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At least 19 recordsLinked to original sources

PubMed2001

Distribution of serpins in the tissues of the tobacco hornworm (Manduca sexta) larvae. Existence of new serpins possibly encoded by a gene distinct from the serpin-1 gene.

Polyclonal antibodies were raised against the isolated hemolymph serine proteinase inhibitors (serpins) of Manduca sexta larvae. Two of these antibodies, MsH49a and MsH49b, displayed characteristic differences in labelling patterns of hemocytes, fat body, integumental epidermis and cuticle on immunoblots, and in light- and electronmicroscopic sections. The serpin composition of the latter three tissue homogenates was determined by native immunoblots and inhibitor binding assays. The results were compared to the hemolymph samples containing all the known inhibitors encoded by the well-characterized serpin-1 gene. The enzyme specificity of the MsH49b-labelled cuticular serpin was similar to serpin-1J, although its electrophoretic mobility on native PAGE was not identical with any of the known proteinase inhibitors encoded by the serpin-1 gene. Based on these data, we suggest that the cuticle and hemolymph may contain novel serpin(s) encoded by a gene other than the serpin-1 gene. Since the serpin-1J proved to be involved in the activation pathway of the prophenoloxidase system in the hemolymph, the in vivo function of cuticular MsH49b serpin was investigated by prophenoloxidase tests in native cuticular homogenates. Our results demonstrated that the cuticular serpin(s) that are labelled by the MsH49b antibody may play a determinant role in the regulation of the prophenoloxidase system of the integumental cuticle.

Journal Article↗

PubMed2005

The ovalbumin serpins revisited: perspective from the chicken genome of clade B serpin evolution in vertebrates.

Serpin superfamily proteins, most of which are serine protease inhibitors, share an unusual mechanism rooted in their conserved metastable tertiary structure. Although serpins have been identified in isolated members of archea, bacteria, and plants, a remarkable expansion is found in vertebrates. The chicken protein ovalbumin, a storage protein from egg white, lacking protease inhibitory activity, is an historical member of the superfamily and the founding member of the subgroup known as ov-serpins (ovalbumin-related serpins) or clade B serpins. In the human, ov-serpins include 13 proteins involved in the regulation of inflammation, apoptosis, angiogenesis, and embryogenesis. Here, a detailed analysis of the chicken (Gallus gallus) genome identified 10 clade B serpin genes that map to a single approximately 150-kb locus and contain the signature protein sequence of serpins and the gene structure of ov-serpins, with either seven or eight exons. Orthologues of PAI-2 (SERPINB2), MNEI (SERPINB1), PI-6 (SERPINB6), and maspin (SERPINB5) are highly conserved. Comparison with human ov-serpins identified avian-specific and mammal-specific genes. Importantly, a unique model of mammalian ov-serpin evolution is revealed from the comparative analysis of the chicken and human loci. The presence of a subset of ov-serpin genes in zebrafish (Danio rerio) gives insight into the ancestral locus. This comparative genomic study provides a valuable perspective on the evolutionary pathway for the clade B serpins, allowing the identification of genes with functions that may have been conserved since the origin of vertebrates. In addition, it suggests that "newer" serpins, such as ovalbumin, have contributed to vertebrate adaptation.

Amino Acid Sequence↗

PubMed1995

Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes.

The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ability to compete with trypsin for binding to alpha 1-proteinase inhibitor (alpha 1PI), plasminogen activator inhibitor 1 (PAI-1), antithrombin (AT), or AT-heparin complex when present at up to a 100-fold molar excess over trypsin. By contrast, equimolar levels of anhydrotrypsin blocked trypsin binding to non-serpin inhibitors. Equilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of alpha 1PI or AT (KI > 10(-5) M), of low affinity in the case of AT-heparin complex (KI 7-9 x 10(-6) M), and of moderate affinity in the case of PAI-1 (KI 2 x 10(-7) M). This contrasted with the stoichiometric high affinity binding of the serpins to trypsin as well as of the non-serpin inhibitors to both trypsin and anhydrotrypsin. Maximal KI values for serpin-trypsin interactions of 1 to 8 x 10(-11) M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the affinity of serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the characteristic fluorescence changes in a P9 Ser-->Cys PAI-1 variant labeled with a nitrobenzofuran fluorescent probe (NBD) which were shown previously to report the serpin conformational change associated with active enzyme binding. These results demonstrate that a covalent interaction involving the proteinase catalytic serine contributes a major fraction of the binding energy to serpin-trypsin interactions and is essential for inducing the serpin conformational change involved in the trapping of enzyme in stable complexes.

