Serpin Superfamily Of Proteinase Inhibitors Research Articles

Iripin-3, a New Salivary Protein Isolated From Ixodes ricinus Ticks, Displays Immunomodulatory and Anti-Hemostatic Properties In Vitro.

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

Serpin peptidase inhibitor, clade A member 3 (SERPINA3), is overexpressed in glioma and associated with poor prognosis in glioma patients.

Glioma is the most common and aggressive human primary tumor in the central nervous system. Despite present clinical advancements, median survival time remains poor in this malignant tumor. Serpin peptidase inhibitor, clade A member 3 (SERPINA3), is a member of the serpin superfamily of protease inhibitors. Its aberrant expression has been observed in various tumors. However, its clinical significance and biological function in glioma remain unclear, especially for the prognosis of glioma patients. In this study, we investigated SERPINA3 expression in glioma tissue samples and its significance in predicting the prognosis of glioma patients. SERPINA3 protein expression was studied by immunohistochemistry, while real-time polymerase chain reaction was used to study SERPINA3 mRNA expression. We found that SERPINA3 was upregulated in glioma tissue at both mRNA and protein levels, compared with noncancerous brain tissues. We also found that high SERPINA3 expression in glioma tissues correlated significantly with advanced World Health Organization grade. Univariate and multivariate analyses revealed that high SERPINA3 expression was an independent prognostic factor for poor overall survival of glioma patients. Moreover, our findings were further validated by online Oncomine database. Taken together, our results suggest that SERPINA3 plays an oncogenic role in glioma progression and provide an insight into the application of SERPINA3 as a novel predictor of clinical outcomes and a potential biomarker of glioma.

Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity

Exhaustive mutagenesis studies of the reactive centre loop (RCL), a key structural component of proteins belonging to the serpin superfamily of protease inhibitors, are complicated by the size of the RCL, serpin conformational complexity, and, for most serpins, the lack of a serpin-dependent phenotype of expressing cells. Here, we describe a thrombin capture assay that distinguished thrombin-inhibitory recombinant human alpha-1 proteinase inhibitor (API M358R) from non-inhibitory API variants in Escherichia coli lysates prepared from either single clones or pools. Binding of API proteins in the lysates to thrombin immobilized on microtiter plate wells was quantified via colour generated by a peroxidase-coupled anti-API antibody. Bacterial expression plasmids encoding inhibitory API M358R were mixed 1:99 with plasmids encoding non-inhibitory API T345R/M358R and the resulting library screened in pools of 10. All above-background signals arising from pools or subsequently re-probed single clones were linked to the presence of plasmids encoding API M358R. Screening of a portion of another expression library encoding hypervariable API with all possibilities at codons 352–358 also yielded only novel, thrombin-inhibitory variants. Probing a smaller library expressing all possible codons at Ala347 yielded the wild type, 6 different functional variants, one partially active variant, and two variants with no thrombin-inhibitory activity. API antigen levels varied considerably less among Ala347 variants than activity levels, and comparison of rate constants of inhibition of purified API variants to their corresponding thrombin capture assay lysate values was used to establish the sensitivity and specificity of the assay. The results indicate that the approach is sufficiently robust to correctly identify functional versus non-functional candidates in API expression libraries, and could be of value in systematically probing structure/function relationships not only in the API RCL, but also in that of other serpins.

Construction of an engineered alpha 1-antitrypsin with inhibitory activity based on theoretical studies

Background: The elastase inhibitor α-1-antitrypsin (AAT), is a member of the serpin superfamily of protease inhibitors. AAT has a characteristic secondary structure of three-β-sheets, nine-α-helices and a reactive central loop (RCL). This protein inhibits target proteases by forming a stable complex in which the cleaved RCL is inserted into β-sheet-A of the serpin, leading to a conformational change in the AAT protein. Spontaneous polymerization and instability of AAT are challenges with regard to producing drugs against AAT-deficient diseases. Therefore, the purpose of many investigations currently is to produce drugs with lower degrees of polymerization and higher stabilities. In order to investigate the effect of the N-terminal segment (residues 1-43) on AAT structure, molecular dynamic (MD) simulation was used to study structural properties including Root-mean-square deviation (RMSD), internal motions, intramolecular non-bonded interactions and the total accessible surface area (ASA) of native and reduced AAT. These properties were compared in native and truncated AAT. Results: Theoretical studies showed no noticeable differences in the dynamic and structural properties of the two structures. These findings provided the basis for the experimental phase of the study in which sequences from the two AAT constructs were inserted into the expression vector pGAPZ and transformed into Pichia pastoris . Results showed no differences in the activities and polymerization of the two AAT constructs. Conclusions: As small-scale medicines are preferred by lung drug delivery systems, in this study AAT was designed and constructed by decreasing the number of amino acids at the N-terminal region.

