Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.

Integrative identification of human serpin PAI-1 inhibitors from Dracaena dragon blood and molecular implications for inhibitor-induced PAI-1 allosterism.

Human plasminogen activator inhibitor-1 (PAI-1) is an important component of the coagulation system and has been recognized as a potential therapeutic target of diverse cardiovascular disorders. Previously, it was found that the extracts from the Chinese medicine Dracaena dragon blood have potent inhibitory activity against PAI-1, but it is unclear which constituents directly participate in the inhibition and how do they regulate PAI-1 at molecular level. Here, we describe an integrated strategy to identify the dragon blood's chemical constituents that can directly target PAI-1. With the strategy, five compounds 1-5 are hit as promising PAI-1 inhibitor candidates, from which three are measured to have high or moderate activity against PAI-1. In particular, the compound 3 is determined to exhibit the highest potency; this value is roughly comparable with the widely used PAI-1 inhibitor Tiplaxtinin. We further examine the molecular effect of compound 3 on PAI-1 conformation at structural level. It is supposed that small-molecule inhibitor regulates the reactive center loop (RCL) of PAI-1 through an allosterism, that is, binding of compound 3 to PAI-1 can allosterically stabilize RCL in latent form, thus promoting PAI-1 conformational conversion from metastable active form to the inactive latent form. Long-term atomistic simulations also demonstrate that removal of compound 3 can destabilize the structured ß-stranded conformation of RCL in latent form, although the current simulations are still not sufficient to characterize the full conversion dynamics trajectory.

Molecular identification, characterization, and expression analysis of a serine protease inhibitor gene from cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) / Identificação molecular, caracterização e análise de expressão de um gene inibidor de serina protease da lagarta Helicoverpa armigera (Lepidoptera: noctuidae) na cultura de algodão

Abstract Serine protease inhibitors (serpins), a superfamily of protease inhibitors, are known to be involved in several physiological processes, such as development, metamorphosis, and innate immunity. In our study, a full-length serpin cDNA, designated Haserpin1, was isolated from the cotton bollworm Helicoverpa armigera. The cDNA sequence of Haserpin1 is 1176 nt long, with an open reading frame encoding 391 amino acids; there is one exon and no intron. The predicted molecular weight of Haserpin1 is 43.53 kDa, with an isoelectric point of 4.98. InterProScan was employed for Haserpin1 functional characterization, which revealed that Haserpin1 contains highly conserved signature motifs, including a reactive center loop (RCL) with a hinge region (E341-N350), the serpin signature, (F367-F375) and a predicted P1-P1′ cleavage site (L357-S358), which are useful for identifying serpins. Transcripts of Haserpin1 were constitutively expressed in the fat body, suggesting that it is the major site for serpin synthesis. During the developmental stages, a fluctuation in the expression level of Haserpin1 was observed, with low expression detected at the 5th-instar larval stage. In contrast, relatively high expression was detected at the prepupal stage, suggesting that Haserpin1 might play a critical role at the H. armigera wandering stage. Although the detailed function of this serpin (Haserpin1) needs to be elucidated, our study provides a perspective for the functional investigation of serine protease inhibitor genes.

