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PubMed1992

High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor.

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.

Amino Acid Sequence↗
PubMed1990

Sheep plasma protease inhibitors influencing protease activity and growth of Lucilia cuprina larvae in vitro.

The enzyme inhibitors alpha 2-macroglobulin (alpha 2M), anti-thrombin III (AT III) and alpha 1-proteinase inhibitor (alpha 1PI) were isolated from sheep plasma and tested for their ability to affect L. cuprina larval proteases and larval growth in vitro. Casein radial diffusion gels indicated that both alpha 2M and alpha 1PI completely inhibited the protease activity of a larval excretory-secretory preparation, while AT III had a partial effect. Casein zymograms revealed that alpha 2M inhibited all of the larval proteases, while AT III was able to modify the normal plaque pattern; alpha 1PI inhibited all plaques except a doublet present at pI 8.5. Larval growth in vitro was significantly inhibited by alpha 2M and AT III (P less than 0.05) when compared to albumin controls but was not affected by alpha 1PI. The levels of alpha 2M in sheep serum were monitored over the course of a larval fly infection. A significant increase in alpha 2M (P less than 0.05) was recorded in the serum of flystruck sheep. It is suggested that, under certain circumstances, these inhibitors may be involved in influencing flystrike through reducing the activity of larval proteases necessary for wound formation and larval nutrition.

Animals↗
PubMed1985

The 1.8 A structure of the complex between chymostatin and Streptomyces griseus protease A. A model for serine protease catalytic tetrahedral intermediates.

The naturally occurring serine protease inhibitor, chymostatin, forms a hemiacetal adduct with the catalytic Ser195 residue of Streptomyces griseus protease A. Restrained parameter least-squares refinement of this complex to 1.8 A resolution has produced an R index of 0 X 123 for the 11,755 observed reflections. The refined distance of the carbonyl carbon atom of the aldehyde to O gamma of Ser195 is 1 X 62 A. Both the R and S configurations of the hemiacetal occur in equal populations, with the end result resembling the expected configuration for a covalent tetrahedral product intermediate of a true substrate. This study strengthens the concept that serine proteases stabilize a covalent, tetrahedrally co-ordinated species and elaborates those features of the enzyme responsible for this effect. We propose that a major driving force for the hydrolysis of peptide bonds by serine proteases is the non-planar distortion of the scissile bond by the enzyme, which thereby lowers the activation energy barrier to hydrolysis by eliminating the resonance stabilization energy of the peptide bond.

Amino Acid Sequence↗
PubMed2006

Closing of the flaps of HIV-1 protease induced by substrate binding: a model of a flap closing mechanism in retroviral aspartic proteases.

The active site of aspartic proteases is covered by one or more flaps, which control access to the active site and play a significant role in the binding of the substrate. An extensive conformational change of the flaps takes place upon binding of substrate to the active site. A long molecular dynamics simulation was performed on the complex consisting of a peptide (CA-p2) from a natural substrate cleavage site of the gag/pol polyprotein placed in the active site of HIV-1 protease (PR) with an open flap conformation. During the simulation, the substrate induced the closing of the flaps into the closed conformation in an asymmetrical way through a hydrophobic intermediate state cluster. The nature of the residues of HIV-1 PR identified to be important in the flap closing mechanism is conserved across known structures of retroviral aspartic proteases family. The flap closing mechanism described in HIV-1 PR is proposed to be a general model for flap closing in retroviral aspartic proteases.

HIV Protease↗
PubMed1998

Tumor-associated proteases and inhibitors in gastric cancer: analysis of prognostic impact and individual risk protease patterns.