Protein Z and protein Z-dependent protease inhibitor and renal tubules

Dear Sirs, Protein Z (PZ) is a vitamin K-dependent, plasma protein that serves as a cofactor for the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI) in the presence of calcium ions and phospholipids. ZPI is a member of the serpin superfamily of proteinase inhibitors and not only inhibits factor Xa in a PZ-dependent fashion, but also inhibits factor XIa in the absence of cofactors. Both PZ and ZPI are thought to be produced predominantly by the liver (1). To assess other potential sources of PZ besides the liver, tissue northern analysis was performed and showed substantial PZ mRNA expression (~1.4 kb) in the kidney (▶Fig. 1A). Subsequent immunohistochemistry studies using two monoclonal anti-PZ antibodies (Mab 2048, Mab 2306.BF12.2) (2) demonstrated PZ staining in the renal distal and collecting tubules based on tubular morphology and location in paraffin and frozen sections of the renal cortex (not shown) and the renal medulla (Fig. 1C). ZPI was also detected in kidney tubules by immunohistochemical analysis using two monoclonal antibodies (Mab 4242.2, Mab 4336.1E5) (3) in the same location as PZ (Fig. 1D). As Northern analysis has shown little if any ZPI expression in the kidney, compared to that in the liver (4), it is conceivable that the ZPI detected by immunohistochemistry was produced elsewhere and then bound to tubular PZ (2). Notably, PZ and ZPI immune-reactivity was not seen in glomeruli or other renal vascular structures. PZ and ZPI expression was also detected in the liver by immunohistochemistry, but not in lung, heart, spleen, or vasculature (not shown). Figure 1 PZ and ZPI in human kidney tubules Madin-Darby canine kidney cells (MDCK, CCL34, ATCC, Manassas, VA, USA) are derived from canine distal tubule cells, have retained a polarised phenotype, and have been used extensively to examine the sorting of proteins and other molecules. Studies of MDCK cells stably expressing recombinant human PZ, ZPI and both PZ and ZPI cultured on Transwell filter units (0.4 μm, Fisher Scientific, Pittsburgh, PA, USA) (5) demonstrated that these proteins were secreted apically, i.e. toward the tubular lumen (not shown). Random urine testing of six laboratory personnel showed the presence of PZ (61.3 ± 9.3 ng/ml; 2.4 ± 0.4% of the plasma level), but no ZPI by monoclonal sandwich immunoassays (2, 3). Western blotting using rabbit polyclonal antibodies of concentrated (YM 10, Millipore, Billerica, MA, USA) urine specimens demonstrated apparent full-length PZ (62,000 molecular weight [MW]), with little evidence of proteolytic degradation and a trace of ZPI reactivity at 72,000 and 50,000 MW. These results suggest that at least some of the PZ expressed by tubular cells is secreted into the urine, whereas the bulk of ZPI is either not secreted into the urine or is degraded to forms of low molecular weight. Glomerular filtration appears less likely as a source of urinary PZ, since PZ and ZPI circulate as a relatively high-molecular-weight complex in plasma (2). Prothrombin, factor XI and prekallikrein are also produced by kidney tubules (6–10) and the F1 fragment of prothrombin (PTF1) and PZ (11, 12) have been detected in kidney stones. Northern analysis of mouse tissues demonstrates kidney expression of PZ (not shown) and mouse urine (n=5) contains PZ at 1.9 ± 0.6% the PZ level of mouse plasma. Therefore the mouse will likely provide an appropriate model to investigate the physiologic relevance of urinary PZ.