Resumo Sabe-se que os inibidores de serina protease (serpinas), uma superfamília de inibidores de protease, estão envolvidos em vários processos fisiológicos, como desenvolvimento, metamorfose e imunidade inata. Neste estudo, um cDNA de serpina de comprimento total, denominado Haserpin1, foi isolado da lagarta Helicoverpa armigera na cultura de algodão. A sequência de ADNc de Haserpin1 tem 1.176 nt de comprimento, com uma grelha de leitura aberta que codifica 391 aminoácidos; existe um éxon, mas nenhum íntron. O peso molecular previsto de Haserpin1 é de 43,53 kDa, com um ponto isoelétrico de 4,98. O InterProScan foi empregado para a caracterização funcional do Haserpin1, que revelou que o Haserpin1 contém motivos de assinatura altamente conservados, incluindo um loop central reativo (RCL) com uma região de dobradiça (E341-N350), a assinatura da serpina (F367-F375) e um local de clivagem previsto de P1-P1' (L357-S358), que são úteis para identificar serpinas. As transcrições de Haserpin1 foram expressas constitutivamente no corpo gordo, sugerindo que é o principal local para a síntese de serpinas. Durante os estágios de desenvolvimento, observou-se uma flutuação no nível de expressão de Haserpin1, com baixa expressão detectada no estágio larval do 5º ínstar. Por outro lado, detectou-se uma expressão relativamente alta no estágio pré-pupal, sugerindo que o Haserpin1 pode desempenhar um papel crítico no estágio errante de H. armigera. Embora a função detalhada dessa serpina (Haserpin1) precise ser elucidada, este estudo fornece uma perspectiva para a investigação funcional dos genes inibidores da serina protease.

Therapeutic SERPINs: Improving on Nature.

Serine proteases drive important physiological processes such as coagulation, fibrinolysis, inflammation and angiogenesis. These proteases are controlled by serine protease inhibitors (SERPINs) that neutralize their activity. Currently, over 1,500 SERPINs are known in nature, but only 37 SERPINs are found in humans. Thirty of these are functional protease inhibitors. The inhibitory potential of SERPINs is in perfect balance with the proteolytic activities of its targets to enable physiological protease activity. Hence, SERPIN deficiency (either qualitative or quantitative) can lead to disease. Several SERPIN resupplementation strategies have been developed to treat SERPIN deficiencies, including concentrates derived from plasma and recombinant SERPINs. SERPINs usually inhibit multiple proteases, but only in their active state. Over the past decades, considerable insights have been acquired in the identification of SERPIN biological functions, their inhibitory mechanisms and specificity determinants. This paves the way for the development of therapeutic SERPINs. Through rational design, the inhibitory properties (selectivity and inhibitory potential) of SERPINs can be reformed and optimized. This review explores the current state of SERPIN engineering with a focus on reactive center loop modifications and backbone stabilization. We will discuss the lessons learned from these recombinant SERPINs and explore novel techniques and strategies that will be essential for the creation and application of the future generation of therapeutic SERPINs.

Detection of truncated isoforms of human neuroserpin lacking the reactive center loop: Implications in noninhibitory role.

Neuroserpin is a serine protease inhibitor expressed mainly in the brain and at low levels in other tissues like the kidney, testis, heart, and spinal cord. It is involved in the inhibition of tissue plasminogen activator (tPA), plasmin, and to a lesser extent, urokinase-type plasminogen (uPA). Neuroserpin has also been shown to plays noninhibitory roles in the regulation of N-cadherin-mediated cell adhesion. It is involved in neuroprotection from seizure and stroke through tPA-mediated inhibition and also through its other protease targets. Mutations in critical domains of neuroserpin lead to its polymerization and neuronal death. In this study, a novel truncated isoform of human neuroserpin was identified in the brain and liver, which was confirmed by reverse transcriptase-PCR and DNA sequencing using exon-specific primers. Structural characterization of novel isoform using MD simulations studies indicated that it lacks the reactive center loop (RCL) but largely maintains its secondary structure fold. The novel truncated variant was cloned, expressed, and purified. A comparative intrinsic fluorescence and 4,4'-bis-1-anilino naphthalene 8-sulfonate studies revealed a decrease in fluorescence emission intensity and a more exposed hydrophobic surface as compared to the reported isoform. However, the novel isoform has lost its ability for tPA inhibition and complex formation. The absence of RCL indicates a noninhibitory role for the truncated isoform, prompting a detailed search and identification of two smaller isoforms in the human brain. With indications of the noninhibitory role of neuroserpin, identifying novel isoforms that appear to be without the tPA recognition domain is significant.

Deriving Immune-Modulating Peptides from Viral Serine Protease Inhibitors (Serpins).

