Serine Protease Inhibitor Superfamily Research Articles

Molecular analysis of the intergenic region of the duplicated Amy genes of Drosophila melanogaster and Drosophila teissieri.

The intergenic regions between the duplicated amylase coding regions (Amy) of D. melanogaster and D. teissieri were sequenced. Their lengths in D. melanogaster and D. teissieri were 4,536 bp and 4,621 bp, respectively. Since homology between the upstream regions of the two duplicated genes was found up to 450 bp from the initiation codon of the Amy genes, the ancestral Amy coding region duplicated together with at least 450 bp of the 5'-flanking region as one unit. Comparison of the regions between the two species revealed that the level of divergence was very heterogeneous. Although the mean level of the nucleotide difference in this region was 0.107, no nucleotide substitution was found in four subregions whose sizes were more than 100 bp. Since the probability of these four subregions being completely conserved between D. melanogaster and D. teissieri was very low, these subregions were considered to have relatively important roles in evolution. Large insertions and deletions were not observed in this region but small ones were observed all over the region except for an about 1-kb subregion. This 1-kb region corresponded to an open reading frame encoding a protein which had some sequence identity with the proteins of the serine protease inhibitor superfamily (serpin). Since we could find a transcript of this gene and the synonymous substitution rate was higher than the replacement substitution rate, we suggest that this gene encodes an active serpin in Drosophila.

Purification, cDNA Cloning, and Characterization of a New Serpin with Megakaryocyte Maturation Activity

A new member of the serine protease inhibitor (serpin) superfamily with megakaryocyte maturation activity was purified, and its cDNA was cloned and characterized. The predicted amino acid sequence consisting of 380 residues was unique and was 38% identical to the serpin plasminogen activator inhibitor type 2 (PAI-2). The recombinant factor expressed in Chinese hamster ovary cells showed species-specific activity on the induction of megakaryocyte maturation in vitro. When injected into mice, the factor indeed elicited an increase in the number of platelets in plasma. The sequence alignment indicated that the factor possessed a lysine residue at the P1 position, suggesting that it might function as an inhibitor of Lys-specific proteases. Although we could not show any inhibitory activities toward several known Lys-specific proteases, we detected the activity toward protease activity present in the culture supernatant of COLO 201 cells. These results suggested that the protein might influence the maturation of megakaryocytes via action as a serpin.

Introduction of an RRHR motif into chicken urokinase-type plasminogen activator (ch-uPA) confers sensitivity to plasminogen activator inhibitor (PAI)-1 and PAI-2 and allows ch-uPA-mediated extracellular matrix degradation to be controlled by PAI-1.

Comparison of the amino acid sequence of the chicken and human urokinase-type plasminogen activators (uPAs) revealed that the putative PAI-binding site found in the variable region 1 (VR1) loop of mammalian PAs is absent in the homologous region of ch-uPA. ch-uPA, unlike mammalian PAs, also appears to be refractory to inhibition by human PAIs and as a naturally occurring PAI-resistant variant, constitutes a unique model system for assessing the functional relevance of the PAI-binding site. Therefore, we molecularly constructed a ch-uPA, ch-uPA(RRHR), which contains the putative PAI-binding motif RRHR (residues 192-195) in its VR1 loop. As a result of this substitution, the second-order rate constant of inhibition of PAI-1 increased approximately 700-fold from 4.50 x 10(4) M(-1) x s(-1) for wild-type ch-uPA to 3.02 x 10(7) M(-1) x s(-1) for ch-uPA(RRHR), and the ability to form SDS-stable, uPA-PAI-1 complexes increased approximately 1000-fold. Furthermore, the interaction of ch-uPA(RRHR) with PAI-2 was also substantially enhanced, while the interaction with other members of the serine proteinase inhibitor superfamily, protein nexin 1, alpha1-PI, and C1-inhibitor, was unaffected indicating that the RRHR motif is not a general serine proteinase inhibitor binding site. Finally, we show that extracellular matrix degradation by cells expressing ch-uPA(RRHR) is inhibited by PAI-1 in a dose-dependent manner, while matrix breakdown by cells expressing wild-type ch-uPA is unaffected by PAI-1. Thus acquisition of sensitivity to PAI-1 through a structural motif that enhances the specificity of the protease-inhibitor interaction confers to ch-uPA an added level of regulation in the context of the degradative cellular phenotype.

