Comparison of the effects of serpin A1, a recombinant serpin A1-IGF chimera and serpin A1 C-terminal peptide on wound healing

Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor), a potent neutrophil elastase inhibitor, has therapeutic potential as a wound-healing agent. We compared the in vitro wound-healing action of serpin A1-IGF, a recombinant fusion protein of serpin A1(M351E-M358L) and insulin-like growth factor I with that observed in the presence of natural serpin A1 or A1-C26, the synthetic C-terminal 26 residue peptide of serpin A1, previously shown to have mitogenic and antiviral activities. All agents reduced wound sizes in monolayers of the kidney epithelial cell line LLC-PK1 and in primary cultures of human skin fibroblasts. Wound reduction in primary human keratinocytes was only observed with the serpin A1-IGF chimera. None of the factors stimulated cell proliferation using a colorimetric assay, with the exception of the serpin A1-IGF chimera, which caused a significant increase of cell proliferation and thymidine incorporation in human skin fibroblasts. However, wound healing by the A1-IGF chimera was reduced in keratinocytes in the presence of mitomycin C, suggesting a role of cell proliferation in wound reduction. The hydrophobic A1-C26 peptide significantly increased the production of collagen I in skin fibroblasts, an appealing asset for skin care applications.     

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.     

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.     

Identification and cloning of caprine uterine serpin

The uterine serpins have been described in sheep, cattle, and pigs as a highly diverged group of the large superfamily of serpin proteins that typically function as serine proteinase inhibitors. Here, the range of species that possess and express a uterine serpin gene is extended to the goat. Sequencing of cDNA amplified from total RNA from a pregnant goat at day 25 of pregnancy resulted in a 1,292 bp full-length consensus cDNA sequence for caprine uterine serpin (CaUS). The predicted amino acid sequence of the caprine precursor showed 96%, 82%, 55%, and 56% identity to OvUS, BoUS, PoUS1, and PoUS2, respectively. The signal peptide extends from amino acids 1 to 25, resulting in a secreted protein of 404 amino acids and 46,227 Mr (excluding carbohydrate). Both the goat and sheep uterine serpins have a nine amino acid insert in the Helix I region that is not found in bovine or porcine uterine serpins. A total of 13 amino acids in CaUS are different than those for the nearest homologue, ovine uterine serpin. One of these is in the site of cleavage of the signal sequence, where a single nucleotide substitution (G --> C) changed the cysteine for the sheep, bovine, and porcine genes to a serine. In addition, the amino acid at the putative P1-P1' site (the scissile bond for antiproteinase activity) is a valine for CaUS, BoUS, PoUS1, and PoUS2 versus an alanine for OvUS. The hinge region of all five of the uterine serpins (P17-P9) is distinct from the consensus pattern for inhibitory sequences and it is unlikely, therefore, that the uterine serpins possess prototypical proteinase inhibitory activity. The goat uterine serpin was immunolocalized to the glandular epithelium of the endometrium from a pregnant nanny at day 25 of pregnancy. There was also immunoreactive product in scattered luminal epithelial cells. No immunoreaction product was detected in endometrium from a nanny at day 5 of the estrous cycle. Western blotting of uterine fluid collected from the pregnant uterine horn of a unilaterally-pregnant goat revealed the presence of a protein band at Mr approximately 56,000 that reacted with monoclonal antibody to OvUS. In conclusion, the range of species in which uterine serpins are present and expressed in the uterus includes the goat in addition to the previously described sheep, cow, and pig. In all of these species, the uterine serpin is derived primarily from glandular epithelium, is secreted into the uterine lumen, and contains sequence characteristics suggesting it is not an inhibitory serpin.     

New insight into serpin polymerization and aggregation

We recently solved the crystallographic structure of a dimeric form of the serpin antithrombin which has fundamentally changed the way we think about serpin polymerization. Like for other diseases that have protein deposition as a hallmark, the serpinopathies are associated with discrete inter-protomer linkage followed by subsequent association into larger fibrils and aggregates. Polymerization of the serpins is an off-pathway event that occurs during folding in the endoplasmic reticulum. Our structure reveals the nature of the polymerogenic folding intermediate, the reason that the inter-protomer linkage is hyperstable, and suggests a mechanism of lateral association of polymers into soluble fibrils and insoluble aggregates. While the basis of cellular toxicity is still unclear, novel therapeutic approaches targeting the folding intermediate or the lateral association event are now conceivable.     