Expression of proteolytic parameters of the urokinase-type plasminogen activator (uPA) system [uPA receptor (uPA-R), plasminogen activator inhibitor (PAI)-1] has been proven to be an independent prognostic parameter in cancer. However, it has not been considered that the uPA system is interacting with several other protease/inhibitor systems, neither has a comparable prognostic role of these factors been investigated. Moreover, studies evaluating specific protease patterns indicating high individual risk are missing completely. Therefore, in a consecutive prospective series of 203 gastric cancer patients, the expression of activators (plasminogen, tPA, MMP-2, cathepsin D, antithrombin 3) and inhibitors (alpha-2-antiplasmin, alpha-2-macroglobulin, alpha-1-antitrypsin, alpha-1-antichymotrypsin) of proteolysis was studied immunohistochemically in the tumor epithelium semiquantitatively (score 0-3) in addition to the uPA system. Kaplan-Meier analysis (median time of follow-up 31 months) revealed a significant association of cathepsin D (P=0.0042), alpha-2-macroglobulin (P=0.0281) and antitrypsin (P=0.0372) with disease-free survival and of cathepsin D (P=0.0018), antitrypsin (P=0.0112) and antichymotrypsin (P=0.0002) with overall survival. Multivariate Cox analysis performed to correct these results for relative impact of the uPA system and established prognostic factors showed PAI-1 (disease-free survival: P=0.002, relative risk 1.86; overall survival: P=0.005, relative risk 1.39), pT and pN as independent parameters. Cathepsin D was shown to have an independent impact on disease-free survival (P=0.020, relative risk 2.98). Comparative chi-square analysis of cases with poor and good prognoses revealed that in patients with good clinical outcome, inhibitors of proteolysis are correlated significantly, whereas in patients with poor prognosis activators of proteolysis are significantly associated preferentially and significant correlations with the uPA-R are dominant. For detailed pattern analysis, stepwise overall Kaplan-Meier analyses were performed in subgroups of high uPA-R-, uPA-, PAI1- and cathepsin D expression for two additional proteases each. From these analyses, the combination of high (score 2/3) expression of uPA-R, PAI-1, antichymotrypsin and alpha-2-macroglobulin was identified as a high-risk pattern, representing parameters known to be essential for uPA-R internalization and recycling. This suggests some of the uPA-associated proteases and inhibitors investigated as univariate prognostic parameters in gastric cancer. Cathepsin D is a new independent parameter for disease-free survival. The study further demonstrates that a protease pattern promoting uPA-R recycling in tumor cells especially indicates high individual risk tumors in gastric cancer.

Adult↗
PubMed1998

A trypsin-like platelet protease propagates protease-activated receptor-1 cleavage and platelet activation.

Protease-activated receptor-1 (PAR-1) is a G-protein-linked receptor on platelets and perivascular cells activated by alpha-thrombin and the PAR-1-activating peptide, SFLLRN. alpha-Thrombin activates PAR-1 by cleaving it at R41-S42 to release the 41-residue peptide TR(1-41). Unexpectedly, platelet activation with SFLLRN was also associated with PAR-1 cleavage and the release of TR(1-41). Both PAR-1 cleavage and platelet activation resulting from SFLLRN addition to platelets were markedly inhibited by the serine protease inhibitor 4, 2-(aminoethyl)-benzene sulphonylfluoride.HCl (pefabloc SC) and soybean trypsin inhibitor, but not by inhibitors of calpain, cysteine proteases or metalloproteases. Thus, a trypsin-like platelet protease propagates SFLLRN-dependent PAR-1 cleavage and platelet activation.

Collagen↗
PubMed1995

A serine protease inhibitor, protease nexin I, rescues motoneurons from naturally occurring and axotomy-induced cell death.

Protease nexin I (PNI) is a member of the family of serine protease inhibitors (serpins) that have been shown to promote neurite outgrowth in vitro from different neuronal cell types. These include neuroblastoma cells, hippocampal neurons, and sympathetic neurons. Free PNI protein is markedly decreased in various anatomical brain regions, including hippocampus, of patients with Alzheimer disease. Here, we report that PNI rescued spinal motoneurons during the period of naturally occurring (programmed) cell death in the chicken in a dose-dependent fashion. Furthermore, PNI prevented axotomy-induced spinal motoneuron death in the neonatal mouse. The survival effect of PNI on motoneurons during the period of programmed cell death was not associated with increased intramuscular nerve branching. PNI also significantly increased the nuclear size of motoneurons during the period of programmed cell death and prevented axotomy-induced atrophy of surviving motoneurons. These results are consistent with the possible role of PNI as a neurotrophic agent. They also support the idea that serine proteases or, more precisely, the balance of proteases and serpins may be involved in regulating the fate of neuronal cells during development.

Amyloid beta-Protein Precursor↗
PubMed2005

Functional characterization of complement proteases C1s/mannan-binding lectin-associated serine protease-2 (MASP-2) chimeras reveals the higher C4 recognition efficacy of the MASP-2 complement control protein modules.

