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.     

The structure of active serpin 1K from Manduca sexta.

BACKGROUND: The reactive center loops (RCL) of serpins undergo large conformational changes triggered by the interaction with their target protease. Available crystallographic data suggest that the serpin RCL is polymorphic, but the relevance of the observed conformations to the competent active structure and the conformational changes that occur on binding target protease has remained obscure. New high-resolution data on an active serpin, serpin 1K from the moth hornworm Manduca sexta, provide insights into how active serpins are stabilized and how conformational changes are induced by protease binding. RESULTS: The 2.1 A structure shows that the RCL of serpin 1K, like that of active alpha1-antitrypsin, is canonical, complimentary and ready to bind to the target protease between P3 and P3 (where P refers to standard protease nomenclature),. In the hinge region (P17-P13), however, the RCL of serpin 1K, like ovalbumin and alpha1-antichymotrypsin, forms tight interactions that stabilize the five-stranded closed form of betasheet A. These interactions are not present in, and are not compatible with, the observed structure of active alpha1-antitrypsin. CONCLUSIONS: Serpin 1K may represent the best resting conformation for serpins - canonical near P1, but stabilized in the closed conformation of betasheet A. By comparison with other active serpins, especially alpha1-antitrypsin, a model is proposed in which interaction with the target protease near P1 leads to conformational changes in betasheet A of the serpin.     

Serpins Flex Their Muscle: II. STRUCTURAL INSIGHTS INTO TARGET PEPTIDASE RECOGNITION,POLYMERIZATION, AND TRANSPORT FUNCTIONS*

Inhibitory serpins are metastable proteins that undergo a substantial conformational rearrangement to covalently trap target peptidases. The serpin reactive center loop contributes a majority of the interactions that serpins make during the initial binding to target peptidases. However, structural studies on serpin-peptidase complexes reveal a broader set of contacts on the scaffold of inhibitory serpins that have substantial influence on guiding peptidase recognition. Structural and biophysical studies also reveal how aberrant serpin folding can lead to the formation of domain-swapped serpin multimers rather than the monomeric metastable state. Serpin domain swapping may therefore underlie the polymerization events characteristic of the serpinopathies. Finally, recent structural studies reveal how the serpin fold has been adapted for non-inhibitory functions such as hormone binding.     

Serpinopathies and the conformational dementias

The serpin superfamily of serine proteinase inhibitors has a central role in controlling proteinases in many biological pathways in a wide range of species. The inhibitory function of the serpins involves a marked conformational transition, but this inherent molecular flexibility also renders the serpins susceptible to point mutations that result in aberrant intermolecular linkage and polymer formation. The effects of such protein aggregation are cumulative, with a progressive loss of cellular function that results in diseases as diverse as cirrhosis and emphysema. The recent recognition that mutations in a serpin can also result in late-onset dementia provides insights into changes that underlie other conformational diseases, such as the amyloidoses, the prion encephalopathies and Huntington and Alzheimer diseases.     

The native metastability and misfolding of serine protease inhibitors

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.     

Serpin protease inhibitors in plant biology

Protease inhibitors of the serpin family are ubiquitous in the plant kingdom but relatively little is known about their biological functions in comparison with their counterparts in animals. X-ray crystal structures have provided crucial insights into animal serpin functions. The recently solved structure of AtSerpin1 from Arabidopsis thaliana, which has the highly conserved reactive center P2-P1' Leu-Arg-Xaa (Xaa = small residue), displays both conserved and plant-specific serpin features. Sequence homology suggests that AtSerpin1 belongs to serpin Clade B, composed of intracellular mammalian serpins, which is consistent with the lack of strong evidence for secretion of serpins from plant cells. The major in vivo target protease for AtSerpin1 is the papain-like cysteine RD21 protease, a match reminiscent of the inhibition of cathepsins K, L and S by the Clade-B mammalian serpin, SCCA-1 (SERPINB3). The function of AtSerpin1 and other serpins that contain P2-P1' Leu-Arg-Xaa (the 'LR' serpins) in plants remains unknown. However, based on its homology and interactive partners, AtSerpin1 and perhaps other serpins are likely to be involved in regulating programmed cell death or associated processes such as senescence. Abundant accumulation of serpins in seeds and their presence in phloem sap suggest additional functions in plant defense by irreversible inhibition of digestive proteases from pests or pathogens. Here we review the most recent findings in plant serpin biology, focusing on advances in describing the structure and inhibitory specificity of the LR serpins.     

A serpin regulates dorsal-ventral axis formation in the Drosophila embryo

Extracellular serine protease cascades have evolved in vertebrates and invertebrates to mediate rapid, local reactions to physiological or pathological cues. The serine protease cascade that triggers the Toll signaling pathway in Drosophila embryogenesis shares several organizational characteristics with those involved in mammalian complement and blood clotting. One of the hallmarks of such cascades is their regulation by serine protease inhibitors (serpins). Serpins act as suicide substrates and are cleaved by their target protease, forming an essentially irreversible 1:1 complex. The biological importance of serpins is highlighted by serpin dysfunction diseases, such as thrombosis caused by a deficiency in antithrombin. Here, we describe how a serpin controls the serine protease cascade, leading to Toll pathway activation. Female flies deficient in Serpin-27A produce embryos that lack dorsal-ventral polarity and show uniform high levels of Toll signaling. Since this serpin has been recently shown to restrain an immune reaction in the blood of Drosophila, it demonstrates that proteolysis can be regulated by the same serpin in different biological contexts.     