New insight into serpin polymerization and aggregation

We recently solved the crystallographic structure of a dimeric form of the serpin antithrombin which has fundamentally changed the way we think about serpin polymerization. Like for other diseases that have protein deposition as a hallmark, the serpinopathies are associated with discrete inter-protomer linkage followed by subsequent association into larger fibrils and aggregates. Polymerization of the serpins is an off-pathway event that occurs during folding in the endoplasmic reticulum. Our structure reveals the nature of the polymerogenic folding intermediate, the reason that the inter-protomer linkage is hyperstable, and suggests a mechanism of lateral association of polymers into soluble fibrils and insoluble aggregates. While the basis of cellular toxicity is still unclear, novel therapeutic approaches targeting the folding intermediate or the lateral association event are now conceivable.Key words: aggregation, conformation, folding, polymerization, serpin, structure, toxicityThe serpins are serine protease inhibitors that utilize a unique and well characterized β-sheet expansion event as a necessary part of their mechanism.^1–3 This conformational/topological change from a five-stranded (N in Fig. 1) to a six-stranded β-sheet A (L in Fig. 1) results in a doubling of the protein''s stability, and is driven by a free-energy term of around −32 kcal/mol.⁴ Thus, the native form of serpins is metastable in defiance of the Anfinsen principle,⁵ requiring a folding pathway that kinetically traps the five-stranded state. This unusual protein folding requirement allows for an off-pathway event known as polymerization, where one protomer completes the A sheet of another.⁶ For secreted serpins, this occurs in the endoplasmic reticulum (ER) where polymers are seen to accumulate as insoluble proteinaceous inclusions. Polymerization has been described for several serpins and is always associated with a loss of functional levels due to accumulation within the cell, and occasionally it is associated with the death of the secretory cells through a poorly understood mechanism.^7–9 The best examples of the latter are provided by the Z variant of α₁-antitrypsin (α₁AT) leading to liver disease and the Syracuse variant of neuroserpin that causes early onset dementia.Open in a separate windowFigure 1Serpin folding and polymerization. The pathway of serpin folding proceeds from the unfolded state (U) to the native state (N) via a stable intermediate (M*). The native conformation is the only active state, and is composed of a five-stranded A sheet (red) and a 20 residue reactive centre loop (RCL, yellow). Serpin inhibitory function requires the native conformation to be a kinetically trapped metastable state. Completion of sheet A by incorporation of the RCL as strand 4, to form the latent (L) state, results in the doubling of the serpin''s thermodynamic stability (the six strands are labelled on L). Folding and unfolding of native serpins is known to proceed via a stable intermediate denoted M*, which also corresponds to the polymerogenic form.^24–26 The key feature of the M* state is that strand 5 is not yet incorporated into sheet A, and can thus insert in an intermolecular fashion to form off-pathway polymers (P, each protomer of the pentamer is in a different colour). The polymers have complete A sheets and are thus hyperstable. As a consequence of polymerization, the linker region (cyan), containing helix I, remains unfolded. We hypothesize that the hydrophobic linker (indicated by the oval) is responsible for the lateral association of polymers into insoluble aggregates.We recently solved a crystal structure of a self-terminating (closed) serpin dimer that revealed a large domain swap including the fourth and fifth strands of β-sheet A.¹⁰ We then modelled an open polymerization competent polymer based on the structure (P in Fig. 1) that explained their facile propagation, the hyperstability and flexibility of the inter-protomer linkage, and also suggested a structure for the polymerogenic folding intermediate (M* in Fig. 1). We proposed that the final step in folding to the native state is the insertion of strand 5 into β-sheet A and the association of the coiled linker domain to the ‘bottom’ of the molecule. This event would leave the fourth strand (the reactive centre loop) accessible to serve as bait for proteolytic attack, necessary for the functioning of the serpin mechanism. While many details are yet to be confirmed, the position of certain polymerogenic mutations on and underlying strand 5A supports the proposal.