Viruses have devised highly effective approaches that modulate the host immune response, blocking immune responses that are designed to eradicate viral infections. Over millions of years of evolution, virus-derived immune-modulating proteins have become extraordinarily potent, in some cases working at picomolar concentrations when expressed into surrounding tissues and effectively blocking host defenses against viral invasion and replication. The marked efficiency of these immune-modulating proteins is postulated to be due to viral engineering of host immune modulators as well as design and development of new strategies (i.e., some derived from host proteins and some entirely unique). Two key characteristics of viral immune modulators confer both adaptive advantages and desirable functions for therapeutic translation. First, many virus-derived immune modulators have evolved structures that are not readily recognized or regulated by mammalian immune pathways, ensuring little to no neutralizing antibody responses or proteasome-mediated degradation. Second, these immune modulators tend to target early steps in central immune responses, producing a powerful downstream inhibitory "domino effect" which may alter cell activation and gene expression.We have proposed that peptide metabolites of these immune-modulating proteins can enhance and extend protein function. Active immunomodulating peptides have been derived from both mammalian and viral proteins. We previously demonstrated that peptides derived from computationally predicted cleavage sites in the reactive center loop (RCL) of a viral serine proteinase inhibitor (serpin ) from myxoma virus, Serp-1 , can modify immune response activation. We have also demonstrated modulation of host gut microbiota produced by Serp-1 and RCL-derived peptide , S7, in a vascular inflammation model. Of interest, generation of derived peptides that maintain therapeutic function from a serpin can act by a different mechanism. Whereas Serp-1 has canonical serpin-like function to inhibit serine proteases, S7 instead targets mammalian serpins. Here we describe the derivation of active Serp- RCL peptides and their testing in inflammatory vasculitis models.

Truncated reactive center loop decrease the inhibitory activity of Antheraea pernyi serine protease inhibitor 6.

Here, we assessed the effect of a systematic change in reactive center loop (RCL) length, N-terminal to the reactive center, on the inhibitory activity of the recombinant Apserpin-6. The domain prediction results indicated that the RCL is located between the amino acid numbered 359-379 at the C-terminal of Apserpin-6. The N-terminal variable region for amino acid positions P7-P1 of the RCL of Apserpin-6 was truncated or extended by residue deletion or insertion using site-directed mutagenesis. The recombinant Apserpin-6 with one or two residues insertion in RCL had no effect on prophenoloxidase (proPO) activity, whereas deletion of one or two residues in RCL lowered the efficiency of inhibition of Apserpin-6. The results of this study will facilitate the understanding of inhibition mechanism of RCL on proPO activity.

Cloning and high-level SUMO-mediated fusion expression of a serine protease inhibitor from Hyphantria cunea Drury that exhibits activity against papain.

Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities. Todate some functional roles for serpins in Hyphantria cunea Drury have been identified. Here, new functional features of the H. cunea serine protease inhibitor (dHC-serpin) were characterized. In this study, the cDNA encoding serpin was amplified from H. cunea (dHC) pupa fat body total RNA using RT-PCR. The full-length dHC-serpin cDNA encoded a protein of 440 amino acids with a predicted 19-amino acid signal peptide and a 421-amino acid functional domain. The functional domain was cloned into a pSUMO vector and transformed into Escherichia coli, resulting in the production of a pSUMO-dHC-serpin fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography. The SUMO-dHC-serpin fusion protein was then cleaved using a SUMO protease and purified again by Ni-IDA chromatography. dHC-serpin did not inhibit trypsin, elastase, proteinase K or cathepsin B, but strongly inhibited papain. The inhibitor retained its inhibitory activity over a broad range of pH (pH 2-12), temperature (20-50 °C), and DTT concentration (up to 100 mM). A complete loss of inhibitory activity was observed at pH 13 and 70 °C. Serpins generally serve as inhibitors that use a mobile reactive center loop (RCL) as bait to trap protease targets. dHC-serpin, like others serpins, binds papain using the RCL structure.