Animals↗

PubMed2005

Identification of plasma proteases inhibited by Manduca sexta serpin-4 and serpin-5 and their association with components of the prophenol oxidase activation pathway.

One innate immune response pathway of insects is a serine protease cascade that activates prophenol oxidase (pro-PO) in plasma. However, details of this pathway are not well understood, including the number and order of proteases involved. Protease inhibitors from the serpin superfamily appear to regulate the proteases in the pathway. Manduca sexta serpin-4 and serpin-5 suppress pro-PO activation in plasma, apparently by inhibiting proteases upstream of the direct activator of pro-PO. To identify plasma proteases inhibited by these serpins, we used immunoaffinity chromatography with serpin antibodies to isolate serpin-protease complexes that formed after activation of the cascade by exposure of plasma to bacteria or lipopolysaccharide. Covalent complexes of serpin-4 with hemolymph proteases HP-1 and HP-6 appeared in plasma activated by Gram-positive or Gram-negative bacteria, whereas serpin-4 complexes with HP-21 and two unidentified proteases were unique to plasma treated with Gram-positive bacteria. HP-1 and HP-6 were also identified as target proteases of serpin-5, forming covalent complexes after bacterial activation of the cascade. These results suggest that HP-1 and HP-6 may be components of the pro-PO activation pathway, which are activated in response to infection and regulated by serpin-4 and serpin-5. HP-21 and two unidentified proteases may participate in a Gram-positive bacteria-specific branch of the pathway. Several plasma proteins that co-purified with serpin-protease complexes, most notably immulectins and serine protease homologs, are known to be components of the pro-PO activation pathway. Our results suggest that after activation by exposure to bacteria, components of the pro-PO pathway associate to form a large noncovalent complex, which localizes the melanization reaction to the surface of invading microorganisms.

Amino Acid Sequence↗

PubMed2000

Formation of a noncovalent serpin-proteinase complex involves no conformational change in the serpin. Use of 1H-15N HSQC NMR as a sensitive nonperturbing monitor of conformation.

A structural understanding of the nature and scope of serpin inhibition mechanisms has been limited by the inability so far to crystallize any serpin-proteinase complex. We describe here the application of [(1)H-(15)N]-HSQC NMR on uniformly and residue-selectively (15)N-labeled serpin alpha(1)-proteinase inhibitor (Pittsburgh variant with stabilizing mutations) to provide a nonperturbing and exquisitely sensitive means of probing the conformation of the serpin alone and in a noncovalent complex with inactive, serine 195-modified, bovine trypsin. The latter should be a good model both for the few examples of reversible serpin-proteinase complexes and for the initial Michaelis-like complex formed en route to irreversible covalent inhibition. Cleavage of the reactive center loop, with subsequent insertion into beta-sheet A, caused dramatic perturbation of most of the NMR cross-peaks. This was true for both the uniformly labeled and alanine-specifically labeled samples. The spectra of uniformly or leucine- or alanine-specifically labeled alpha(1)-proteinase inhibitor in noncovalent complex with unlabeled inactive trypsin gave almost no detectable chemical shift changes of cross-peaks, but some general increase in line width. Residue-specific assignments of the four alanines in the reactive center loop, at P12, P11, P9, and P4, allowed specific examination of the behavior of the reactive center loop. All four alanines showed higher mobility than the body of the serpin, consistent with a flexible reactive center loop, which remained flexible even in the noncovalent complex with proteinase. The three alanines near the hinge point for insertion showed almost no chemical shift perturbation upon noncovalent complex formation, while the alanine at P4 was perturbed, presumably by interaction with the active site of bound trypsin. Reporters from both the body of the serpin and the reactive center loop therefore indicate that noncovalent complex formation involves no conformational change in the body of the serpin and only minor perturbation of the reactive center loop in the region which contacts proteinase. Thus, despite the large size of serpin and serpin-proteinase complex, 45 and 69 kDa respectively, NMR provides a very sensitive means of probing serpin conformation and mobility, which should be applicable both to noncovalent and to covalent complexes with a range of different proteinases, and probably to other serpins.

Alanine↗

PubMed2004

Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis.

Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement. Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses. The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades. Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems. Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells. Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury. Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life. As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.

Amino Acid Sequence↗

PubMed2005

Manduca sexta serpin-4 and serpin-5 inhibit the prophenol oxidase activation pathway: cDNA cloning, protein expression, and characterization.