¹⁰One unexpected implication of our model is the requisite unfolding and exposure of helix I and the following coiled region in linear serpin polymers. Exposure of this ‘linker region’ was verified in linear polymers of serpins antithrombin and α₁AT through limited proteolysis and fluorescence studies, and explains the observation that polymers are hydrophobic and exhibit an increased propensity towards aggregation. Unglycosylated serpins typically aggregate when polymerized in vitro (with heat or low concentrations of chaotrophes), even at vanishingly low concentrations, whereas high concentrations are required to observe aggregation of glycosylated serpin polymers. We hypothesized that aggregation/precipitation occurs via lateral association of linear polymers, either through specific β-strand linkage or non-specific hydrophobic interactions involving the linker region. Sequence analysis of helix I suggest that it is a ‘frustrated’ β-strand¹¹ for several serpins including α₁AT, supporting the idea that aggregates of serpin polymers form through an extended β-sheet mechanism akin to other ‘conformational diseases.’¹²While there are clear parallels between the ‘serpinopathies’ and conformational diseases such as Alzheimer, Huntington and the prion encephalopathies (e.g., ordered intermolecular linkage, β-sheet expansion, cell death, dementia, accumulation of insoluble aggregates, domain-swapping),^12,13 the detailed molecular mechanism revealed by our crystal structure is unique to the serpins. Domain swaps in other proteins are generally characterized by normal activity and stability, and may not play a role in the secondary association event that leads to the toxic species.^14,15 For serpins the domain swap leads to hyperstability and the exposure of hydrophobic regions not seen in the monomeric state. Another key difference is the manner of cellular toxicity and the nature of the toxic species. It is becoming clear that for Alzheimer, Huntington and other conformational diseases the toxic fragments are likely to be the soluble (proto)-fibrils, not the insoluble aggregates (inclusions).^16,17 Serpin polymerization generally leads to disease through loss of secretion of the active species, and only in two special cases is it through gain-of-function cellular toxicity, and although the toxic mechanisms are incompletely resolved, they appear to involve the insoluble aggregates.The most common cause of cirrhosis among children is the homozygous Z mutation in α₁AT.¹⁸ Antitrypsin is expressed at high levels by hepatocytes (1.3–3.5 g/l in blood plasma) and its expression can increase in response to infection and other stimuli.¹⁹ However, only about one-third of the homozygous carriers ever manifest liver disease¹⁸ and it never occurs in carriers of a single Z allele, indicating that hepatocytes are generally well equipped to deal with the mutant protein. Soluble Z α₁AT in the ER binds to chaperones and is subsequently targeted to the ERAD pathway for clearance by the proteosome, and insoluble polymers and aggregates are thought to activate autophagy for degradation in the lysosomes.^20,21 In such a model, accumulation of polymers and cellular toxicity only occur when the proteosomal and autophagic pathways have been saturated by the high level of expression of mutant α₁AT in the liver.²¹ In contrast, neuroserpin is expressed at low levels in neurons and mutation leads to polymerization and dementia in an autosomal dominant fashion.^22,23 However, cell death and disease are still associated with accumulation of inclusion bodies within the ER, and the toxic mechanisms are likely to be similar.⁹In summary, we have elucidated a novel mechanism of serpin polymerization that involves a hyperstable domain swap of a folding intermediate. Formation of linear polymers exposes hydrophobic regions that mediate lateral polymer association and eventually leads to intra ER accretion and cellular toxicity. Our proposal suggests new avenues for the rational design of compounds to combat the diseases caused by serpin polymerization, either through targeting the folding intermediate or the lateral association of soluble polymers.     