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s(MASP-2 CCP1/2) and C1s(MASP-2 SP), respectively) were expressed and characterized chemically and functionally. Whereas C1s(MASP-2 SP) was recovered as an active enzyme, C1s(MASP-2 CCP1/2) was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s(MASP-2 SP) and C1s had Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21-27-fold higher kcat/Km ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s(MASP-2 CCP1/2) retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.

Amino Acid Sequence↗
PubMed2006

Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies.

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

Amino Acid Sequence↗
PubMed2003

Interactions of proteases, protease inhibitors, and the beta1 integrin/laminin gamma3 protein complex in the regulation of ectoplasmic specialization dynamics in the rat testis.

During spermatogenesis, developing germ cells migrate progressively across the seminiferous epithelium. This event requires extensive restructuring of cell-cell actin-based adherens junctions (AJs), such as the ectoplasmic specialization (ES, a testis-specific AJ type), between Sertoli cells and elongating/elongate spermatids. It was postulated that proteases and protease inhibitors worked in a yin-yang relationship to regulate these events. If this is true, then it is anticipated that both proteases and protease inhibitors are found at the ES. Indeed, matrix metalloprotease (MMP)-2, membrane-type 1 (MT1)-MMP and their inhibitor, tissue-inhibitor of metalloproteases (TIMP)-2, were shown to localize at the apical ES. In order to identify the putative MMP substrate as well as the unknown binding ligand for alpha6beta1 integrin in the ES, immunofluorescent microscopy coupled with immunoprecipitation techniques were used to demonstrate that laminin gamma3, largely a germ cell product, was present at the apical ES and could form a bona fide complex with beta1-integrin. Furthermore, the structural interactions of MMP-2 and MT1-MMP with laminin gamma3 and beta1-integrin, but not with N-cadherin or nectin-3, have implicated the crucial role of MMP-2/MT1-MMP in the regulation of integrin/laminin-based ES dynamics. Using an in vivo model to study AJ dynamics where adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to disrupt Sertoli-germ cell adhesive function, an induction of active MMP-2, active MT1-MMP and TIMP-2 but not active MMP-9 was detected between 0.5 and 8 h after AF-2364 treatment. This time frame coincided with the depletion of elongating/elongate spermatids from the epithelium, illustrating the synergistic relationships between MMP-2, MT1-MMP, and TIMP-2 in AJ disassembly. Perhaps the most important of all, the use of a specific MMP-2 and MMP-9 inhibitor, (2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid, could effectively delay the AF-2364-induced elongating/elongate spermatid loss from the epithelium, demonstrating the pivotal role of MMP-2 activation in ES disassembly. Collectively, these studies illustrate that the beta1-integrin/laminin gamma3 complex is a putative ES-structural protein complex, which is regulated, at least in part, by the activation of MMP-2 involving MT1-MMP and TIMP-2 at the apical ES. The net result of this interaction likely regulates germ cell movement in the seminiferous epithelium.

Adherens Junctions↗
PubMed1989

Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease.

The rational design of drugs that can inhibit the action of viral proteases depends on obtaining accurate structures of these enzymes. The crystal structure of chemically synthesized HIV-1 protease has been determined at 2.8 angstrom resolution (R factor of 0.184) with the use of a model based on the Rous sarcoma virus protease structure. In this enzymatically active protein, the cysteines were replaced by alpha-amino-n-butyric acid, a nongenetically coded amino acid. This structure, in which all 99 amino acids were located, differs in several important details from that reported previously by others. The interface between the identical subunits forming the active protease dimer is composed of four well-ordered beta strands from both the amino and carboxyl termini and residues 86 to 94 have a helical conformation. The observed arrangement of the dimer interface suggests possible designs for dimerization inhibitors.

Amino Acid Sequence↗
PubMed2006

Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors.

