinase inhibitors, as determined by rocket immunoelectrophoresis, are suggested to be responsible for the observed differences in inhibition. The highest increases in plasma of tumour-bearing rats were observed for alpha 2-macroglobulin and alpha 1-acute-phase globulin. The synthesis and secretion of six proteinase inhibitors: antithrombin III, alpha 1-proteinase inhibitor, alpha 1-macroglobulin, alpha 2-macroglobulin, alpha 1-acute-phase globulin and haptoglobin, as well as albumin, were measured in tissue slices from rat liver and Morris hepatoma after incubation with [14C]leucine. Local inflammation inflicted upon the tumour-bearing rats increased formation of acute-phase proteins in liver slices but not in hepatoma slices.     

Selective oxidation of methionine residues in Kunitz-type protease inhibitors

Bovine pancreatic trypsin inhibitor (BPTI, also known as aprotinin or Kunitz inhibitor, a mini-protein composed of 58 amino-acid residues, containing a single methionine residue at position 52) has been selectively oxidized by treatment with chloramine T, under mild conditions, to the methionyl sulfoxide derivative. Spleen inhibitor II (SI II, an isoform of BPTI containing two methionine residues at positions 18 and 52) has been oxidized under the same conditions. Oxidation affects the functional properties of the two inhibitors differently: the antiproteolytic activity of BPTI towards bovine trypsin and chymotrypsin, porcine kallikrein and human leukocyte elastase is not changed upon oxidation, while in the oxidized SI II, the affinity for both chymotrypsin and elastase decreases, with respect to the native protein. These results have been directly related to the oxidation of Met18 in SI II, located at the P'3 site in the contact area with the proteases.     

Molecular cloning and sequence analysis of cDNAs coding for guinea pig alpha 1-antiproteinases S and F and contrapsin

The cDNAs encoding two isoforms, S (slow) and F (fast), of alpha 1-antiproteinase (also referred to as alpha 1-antitrypsin or alpha 1-proteinase inhibitor) as well as contrapsin were obtained by screening lambda gt11 cDNA library prepared fro inflamed guinea pig liver. The sequence analyses of these cDNAs and NH2-terminal peptides of the purified proteins revealed that both isoforms of alpha 1-antiproteinase consist of 405 amino acid residues including a signal peptide of 24 residues and that contrapsin consists of 410 amino acid residues with the same length of the signal peptide. Guinea pig contrapsin had 89, 88, 62, 42, and 41% homology to its own alpha 1-antiproteinases F and S, rat alpha 1-antiproteinase, mouse and rat contrapsins, respectively. This suggests that guinea pig contrapsin is not orthologous to mouse and rat contrapsins and that it developed from a much later duplication of alpha 1-antiproteinase gene after the guinea pig had diverged from the murine lineage. The available data suggest that the reactive site region of alpha 1-antiproteinase can be categorized into orthodox and unorthodox types: the former has P3-P'3 consensus sequence of Xaa-Pro-Met-Ser-Xaa-Pro, where Xaa is Leu, Ile, Val, or Met, while the latter, which occurs in species having multiple alpha 1-antiproteinase isoforms, has the sequence whose P1 Met has changed to other amino acids. Thus, the reactive site region of the orthodox type, which occurs in all seven mammals examined to date, is highly conserved. This is in marked contrast to the fact that the same region is hypervariable among the paralogous proteins belonging to the serpin superfamily.     

Differential interactions of rabbit plasma alpha-1-antiproteinases S and F with porcine trypsin

Two major forms of rabbit plasma alpha-1-antiproteinase, S and F, were separated by affinity chromatography on Red Sepharose, and their modes of interaction with porcine trypsin were studied. The S form interacted with trypsin much more slowly than the F form, and the resulting complex partially retained the amidolytic and proteolytic activities towards benzoyl-L-arginine p-nitroanilide and remazol brilliant blue hide powder, respectively. This S form-trypsin complex also prevented the inactivation of bound trypsin by soybean trypsin inhibitor. In marked contrast, an equimolar complex of trypsin and the F form retained neither amidolytic nor proteolytic activity. These results suggest that the F form blocks the active site of trypsin while the S form does not bind directly to the active site, thereby preserving the catalytic potential of trypsin. No similar interaction was observed, however, between the S form and either bovine chymotrypsin or porcine pancreatic elastase. Both the S and F forms inactivated these proteinases in a stoichiometric manner with differing inhibitor/proteinase binding ratios. The S form showed about twofold greater capacity to inhibit elastase than the F form, whereas the reverse was the case for chymotrypsin.     