Squamous Cell Carcinoma Antigen 2 Is a Novel Serpin That Inhibits the Chymotrypsin-like Proteinases Cathepsin G and Mast Cell Chymase

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for more advanced squamous cell tumors. Molecular cloning of the SCCA genomic region revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. Analysis of the primary amino acid sequences shows that both genes are members of the high molecular weight serpin superfamily of serine proteinase inhibitors. Although SCCA1 and SCCA2 are nearly identical in primary structure, the reactive site loop of each inhibitor suggests that they may differ in their specificity for target proteinases. SCCA1 has been shown to be effective against papain-like cysteine proteinases. The purpose of this study was to determine whether SCCA2 inhibited a different family of proteolytic enzymes. Using recombinant DNA techniques, we prepared a fusion protein of glutathione S-transferase and full-length SCCA2 . The recombinant SCCA2 was most effective against two chymotrypsin-like proteinases from inflammatory cells, but was ineffective against papain-like cysteine proteinases. Serpin-like inhibition was observed for both human neutrophil cathepsin G and human mast cell chymase. The second order rate constants for these associations were on the order of approximately 1 x 10(5) M-1 s-1 and approximately 3 x 10(4) M-1 s-1 for cathepsin G and mast cell chymase, respectively. Moreover, SCCA2 formed SDS-stable complexes with these proteinases at a stoichiometry of near 1:1. These data showed that SCCA2 is a novel inhibitor of two physiologically important chymotrypsin-like serine proteinases.

The inhibition of human factor Xa by plasminogen activator inhibitor type 1 in the presence of calcium ion, and its enhancement by heparin and vitronectin

Plasminogen activator inhibitor type 1 (PAI-1), a member of serine proteinase inhibitor superfamily, is known to inhibit thrombin in the presence of either heparin or vitronectin. We analyzed possible inhibitory activity of PAI-1 on human factor Xa. PAI-1 inhibited factor Xa in the presence of calcium ion (Ca 2+), whereas no inhibition was observed in the absence of Ca 2+. Half maximal enhancement by Ca 2+ was obtained at 0.8 mM. An equimolar complex formation between factor Xa and PAI-1 in the presence of Ca 2+ was observed by SDS polyacrylamide gel electrophoresis. Both unfractionated heparin and vitronectin enhanced the inhibition only in the presence of Ca 2+. Apparent second-order rate constant ( k i) for the inhibition of factor Xa by PAI-1 at 5 mM Ca 2+ was 1.6 × 104 M −1 s −1, and was enhanced 3-fold by 2 u/ml of heparin (4.6 × 104 M −1 s −1[ and 10-fold by 100 nM vitronectin (1.6 × 105 M −1 s −1, respectively. The interaction between Ca 2+-bound factor Xa and PAI-1 could be important from the view of PAI-1 neutralization and enhancement of fibrinolysis.

New insights into the structural basis of alpha 1-antitrypsin deficiency.

The serpin superfamily of serine proteinase inhibitors contains many members but the best-characterized is the plasma protein alpha 1-antitrypsin. its genetic deficiency is associated, in the homozygote, with hepatic damage that may progress to cirrhosis and hepatocellular carcinoma. Low levels of circulating alpha 1-antitrypsin fail to protect the lungs against proteolytic attack and predispose the homozygote to early onset pan-lobular emphysema, bronchiectasis and asthma. The major cause of alpha 1-antitrypsin deficiency, the Z mutation (Glu342Lys), results in the accumulation of protein in the endoplasmic reticulum of the liver. Using a structural approach, we have shown that the hepatic inclusions result from a protein-protein interaction between the reactive centre loop of one molecule and the beta-pleated sheet of a second. This loop-sheet polymerization is now also recognized to be the basis of deficiencies associated with mutations of C1-inhibitor, antithrombin and alpha 1-antichymotrypsin. Our recent solution of a crystal structure of a thermostable mutant of alpha 1-antitrypsin shows the detailed interactions that result in loop-sheet linkage and helps to explain the mechanism of action of this family of proteinase inhibitors.