Infection stimulates the innate immune responses of insects, including activation of prophenol oxidase (pro-PO) in plasma as the last step of a serine protease cascade. To investigate the roles of protease inhibitors in regulating this pathway, we cloned cDNAs for two new serpins (serpin-4 and serpin-5) from the tobacco hornworm, Manduca sexta. Serpin-4 and serpin-5 mRNAs are constitutively expressed at a low level in larval hemocytes and fat body and increased dramatically upon bacterial challenge. These serpins are present in larval plasma at approximately 3 (serpin-4) and approximately 1 mug/ml (serpin-5) and increased 3-8-fold by 24 h after injection of bacteria or fungi. Recombinant serpin-4 and serpin-5 decreased pro-PO activation when added to plasma, but they did not directly inhibit the pro-PO-activating proteases. Instead, they apparently regulate the pathway by inhibiting one or more target proteases upstream of the pro-PO-activating proteases.

Amino Acid Sequence↗

PubMed2004

Identification and activity of a lower eukaryotic serine proteinase inhibitor (serpin) from Cyanea capillata: analysis of a jellyfish serpin, jellypin.

Delineating the phylogenetic relationships among members of a protein family can provide a high degree of insight into the evolution of domain structure and function relationships. To identify an early metazoan member of the high molecular weight serine proteinase inhibitor (serpin) superfamily, we initiated a cDNA library screen of the cnidarian, Cyanea capillata. We identified one serpin cDNA encoding for a full-length serpin, jellypin. Phylogenetic analysis using the deduced amino acid sequence showed that jellypin was most similar to the platyhelminthe Echinococcus multiocularis serpin and the clade P serpins, suggesting that this serpin evolved approximately 1000 million years ago (MYA). Modeling of jellypin showed that it contained all the functional elements of an inhibitory serpin. In vitro biochemical analysis confirmed that jellypin was an inhibitor of the S1 clan SA family of serine proteinases. Analysis of the interactions between the human serine proteinases, chymotrypsin, cathepsin G, and elastase, showed that jellypin inhibited these enzymes in the classical serpin manner, forming a SDS stable enzyme/inhibitor complex. These data suggest that the coevolution of serpin structure and inhibitory function date back to at least early metazoan evolution, approximately 1000 MYA.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed2005

Serpins 2005 - fun between the beta-sheets. Meeting report based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia).

Serpins are the largest family of protease inhibitors and are fundamental for the control of proteolysis in multicellular eukaryotes. Most eukaryote serpins inhibit serine or cysteine proteases, however, noninhibitory members have been identified that perform diverse functions in processes such as hormone delivery and tumour metastasis. More recently inhibitory serpins have been identified in prokaryotes and unicellular eukaryotes, nevertheless, the precise molecular targets of these molecules remains to be identified. The serpin mechanism of protease inhibition is unusual and involves a major conformational rearrangement of the molecule concomitant with a distortion of the target protease. As a result of this requirement, serpins are susceptible to mutations that result in polymerization and conformational diseases such as the human serpinopathies. This review reports on recent major discoveries in the serpin field, based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia).

Animals↗

PubMed2001

A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.

Amino Acid Sequence↗

PubMed1995

Pigment epithelium-derived factor behaves like a noninhibitory serpin. Neurotrophic activity does not require the serpin reactive loop.

Pigment epithelium-derived factor (PEDF), a neurite-promoting factor, has an amino acid primary structure that is related to members of the serine protease inhibitor (serpin) family. Controlled proteolysis of native PEDF (50 kDa) with either trypsin, chymotrypsin, elastase, or subtilisin yields in each case one major limited product of 46 kDa as analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analysis of the isolated 46-kDa products indicates a favored cleavage region located toward the C-terminal end of PEDF. A proteolyzed PEDF protein reaction mixture reveals two overlapping sequences: that of the N terminus of intact PEDF and that of an internal region, consistent with cleavage of PEDF about position 382. These data indicate that PEDF protein has a globular conformation with one protease-sensitive exposed loop that contains the homologous serpin-reactive site. Cleavage within the reactive-site loop of PEDF does not cause a conformational change in the molecules (the stressed (S)-->relaxed (R) transition) and results in heat denaturation identical to its native counterpart. This lack of conformational change is also seen upon cleavage within the reactive-site loop of the noninhibitory serpin ovalbumin. Furthermore, the PEDF neurite-promoting function is not lost with cleavage of the exposed loop. Recombinant PEDF polypeptide fragments with larger truncations from the C-terminal end show neurotrophic activity. Our results clearly indicate that integrity of the PEDF homologous serpin reactive center is dispensable for neurotrophic activity. Thus, the PEDF induction of neurites must be mediated by a mechanism other than serine protease inhibition. Altogether our data indicate that PEDF belongs to the subgroup of noninhibitory serpins and that its N-terminal region confers a neurite-promoting activity to the protein. The neurotrophic active site of PEDF is separated from the serpin reactive-site loop, not only in the primary structure, but also in the folded protein structure.

