The reaction between plasmin and C1-inhibitor results in plasmin inhibition by the serpin mechanism.

C1-inhibitor is an important inhibitor of plasma kallikrein and C1, but also has inhibitory activity against numerous other plasma proteinases such as plasmin. The relevance of plasmin inhibition by the C1-inhibitor has been debated, with some evidence showing that plasmin causes significant proteolysis of C1-inhibitor. In the present study, we show that C1-inhibitor in its native state will inhibit plasmin without being significantly degraded, in a manner typical of all serpin reactions. However, if C1-inhibitor is in a denatured polymeric state (as can easily occur during storage, or as produced by heating of the native protein), it will be extensively degraded by plasmin. In addition, we show that hydrophobic interaction chromatography is an effective method to remove trace contaminants of inactive C1-inhibitor polymers.

The inhibition of TNK-t-PA by C1-inhibitor.

TNK-t-PA is a recombinant mutant of tissue plasminogen activator that has a longer half-life and higher selectivity for fibrin than normal tissue plasminogen activator (t-PA). In addition, it is reported to be serpin resistant because of reduced inhibition by plasminogen activator inhibitor-1. In this study, we have investigated the inhibition of TNK-t-PA by the serpin C1-inhibitor. TNK-t-PA is inhibited with a second-order rate constant of 7.5 per mol/l per s compared with 4.5 per mol/l per s for t-PA. In both cases, the stoichiometry was close to 20, indicating that C1-inhibitor was predominantly a substrate for both forms of t-PA. The formation of cleaved C1-inhibitor was seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the t-PA-C1-inhibitor (or TNK-t-PA-C1-inhibitor) complex seen on SDS-PAGE and by zymography. Although the rates of inhibition are very slow in vitro, the fact that in vivo formation of the t-PA-C1-inhibitor complex after infusion of t-PA has been well documented suggests that TNK-t-PA will also be inhibited by C1-inhibitor in vivo and, perhaps more importantly, could cause a significant reduction in C1-inhibitor concentration.

The native metastable fold of C1-inhibitor is stabilized by disulfide bonds.

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A.

C1 inhibitor cross-linking by tissue transglutaminase.

C1 inhibitor, a plasma proteinase inhibitor of the serpin superfamily involved in the regulation of complement classical pathway and intrinsic blood coagulation, has been shown to bind to several components of the extracellular matrix. These reactions may be responsible for C1 inhibitor localization in the perivascular space. In the study reported here, we have examined whether C1 inhibitor could function as a substrate for plasma (factor XIIIa) or tissue transglutaminase. We made the following observations: 1) SDS-polyacrylamide gel electrophoresis and autoradiography showed that C1 inhibitor exposed to tissue transglutaminase (but not to factor XIIIa) incorporated the radioactive amine donor substrate [(3)H]putrescine in a calcium-dependent manner; 2) the maximum stoichiometry for the uptake of [(3)H]putrescine by C1 inhibitor was 1:1; 3) proteolytic cleavage and peptide sequencing of reduced and carboxymethylated [(3)H]putrescine-C1 inhibitor identified Gln(453) (P'9) as the single amine acceptor residue; 4) studies with (125)I-labeled C1 inhibitor showed that tissue transglutaminase was also able to cross-link C1 inhibitor to immobilized fibrin; and 5) C1 inhibitor cross-linked by tissue transglutaminase to immobilized fibrin had inhibitory activity against its target enzymes. Thus, tissue transglutaminase-mediated cross-linking of C1 inhibitor to fibrin or other extracellular matrix components may serve as a mechanism for covalent serpin binding and influence local regulation of the proteolytic pathways inhibited by C1 inhibitor.

Linkage between the hormone binding site and the reactive center loop of thyroxine binding globulin.

Thyroxine binding globulin (TBG) is the major carrier of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in plasma. TBG is member of the serpin family of proteins although it has no proteinase inhibitory activity. In this study we show that TBG has properties typical of a metastable serpin and provide evidence that occupancy of the hormone binding site alters the conformation of the reactive center loop. After reactive center loop cleavage by endoproteinase Asp-N or neutrophil elastase the protein became more stable to guanidine hydrochloride denaturation compared to the native protein, as a result of loop insertion. In addition, incubation of the native protein with a reactive center loop peptide, caused a change in mobility on a native gel. This is consistent with the idea that thyroxine binding globulin is able to form a binary complex with the peptide as a result of beta-sheet A expansion. To assess the effect of cleavage and loop insertion on the hormone binding site we used the specific binding of a fluorophore, 1,8-anilinonaphthalene sulfonic acid (ANS). Loop insertion itself had no effect on ANS affinity, but cleavage with elastase at the P4'-P5' bond caused a reduction in affinity, presumably because this cleavage site is located within the hormone binding site. These data support the concept that cleavage of TBG by proteinases released in inflammation is a mechanism to deliver thyroid hormones to target tissues. A linkage between the occupancy state of the hormone binding site and the conformation of the reactive center loop was indicated by the observation that binding of T3 to native TBG reduced proteolytic susceptibility by both endoproteinase Asp-N and elastase.

