Matriptase Activation, an Early Cellular Response to Acidosis

Extracellular acidosis often rapidly causes intracellular acidification, alters ion channel activities, and activates G protein-coupled receptors. In this report, we demonstrated a novel cellular response to acidosis: induction of the zymogen activation of matriptase. Acid-induced matriptase activation is ubiquitous among epithelial and carcinoma cells and is characterized by rapid onset, fast kinetics, and the magnitude of activation seen. Trace amounts of activated matriptase can be detected 1 min after cells are exposed to pH 6.0 buffer, and the vast majority of latent matriptase within the cells is converted to activated matriptase within 20 min. Matriptase activation may be a direct response to proton exposure because acid-induced matriptase activation also occurs in an in vitro, cell-free setting in which intracellular signaling molecules and ion channel activities are largely absent. Acid-induced matriptase activation takes place both on the cell surface and inside the cells, likely due to the parallel intracellular acidification that activates intracellular matriptase. Following matriptase activation, the active enzyme is immediately inhibited by binding to hepatocyte growth factor activator inhibitor 1, resulting in stable matriptase-hepatocyte growth factor activator inhibitor 1 complexes that are rapidly secreted. As an early response to acidosis, matriptase activation can also be induced by perturbation of intracellular pH homeostasis by 5-(N-methyl-N-isobutyl)-amiloride and 5-(N-ethyl-N-isopropyl)-amiloride, both of which inhibit Na⁺/H⁺ exchangers, and diisothiocyanostilbene-2,2′-disulfonic acid, which can inhibit other acid-base ion channels. This study uncovers a novel mechanism regulating proteolysis in epithelial and carcinoma cells, and also demonstrates that a likely function of matriptase is as an early response to acidosis.     

Interactions Outside the Proteinase-binding Loop Contribute Significantly to the Inhibition of Activated Coagulation Factor XII by Its Canonical Inhibitor from Corn

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (K_i = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a K_i of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20–45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (K_i = 11.7 ± 1.2 μm) and activated factor XI (K_i = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a K_i of 1.3 ± 0.2 nm and activated factor XI with a K_i of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.     

X-ray Structure Analysis and Characterization of AFUEI,an Elastase Inhibitor from Aspergillus fumigatus

Elastase from Aspergillus sp. is an important factor for aspergillosis. AFUEI is an inhibitor of the elastase derived from Aspergillus fumigatus. AFUEI is a member of the I78 inhibitor family and has a high inhibitory activity against elastases of Aspergillus fumigatus and Aspergillus flavus, human neutrophil elastase and bovine chymotrypsin, but does not inhibit bovine trypsin. Here we report the crystal structure of AFUEI in two crystal forms. AFUEI is a wedge-shaped protein composed of an extended loop and a scaffold protein core. The structure of AFUEI shows remarkable similarity to serine protease inhibitors of the potato inhibitor I family, although they are classified into different inhibitor families. A structural comparison with the potato I family inhibitors suggests that the extended loop of AFUEI corresponds to the binding loop of the potato inhibitor I family, and AFUEI inhibits its cognate proteases through the same mechanism as the potato I family inhibitors.     

Molecular Basis for Auto- and Hetero-catalytic Maturation of a Thermostable Subtilase from Thermophilic Bacillus sp. WF146

The proform of the WF146 protease, an extracellular subtilase produced by thermophilic Bacillus sp. WF146, matures efficiently at high temperatures. Here we report that the proform, which contains an N-terminal propeptide composed of a core domain (N*) and a linker peptide, is intrinsically able to mature via multiple pathways. One autocatalytic pathway is initiated by cis-processing of N* to generate an autoprocessed complex N*-I^WT, and this step is followed by truncation of the linker peptide and degradation of N*. Another autocatalytic pathway is initiated by trans-processing of the linker peptide followed by degradation of N*. Unlike most reported subtilases, the maturation of the WF146 protease occurs not only autocatalytically but also hetero-catalytically whereby heterogeneous proteases accelerate the maturation of the WF146 protease via trans-processing of the proform and N*-I^WT. Although N* acts as an intramolecular chaperone and an inhibitor of the mature enzyme, the linker peptide is susceptible to proteolysis, allowing the trans-processing reaction to occur auto- and hetero-catalytically. These studies also demonstrate that the WF146 protease undergoes subtle structural adjustments during the maturation process and that the binding of Ca²⁺ is required for routing the proform to mature properly at high temperatures. Interestingly, under Ca²⁺-free conditions, the proform is cis-processed into a unique propeptide-intermediate complex (N*-I^E) capable of re-synthesis of the proform. Based on the basic catalytic principle of serine proteases and these experimental results, a mechanism for the cis-processing/re-synthesis equilibrium of the proform and the role of the linker peptide in regulation of this equilibrium has been proposed.     