Serine proteinase inhibitors from insect hemolymph.

Insect hemolymph, like vertebrate serum, contains several different types of polypeptides that are able to inhibit the catalytic function of proteolytic enzymes, however studies on proteins possessing this capability have been limited to a relatively few species. A comparative examination of the inhibition of trypsin, chymotrypsin, neutrophil elastase and cathepsin G and pancreatic elastase by the hemolymph of 14 insect species belonging to six orders showed great diversity in terms of both total proteinase inhibitory capacity and specificity. Most of the inhibitors examined fall into two groups: low molecular mass proteins (below 10 kDa) related to Kunitz type inhibitors, and proteins of about 45 kDa which belong to the serpin superfamily of serine proteinase inhibitors. This minireview describes the properties, characteristics and possible biological significance of selected inhibitors.

Complex regulation of plasminogen activator inhibitor type-1 (PAI-1) gene expression by serum and substrate adhesion

Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the serine protease inhibitor (SERPIN) superfamily that functions to negatively regulate the plasmin-based pericellular proteolytic cascade, was induced early after exposure of growth-arrested normal rat kidney (NRK) cells to serum-containing medium. Increased PAI-1 transcription was rapid (evident within 10 min of serum addition) and involved immediate-early response kinetics. [3H]Thymidine autoradiography was used to map the time frame of PAI-1 expression during a synchronous growth cycle. PAI-1 transcript accumulation peaked in mid-G1 phase (approx. 4-6 h post-stimulation) and declined prior to, or concomitant with, the onset of DNA synthetic phase. Serum increased PAI-1 expression in NRK cells in agarose suspension, as well as monolayer, culture; induction in suspended cells (similar to monolayer culture conditions) also occurred in the presence of cyclohexamide or puromycin. The serum-inductive pathway leading to PAI-1 gene activation is thus functional regardless of adhesive conditions or capacity for de novo protein synthesis. The amplitude of induction and maintenance of expression in later stages of G1, however, were subject to adhesive influences. PAI-1 transcript accumulation at 4 and 8 h post-stimulation in newly adherent cells, moreover, was blocked by puromycin, indicating that both immediate-early and secondary mechanisms regulate PAI-1 mRNA levels during progression of NRK cells through an 'activated' G1 growth phase.

Characterization of common neoantigenic epitopes generated in plasminogen activator inhibitor-1 after cleavage of the reactive center loop or after complex formation with various serine proteinases

Plasminogen activator inhibitor-1 (PAI-1), an important risk factor for thrombotic diseases, is a member of the superfamily of serine proteinase inhibitors. To define structural rearrangements occurring during interaction between PAI-1 and its target proteinases we have raised monoclonal antibodies against the PAI-1/t-PA complex. Thirteen out of 401 monoclonal antibodies reacted preferentially with the PAI-1/t-PA complex as compared to free PAI-1 or free t-PA. Detailed characterization revealed the presence of two non-overlapping neoantigenic epitopes in the PAI-1/t-PA complex. Both neoantigenic epitopes were also exposed after complex formation between PAI-1 and either urokinase-type plasminogen activator, plasmin or thrombin as well as after cleavage of the reactive site loop of non-inhibitory substrate type PAI-1 variants. Thus, we have identified two neoantigenic epitopes, localized entirely in PAI-1, and commonly exposed after complex formation of active PAI-1 with various proteinases or after cleavage of substrate PAI-1. These monoclonal antibodies should facilitate further studies on the mechanism of interaction between various PAI-1 forms and its target proteinases.