A computational modeling and molecular dynamics study of the Michaelis complex of human protein Z-dependent protease inhibitor (ZPI) and factor Xa (FXa)

Protein Z-dependent protease inhibitor (ZPI) and antithrombin III (AT3) are members of the serpin superfamily of protease inhibitors that inhibit factor Xa (FXa) and other proteases in the coagulation pathway. While experimental structural information is available for the interaction of AT3 with FXa, at present there is no structural data regarding the interaction of ZPI with FXa, and the precise role of this interaction in the blood coagulation pathway is poorly understood. In an effort to gain a structural understanding of this system, we have built a solvent equilibrated three-dimensional structural model of the Michaelis complex of human ZPI/FXa using homology modeling, protein-protein docking and molecular dynamics simulation methods. Preliminary analysis of interactions at the complex interface from our simulations suggests that the interactions of the reactive center loop (RCL) and the exosite surface of ZPI with FXa are similar to those observed from X-ray crystal structure-based simulations of AT3/FXa. However, detailed comparison of our modeled structure of ZPI/FXa with that of AT3/FXa points to differences in interaction specificity at the reactive center and in the stability of the inhibitory complex, due to the presence of a tyrosine residue at the P1 position in ZPI, instead of the P1 arginine residue in AT3. The modeled structure also shows specific structural differences between AT3 and ZPI in the heparin-binding and flexible N-terminal tail regions. Our structural model of ZPI/FXa is also compatible with available experimental information regarding the importance for the inhibitory action of certain basic residues in FXa.

Maspin Expression in Oral Squamous Cell Carcinoma

Maspin (mammary serine protease inhibitor) is a member of the serpin superfamily of protease inhibitors and it has a role as a tumor suppressor. Maspin has been reported to be important in processes relevant to tumor growth and metastasis such as cell invasion, angiogenesis, and apoptosis. A high expression of maspin was correlated with better rates of survival and absence of nodal metastases in head and neck squamous cell carcinoma. In contrast, some studies have shown that maspin overexpression is correlated with a poor prognosis in pancreatic and ovarian cancers and in lung adenocarcinoma. The aim of this study was an immunohistochemical evaluation of the maspin expression in oral squamous cell carcinoma and thus 89 patients were evaluated. Maspin expression in oral squamous cell carcinoma was significantly associated with the tumor differentiation grade (chi test: P = 0.0318) and the lymph node status (chi test: P < 0.005), but not with the tumor stage (chi test: P = 0.666). Metastatic involvement of lymph nodes was observed more frequently in maspin-negative cases than in tumors with more than 5% of positive cells (P = 0.0024). The present results confirm that maspin expression predicts a better prognosis in oral squamous cell carcinoma and that maspin probably plays a role in tumor progression.

Additivity in Effects of Vitronectin and Monoclonal Antibodies against α-Helix F of Plasminogen Activator Inhibitor-1 on Its Reactions with Target Proteinases

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against alpha-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to alpha-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo.

Protein Z-Dependent Regulation of Coagulation

Protein Z (PZ) is a 62 kDa vitamin K-dependent plasma protein that serves as a cofactor for the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI). ZPI is a recently identified 72 kDa member of the serpin superfamily of proteinase inhibitors that contains a tyrosine at its reactive center. PZ circulates in plasma in a complex with ZPI. Inhibition of factor Xa by ZPI in the presence of phospholipids and Ca++ is enhanced 1000-fold by PZ, but ZPI also inhibits factor XIa in a process that does not require PZ, phospholipids or Ca++. ZPI activity is consumed during coagulation through proteolysis mediated by factor Xa with PZ and factor Xla. Concomitant PZ deficiency dramatically increases the severity of the prothrombotic phenotype of factor VLeiden mice. Studies to determine the potential roles of PZ and ZPI deficiency in human thrombosis are in progress.

Characterization of the protein Z–dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca++ (t1/2 less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa–ZPI complex is not stable to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but is detectable by alkaline–polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca++. In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca++. Heparin (0.2 U/mL) enhances the rate (t1/2 = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P1 residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.

Characterization of the protein Z–dependent protease inhibitor

AbstractProtein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca++ (t1/2 less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa–ZPI complex is not stable to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but is detectable by alkaline–polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca++. In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca++. Heparin (0.2 U/mL) enhances the rate (t1/2 = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P1 residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.