Influence of the P5 residue on alpha1-proteinase inhibitor mechanism.

The reactive center loop of native alpha1-proteinase inhibitor has been reported to be in a helical conformation and in a beta-strand conformation by two different studies. In the beta-strand loop structure the P5 glutamic acid plays a unique role by stabilizing the loop in the predicted optimal conformation for the interaction with target proteinases and insertion into beta-sheet A. We hypothesize here that disrupting the interactions that stabilize the beta-strand conformation of the loop would result in changes in the inhibitory properties of the serpin. In addition, our earlier studies on reactive center loop mutants of alpha1-proteinase inhibitor suggested that the P5 residue was important in stabilizing the alpha1-proteinase inhibitor-proteinase complexes. To address these issues we made mutants of alpha1-proteinase inhibitor with glycine, glutamine, or lysine at the P5 position and measured the rates and stoichiometries of inhibition with trypsin and human neutrophil elastase and the stabilities of the resulting complexes. In most cases the rate of inhibition was reduced by about half and the stoichiometry increased between 2- and 4-fold. The only exception was for trypsin with the lysine variant where the P5 was now the favored site of cleavage. These data show that the P5 Glu is important in maintaining the reactive center loop in a conformation optimal for interaction with the proteinase and for a fast rate of loop insertion. The complexes formed with trypsin and the variant serpins were less stable than that formed with wild-type serpin and resulted in up to 33% regeneration of trypsin activity over a period of 6 days, compared with 17% with wild type. Thus, the P5 residue of alpha1-proteinase inhibitor is important in all steps of the inhibitory mechanism in a manner consistent with the structural role played by this residue in the beta-strand loop structure of native alpha1-proteinase inhibitor.

The P6-P2 region of serpins is critical for proteinase inhibition and complex stability.

Two of the prototypic serpins are alpha1-proteinase inhibitor and ovalbumin. alpha1-Proteinase inhibitor is a rapid inhibitor of a number of proteinases and undergoes the characteristic serpin conformational change on cleavage within the reactive center loop, whereas ovalbumin is noninhibitory and does not undergo the conformational change. To investigate if residues from P12 to P2 in the reactive center loop of ovalbumin are intrinsically incapable of being in an inhibitory serpin, we have made chimeric alpha1-proteinase inhibitor variants containing residues P12-P7, P6-P2, or P12-P2 of ovalbumin and determined their inhibitory properties with trypsin and human neutrophil elastase. With the P12-P7 and P6-P2 variants, the steps before and after the fork of the branched suicide-substrate pathway were affected as reflected by changes in rates and stoichiometries of inhibition with both proteinases. The P12-P2 variant showed that those effects were nonadditive, with exclusive substrate behavior for elastase and only residual inhibitory activity against trypsin. The properties of the variants were consistent with them obeying the suicide-substrate mechanism characteristic of serpins. Enzyme activity was regenerated from complexes formed with the P6-P2 variant faster than with wild-type indicating that the rate of turnover of the complex was increased. Based on proteinase susceptibility in the reactive center loops of the P6-P2 and P12-P2 variants, and on an increase in heat stability of the cleaved P12-P2 variant, it was concluded that the variants had undergone complete loop insertion on cleavage. These results show that the reactive center loop residues P12-P2 of ovalbumin can be present in inhibitory serpins although decreasing the inhibitory properties. These data also demonstrate that the residues in the P6-P2 region of serpins are critical for rapid inhibition of proteinases and formation of stable serpin-proteinase complexes.

Reactivation of C1-inhibitor polymers by denaturation and gel-filtration chromatography.

C1-inhibitor is a proteinase inhibitor in the serpin family. It is an important inhibitor of complement C1, plasma kallikrein, and factor XIIa, and as such is involved in regulating inflammatory pathways. Studies on the plasma-derived protein are hampered by the relative ease with which the protein converts to an inactive state on storage, under mild denaturing conditions, or by incubating in some unfavorable buffers. This inactivation is caused by formation of soluble polymers which can be visualized on native electrophoresis. In order to facilitate studies on both the plasma-derived protein and recombinant variants planned for the future, it was necessary to devise a method for the rapid reactivation of the polymers in high yield. It was found that nonionic detergents did not dissociate the polymers, but they were readily dissociated in 0.1% SDS. Treatment with 0.1% SDS followed by rapid removal of the SDS and refolding on an FPLC Superose 6 column allowed for recovery of about 15% of the protein in the active monomeric form. Eighty-five percent eluted as a range of higher order polymers. Using 8 M urea as the denaturant a 25% yield of active monomer was recovered. However, with 6 M guanidine hydrochloride as the denaturant, the yield of active monomer was almost 50%. The remaining material was not present as a range of polymeric species but was probably a dimer. Therefore this method is a useful technique to facilitate studies on C1-inhibitor. Moreover, the ability to produce monomer, dimer, and polymer forms of C1-inhibitor is useful for studies investigating the conformational changes which have occurred in the different forms.