Modeling the Three-Dimensional Structure of Serpin/Molecular Chaperone HSP47

Heat shock protein 47 (HSP47) is a molecular chaperone which assists procollagen triple helix assembly and secretion. As a molecular chaperone it is unique in that it binds specifically to a very narrow range of protein "substrates" (i.e., procollagen and collagen only), and it is also a member of the well-characterized serine protease inhibitor (serpin) superfamily. In the absence; of any X-ray crystallographic data, a novel tandem-modeling procedure is used to obtain three-dimensional structural information on mature recombinant mouse HSP47 (mrmHSP47). MrmHSP47 is shown to have 30% amino acid sequence identity and 70% sequence similarity with human protein C inhibitor (hPCI). Therefore, molecular models of inhibitory and latent states of hPCI are generated, using the X-ray crystal structure coordinates of proteolytically cleaved hPCI, and used as templates for the homology modeling of inhibitory and latent states of mrmHSP47. The validity of the models is discussed and the latent state model of mrmHSP47 is shown to have a suitable candidate binding groove for procollagen/collagen peptides, which appears to account for experimental observations made with aminoacid deletion experiments.

Mitogenic stimulation of human lymphocytes mediated by a cell surface elastase

A ca. 45-kDa protein which was recently identified and purified to homogeneity from a solid tumor cell line as a T-cell mitogen was found to have significant sequence similarities with human monocyte/neutrophil elastase inhibitor (EI)[1]. Since EI is a known substrate for elastase, a determination of whether a cell surface expressed elastase-like molecule might be the binding protein for this 45-kDa factor and mediate mitogenic signal transduction was undertaken. First, the surface of tumor infiltrating lymphocytes, TIL 660, the indicator cell line used for the purification of this mitogen, was shown to stain positively with an anti-elastase antibody using flow cytofluorometry for quantitation. Then, after observing an inverse correlation between cell surface staining and the proliferative status of the TILs, behavior which might be expected of a growth factor receptor upon activation, mitogenic signal transduction was attempted through the elastase-like molecules of the lymphocytes' plasma membrane with the anti-elastase antibody in the role of mitogen. A greater than 4-fold mitogenic stimulation was observed when this antibody was covalently linked to latex beads; in contrast, addition of the soluble form of the same antibody did not result in any increase in [ 3H]thymidine incorporation into the cells' DNA. Hence, these data support induced clustering of an elastase-like molecule on the lymphocyte surface as a mediator of mitogenesis and suggest that the binding protein for mitogenic signal transduction induced by the 45-kDa protein, a member of the serine protease inhibitor (serpin) superfamily of proteins, is a molecule with structure similar to a serine protease.

A serpin from human tumor cells with direct lymphoid immunomodulatory activity: mitogenic stimulation of human tumor-infiltrating lymphocytes

A serum-free supernatant from an epidermal carcinoma cell line has previously been shown to contain mitogenic activity for human tumor infiltrating lymphocytes in culture [1]. From this conditioned medium we have now purified to homogeneity, as determined by SDS-PAGE analysis, a ca. 45 kDa protein which stimulates [ 3H]thymidine incorporation into the DNA of these human T-lymphocytes. Amino acid composition data and immunoreactivity of the purified protein as well as sequence analyses of 7 tryptic fragments obtained therefrom suggest a strong similarity with human monocyte/neutrophil elastase inhibitor, which is a member of the serine protease inhibitor (serpin) superfamily. We have previously identified and purified from the same conditioned medium a 36 kDa protein with myeloid immunomodulatory activity [2]. Taken together, these two reports support the role of tumor-derived soluble factors in tumor immunosurveillance.

Identification of Pigment Epithelium-Derived Factor in the Interphotoreceptor Matrix of Bovine Eyes