Physical characterization of serpin conformations

The native serpin fold is characterized by being metastable. This thermodynamic characteristic is manifested in the conversion of the native state to other more stable conformations. Whilst this structural transition is required for proteinase inhibition and regulation of a range of biological phenomena, inappropriate structural changes can result in a number of disease states. Identification of these alternative conformations has been essential in our understanding of serpin structure and function. However, identifying these alternative forms is also important if we are not to misinterpret data due to the formation of these states during in vitro studies. The different physical properties of these alternative serpin conformational states make it possible to use a range of standard laboratory techniques to identify these structures. In this chapter, we will outline these general approaches that can be used routinely to identify the alternative serpin conformational states.     

Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation

The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A.The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered.The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta-sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A.The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin.     

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.     

Ancestry and evolution of a secretory pathway serpin

Background  

The serpin (serine protease inhibitor) superfamily constitutes a class of functionally highly diverse proteins usually encompassing several dozens of paralogs in mammals. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. The aim of this study was to elucidate the phylogenetic relationships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity.     

Structural basis for serpin inhibitor activity

The mechanism of formation and the structures of serpin–inhibitor complexes are not completely understood, despite detailed knowledge of the structures of a number of cleaved and uncleaved inhibitor, noninhibitor, and latent serpins. It has been proposed from comparison of inhibitor and noninhibitor serpins in the cleaved and uncleaved forms that insertion of strand s4A into preexisting β-sheet A is a requirement for serpin inhibitor activity. We have investigated the role of this strand in formation of serpin–proteinase complexes and in serpin inhibitor activity through homology modeling of wild type inhibitor, mutant substrate, and latent serpins, and of putative serpin–proteinase complexes. These models explain the high stability of the complexes and provide an understanding of substrate behavior in serpins with point mutations in s4A and of latency in plasmingoen activator inhibitor I. © 1995 Wiley-Liss, Inc.     

How small peptides block and reverse serpin polymerisation

Many of the late-onset dementias, including Alzheimer's disease and the prion encephalopathies, arise from the aberrant aggregation of individual proteins. The serpin family of serine protease inhibitors provides a well-defined structural example of such pathological aggregation, as its mutant variants readily form long-chain polymers, resulting in diseases ranging from thrombosis to dementia. The intermolecular linkages result from the insertion of the reactive site loop of one serpin molecule into the middle strand (s4A) position of the A beta-sheet of another molecule. We define here the structural requirements for small peptides to competitively bind to and block the s4A position to prevent this intermolecular linkage and polymerisation. The entry and anchoring of blocking-peptides is facilitated by the presence of a threonine which inserts into the site equivalent to P8 of s4A. But the critical requirement for small blocking-peptides is demonstrated in crystallographic structures of the complexes formed with selected tri- and tetrapeptides. These structures indicate that the binding is primarily due to the insertion of peptide hydrophobic side-chains into the P4 and P6 sites of s4A. The findings allow the rational design of synthetic blocking-peptides small enough to be suitable for mimetic design. This is demonstrated here with a tetrapeptide that preferentially blocks the polymerisation of a pathologically unstable serpin commonly present in people of European descent.     

Leukocyte chemoattractant peptides from the serpin heparin cofactor II

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the coagulant proteinase alpha-thrombin. We have recently demonstrated that proteolysis of HC by catalytic amounts of polymorphonuclear leukocyte proteinases (elastase or cathepsin G) generates leukocyte chemotaxins (Hoffman, M., Pratt, C. W., Brown, R. L., and Church, F. C. (1989) Blood 73, 1682-1685). One of four peptides produced when HC is degraded by neutrophil elastase has chemotactic activity for both monocytes and neutrophils with maximal migration comparable to formyl-Met-Leu-Phe, the "gold standard" bacterially derived chemotaxin. The amino-terminal sequence of this HC peptide is Asp-Phe-His-Lys-Glu-Asn-Thr-Val-... and the peptide corresponds to Asp-39 to Ile-66 of HC. A variety of synthetic peptides derived from this sequence were evaluated for leukocyte migration activity, and a dodecapeptide from Asp-49 to Tyr-60 (Asp-Trp-Ile-Pro-Glu-Gly-Glu-Glu-Asp-Asp-Asp-Tyr) was identified as the active site for leukocyte chemotactic action. The 12-mer synthetic peptide possesses significant neutrophil chemotactic action at 1 nM (60% of the maximal activity of formyl-Met-Leu-Phe), while a peptide with the reverse sequence has essentially no chemotactic activity. Cross-desensitization experiments also show that pretreatment of neutrophils with a 19-mer peptide (Asn-48 to Ile-66) greatly reduces subsequent chemotaxis to HC-neutrophil elastase proteolysis reaction products. When injected intraperitoneally in mice, the HC-neutrophil elastase digest elicits neutrophil migration. Our results demonstrate that not only does HC function as a thrombin inhibitor, but that limited proteolysis of HC near the amino terminus yields biologically active peptide(s) which might participate in inflammation and in wound healing and tissue repair processes.     