In vitro interaction of porcine serum and colostrum protease inhibitors with pancreatic trypsin, chymotrypsin and elastase

The partition of 125I-labelled pancreatic trypsin, chymotrypsin and elastase between the inhibitors, alpha 2-macroglobulin f and s, alpha 1-protease inhibitor, alpha 2-antitrypsin, inter-alpha-trypsin inhibitor and the specific sow colostrum protease inhibitor, was studied in vitro by gradually increasing the concentration of these proteases in blood serum from adult and newborn pigs. As revealed by immunoelectrophoresis in combination with autoradiography, differences were noted in the abilities of the various protease inhibitors to interact with and to form complexes with the three proteases, resulting in changes in location, height and numbers of precipitates. Among the serum inhibitors, alpha 2-macroglobulins showed the highest relative affinity to all three proteases, while alpha 1-protease inhibitor showed a high relative affinity only for chymotrypsin. Serum alpha 2-antitrypsin complexed only with trypsin, with a low relative affinity. alpha 2-Antitrypsin also interacted with chymotrypsin and elastase, but without forming complexes. When complexes of sow colostrum protease inhibitor and trypsin were added to the serum from neonatal pigs, these complexes remained stable. The results obtained from these in vitro studies, indicating differences in the relative affinities of the inhibitors to the various proteases, give some information about the role of the inhibitors in vivo, both in adult and in neonatal pigs.     

Concentrations of murinoglobulin and alpha-macroglobulin in the mouse serum: variations with age, sex, strain, and experimental inflammation

Serum levels of murinoglobulin and alpha-macroglobulin were low in newborn mice but increased during the prepubertal development. The adult levels of the two inhibitors were higher in males than in females in 8 inbred strains tested. Gonadectomy at 2 w of age did not significantly affect the prepubertal rise in the levels not only of the above two inhibitors but of contrapsin and alpha-1-antiprotease. The gonadectomy, however, abolished the sex differences seen in the adult levels of these inhibitors. The levels of murinoglobulin and alpha-macroglobulin reached their minimum at 12 h after inducing inflammation and returned to normal at 24 h. Little change was observed in the levels of contrapsin and alpha-1-antiprotease under the same inflammatory conditions.     

Proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin

The interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. Mouse alpha-macroglobulin formed complexes with thrombin, clotting factor Xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pancreatic elastase. These complexes lost the proteolytic activities against high-molecular-weight substrates, but protected the active sites of the enzymes from inactivation by their proteinaceous inhibitors. Mouse murinoglobulin showed essentially the same properties except (i) that it did not form a complex with the clotting factor Xa, and (ii) that it did not protect plasma kallikrein, neutrophil elastase or submaxillary proteinase from inactivation by their proteinaceous inhibitors, although it formed complexes with these proteinases. No interaction was detected between Clostridium histolyticum collagenase and murinoglobulin or alpha-macroglobulin. These results indicate (i) that murinoglobulin has a proteinase-binding spectrum similar to that of alpha-macroglobulin, but is weaker in the ability to protect the bound proteinases from inactivation by the proteinaceous inhibitors than alpha-macroglobulin and (ii) that mouse alpha-macroglobulin has essentially the same inhibitory spectrum as the human homologue.     

Synthesis of contrapsin and alpha-1-antiproteinase in inflamed and tumor-bearing mice

In mice bearing ascites tumors, such as Ehrlich, L1210, Meth-A, and P-815, the plasma levels of contrapsin and alpha-1-antiproteinase remained virtually unchanged. However, the total body pools of these proteins as well as their hepatic mRNA levels increased severalfold, and isoelectrofocusing patterns of these proteins shifted to the lower pH side. Under the same conditions, plasma albumin level decreased by 25%, but its total body pool and hepatic mRNA level increased severalfold. On the other hand, induction of acute phase reaction by injection of bacterial lipopolysaccharide caused differential effects on the two proteinase inhibitors: hepatic translatable mRNA for contrapsin was doubled while that for alpha-1-antiproteinase remained unchanged.     

Inhibition of human and dog serine pancreatic proteinases by naturally occurring high-molecular-weight inhibitors from plant or animal origin

Mixtures of human and dog pancreatic proteinases obtained from human pancreatic juice or extracts of canine pancreas were selectively inhibited using nine naturally occurring inhibitors of serine proteinases. Inhibition of trypsin, chymotrypsin, and elastase was monitored using specific synthetic substrates.Dog trypsin was inhibited by all nine inhibitors, while only four inhibitors significantly inhibited dog chymotrypsin and elastase. Inhibition spectra of human enzymes were completely different. Only three inhibitors exhibited significant inhibition of human trypsin. Human chymotrypsin and protease E (elastase) were inhibited only by 2 to 3 inhibitors and, in most cases, the amount of the inhibitor required to achieve full or 80% inhibition greatly exceeded the amount needed for the respective dog enzymes. Trasylol, the most widely used, commercially available inhibitor, inhibited both human and dog trypsins, but was totally ineffective against human and dog chymotrypsins and elastases. It was found that the most effective polyvalent inhibitors of human enzymes were chickpea and lima bean trypsin inhibitors. It was indicated by these results that strong species specificity exists and the invalidity of using animal models for the study of inhibition of human pancreatic proteinases was emphasized.     