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein and a member of the serine protease inhibitor superfamily. Here we describe the identification of PEDF in bovine eyes and optimization of its purification from this natural source. We have developed a polyclonal antibody to recombinant human PEDF, Ab-rPEDF, that immunoreacts in a specific, sensitive, and linear fashion with PEDF protein, and furthermore, blocks its neurotrophic activity. We show that Ab-rPEDF specifically recognizes a 49,500- M r polypeptide on Western transfers of a wash of the extracellular matrix between the retinal pigment epithelium and the neural retina-termed interphotoreceptor matrix (IPM). PEDF is present as approximately 1% of total soluble IPM protein. Starting with an IPM wash, PEDF protein is purified 164-fold to near homogeneity by ammonium sulfate fractionation and cation-exchange chromatography, with a recovery of 47%. The highly purified protein has an apparent M r of 49,500 ± 1,500 as assessed by SDS-polyacrylamide gel electrophoresis, and a native p I of 7.0-7.7. It elutes as a single peak on gel-filtration chromatography with a retention time immediately behind that of ovalbumin (43,000 M r ). N-glycosidase treatment indicates that each PEDF molecule has a 5% carbohydrate content attached to internal asparagine residue(s). Amino terminal sequence of the purified PEDF reveals removal of an amino-terminal peptide region for the mature protein. Purified PEDF has neurotrophic activity on human retino-blastoma cells, as previously observed for IPM. The neurotrophic activities of both PEDF and IPM are blocked by antiserum Ab-rPEDF. Altogether, PEDF is present in the bovine IPM as a soluble, extracellular, monomeric glycoprotein that by itself confers neurotrophic activity to the IPM. Thus, native PEDF isolated and purified as described here should prove useful for biochemical studies as well as other approaches.

Effects of endometrial serpin-like proteins on immune responses in sheep.

The uterine milk proteins (UTMP) are a pair of related glycoproteins that are the major secretory products of the endometrium of the pregnant ewe. UTMP are members of the serpin superfamily of serine protease inhibitors but have no known antiprotease activity. One possible role for UTMP is to inhibit uterine immune responses--UTMP inhibit mitogen and mixed lymphocyte-induced proliferation of peripheral blood lymphocytes, natural killer (NK) cell activity and abortion caused by NK cell activation. Present objectives were to further evaluate the lymphocyte-inhibitory activity of UTMP and test whether UTMP modify immune responses in vivo. One experiment demonstrated that UTMP inhibited antigen-induced lymphocyte proliferation induced by Candida albicans extract. In another experiment, ewes were immunized with OVA mixed with 3.75 mg/ml of UTMP or ovine serum albumin (OSA control). Injections of 1 mg OVA+UTMP or OSA in incomplete adjuvant were administered 6 wk later. Titers of antibody to OVA were lower (P < 0.001) for ewes administered UTMP than for ewes administered OSA. Effects of UTMP on delayed hypersensitivity reactions were evaluated in three experiments using skin-fold thickness assays. UTMP did not inhibit the increase in skin-fold thickness caused by PHA and Mycobacterium tuberculosis but rather tended to increase the response to PHA. Results strengthen the thesis that UTMP are physiologically relevant immunoregulatory molecules. Nonetheless, effects on skin-fold responses indicate that actions of UTMP can be more complex than would be predicted based on the proteins only having a single biological effect.

Vaccinia virus serpins B13R (SPI-2) and B22R (SPI-1) encode Mr 38.5 and 40K, intracellular polypeptides that do not affect virus virulence in a murine intranasal model

A characterization of genes B13R (SPI-2) and B22R (SPI-1) from vaccinia virus strain Western Reserve (WR) is presented. These genes are transcribed early during infection and the predicted encoded proteins show similarity to the superfamily of serine protease inhibitors (serpins). The 5′ transcriptional initiation site of each gene was mapped by primer extension experiments to 71–72 and 31 nucleotides upstream of the B13R and B22R open reading frames (ORFs), respectively. Each ORF was expressed in Escherichia coli and specific antisera were raised against the protein produced. These antisera were used to identify the B13R- and B22R-encoded proteins in vaccinia virus-infected cells as stable, intracellular, nonglycosylated proteins of M r 38.5K and M r 40K, respectively. The B22R gene product was detected in all orthopoxviruses tested including cowpox, rabbitpox, and vaccinia strains WR, Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and IHD-W. In contrast, the B13R gene product had a more limited distribution and was not detected in Copenhagen, Tashkent, Lister, and Tian Tan. Viable virus deletion mutants that lacked only B13R or B22R coding sequences (ΔB13R and ΔB22R) and revertant viruses in which the deleted gene was restored were constructed by transient dominant selection. The growth of the deletion mutants in cell culture was indistinguishable from that of wild-type virus. Additionally the virulence of each deletion mutant was indistinguishable from wild-type and revertant viruses in a murine intranasal model.