The protein Z-dependent protease inhibitor is a serpin.

In the presence of phospholipid vesicles and calcium ions, protein Z (PZ) serves as a cofactor for the inhibition of coagulation factor Xa by a plasma protein called PZ-dependent protease inhibitor (ZPI). To further characterize ZPI, its cDNA has been isolated and cloned from a human liver cDNA library. The ZPI cDNA is 2.44 kb in length and has a relatively long 5' region (466 nt) that contains six potential ATG translation start codons. ATG's 1-4 are followed by short open reading frames, whereas ATG(5) and ATG(6) are in an uninterrupted open reading frame that includes the encoded ZPI protein. In vitro experiments show that ATG(6) is sufficient for the expression of rZPI in cultured Chinese hamster ovary cells. Northern analysis suggests the liver is a major site of ZPI synthesis. The predicted 423 residue amino acid sequence of the mature ZPI protein is 25-35% homologous with members of the serpin superfamily of protease inhibitors and is 78% identical to the amino acid sequence predicted by a previously described cDNA isolated from rat liver, regeneration-associated serpin protein-1 (rasp-1). Thus, ZPI is likely the human homologue of rat rasp-1. Alignment of the amino acid sequence of ZPI with those of other serpins predicts that Y387 is the P(1) residue at the reactive center of the ZPI molecule. Consistent with this notion, rZPI(Y387A), an altered form of ZPI in which tyrosine 387 has been changed to alanine, lacks PZ-dependent factor Xa inhibitory activity.

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus.

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1-PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.

Deletion mutagenesis of heparin cofactor II: defining the minimum size of a thrombin inhibiting serpin

Heparin cofactor II (HCII) is a 66 kDa plasma glycoprotein that belongs to the serpin superfamily of protease inhibitors. Its natural target is thrombin. HCII inhibits thrombin in both a progressive reaction, and in an accelerated reaction catalyzed by a glycosaminoglycan, dermatan sulphate (DS). Both modes of inhibition result in the formation of a stable, denaturation-resistant complex. Using a cDNA clone encoding rabbit HCII recently isolated and characterized in our laboratory, we have employed deletion mutagenesis to identify amino-terminal regions of the molecule which are essential to the progressive reaction. PCR was employed to produce four deletion constructs: Δ58, Δ81, Δ106, and Δ169, all in an in vitro transcription vector plasmid background. Transcription of the full-length construct, and of the four deletion constructs, followed by in vitro translation in rabbit reticulocyte lysate, was used to produce the corresponding HCII-related polypeptides. The Δ106 and Δ169 mutants failed to react with thrombin, even in the presence of DS. In contrast, the Δ58 and Δ82 mutants retained the ability to form complexes with thrombin, although the rate of complex formation was decreased for the latter mutant compared to the full-length recombinant HCII; no acceleration of complex formation in the presence of 20 μg/ml DS was noted for either truncated recombinant HCII. Alignment of the rabbit HCII primary structure with secondary structural elements found in α 1antitrypsin and other serpins showed that the non-functional Δ106 mutant lacks helix A, while the functional Δ82 mutant contains this element. Our results suggest that helix A is an essential part of a functional serpin, and define the limits of the amino-terminal region of HCII which is not essential for thrombin inhibition.

A Drosophila male accessory gland protein that is a member of the serpin superfamily of proteinase inhibitors is transferred to females during mating

Accessory gland peptide 76A, (Acp76A), belongs to the serpin superfamily of proteins ( serine protease inhibitors). Acp76A is a secreted protein synthesized only in the Drosophila melanogaster adult male accessory gland. Accumulation of the protein in males is first detected with polyclonal antibodies at 1 day after eclosion. The level of the protein in virgin males reaches a peak 5–8 days post-eclosion, and remains constant for at least 20 days. Upon mating the amount of Acp76A in males drops dramatically, but recovers by 24 h after mating. Immediately after mating the Acp76A is found in the female uterus. By 6 h after mating the amount of Acp76A detected in females is drastically reduced.

The serpin superfamily of proteinase inhibitors: structure, function, and regulation.

The serpin superfamily of proteinase inhibitors: structure, function, and regulation.