Viral Serpin Reactive Center Loop (RCL) Peptides: Design and Testing.

Serpins function as a trap for serine proteases, presenting the reactive center loop (RCL) as a target for individual proteases. When the protease cleaves the RCL, the serpin and protease become covalently linked leading to a loss of function of both the protease and the serpin; this suicide inhibition is often referred to as a "mouse trap." When the RCL P1-P1' scissile bond is cut by the protease, the resulting bond between the protease and the RCL leads to insertion of the cleaved RCL into the ß-sheet A and relocation of the protease to the opposite pole of the serpin, forming a suicide complex. Only a relatively small part of the serpin molecule can be removed in deletion mutations before the serpin RCL inhibitory function is lost. Serpin RCL peptides have been developed to block formation of serpin aggregates in serpinopathies, genetic serpin mutations wherein the abnormal serpins insert their RCL into adjacent serpins forming aggregates of inactive serpins.We have further posited that this natural cleavage site in the serpin RCL may form active serpin metabolites with potential to add to the serpin's inhibitory functions. We have developed RCL peptides based upon predicted serpin RCL cleavage (or metabolism) sites and tested these serpins for inhibitory function. In this chapter we describe the development of RCL-derived peptides, peptides derived based upon the RCL sequences of two myxomaviral serpins. Methods used to develop peptides are described for RCL-derived peptides from Serp-1, a thrombotic and thrombolytic serine protease inhibitor, and Serp-2, a cross class serine and cysteine protease inhibitor (Subheadings 2.1 and 3.1). Approaches to testing RCL peptide functions, in vitro by molecular assays and in vivo in models of cell migration, MHV-68 infection, and aortic allograft transplant are described (Subheadings 2.2 and 3.2).

Point Mutation of a Non-Elastase-Binding Site in Human α1-Antitrypsin Alters Its Anti-Inflammatory Properties.

Introduction: Human α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357-366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro, and in vivo. Methods: His-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo. Results: Compared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding. Conclusion: Our data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes.

On the folding of a structurally complex protein to its metastable active state.

For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a ß-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a ß-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCL modulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state.

Serpin-9 and -13 regulate hemolymph proteases during immune responses of Manduca sexta.

Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R366 at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R410 as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg366 by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg410 for trypsin-like proteases, Gly406 and Ala409 for the elastase and Thr404 for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0-50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae.

Molecular identification, characterization, and expression analysis of a serine protease inhibitor gene from cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract Serine protease inhibitors (serpins), a superfamily of protease inhibitors, are known to be involved in several physiological processes, such as development, metamorphosis, and innate immunity. In our study, a full-length serpin cDNA, designated Haserpin1, was isolated from the cotton bollworm Helicoverpa armigera. The cDNA sequence of Haserpin1 is 1176 nt long, with an open reading frame encoding 391 amino acids; there is one exon and no intron. The predicted molecular weight of Haserpin1 is 43.53 kDa, with an isoelectric point of 4.98. InterProScan was employed for Haserpin1 functional characterization, which revealed that Haserpin1 contains highly conserved signature motifs, including a reactive center loop (RCL) with a hinge region (E341N350), the serpin signature, (F367F375) and a predicted P1P1 cleavage site (L357S358), which are useful for identifying serpins. Transcripts of Haserpin1 were constitutively expressed in the fat body, suggesting that it is the major site for serpin synthesis. During the developmental stages, a fluctuation in the expression level of Haserpin1 was observed, with low expression detected at the 5th-instar larval stage. In contrast, relatively high expression was detected at the prepupal stage, suggesting that Haserpin1 might play a critical role at the H. armigera wandering stage. Although the detailed function of this serpin (Haserpin1) needs to be elucidated, our study provides a perspective for the functional investigation of serine protease inhibitor genes.

