The structural puzzle of how serpin serine proteinase inhibitors work

Serine proteinase cleavage of proteins is essential to a wide variety of biological processes and is primarily regulated by protein inhibitors. Many inhibitors are conformationally rigid simulations of optimal serine proteinase substrates, which makes them highly efficient competitive inhibitors of target proteinases. In contrast, members of the serpin family of serine proteinase inhibitors display extensive flexibility and polymorphism, particularly in their reactive site segments and in β-sheet secondary structure, which can take up and expel strands. Reactive site and β-sheet polymorphism appear to be coupled in the serpins and may account for the extreme stability of serpinproteinase complexes through the insertion of the reactive site strand into a β-sheet. These unusual properties may have opened an adaptive pathway of proteinase regulation that was unavailable to the conformationally rigid proteinase inhibitors.     

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.     

Plant serine proteinase inhibitors

Evidence that establishes the mechanism of the classes of plant proteinase inhibitors (PIs) is evaluated. Of the eight classes of PIs, six are unique to plants. Except for plant serpins, there is evidence that PIs from all other classes form tight binding complexes with their target proteinases, and that they follow the standard mechanism of inhibition.     

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.     

The distribution of serine proteinase inhibitors in seeds of the Asteridae

The Asteridae is one of the most successful clades of flowering plants comprising some 80,000 species. Despite this diversity, analysis of seeds from 398 species (representing 8 orders, 32 families and 181 genera) showed just two major types of serine proteinase inhibitors (PI). PIs of the potato inhibitor I family were widely distributed. These had M(r) of 7000-7500 and were inhibitory to subtilisin and one or more other proteinases (but only rarely elastase). The second major group was TI related to the well-characterised Bowman-Birk inhibitors of legume seeds but these varied widely in their sequences and structure. In addition to these two groups of inhibitors, seeds of the Solanaceae also often contained PI of the potato inhibitor II family while some other asterids contained inhibitors whose relationships were not established.     

The role of scaffolding in standard mechanism serine proteinase inhibitors

In single domain, "standard mechanism" protein inhibitors of serine proteinases, about a dozen residues make contact with the cognate enzyme. The remainder of the molecule, the scaffolding, holds the reactive site region of the inhibitor in a canonical conformation, improves the binding by about six orders of magnitude and protects it from proteolysis. However, the stability and global structure of the scaffolding is irrelevant to inhibition, provided that inhibition is measured much below the melting temperature, Tm.     

An overview of the serpin superfamily

Serpins are a broadly distributed family of protease inhibitors that use a conformational change to inhibit target enzymes. They are central in controlling many important proteolytic cascades, including the mammalian coagulation pathways. Serpins are conformationally labile and many of the disease-linked mutations of serpins result in misfolding or in pathogenic, inactive polymers.     

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.     

Four serine proteinase inhibitors (serpin) from the brown ear tick,Rhiphicephalus appendiculatus; cDNA cloning and preliminary characterization

While development of an anti-Boophilus microplus vaccine is advanced and practical, work on other economically important ticks such as Rhipicephalus appendiculatus is still in its infancy. Guess PCR primers, designed from a consensus amino acid sequence (NAVYKFG) motif were used with rapid amplification of cDNA ends (RACE) to clone four cDNAs encoding serine proteinase inhibitors (serpin) from the brown ear tick Rhipicephalus appendiculatus. The four genes designated as R. appendiculatus serpin (RAS) -1 to -4 encode polypeptides of 378, 380, 398 and 486 amino acids long, respectively. Sequence comparison of RAS-1 to -4 predicted amino acid sequences to the serpin-like hypothetical protein from Ixodes ricinus (Leboulle et al., 2002) revealed closer structural similarities among tick serpins. Expression analysis by RT-PCR showed that RAS-1 to -4 are expressed in other tick organs in addition to salivary glands and midguts. Except for RAS-3 whose expression level appears to be equivalent in all tick organs, RAS-1, -2 and -4 are predominantly expressed in the salivary glands. We have discussed our findings with reference to development of vaccines against R. appendiculatus.     