Apparent formation of sodium dodecyl sulfate-stable complexes between serpins and 3,4-dichloroisocoumarin-inactivated proteinases is due to regeneration of active proteinase from the inactivated enzyme.

Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3, 4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thogersen, I. B. , Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859-14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and beta-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, alpha1-proteinase inhibitor (alpha1PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a rate-limiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the alpha1PI reactive center P1-P1' bond. Quantitation of kinetic constants for DCI and alpha1PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by alpha1PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.

Regulation of C1-inhibitor function by binding to type IV collagen and heparin.

Serpins inhibit proteinases by a branched pathway, in which an intermediate serpin-proteinase complex can either form a stable covalent serpin-proteinase complex or produce reactive center cleaved serpin in a substrate reaction. It was tested whether these competing reactions could be regulated for the serpin C1-inhibitor by ligand binding. C1-inhibitor bound to type IV collagen, laminin, and entactin. Type IV collagen (10 microg/ml) caused an increase in the stoichiometry of inhibition for C1s inhibition by C1-inhibitor to 1.48 from 1.09 in the absence of ligand. A dose-dependent increase in the stoichiometry up to 1.27 in the presence of 100 microg/ml heparin was also observed. At low ionic strength the stoichiometry increased to 2.55. These data provide the first report that C1-inhibitor can bind to type IV collagen and also show that C 1-inhibitor can be regulated by ligand binding.

Significance of secondary structure predictions on the reactive center loop region of serpins: a model for the folding of serpins into a metastable state.

To address how serpins might fold so as to adopt the mechanistically required metastable conformation we have compared the predicted secondary structures of the reactive center loops (RCLs) of a large number of serpins with those of the equivalent regions of other non-serpin protein proteinase inhibitors. Whereas the RCLs of non-serpin inhibitors are predicted to be loop or beta-strand, those of inhibitory serpins are strongly predicted to be alpha-helical. However, non-inhibitory serpins, which also adopt the metastable conformation, show no consistent preference for alpha-helix. We propose that the RCL primary structure plays little role in promoting the metastable serpin conformation. Instead we hypothesize that preference for the metastable state results from the incorporation of part of the RCL into beta-sheet C, which as a consequence precludes incorporation of the RCL into beta-sheet A to give the most stable conformation. Consequently the RCL must be exposed and by default will adopt the most stable conformation in this particular context, which is likely to be an alpha-helix irrespective of the primary structure. Thus the observed correlation between inhibitory properties in serpins and prediction of alpha-helix in the RCL may instead reflect a need for alanine residues between positions P12 and P9 for functioning as an inhibitor rather than a structural or mechanistic requirement for alpha-helix.

Formation and properties of C1-inhibitor polymers.

Heating of the serpin C1-inhibitor above 55 degrees C induced the formation of inactive polymers. Western blotting of non-denaturing gels showed that the polymers bound to the conformation specific monoclonal antibody 4C3, suggesting that a similar conformational change to that occurring in complexed or cleaved inhibitor had taken place. N-Terminal analysis of tryptic peptides which bound to 4C3 showed that the epitope resides within residues 288-444, a region which includes parts of beta-sheets A and C. alpha 1-Antichymotrypsin, alpha 2-antiplasmin, angiotensinogen and thyroxine binding globulin also polymerised on heating, indicating that this is a property of many serpins.

The effect of sex and age on antithrombin biosynthesis in the rat.

Antithrombin is the primary inhibitor of Factor Xa and thrombin. Numerous reports have indicated that age and sex can influence antithrombin levels, but details of the regulation of antithrombin biosynthesis are not known. Thus, a characterization of antithrombin mRNA in eight tissues of young and old male and female rats was carried out. Liver produced the most mRNA, and hence contributes the majority of the plasma antithrombin, followed by the kidneys, with no age or sex related differences in mRNA levels being observed. Elevated amounts of mRNA were detected in aortas of old male rats compared to young ones. No antithrombin mRNA was detected in brain, lung, heart or skeletal muscle, and spleen showed low but variable levels. Plasma antithrombin protein was elevated in old female rats compared to young female or old male rats. These results show that the rat provides a potentially useful system to study the in vivo regulation of antithrombin biosynthesis.