Purification and characterization of active and stable recombinant plasminogen-activator inhibitor accumulated at high levels in Escherichia coli.

Plasminogen-activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high-level expression system that leads to the accumulation of PAI-1 at 30-50% total microbial protein. We have developed a single-step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI-1/litre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37 degrees C with a half-life of approximately 48 h. At 20 degrees C, PAI-1 activity was stable for a week and at 4 degrees C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI-1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of high-level expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI-1 in high yield.

Structure and chromosomal location of the gene encoding mouse corticosteroid-binding globulin: strain differences in coding sequence and steroid-binding activity

Corticosteroid-binding globulin (CBG) is a member of the serine proteinase inhibitor superfamily and is responsible for the plasma transport of glucocorticoids. The mouse Cbg gene structure has been deduced from two non-overlapping DNA fragments of a λEMBL-3 genomic library, as well as PCR amplification of the approx. 2 kb of genomic DNA that lies between them. Mouse Cbg comprises five exons that span a region of approx. 10.5 kb, and has been localized in tight linkage with the Aat (α-antitrypsin) and Spi (serine proteinase inhibitor) gene complex on chromosome 12, in a region syntenic with this genetic locus on human chromosome 14. Intron-specific oligodeoxyribonucleotide primers were also used to PCR-amplify Cbg coding regions from several mouse strains. No differences were found in the Cbg coding sequences of BALB/c and C57BL/6J- cpk/cpk mice, while two mutations were found within RIIIS/J Cbg that result in Lys 201→ Glu and Ala 357→Thr substitutions in the mature mouse CBG polypeptide. To assess what impact these substitutions might have on the steroid-binding activity of RIIIS/J CBG, these mutations were introduced separately or together into a BALB/c mouse Cbg cDNA. Expression of these mutants in the MDCK cell line indicated that the Lys 201 →Glu substitution accounts for the abnormal steroid-binding affinity of CBG m RIIIS/J mice.

Regulation of protease nexin-1 target protease specificity by collagen type IV.

Recent studies have shown that serine protease inhibitors can be regulated in their activity, specificity, and location by glycoprotein or extracellular matrix (ECM) co-factors. Protease nexin-1 (PN-1) is a member of the serpin superfamily of serine protease inhibitors which can rapidly inhibit thrombin, urokinase, and plasmin. PN-1 binds tightly to and is regulated by the ECM. This interaction accelerates the inhibition of thrombin by PN-1 and blocks urokinase and plasmin inhibition by PN-1. Previous work showed that heparan sulfate proteoglycan is largely responsible for the acceleration of thrombin inhibition by PN-1. Our current studies were directed at identifying ECM component(s) that decreased the ability of PN-1 to inhibit urokinase and plasmin. These studies showed that collagen type IV decreased the formation of SDS-stable complexes between urokinase or plasmin and PN-1 without affecting formation of complexes between thrombin and PN-1. The second order rate constant for inhibition of urokinase by PN-1 was markedly decreased with increasing collagen type IV, whereas the second order rate constant for inhibition of thrombin by PN-1 was unaffected by addition of collagen type IV. Other ECM components (collagen type I, vitronectin, fibronectin, and heat-denatured collagen type IV) did not affect complex formation or the rate of inhibition of proteases by PN-1, indicating that these effects were specific to collagen type IV. Binding of PN-1 to immobilized collagen type IV was demonstrated using an enzyme-linked immunosorbent assay; the concentration of PN-1 necessary to obtain 50% saturation of the immobilized collagen type IV binding sites was approximately 15 nM. Collagen type IV was also copurified with PN-1 from fibroblast-conditioned medium. These results demonstrate a novel regulation of serpin specificity in which an ECM co-factor decreased the inhibition of certain proteases by the serpin without affecting the inhibition of its target protease.