The Major Progesterone-modulated Proteins Secreted into the Sheep Uterus Are Members of the Serpin Superfamily of sErine Protease Inhibitors

The uterine milk proteins (UTMP) are a pair of structurally related basic glycoproteins that when newly synthesized carry phosphorylated mannosyl residues on their carbohydrate chains. They are the major proteins secreted by ovine endometrium under the influence of progesterone. RNA from a late pregnant ewe endometrium was isolated for use in in vitro translation assays and for constructing cDNA libraries. Translation experiments, initially with total cellular RNA and subsequently with RNA selected by hybridization with a specific cDNA, demonstrated the production of two polypeptides (Mr = 47,000 and 55,000) that were precipitated with antiserum to the UTMP. With microsomal membranes in the translation assay, there was increased production of an Mr = 57,000 form that was protected from protease digestion. Antibody screening of a cDNA library in lambda gt11 identified a short clone representing the 3' terminus of the mRNA that was shown by epitope selection experiments to be UTMP specific. This clone was then used to screen a lambda gt10 library. A longer clone (1.3 kilobases) was isolated and sequenced but lacked the 5' terminus to the mRNA. The latter sequence was obtained directly from the mRNA. Interesting features of the UTMP mRNA sequence, which was 1,352 bases long and contained a 1,287-base open reading frame, were two strong start codons, two potential sites for N-glycosylation and a repeat of 21 bases, six bases apart, that resulted in a repeat of seven amino acids. The inferred amino acid sequence agreed closely with the NH2-terminal amino acid sequence obtained directly from the UTMP and clearly placed the UTMP in the serpin superfamily of protease inhibitors. However, we have been unable to demonstrate inhibitory activity toward any serine protease so far tested.

Primary Structure of a Member of the Serpin Superfamily of Proteinase Inhibitors from an Insect, Manduca sexta

A cDNA clone isolated from a fat body cDNA library from an insect, Manduca sexta, has been sequenced and shown to code for a member of the serpin family of proteinase inhibitors. The cDNA has an open reading frame which codes for a 392-residue polypeptide of Mr = 43,500 with a hydrophobic NH2-terminal sequence which appears to be a signal peptide. An alignment of this amino acid sequence with 11 members of the serpin superfamily reveals that the insect protein is 25-30% identical with most members of the superfamily. The alignment was used to construct an evolutionary tree of the serpin sequences analyzed, which indicates that the progenitor of the M. sexta serpin and the human serpins most closely related to it diverged from other serpin genes prior to the divergence of the vertebrates and invertebrates. The M. sexta serpin is predicted to inhibit elastase due to the presence of alanine at the P1 position of its reactive center and is classified as an alaserpin. A glycoprotein of Mr = 47,000 isolated from hemolymph of M. sexta larvae has an NH2-terminal sequence identical to that deduced from the alaserpin cDNA clone and inhibits porcine pancreatic elastase and bovine chymotrypsin.

An elastase inhibitor from equine leukocyte cytosol belongs to the serpin superfamily. Further characterization and amino acid sequence of the reactive center.

Horse leukocyte elastase inhibitor rapidly forms stable, equimolar complexes with both human leukocyte elastase and cathepsin G, porcine pancreatic elastase, and bovine alpha-chymotrypsin. Formation of the inhibitor-pancreatic elastase complex results in peptide bond cleavage at the reactive site of the inhibitor so that a small peptide fragment representing the carboxyl-terminal sequence of the inhibitor is released. Sequence analysis of both this peptide, as well as that of an overlapping peptide obtained by enzymatic inactivation of native inhibitor with either Staphylococcus aureus metalloproteinase, Pseudomonas aeruginosa elastase, or cathepsin B, yields data which indicate that the reactive site encompasses a P1-P1' Ala-Met sequence. However, unlike the human endothelial plasminogen activator inhibitor, which also has a Met residue in the P1' position, oxidation of the horse inhibitor only slightly reduces its association rate constant with either of the elastolytic enzymes tested or with chymotrypsin. Comparison of the amino acid sequence at or near the reactive site of the horse inhibitor (P2-P18') with members of the serpin superfamily of proteinase inhibitors indicates that it not only belongs in this class but also represents the first example of a functionally active intracellular serpin.