Amino Acid Sequence↗

PubMed2000

Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A.

The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the serpin structure involving rapid insertion of the RCL into the center of an existing beta-sheet, serpin beta-sheet A. The present study demonstrates that partitioning between inhibitor and substrate modes of reaction can be altered by varying either the rates of RCL insertion or deacylation using a library of serpin RCL mutants substituted in the critical P(14) hinge residue and three different proteinases. We further correlate the changes in partitioning with the actual rates of RCL insertion for several of the variants upon reaction with the different proteinases as determined by fluorescence spectroscopy of specific RCL-labeled inhibitor mutants. These data demonstrate that the serpin mechanism follows a branched pathway, and that the formation of a stable inhibited complex is dependent upon both the rate of the RCL conformational change and the rate of enzyme deacylation.

Amino Acids↗

PubMed1998

Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length.

The exposed Serpin reactive centre loop controls the specificity of the serpin proteinase interaction. Mutations within this region have been used to generate novel potentially therapeutic inhibitors. In this study we examine the effect of the serpin scaffold and reactive centre loop length upon the generation of such inhibitors. The reactive centre loop regions, P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and ACT/Serp1, respectively. AT/Serp1 formed SDS stable complexes with a range of proteinases with association rate constants for plasmin, tissue plasminogen activator, urokinase, thrombin and factor Xa of approximately 10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for all of them. ACT/Serp1, however, formed SDS-stable complexes with only plasmin and thrombin with association rate constant 100-fold slower than AT/Serp1 and an increased stoichiometry of inhibition. The reactive centre loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1. These four additional residues (VETR) were inserted into AT/Serp1 to form AT/Serp1(VETR). AT/Serp1(VETR) formed SDS stable complexes with plasmin, thrombin and tissue plasminogen activator similar to AT/Serp1, however, the association rate constants were 10-fold slower than those observed with AT/Serp1, while the stoichiometry of inhibition remained around 1. These results suggest that the additional reactive centre loop residues effect the rate of initial complex formation by placing the reactive centre loop in a non-ideal conformation. This study demonstrates that both reactive centre loop length and serpin scaffold are important in defining the inhibitory characteristics of a serpin.

Amino Acid Sequence↗

PubMed1994

Complexes between serpins and inactive proteinases are not thermodynamically stable but are recognized by serpin receptors.

The serpin mechanism of action may resemble the "standard mechanism" described for small protein inhibitors of serine proteinases. Since these inhibitors are able to bind active site-modified target proteinases, we have investigated the interactions between two serpins and their 3,4-dichloroisocoumarin (DCI)-inactivated target proteinases. alpha 2-Antiplasmin and alpha 1-proteinase inhibitor bound stoichiometrically to DCI-inactivated chymotrypsin (EC 3.4.21.1) and DCI-inactivated human neutrophil elastase, respectively. Similar to active proteinases, the DCI-inactivated proteinases failed to bind complexes between serpins and synthetic reactive site loop peptides. Thus, the abilities of active and inactive proteinases to bind the serpins probably depend on the same structural characteristics. The thermodynamic stability of the alpha 2-antiplasmin-DCI/chymotrypsin and alpha 1-proteinase inhibitor-DCI/human neutrophil elastase complexes was similar to that of virgin serpins. However, in mouse plasma elimination studies the two complexes were removed rapidly from the circulation, suggesting that they have adopted the receptor recognized conformation. Consequently, cleavage of the reactive center peptide bond and formation of an inhibitor-acyl enzyme complex is neither obligatory to serpin-proteinase complex formation nor essential for the conformational change responsible for receptor mediated endocytosis.

Amino Acid Sequence↗

PubMed1993

Cross-competition for binding of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes to the serpin-enzyme complex receptor by other serpin-enzyme complexes and by proteolytically modified alpha 1 AT.