Serpins in prokaryotes

Members of the serpin (serine proteinase inhibitor) superfamily have been identified in higher multicellular eukaryotes (plants and animals) and viruses but not in bacteria, archaea, or fungi. Thus, the ancestral serpin and the origin of the serpin inhibitory mechanism remain obscure. In this study we characterize 12 serpin-like sequences in the genomes of prokaryotic organisms, extending this protein family to all major branches of life. Notably, these organisms live in dramatically different environments and some are evolutionarily distantly related. A sequence-based analysis suggests that all 12 serpins are inhibitory. Despite considerable sequence divergence between the proteins, in four of the 12 sequences the region of the serpin that determines proteinase specificity is highly conserved, indicating that these inhibitors are likely to share a common target. Inhibitory serpins are typically prone to polymerization upon heating; thus, the existence of serpins in the moderate thermophilic bacterium Thermobifida fusca, the thermophilic bacterium Thermoanaerobacter tengcongensis, and the hyperthermophilic archaeon Pyrobaculum aerophilum is of particular interest. Using molecular modeling, we predict the means by which heat stability in the latter protein may be achieved without compromising inhibitory activity.     

Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.     

The role of conformational change in serpin structure and function

Serpins are members of a family of structurally related protein inhibitors of serine proteinases, with molecular masses between 40 and 100kDa. In contrast to other, simpler, proteinase inhibitors, they may interact with proteinases as inhibitors, as substrates, or as both. They undergo conformational interconversions upon complex formation with proteinase, upon binding of some members to heparin, upon proteolytic cleavage at the reactive center, and under mild denaturing conditions. These conformational changes appear to be critical in determining the properties of the serpin. The structures and stabilities of these various forms may differ significantly. Although the detailed structural changes required for inhibition of proteinase have yet to be worked out, it is clear that the serpin does undergo a major conformational change. This is in contrast to other, simpler, families of protein inhibitors of serine proteinases, which bind in a substrate-like or product-like manner. Proteolytic cleavage of the serpin can result in a much more stable protein with new biological properties such as chemo-attractant behaviour. These structural transformations in serpins provide opportunities for regulation of the activity and properties of the inhibitor and are likely be important in vivo, where serpins are involved in blood coagulation, fibrinolysis, complement activation and inflammation.     

Serpins: finely balanced conformational traps

Serine protease inhibitors (serpins) play very important roles in the maintenance of various physiologically important systems. As knowledge of the workings of proteins of this family grows, new understanding is gained of the mechanisms by which they inhibit target proteases, using conformational changes for which the structure of serpins is uniquely adapted. This finely balanced system is utilized to healthy benefit in the control of serpin function by modulators, arguably the most striking examples of which occur in the control of proteolytic cascades, such as the coagulation system. Serpins also play very important intracellular roles: one example is the protection of immune cells from their own cytotoxic proteases. The finely balanced serpin mechanism also means that it is prone to disastrous consequences if mutations should occur in vital positions in the serpin structure. Many examples of disease-associated mutations have been shown, which has the dual effect of highlighting how important these molecules are in the maintenance of health and the fine balance that must be maintained in order to preserve their active, inhibitory conformation.     

Understanding the specificity of serpin–protease complexes through interface analysis

Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.     

DNA accelerates the inhibition of human cathepsin V by serpins

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.     

The structural basis of serpin polymerization studied by hydrogen/deuterium exchange and mass spectrometry

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type alpha(1)-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the alpha(1)-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of beta-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into beta-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of beta-sheet C. On the basis of our results, we propose a mechanism for polymerization in which beta-strand 1C is displaced from the rest of beta-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of beta-sheet A, and allows for subsequent polymerization.     

The expanding superfamily of serpins: searching for the real targets

The serpin superfamily of proteins has expanded rapidly in recent years as represented by the ovalbumin-type serpin subfamily. PCR methods have been used to identify new ovalbumin-type serpins, including leupin or SCCA-2, a close relative of SCCA. Although in vitro inhibition of proteases by these serpins has been shown, and we have evidence that leupin can protect cells against apoptosis, the exact role of these serpins is not well understood. Finding relevant targets is a major challenge in serpin biology, and we have investigated the yeast-2-hybrid trap for identification of new interactions. Preliminary studies suggest that serpins are at the upper limit for bait size, but this technique may be useful for identifying interactions where full-length serpin protein is not required.     