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement C1 inhibitor is produced by brain tissue and is cleaved in Alzheimer disease.

C1 inhibitor was identified in human brain tissue by Western blotting and by immunohistochemistry using multiple antibodies to the native protein. The presence of C1 inhibitor mRNA was identified by reverse transcriptase-polymerase chain reaction analysis of brain mRNA extracts. The mRNA was also detected in cultured postmortem human microglia and in the IMR-32 human neuroblastoma cell line. Immunohistochemically, the native protein was detected in residual serum of capillaries and pyramidal neurons of both control and Alzheimer disease cases, as well as in occasional senile plaques of Alzheimer tissue. The reacted protein was detected on dystrophic neurites and neuropil threads in Alzheimer tissue by 4C3 monoclonal antibody, which recognizes a neoepitope following suicide inhibition. These data indicate that C1 inhibitor, a regulatory molecule controlling multiple inflammatory proteolytic cascades, is produced in normal brain. In Alzheimer disease, C1 inhibitor undergoes a prominent reaction in abnormal neuronal processes, such as dystrophic neurites and neuropil threads.

Studies on inhibition of neutrophil cathepsin G by alpha 1-antichymotrypsin.

alpha 1-Antichymotrypsin, a member of the serpin family of serine proteinase inhibitors, has been reported to inhibit chymotrypsin by a modified version of the suicide substrate reaction mechanism operative for other serpins. To investigate if this mechanism is also applicable to the inhibition of cathepsin G by this serpin, the effect of temperature on the reaction between cathepsin G and alpha 1-antichymotrypsin has been examined by SDS-PAGE and stoichiometric titrations. At 0 degree C, a cathepsin G-alpha 1-antichymotrypsin complex of M(r) 89,250 is formed which, at 38 degrees C, was cleaved by free enzyme to give a stable complex of M(r) 80,250. The reaction stoichiometry at 0 degree C was 1.53, which decreased to 1.30 at 38 degrees C, consistent with an decrease in the substrate pathway. These data are compatible with the modified suicide substrate reaction scheme. The formation of three products (cleaved inhibitor and two forms of complex) from the reaction and the potential for differential product formation suggests that modulation of the suicide substrate mechanism may play a role in the regulation of inflammatory processes mediated by cathepsin G.

The effect of cleavage by a Crotalus atrox alpha-proteinase fraction on the properties of C1-inhibitor.

The effect of cleavage of C1-inhibitor at Pro36 by a Crotalus atrox alpha-proteinase fraction on the properties of this serpin was studied. This truncated C1-inhibitor (des 1-36) was fully active as an inhibitor of kallikrein, beta-factor XIIa, and C1s, and modulated the functions of C1 in a normal manner. Also, the half-life of the truncated protein in the circulation of rabbits, both alone and in complex with C1, was not altered. These results show that shock-like symptoms caused by C. atrox envenomation are not attributable to a deficiency in C1-inhibitor caused by the action of the metalloproteinase in the alpha-proteinase fraction.

Low-affinity heparin stimulates the inactivation of plasminogen activator inhibitor-1 by thrombin.

The influence of heparin on the reaction between thrombin and plasminogen activator inhibitor-1 (PAI-1) has been examined. With a 50-fold excess of PAI-1, the rate constant for the inhibition of thrombin was 458 mol/L-1s-1, which increased to 5,000 mol/L-1s-1 in the presence of 25 micrograms/mL unfractionated heparin or heparin with low affinity for antithrombin. The effect of low affinity heparin was then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using close to equimolar concentrations of reactants. Thrombin and PAI-1 formed a stable stoichiometric complex in the absence of heparin, which did not dissociate after the addition of 25 micrograms/mL low-affinity heparin. In contrast, when low-affinity heparin was added at the beginning of the reaction, there was an initial increase in PAI-1-thrombin complex formation, but this was rapidly followed by substantial proteolytic cleavage of unreacted PAI-1 and of the thrombin-PAI-1 complex. The idea that the relative concentrations of thrombin and PAI-1, and the presence of low affinity heparin, could influence the products of the reaction was examined in detail. Quantitative zymographic analysis of tissue plasminogen activator and PAI-1 activities and chromogenic substrate assay of thrombin activity showed that low-affinity heparin stimulated the inactivation of PAI-1 by an equimolar amount of thrombin, but caused only a minimal stimulation of thrombin inhibition. It is concluded that low-affinity heparin stimulates thrombin inhibition when PAI-1 is in excess, but, unexpectedly, that low-affinity heparin enhances PAI-1 inactivation when thrombin is equimolar to PAI-1.