The serpin-enzyme complex (SEC) receptor recognizes a pentapeptide neo-domain of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes and, in so doing, mediates internalization and intracellular catabolism of the macromolecular complex, mediates an increase in synthesis of alpha 1 AT, and elicits neutrophil chemotactic activity. In previous studies we have shown that this pentapeptide domain is highly conserved among members of the serpin family and that binding of a synthetic peptide corresponding to this region (125I-peptide 105Y, SIP-PEVKFNKPFVYLI, based on alpha 1 AT sequence 359-374) to HepG2 cells is blocked by several serpin-enzyme complexes. To determine whether the SEC receptor is the primary HepG2 cell surface binding site for these serpin-enzyme complexes, we examined the capacity for serpin-enzyme complexes to compete with each other for binding to the SEC receptor. The results indicate that binding of 125I-elastase-alpha 1 AT complexes is blocked by thrombin-antithrombin III (ATIII), thrombin-heparin cofactor II, and cathepsin G-alpha 1-antichymotrypsin (alpha 1 ACT) complexes. Moreover, unlabeled elastase-alpha 1 AT complexes compete for binding of 125I-thrombin-ATIII, 125I-thrombin-heparin cofactor II, and 125I-cathepsin G-alpha 1 ACT complexes. Preformed soluble tissue plasminogen activator-plasminogen activator inhibitor 1 complexes also compete for binding of elastase-alpha 1 AT complexes to the SEC receptor but do so to a less effective extent, probably because of a less favorable pentapeptide sequence for binding to the SEC receptor. Under conditions in which these serpin-enzyme complexes would be expected to bind to the SEC receptor there is an increase in synthesis of alpha 1 AT but not in synthesis of ATIII or alpha 1 ACT. Proteolytically modified alpha 1 AT also competes for binding of 125I-elastase-alpha 1 AT complexes to the SEC receptor and vice versa. The purified 51-kDa amino-terminal fragment of alpha 1 AT does not compete for binding of 125I-elastase-alpha 1 AT complexes, indicating that the pentapeptide neodomain in the 4-kDa carboxyl-terminal fragment is sufficient for binding to the SEC receptor.

Amino Acid Sequence↗

PubMed2003

Serp-1, a viral anti-inflammatory serpin, regulates cellular serine proteinase and serpin responses to vascular injury.

Complex DNA viruses have tapped into cellular serpin responses that act as key regulatory steps in coagulation and inflammatory cascades. Serp-1 is one such viral serpin that effectively protects virus-infected tissues from host inflammatory responses. When given as purified protein, Serp-1 markedly inhibits vascular monocyte invasion and plaque growth in animal models. We have investigated mechanisms of viral serpin inhibition of vascular inflammatory responses. In vascular injury models, Serp-1 altered early cellular plasminogen activator (tissue plasminogen activator), inhibitor (PAI-1), and receptor (urokinase-type plasminogen activator) expression (p < 0.01). Serp-1, but not a reactive center loop mutant, up-regulated PAI-1 serpin expression in human endothelial cells. Treatment of endothelial cells with antibody to urokinase-type plasminogen activator and vitronectin blocked Serp-1-induced changes. Significantly, Serp-1 blocked intimal hyperplasia (p < 0.0001) after aortic allograft transplant (p < 0.0001) in PAI-1-deficient mice. Serp-1 also blocked plaque growth after aortic isograft transplant and after wire-induced injury (p < 0.05) in PAI-1-deficient mice indicating that increase in PAI-1 expression is not required for Serp-1 to block vasculopathy development. Serp-1 did not inhibit plaque growth in uPAR-deficient mice after aortic allograft transplant. We conclude that the poxviral serpin, Serp-1, attenuates vascular inflammatory responses to injury through a pathway mediated by native uPA receptors and vitronectin.

Angioplasty↗

PubMed2006

Serpin crystal structure and serpin polymer structure.

Serpins are a family of structurally homologous proteins having metastable native structures. As a result, a serpin variant destabilized by mutation(s) has a tendency to undergo conformational changes leading to inactive forms, e.g., the latent form and polymer. Serpin polymers are involved in a number of conformational diseases. Although several models for polymer structure have been proposed, the actual structure remains unknown. Here, we provide a comprehensive list of serpins, both free and in complexes, deposited in the Protein Data Bank. Our discussion focuses on structures that potentially can contribute to a better understanding of polymer structure.

Amino Acid Sequence↗