Serine proteinase inhibitors from Vipera ammodytes venom. Isolation and kinetic studies

Three protein inhibitors of serine proteinases were isolated from the crude venom of the long-nosed viper Vipera ammodytes ammodytes by ion-exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki = 3.4 X 10(-10) and 5.6 X 10(-10) M), while the third one primarily inhibits chymotrypsin (Ki = 4.3 X 10(-9) M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N-terminal group in the former inhibitors is blocked; arginine is the N-terminal amino acid in the latter. Besides trypsin and alpha-chymotrypsin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design

Isocoumarins are potent mechanism-based heterocyclic irreversible inhibitors for a variety of serine proteases. Most serine proteases are inhibited by the general serine protease inhibitor 3,4-dichloroisocoumarin, whereas isocoumarins containing hydrophobic 7-acylamino groups are potent inhibitors for human leukocyte elastase and those containing 7-alkylureidogroups are inhibitors for procine pancreatic elastase. Isocoumarins containing basic side chains that resemble arginine are potent inhibitors for trypsin-like enzymes. A number of 3-alkoxy-4-chloro-7-guanidinoisocoumarins are potent inhibitors of bovine thrombin, human factor Xa, human factor XIa, human factor XIIa, human plasma kallikrein, porcine pancreatic kallikrein, and bovine trypsin. Another cathionic derivative, 4-chloro-3-(2-isothiureidoethoxy) isocoumarin, is less reactive toward many of these enzymes but is an extremely potent inhibitor of human plasma kallikrein. Several guanidinoisocoumarins have been tested as anticoagulants in human plasma and are effective at prolonging the prothrombin time. The mechanism of inhibition by this class of heterocyclic inactivators involves formation of an acyl enzyme by reaction of the active site serine with the isocoumarin carbonyl group. Isocoumarins with 7-amino or 7-guanidino groups will then decompose further to quinone imine methide intermediates, which react further with an active site residue (probably His-57) to form stable inhibited enzyme derivatives. Isocoumarins should be useful in further investigations of the physiological function of serine proteases and may have future therapeutic utility for the treatment of emphysema and coagulation disorders.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Pancreatic proteolytic enzymes of ostrich purified on immobilized protein inhibitors. Characterization of a new form of chymotrypsin (Chtr1)

Four forms of chymotrypsin (Chtr1, Chtr2, Chtr3, Chtr4), one form of trypsin and one form of elastase were purified from a slightly alkaline extract of ostrich (Struthio camelus) pancreas. The zymogens in the crude extract were activated with immobilized trypsin and then separated by affinity chromatography using immobilized inhibitors and ion exchange chromatography. One of the purified forms of chymotrypsin (Chtr1) exhibited an unusual interaction with the highly selective protein trypsin inhibitor from Cucurbita maxima (CMTI). Interactions with other protein trypsin inhibitors such as basic pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (STI), trypsin inhibitors from Cyclanthera pedata (CyPTI), Cucurbita pepo (CPTI), Cucurbita pepo var. giramontia (CPGTI) and Linum usitatissimum (LUTI) were also investigated. This study demonstrated the affinity of Chtr1 to inhibitors containing Arg at P1 position. Studies of substrate specificity of Chtr1 using oxidized B-chain of insulin revealed four susceptible bonds: Tyr15-Leu16, Phe24-Phe25, Phe25-Tyr26 and, surprisingly, Arg22-Gly23. The amino acid composition, as well as the first 13 residues of the N-terminal amino acid sequence, was determined. Studies of ostrich elastase showed that it can interact with immobilized CMTI in the presence of 5 M NaCl. This unusual characteristic is reported for the first time and suggests that elastase specificity depends on ionic strength. The kinetic constants K(M), k(cat) and k(cat)/K(M) for purified ostrich trypsin, chymotrypsin 4 and elastase were also determined.     

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors.

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.     

Isolation, characterization, and cDNA sequencing of alpha-1-antiproteinase-like protein from rainbow trout seminal plasma

Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.