Interactions causing the kinetic trap in serpin protein folding

Conformational transition is fundamental to the mechanism of functional regulation in proteins, and serpins (serine protease inhibitors) can provide insight into this process. Serpins are metastable in their native forms, and they ordinarily undergo conformational transition to a stable state only when they form a tight complex with target proteases. The metastable native form is thus considered to be a kinetically trapped folding intermediate. We sought to understand the nature of the serpin kinetic trap as a step toward discovering how conformational transition is regulated. We found that mutations of the B/C beta-barrel of native alpha(1)-antitrypsin, a prototypical serpin, allowed conversion of the molecule into a more stable state. A 2.2 A resolution crystal structure of the stable form (PDB code, ) showed that the reactive site loop is inserted into an A beta-sheet, as in the latent plasminogen activator inhibitor-1. Mutational analyses suggest strongly that interactions not found in the final stable form cause the kinetic trap in serpin protein folding.     

Mechanisms of serpin dysfunction in disease

The serpin superfamily encompasses hundreds of proteins, spread across all kingdoms of life, linked by a common tertiary fold. This review focuses on five diseases caused by serpin dysfunction: variants of antithrombin III lose their ability to interact with heparin; the alpha1-antitrypsin Pittsburgh mutation causes a change in target proteinase; the alpha1-antitrypsin Z mutation and neuroserpin, polymerisation of which lead to cellular cytotoxicity; and a loss of maspin expression resulting in cancer.     

Production, characterization, and use of serpin antibodies

Serine protease inhibitors (serpins) constitute a still expanding superfamily of structural similar proteins, which are localized extracellularly and intracellularly. Serpins play a central role in the regulation of a wide variety of (patho) physiological processes including coagulation, fibrinolysis, inflammation, development, tumor invasion, and apoptosis. Serpins have a unique mechanism of inhibition that involves a profound change in conformational state upon interaction with their protease. This conformational change enables the production of monoclonal antibodies specific for native, complexed, and inactivated serpins. Antibodies, and assays based on these antibodies, have been helpful in elucidating the (patho) physiological function of serpins in the last decade. Serpin-specific antibodies can be used for: (1) structure-function studies such as detection of conformational changes; (2) identification of target-proteases; (3) the detection and quantification of serpin and serpin-protease complexes in bodily fluids by immunoassays such as ELISA, RIA or FACS; (4) detection of serpins in tissues by immunohistochemistry; and (5) possible therapeutical interventions. This review summarizes the techniques we have used to obtain and screen antibodies against extra- and intracellular serpins, as well as the use of these antibodies for some of the above-mentioned purposes.     

A recombinant wheat serpin with inhibitory activity

A full-length clone encoding the wheat (Triticum aestivum L.) serpin WSZ1 was isolated from a cDNA library based on mRNA from immature grain. The 398 amino acid sequence deduced from the cDNA was corroborated by sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 belongs to the subfamily of protein Z-type serpins and the amino acid sequence is 70% identical with the barley serpins BSZ4 and BSZx and 27–33% identical with human serpins such as ₁-proteinase inhibitor, antithrombin III, and plasminogen activator inhibitor. The cDNA was subcloned in the pET3d expression vector, equipped with a histidine affinity tag at the N-terminus and expressed in Escherichia coli BL(21) DE3 pLysS. Recombinant WSZ1 from the soluble fraction was partially purified on Ni-NTA agarose and MonoQ columns and shown to form SDS-stable complexes with -chymotrypsin. Southern blots and amino acid sequencing indicated that only few serpins are encoded by wheat, but at least three distinct genes are expressed in the grain. Cleavage experiments on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins.     

Characterisation of serpin polymers in vitro and in vivo

Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation.     

Identification and characterization of a serpin from Eimeria acervulina

Serpins are serine protease inhibitors that are widely distributed in metazoans but have not been previously characterized in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed and characterized. Random screening of an E.acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame (ORF) consisting of 1,245 nts that encode a peptide 413 amino acids in length with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues in length. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55 kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 kDa and 48 kDa in sporozoites (SZ) and merozoites (MZ) respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa which may correspond to secreted serpin. By immuno-staining the serpin was located in granules distributed throughout both the SZ and MZ but granules appeared to be concentrated in the parasite's anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis the endogenous serpin in MZ extracts did not form the expected high molecular weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.