Resumo Sabe-se que os inibidores de serina protease (serpinas), uma superfamília de inibidores de protease, estão envolvidos em vários processos fisiológicos, como desenvolvimento, metamorfose e imunidade inata. Neste estudo, um cDNA de serpina de comprimento total, denominado Haserpin1, foi isolado da lagarta Helicoverpa armigera na cultura de algodão. A sequência de ADNc de Haserpin1 tem 1.176 nt de comprimento, com uma grelha de leitura aberta que codifica 391 aminoácidos; existe um éxon, mas nenhum íntron. O peso molecular previsto de Haserpin1 é de 43,53 kDa, com um ponto isoelétrico de 4,98. O InterProScan foi empregado para a caracterização funcional do Haserpin1, que revelou que o Haserpin1 contém motivos de assinatura altamente conservados, incluindo um loop central reativo (RCL) com uma região de dobradiça (E341-N350), a assinatura da serpina (F367-F375) e um local de clivagem previsto de P1-P1' (L357-S358), que são úteis para identificar serpinas. As transcrições de Haserpin1 foram expressas constitutivamente no corpo gordo, sugerindo que é o principal local para a síntese de serpinas. Durante os estágios de desenvolvimento, observou-se uma flutuação no nível de expressão de Haserpin1, com baixa expressão detectada no estágio larval do 5º ínstar. Por outro lado, detectou-se uma expressão relativamente alta no estágio pré-pupal, sugerindo que o Haserpin1 pode desempenhar um papel crítico no estágio errante de H. armigera. Embora a função detalhada dessa serpina (Haserpin1) precise ser elucidada, este estudo fornece uma perspectiva para a investigação funcional dos genes inibidores da serina protease.

Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL). However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. We document that RCL-localized Asn(347) glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn(347) glycosylation as a function of digestion time. The site-specific (Val(344)-Thr(345)) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn(347) glycan features (i.e. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn(347) NeuAc-type sialylation (all p < 0.05). In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr(345)-Leu(346) and Asn(347)-Leu(348), was abolished by the presence of Asn(347) glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. Molecular dynamics simulations of various Asn(347) glycoforms of uncleaved CBG indicated that multiple Asn(347) glycan features are modulating the RCL digestion efficiencies by NE/PAE. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system.

Reactive Center Loop (RCL) Peptides Derived from Serpins Display Independent Coagulation and Immune Modulating Activities.

Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.

Distinct encounter complexes of PAI-1 with plasminogen activators and vitronectin revealed by changes in the conformation and dynamics of the reactive center loop.

UNLABELLED: Plasminogen activator inhibitor-1 (PAI-1) is a biologically important serine protease inhibitor (serpin) that, when overexpressed, is associated with a high risk for cardiovascular disease and cancer metastasis. Several of its ligands, including vitronectin, tissue-type and urokinase-type plasminogen activator (tPA, uPA), affect the fate of PAI-1. Here, we measured changes in the solvent accessibility and dynamics of an important unresolved functional region, the reactive center loop (RCL), upon binding of these ligands. Binding of the catalytically inactive S195A variant of tPA to the RCL causes an increase in fluorescence, indicating greater solvent protection, at its C-terminus, while mobility along the loop remains relatively unchanged. In contrast, a fluorescence increase and large decrease in mobility at the N-terminal RCL is observed upon binding of S195A-uPA to PAI-1. At a site distant from the RCL, binding of vitronectin results in a modest decrease in fluorescence at its proximal end without restricting overall loop dynamics. These results provide the new evidence for ligand effects on RCL conformation and dynamics and differences in the Michaelis complex with plasminogen activators that can be used for the development of more specific inhibitors to PAI-1. This study is also the first to use electron paramagnetic resonance (EPR) spectroscopy to investigate PAI-1 dynamics. SIGNIFICANCE: Balanced blood homeostasis and controlled cell migration requires coordination between serine proteases, serpins, and cofactors. These ligands form noncovalent complexes, which influence the outcome of protease inhibition and associated physiological processes. This study reveals differences in binding via changes in solvent accessibility and dynamics within these complexes that can be exploited to develop more specific drugs in the treatment of diseases associated with unbalanced serpin activity.