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with ∼40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31–41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited ∼5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with ∼4-fold decreased k_cat and ∼2-fold decreased K_m, although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.     

Isolation,cDNA Cloning,and Structure-based Functional Characterization of Oryctin,a Hemolymph Protein from the Coconut Rhinoceros Beetle,Oryctes rhinoceros,as a Novel Serine Protease Inhibitor

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant ¹³C,¹⁵N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K_i values of 3.9 × 10⁻¹⁰ m, 6.2 × 10⁻¹⁰ m, 1.4 × 10⁻⁹ m, and 1.2 × 10⁻⁸ m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.     

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg¹⁵-Ala¹⁶ reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.     

Identification of the Matriptase Second CUB Domain as the Secondary Site for Interaction with Hepatocyte Growth Factor Activator Inhibitor Type-1

Matriptase is a type II transmembrane serine protease comprising 855 amino acid residues. The extracellular region of matriptase comprises a noncatalytic stem domain (containing two tandem repeats of complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein (CUB) domain) and a catalytic serine protease domain. The stem domain of matriptase contains site(s) for facilitating the interaction of this protease with the endogenous inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). The present study aimed to identify these site(s). Analyses using a secreted variant of recombinant matriptase comprising the entire extracellular domain (MAT), its truncated variants, and a recombinant HAI-1 variant with an entire extracellular domain (HAI-1–58K) revealed that the second CUB domain (CUB domain II, Cys³⁴⁰–Pro⁴⁵²) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys³⁷⁹ and Val³⁸⁰ within CUB domain II and that the C-terminal residues after Val³⁸⁰ are responsible for facilitating the interaction with HAI-1–58K. A synthetic peptide corresponding to Val³⁸⁰–Asp³⁹⁰ markedly increased the matriptase-inhibiting activity of HAI-1–58K, whereas the peptides corresponding to Val³⁸⁰–Val³⁸⁹ and Phe³⁸²–Asp³⁹⁰ had no effect. HAI-1–58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val³⁸⁰–Pro³⁹² with a biotinylated lysine residue at its C terminus was bound, suggesting direct interaction between CUB domain II and HAI-1. These results led to the identification of the matriptase CUB domain II, which facilitates the primary inhibitory interaction between this protease and HAI-1.     

Degradation of Some Polyubiquitinated Proteins Requires an Intrinsic Proteasomal Binding Element in the Substrates

Lysine 48-linked polyubiquitin chains usually target proteins for 26 S proteasomal degradation; however, this modification is not a warrant for destruction. Here, we found that efficient degradation of a physiological substrate UbcH10 requires not only an exogenous polyubiquitin chain modification but also its unstructured N-terminal region. Interestingly, the unstructured N-terminal region of UbcH10 directly binds the 19 S regulatory complex of the 26 S proteasome, and it mediates the initiation of substrate translocation. To promote ubiquitin- dependent degradation of the folded domains of UbcH10, its N-terminal region can be displaced by exogenous proteasomal binding elements. Moreover, the unstructured N-terminal region can initiate substrate translocation even when UbcH10 is artificially cyclized without a free terminus. Polyubiquitinated circular UbcH10 is completely degraded by the 26 S proteasome. Accordingly, we propose that degradation of some polyubiquitinated proteins requires two binding interactions: a polyubiquitin chain and an intrinsic proteasomal binding element in the substrates (likely an unstructured region); moreover, the intrinsic proteasomal binding element initiates substrate translocation regardless of its location in the substrates.     

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp³²–Asp²¹⁵ diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.     