The evolution of a genetic locus encoding small serine proteinase inhibitors

We previously identified a locus on human chromosome 20 that encompasses 14 genes of postulated WFDC-type proteinase inhibitors with a potential role in innate immunity. In an extended study, homologous loci are here described on mouse chromosome 2, rat chromosome 3, and dog chromosome 24. As in humans, the murine and canine loci are divided into two sub-loci separated by 0.2Mb. The majority of genes are conserved in all species, but there are also species-specific gains and losses of genes, e.g., several duplications have yielded four SLPI genes in the rat and, most surprisingly, there is no murine elafin gene. Two human pseudogenes were identified due to the discovery of functional rodent genes. The conservation of different WFDC domains varies considerably, and it is hypothesized that this reflects a dual role of WFDC inhibitors in natural immunity, which is directed both against microbes and proinflammatory cells.     

Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors

Ingestion of Kunitz soybean trypsin inhibitor (STI) by larval Helicoverpa zea, Agrotis ipsilon, and Trichoplusia ni extended the retention time of food in the digestive tract and increased the level of activity of proteolytic enzymes that were not susceptible to inhibition by STI. The level of enhancement of activity of STI-resistant (STI-R) enzyme(s) was directly influenced by the dosage and timing of exposure to STI. However, not all proteinase inhibitors (PIs) enhanced the level of proteinase inhibitor resistant (PI-R) enzymes, even if those PIs inhibited a significant proportion of enzyme activity. These findings suggest that a complex system may be responsible for the regulation of proteolytic enzymes in the midgut of larval Lepidoptera, and one hypothesis for this regulation is proposed.     

Detection of serine proteinase inhibitors in human cornified cells

A screening test for serine proteinase inhibitors revealed trypsin and urokinase inhibitors in the extract of human cornified cells. No inhibition for α-chymotrypsin, thrombin or plasmin was detected. Characterization of the inhibitors separated with a Sephacryl S-200 gel column demonstrated that: 1) trypsin inhibitor with a molecular weight of 45,000 was labile to heat, acid and alkali and showed temporary inhibition, and 2) urokinase inhibitor with a molecular weight of 35,000 was found relatively stable and exhibited time dependent inhibition. Both were distinct from a known thiol proteinase inhibitor which showed high stability and immediate inhibition. Regulatory roles of serine proteinase inhibitors are postulated.     

Atypical Kunitz-type serine proteinase inhibitors produced by the ruminant placenta

Recently, an unusual family of genes was identified with expression confined to the trophoblast of ruminant ungulate species. The members of this family (the trophoblast Kunitz domain proteins, or TKDPs) are characterized by the presence of one or more similar, approximately 80-residue repeat sequences placed ahead of a Kunitz serine proteinase-inhibitor domain. To examine the specificity of the Kunitz moiety, the Kunitz domains of selected TKDPs and a control Kunitz protein, bovine pancreatic trypsin inhibitor (BPTI), were produced as glutathione S-transferase fusions, and their abilities to inhibit six serine proteinases were examined. Circular dichroism spectroscopy confirmed that the Kunitz fold was intact. Three of the TKDPs had unusual residues at their P1 "warhead" (ovine TKDP-1, Asn; bovine TKDP-3, Thr; and bovine TKDP-5, Ile) and exhibited no measurable inhibitory activity toward any of the proteinases. Three (ovine TKDP-3, bovine TKDP-3, and bovine TKDP-4) lacked the conserved cysteines at residues 14 and 38 that form one of the highly conserved disulfide bonds that are structurally important in all known mammalian Kunitz proteins. Ovine TKDP-3 and bovine TKDP-4 had P1 lysines and inhibited trypsin and plasmin with K(i) values only approximately 10-fold higher than that of BPTI. Bovine TKDP-2 had a P1 lysine and the three conserved disulfides, but it possessed an unusual residue (Asp) at P2. It exhibited no inhibitory activity. These data suggest that the function of the TKDP, like certain Kunitz proteins found in snake venoms, may not be in proteinase inhibition.     

A comparison of three heparin-binding serine proteinase inhibitors.