BACKGROUND: The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis. RESULTS: In this study, we employ state-of-the-art mass spectrometric identification, using both a hybrid linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) mass spectrometer, and high confidence identification by two consecutive stages of peptide fragmentation (MS/MS/MS or MS3), to characterize the protein content of the tear fluid. Low microliter amounts of tear fluid samples were either pre-fractionated with one-dimensional SDS-PAGE and digested in situ with trypsin, or digested in solution. Five times more proteins were detected after gel electrophoresis compared to in solution digestion (320 versus 63 proteins). Ontology classification revealed that 64 of the identified proteins are proteases or protease inhibitors. Of these, only 24 have previously been described as components of the tear fluid. We also identified 18 anti-oxidant enzymes, which protect the eye from harmful consequences of its exposure to oxygen. Only two proteins with this activity have been previously described in the literature. CONCLUSION: Interplay between proteases and protease inhibitors, and between oxidative reactions, is an important feature of the ocular environment. Identification of a large set of proteins participating in these reactions may allow discovery of molecular markers of disease conditions of the eye.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

In-vitro and in-vivo pharmacokinetic interactions of amprenavir, an HIV protease inhibitor, with other current HIV protease inhibitors in rats.

The drug interactions between a new human immune deficiency virus (HIV) protease inhibitor, amprenavir, and four other protease inhibitors which are presently used have been characterized by in-vitro metabolic studies using rat liver microsomal fractions and in-vivo oral administration studies. The metabolic clearance rates (Vmax/Km) of amprenavir, saquinavir, indinavir and nelfinavir in rat liver microsomes were 50.67+/- 3.77, 170.88 +/- 15.34, 73.01 +/- 2.76 and 126.06 +/- 6.23 microLmin(-1) (mg protein)(-1), respectively, and the degree of metabolicclearance was in the order of saquinavir > nelfinavir > indinavir > amprenavir > ritonavir. The inhibition constants (Ki) of ritonavir for amprenavir, indinavir, nelfinavir and saquinavir were 2.29, 0.95, 1.01 and 1.64 microM, respectively, and that of indinavir for amprenavir was 0.67, indicating that amprenavir metabolism in rat liver microsomes was strongly inhibited by indinavir. The Ki values of amprenavir for indinavir, nelfinavir and saquinavir were 7.41, 2.13 and 16.11 microM, respectively, and those of nelfinavirand saquinavirforamprenavirwere 9.15 and 34.57 microM, respectively. The area under the concentration vs time curve (AUC) of amprenavir after oral co-administration with saquinavir, indinavir, nelfinavir or ritonavir (20 mg kg(-1) for each oral dose in rats) was increased by 1.6-, 2.0-, 1.2- and 9.1-fold, respectively. The AUC values of saquinavir, indinavir and nelfinavir by co-administration with amprenavir showed about 7.3-, 1.3-, and 7.9-fold increase, respectively. These observations suggested that the oral bioavailability of amprenavir was not so affected by co-administration with saquinavir, nelfinavir or indinavir, compared with ritonavir, whereas amprenavir markedly affected the oral bioavailability of saquinavir and nelfinavir. In addition, the in-vivo effects after co-administration of two kinds of HIV protease inhibitors cannot always be predicted from in-vitro data, suggesting the presence of other interaction processes besides metabolism in the liver. However, these results provide useful information for the treatment of AIDS patients when they receive a combination therapy with two kinds of HIV protease inhibitor.

Administration, Oral↗
PubMed2003

Inhibition of membrane-type serine protease 1/matriptase by natural and synthetic protease inhibitors.

Membrane-type serine protease 1 (MT-SP1), identical to matriptase, is a recently identified type II transmembrane serine protease. MT-SP1/matriptase is of considerable interest for the development, homeostasis, and cancer invasion and metastasis of epithelial tissues. The administration of inhibitors for MT-SP1/matriptase may be effective to suppress the development of tumors where the enzyme may be involved. In the present study, we produced a secreted form of recombinant MT-SP1/matriptase (ekMT-SP1s) that can be activated by enterokinase in vitro and investigated the inhibitory ability of various protease inhibitors toward the recombinant enzyme. The enterokinase-treated ekMT-SP1s (active ekMT-SP1s) cleaved various peptidyl-4-methylcoumaryl-7-amide (MCA) substrates with arginine (or lysine) residue at position P1, and the best substrate was t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. The specificity for the synthetic and natural substrates of the active ekMT-SP1s was in good agreement with that of the natural enzyme. Endogenous protease inhibitors tested, except for antithrombin III, showed no or little inhibition on the cleavage of Boc-Gln-Ala-Arg-MCA by the active ekMT-SP1s. Aprotinin showed strong inhibitory activity toward the cleavage. Food-derived inhibitors, such as soybean trypsin inhibitor, Bowman-Birk inhibitor, and lima bean trypsin inhibitor inhibited it, while chicken ovomucoid did not. Synthetic inhibitors tested inhibited it, and among them, the inhibitory effect of FOY-305 was strongest. The present findings provide important information for the suppression of cancer invasion and metastasis for which MT-SP1/matriptase is responsible.