Protein Misfolding and the Serpinopathies

The serpins are the largest superfamily of protease inhibitors. They are found in almost all branches of life including viruses, prokaryotes and eukaryotes. They inhibit their target protease by a unique mechanism that involves a large conformational transition and the translocation of the enzyme from the upper to the lower pole of the protein. This complex mechanism, and the involvement of serpins in important biological regulatory processes, make them prone to mutation-related diseases. For example the polymerization of mutant α 1-antitrypsin leads to the accumulation of ordered polymers within the endoplasmic reticulum of hepatocytes in association with cirrhosis. An identical process in the neuron specific serpin, neuroserpin, results in the accumulation of polymers in neurons and the dementia FENIB. In both cases there is a clear correlation between the molecular instability, the rate of polymer formation and the severity of disease. A similar process underlies the hepatic retention and plasma deficiency of antithrombin, C1 inhibitor, α 1-antichymotrypsin and heparin co-factor II. The common mechanism of polymerization has allowed us to group these conditions together as a novel class of disease, the serpinopathies.     

Tip of another iceberg: Drosophila serpins

Serpins are serine protease inhibitors with a conserved structure that have been identified in nearly all species and act as suicide substrates by binding covalently to their target proteases. Serpins regulate various physiological processes and defence mechanisms. In humans, several serpin mutations are linked to diseases. The genome of Drosophila melanogaster encodes 29 serpins and even more serine proteases. To date, three serpins have been investigated in detail. Spn27A controls the Toll pathway during early development and is involved in defence reactions in adult flies. SPN42DaA is an inhibitor of furin, a subtilisin-like convertase that is required for pro-protein maturation. Spn43Ac controls the Toll pathway during the immune response. In each case, Drosophila genetics has shed new light on the function of these serine protease inhibitors.     

Two latent and two hyperstable polymeric forms of human neuroserpin

Human neuroserpin (hNS) is a serine protease inhibitor that belongs to the serpin superfamily and is expressed in nervous tissues. The serpin fold is generally characterized by a long exposed loop, termed the reactive center loop, that acts as bait for the target protease. Intramolecular insertion of the reactive center loop into the main serpin β-sheet leads to the serpin latent form. As with other known serpins, hNS pathological mutants have been shown to accumulate as polymers composed of quasi-native protein molecules. Although hNS polymerization has been intensely studied, a general agreement about serpin polymer organization is still lacking. Here we report a biophysical characterization of native hNS that is shown to undergo two distinct conformational transitions, at 55°C and 85°C, both leading to distinct latent and polymeric species. The latent and polymer hNS forms obtained at 45°C and 85°C differ in their chemical and thermal stabilities; furthermore, the hNS polymers also differ in size and morphology. Finally, the 85°C polymer shows a higher content of intermolecular β-sheet interactions than the 45°C polymer. Together, these results suggest a more complex conformational scenario than was previously envisioned, and, in a general context, may help reconcile the current contrasting views on serpin polymerization.     

Thrombin inhibition by serpins disrupts exosite II

Thrombin uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite I and the exposure of exosite II. The physiological inhibitors of thrombin are all serpins, whose mechanism involves significant conformational change in both serpin and protease. It has been shown that the formation of the thrombin-serpin final complex disorders the active site and exosite I of thrombin, but exosite II is thought to remain functional. It has also been hypothesized that thrombin contains a receptor-binding site that is exposed upon final complex formation. The position of this cryptic site may depend on the regions of thrombin unfolded by serpin complexation. Here we investigate the conformation of thrombin in its final complex with serpins and find that in addition to exosite I, exosite II is also disordered, as reflected by a loss of affinity for the γ'-peptide of fibrinogen and for heparin and by susceptibility to limited proteolysis. This disordering of exosite II occurs for all tested natural thrombin-inhibiting serpins. Our data suggest a novel framework for understanding serpin function, especially with respect to thrombin inhibition, where serpins functionally "rezymogenize" proteases to ensure complete loss of activity and cofactor binding.     

Structural factors affecting the choice between latency transition and polymerization in inhibitory serpins

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) protein family, is unique among the serpins in its conformational lability. This lability allows spontaneous conversion of the active form to a more stable, latent conformation under physiological conditions. In other serpins, polymerization, rather than latency transition, is induced under pathological conditions or upon heat treatment. To identify specific factors promoting latency conversion in PAI-1, we mutated PAI-1 at various positions and compared the effects with those of equivalent mutations in alpha(1)-antitrypsin, the archetypal serpin. Mutations that improved interactions with the turn between helix F and the third strand of beta-sheet A (thFs3A) or the fifth strand of beta-sheet A (s5A), which are near the site of latency transition-associated insertion of the reactive center loop, retarded latency conversion but did not greatly increase structural stability. Mutations that decreased interactions with s2C facilitated conformational conversion, possibly by releasing the reactive center loop from beta-sheet C. Mutations of Thr93 that filled a hydrophobic surface pocket on s2A dramatically increased structural stability but had a negligible effect on the conformational transition. Our results suggest that the structural features controlling latency transition in PAI-1 are highly localized, whereas the conformational strain of the native forms of other inhibitory serpins is distributed throughout the molecule and induces polymerization.