The purpose of this study was to compare three heparin-binding plasma proteinase inhibitors in order to identify common and unique features of heparin binding and heparin-enhanced proteinase inhibition. Experiments with antithrombin, heparin cofactor, and protein C inhibitor were performed under identical conditions in order to facilitate comparisons. Synthetic peptides corresponding to the putative heparin binding regions of antithrombin, heparin cofactor, and protein C inhibitor bound to heparin directly and interfered in heparin-enhanced proteinase inhibition assays. All three inhibitors obeyed a ternary complex mechanism for heparin-enhanced thrombin inhibition, and the optimum heparin concentration was related to the apparent heparin affinity of the inhibitor. The maximum inhibition rate and rate enhancement due to heparin appeared to be unique properties of each inhibitor. In assays with heparin oligosaccharides of known size, only the antithrombin-thrombin reaction exhibited a sharp threshold for rate enhancement at 14-16 saccharide units. Acceleration of antithrombin inhibition of factor Xa, heparin cofactor inhibition of thrombin, and protein C inhibitor inhibition of thrombin, activated protein C, and factor Xa did not require a minimum saccharide size. The differences in heparin size dependence and rate enhancement of proteinase inhibition by these inhibitors might reflect differences in the importance of the ternary complex mechanism and other mechanisms, alterations in inhibitor reactivity, and orientation effects in heparin-enhanced proteinase inhibition.     

The effect of endogeneous proteinase inhibitors on the prophenoloxidase activating enzyme,a serine proteinase from crayfish haemocytes

Three proteinase inhibitors have so far been isolated and purified from crayfish haemolymph. One of these, isolated from crayfish plasma, namely a trypsin inhibitor with a molecular mass of 155 kDa was found to inhibit a serine proteinase, ppA, which is involved in the activation of prophenoloxidase, and is localized in the haemocytes. Another high molecular mass proteinase inhibitor, an α₂-macroglobulin from crayfish plasma, which is a dimer of 190 kDa-subunits, was only inhibitory towards ppA to a lesser extent. A 23 kDa subtilisin inhibitor, purified from haemocytes, did not have any effect on the serine proteinase.We suggest that mainly the trypsin inhibitor, but to some extent also the α₂-macroglobulin, are important in the regulation of the prophenoloxidase activating cascade, as they both inhibit ppA, which in its active form has been shown to mediate prophenoloxidase activation.     

Low molecular weight serine proteinase inhibitors of the human intervertebral disc

Human lumbar disc tissue when extracted with 4M GuHCl and subjected to dissociative CsCl density gradient ultracentrifugation yielded trypsin inhibitor activity in the low bouyant density fractions (rho less than or equal to 1.38 g/ml). Disc proteoglycans sedimented in the high bouyant density fractions (rho greater than or equal to 1.5 g/ml). Sephadex G75F gel filtration of the low bouyant density protein fractions afforded a major low molecular weight (Kav = 0.5) trypsin inhibitor pool which was further purified by trypsin affinity chromatography. This latter step facilitated separation of the trypsin inhibitors from neutral proteinase activity also present. The trypsin inhibitor fraction so isolated was shown to possess potent inhibitory activity against a range of human serine proteinases including leukocyte elastase and cathepsin G, urokinase, kallikrein, plasmin and thrombin. Significantly this serine proteinase inhibitor preparation effectively prevented degradation of proteoglycans by a neutral proteinase also isolated from the human intervertebral disc.     

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.     

Identification of four distinct serine proteinase inhibitors in rat skeletal muscle

The serine proteinase inhibitory capacity in the cytosolic fraction of rat skeletal muscle tissue is accounted for by several discrete inhibitory activities. Three of these activities are identical with the proteinase inhibitors α₁-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor I I respectively, which have been recently characterized as major serine proteinase inhibitors in rat serum (Kuehn, L., Rutschmann, M., Dahlmann, B. and Reinauer, H. (1984) Biochem. J. $\text{218}$, in the press). The other inhibitor molecule, having an M_r of about 15 000, appears to be an endogeneous inhibitor.