Animals↗
PubMed1976

Double-headed protease inhibitors from black-eyed peas. V. Analysis of the energetics of protease-inhibitor interactions.

The half-site reactivity of trypsin and chymotrypsin binding to two double-headed black-eyed pea protease inhibitors a trypsin-chymotrypsin inhibitor (BEPCI) had a trypsin inhibitor (BEPTI), is explained in terms of the energetics of these inhibitor-protease interactions. Free energy diagrams are constructed to facilitate interpretation of the energetics. Coupling-free energies are calculated to reflect the magnitude of the interdependence of protease-binding (alloassociation) and inhibitor subunit interactions (isoassociation). The experiment observation of the predominance of liganded monomer complexes for the lima bean inhibitor and the Bowman-Birk soybean inhibitor and the predominance of half-site-liganded complexes for BEPCI and BEPTI is the direct result of the magnitudes and signs of the coupling free energies which result from these protease-inhibitor interactions.

Binding Sites↗
PubMed1991

Effect of human immunodeficiency virus type 1 protease on the intermediate filament subunit protein vimentin: cleavage, in vitro assembly and altered distribution of filaments in vivo following microinjection of protease.

The intermediate filament (IF) subunit protein vimentin is efficiently cleaved in vitro by purified human immunodeficiency virus type 1 (HIV-1) protease. Immunological data confirm that identical sites are cleaved when vimentin is polymerized into filaments or occurs as protofilaments. Preformed filaments require 10 times more protease to achieve the same extent of cleavage seen with protofilaments, suggesting that the cleavage sites are partially masked in IFs. The primary cleavage gives rise to molecule lacking most of the tail domain and which not only remains in preformed filaments, but also is capable of polymerizing into essentially normal 10 nm filaments. However, these filaments of the vimentin primary cleavage product show a propensity to form large lateral aggregates. The three secondary cleavage products of vimentin additionally lack portions of the head domain, are almost quantitatively released from preformed filaments and are not capable of forming filaments de novo. These results confirm and extend previous data obtained with desmin and provide a limit for that portion of the tail domain of type III IF subunit proteins that play a role in IF formation and stability. Microinjection of HIV-1 protease into cultured human skin fibroblasts resulted in a large increase in the percentage of cells with an altered and abnormal distribution of vimentin IFs. Most commonly, the IFs were observed to have collapsed into a clump with a juxtanuclear localization. The efficient cleavage of vimentin observed in vitro and the ability of microinjected HIV-1 protease to alter IF distribution in vivo suggest that IF proteins may serve as substrates within HIV-1 infected cells and may play a role in viral infection.

Amino Acid Sequence↗
PubMed1991

Protease specificity and heparin binding and activation of recombinant protease nexin I.

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.

Amino Acid Sequence↗
PubMed1990

Location of the protease-inhibitory region of secretory leukocyte protease inhibitor.

Secretory leukocyte protease inhibitor (SLPI) is a two-domain protein that inhibits a wide range of proteases including chymotrypsin, leukocyte elastase, and trypsin. Based on its homology to other protease inhibitors and on x-ray crystallography of an SLPI-chymotrypsin complex it has been proposed that the elastase and chymotrypsin-inhibitory site is in the COOH-terminal domain and that the trypsin-inhibitory site is in the NH2-terminal domain. We have prepared muteins of SLPI by site-directed mutagenesis of a synthetic gene for the protein, followed by expression in Escherichia coli. The protease-inhibitory activities of these muteins indicate that leucine 72 in the COOH-terminal domain is at the inhibitory site for elastase and chymotrypsin. Unexpectedly, our measurements indicate that the trypsin-inhibitory site is not in the NH2-terminal domain. Instead they suggest that leucine 72 is also the inhibitory site for trypsin, even though the amino acid residues at the inhibitory sites of other trypsin inhibitors are almost always either lysine or arginine.

Amino Acid Sequence↗