Corticosteroid-binding globulin: modulating mechanisms of bioavailability of cortisol and its clinical implications.

Corticosteroid-binding globulin (CBG) is the principal transport protein of glucocorticoids. Approximately 80-90% of serum cortisol binds to CBG with high affinity and only about 5% of cortisol remain unbound and is considered biologically active. CBG seems to modulate and influence the bioavailability of cortisol to local tissues. In this review, we will discuss physicochemical properties of CBG and structure of CBG in the mechanisms of binding and release of cortisol. This review describes several factors affecting CBG functions, such as genetic factors or temperature. Furthermore, clinical implications of CBG abnormalities and the measurement of CBG and its use for assessment of free cortisol levels are described in this review.

Dynamic properties of the native free antithrombin from molecular dynamics simulations: computational evidence for solvent- exposed Arg393 side chain.

While antithrombin (AT) has small basal inhibitory activity, it reaches its full inhibitory potential against activated blood coagulation factors, FXa, FIXa, and FIIa (thrombin), via an allosteric and/or template (bridging) mechanism by the action of heparin, heparan sulfate, or heparin-mimetic pentasaccharides (PS). From the numerous X-ray structures available for different conformational states of AT, only indirect and incomplete conclusions can be drawn on the inherently dynamic properties of AT. As a typical example, the basal inhibitory activity of AT cannot be interpreted on the basis of "non-activated" free antithrombin X-ray structures since the Arg393 side chain, playing crucial role in antithrombin-proteinase interaction, is not exposed. In order to reveal the intrinsic dynamic properties and the reason of basal inhibitory activity of antithrombin, 2 µs molecular dynamics simulations were carried out on its native free-forms. It was shown from the simulation trajectories that the reactive center loop which is functioning as "bait" for proteases, even without any biasing potential can populate conformational state in which the Arg393 side chain is solvent exposed. It is revealed from the trajectory analysis that the peptide sequences correspond to the helix D extension, and new helix P formation can be featured with especially large root-mean-square fluctuations. Mutual information analyses of the trajectory showed remarkable (generalized) correlation between those regions of antithrombin which changed their conformations as the consequence of AT-PS complex formation. This suggests that allosteric information propagation pathways are present even in the non-activated native form of AT.

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy.

BACKGROUND: Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern. METHODS: Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy. RESULTS: Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation. CONCLUSIONS: Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein. GENERAL SIGNIFICANCE: Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development.

Antithrombin deficiency in three Japanese families: one novel and two reported point mutations in the antithrombin gene.

INTRODUCTION: Inherited antithrombin (AT) deficiency is associated with a predisposition to familial venous thromboembolic disease. We analyzed the AT gene in three unrelated patients with an AT deficiency who developed thrombosis. MATERIALS AND METHODS: We analyzed the SERPINC1 gene in three patients. Additionally, we expressed the three mutants in the COS-1 cells and compared their secretion rates and levels of AT activity with those of the wild-type (WT). RESULTS: We identified three distinct heterozygous mutations of c.2534C>T: p.56Arginine → Cysteine (R56C), c.13398C>A: p.459Alanine → Aspartic acid (A459D) and c.2703C>G: p.112 Proline → Arginine (P112R). In the in vitro expression experiments, the AT antigen levels in the conditioned media (CM) of the R56C mutant were nearly equal to those of WT. In contrast, the AT antigen levels in the CM of the A459D and P112R mutants were significantly decreased. The AT activity of R56C was decreased in association with a shorter incubation time in a FXa inhibition assay and a thrombin inhibition-based activity test. However, the AT activity of R56C was comparable to that of WT when the incubation time was increased. CONCLUSIONS: We concluded that the R56C mutant is responsible for type II HBS deficiency. We considered that the A459D and P112R mutants can be classified as belonging to the type I AT deficiency.