Engineering Plasminogen Activator Inhibitor 1 Mutants with Increased Functional Stability

Plasminogen activator inhibitor 1 (PAI-1), a member of the serine protease inhibitor (Serpin) superfamily, is the primary inhibitor of the plasminogen activators tPA and uPA. PAI-1 is produced in an active form but converts to an inactive or latent form with a half-life of approximately 1 h at pH 7.5, 37 degrees C. This study describes the construction, expression, and characterization of PAI-1 mutants with increased functional stability. Three mutations that disrupt an ion pair, present in latent PAI-1, between Arg-30 and Glu-350 (P4'), were introduced into recombinant PAI-1. All three mutant proteins maintained normal functional activity against both uPA and tPA. However, the half-life of each purified PAI-1 mutant was extended compared to the 1.1 h observed for wild-type PAI-1 (wtPAI-1) (1.2 h for Glu-350-->Arg, 2.0 h for Glu-350-->Pro, and 2.1 h for the Arg-30-->Glu mutation). An additional PAI-1 variant containing a second mutation designed to potentially reconstitute the ion pair (Arg-30-->Glu, Glu-350-->Arg) failed to restore the wild-type half-life. Circular dichroism spectra analysis indicated that the active and latent forms of wtPAI-1 and all four mutants contained similar secondary structural elements. Thermal stability determinations showed that latent wtPAI-1 was much more structurally stable than the active conformation. However, the latent form for all four mutants was significantly less stable than the corresponding wtPAI-1 conformer. This is the first report of PAI-1 mutants which have been specifically engineered to produce enhanced functional stability.

Complementary DNA cloning and kinetic characterization of a novel intracellular serine proteinase inhibitor: mechanism of action with trypsin and factor Xa as model proteinases.

The full-length cDNA encoding a novel human intracellular serine proteinase inhibitor has been sequenced and found to encode a 376 amino acid protein (M(r) approximately 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the cytoplasmic antiproteinase has the majority of structural motifs conserved among the greater superfamily of serine proteinase inhibitors, or serpins. On the basis of several criteria such as amino acid identity and the absence of a classical N-terminal signal peptide, the cytoplasmic antiproteinase represents a new member of the intracellular serpin family. Further inspection of the cytoplasmic antiproteinase amino acid sequence identified three potential N-glycosylation sites and Arg341-Cys342 as the reactive site P1-P1' residues, respectively. We have also employed the slow binding kinetic approach to detail the mechanism of bovine trypsin and human factor Xa inhibition by the novel cytoplasmic antiproteinase. Inhibition of trypsin by the cytoplasmic antiproteinase was preceded by a two-step mechanism corresponding to the formation of an initial loose complex, followed by an isomerization step to a more stable, tight complex. The binding of the cytoplasmic antiproteinase to trypsin occurred with a second-order association rate constant of 2.8 x 10(6) M-1 s-1 and an overall equilibrium constant of 22.5 pM, demonstrating that the factor is a potent inhibitor of this proteinase. Under the appropriate conditions, the tight complex between trypsin and the cytoplasmic inhibitor was reversible, indicated by an exponential regeneration of proteinase amidolytic activity from the preformed complex. Therefore, the tight complex appears to be stabilized predominantly by reversible bonds that form between trypsin and the cytoplasmic inhibitor. In contrast to the inhibition of trypsin, the inhibition of factor Xa amidolytic activity by the cytoplasmic antiproteinase followed a single-step binding mechanism. The apparent first-order rate constant for factor Xa inhibition was found to increase as a linear function of the inhibitor concentration range studied. Formation of the inhibitory complex between factor Xa and the cytoplasmic antiproteinase occurred with a second-order association rate constant of approximately 1.3 x 10(5) M-1 s-1 and a equilibrium constant of 3.7 nM. These findings suggests that the cytoplasmic inhibitor may initially encounter significant energy barriers for proper alignment with the substrate binding cleft of factor Xa. However, once aligned, the reaction proceeds rapidly to a tight factor Xa.inhibitor complex that dissociates at a slow rate.