Identification of Serpinb6b as a Species-specific Mouse Granzyme A Inhibitor Suggests Functional Divergence between Human and Mouse Granzyme A

The granzyme family serine proteases are key effector molecules expressed by cytotoxic lymphocytes. The physiological role of granzyme (Gzm) A is controversial, with significant debate over its ability to induce death in target cells. Here, we investigate the natural inhibitors of GzmA. We employed substrate phage display and positional proteomics to compare substrate specificities of mouse (m) and human (h) GzmA at the peptide and proteome-wide levels and we used the resulting substrate specificity profiles to search for potential inhibitors from the intracellular serpin family. We identified Serpinb6b as a potent inhibitor of mGzmA. Serpinb6b interacts with mGzmA, but not hGzmA, with an association constant of 1.9 ± 0.8 × 10⁵ m⁻¹ s⁻¹ and a stoichiometry of inhibition of 1.8. Mouse GzmA is over five times more cytotoxic than hGzmA when delivered into P815 target cells with streptolysin O, whereas transfection of target cells with a Serpinb6b cDNA increases the EC₅₀ value of mGzmA 13-fold, without affecting hGzmA cytotoxicity. Unexpectedly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric but not monomeric mGzmA. The identification of an intracellular inhibitor specific for mGzmA only indicates that a lineage-specific increase in GzmA cytotoxic potential has driven cognate inhibitor evolution.     

Molecular Mechanism by Which a Potent Hepatitis C Virus NS3-NS4A Protease Inhibitor Overcomes Emergence of Resistance

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes. In turn, these structural studies revealed a complex molecular basis of resistance and rationalized how such compounds are able to circumvent these mechanisms.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.     

The Allosteric Mechanism of Activation of Antithrombin as an Inhibitor of Factor IXa and Factor Xa: HEPARIN-INDEPENDENT FULL ACTIVATION THROUGH MUTATIONS ADJACENT TO HELIX D*

Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa but not of thrombin. These are accompanied by CD, fluorescence, and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131–136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop. To examine the importance of helix D extension, we have introduced strong helix-promoting mutations in the 131–136 region of antithrombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114- and 110-fold, respectively), but not of thrombin, together with changes in near- and far-UV CD and ¹H NMR spectra. Heparin binding gives only ∼3–4-fold further rate enhancement but increases tryptophan fluorescence by ∼23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin, again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr-131 and Ala-134 with core hydrophobic residues, whereas the reactive center loop hinge expulsion plays only a minor additional role.     

Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction ∼2000-fold in the presence of phospholipid and Ca²⁺. To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ_ΔGD) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing ∼5–10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only ∼2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional ∼1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.     

Blastocystis Legumain Is Localized on the Cell Surface, and Specific Inhibition of Its Activity Implicates a Pro-survival Role for the Enzyme

Programmed cell death (PCD) is crucial for cellular growth and development in multicellular organisms. Although distinct PCD features have been described for unicellular eukaryotes, homology searches have failed to reveal clear PCD-related orthologues among these organisms. Our previous studies revealed that a surface-reactive monoclonal antibody (mAb) 1D5 could induce multiple PCD pathways in the protozoan Blastocystis. In this study, we identified, by two-dimensional gel electrophoresis and mass spectrometry, the target of mAb 1D5 as a surface-localized legumain, an asparagine endopeptidase that is usually found in lysosomal/acidic compartments of other organisms. Recombinant Blastocystis legumain displayed biphasic pH optima in substrate assays, with peaks at pH 4 and 7.5. Activity of Blastocystis legumain was greatly inhibited by the legumain-specific inhibitor carbobenzyloxy-Ala-Ala-AAsn-epoxycarboxylate ethyl ester (APE-RR) (where AAsn is aza-asparagine) and moderately inhibited by mAb 1D5, cystatin, and caspase-1 inhibitor. Interestingly, inhibition of legumain activity induced PCD in Blastocystis, observed by increased externalization of phosphatidylserine residues and in situ DNA fragmentation. In contrast to plants, in which legumains have been shown to play a pro-death role, legumain appears to display a pro-survival role in Blastocystis.     

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli: STRUCTURAL AND FUNCTIONAL STUDIES*

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA′ (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.     

Apical Surface Expression of Aspartic Protease Plasmepsin 4, a Potential Transmission-blocking Target of the Plasmodium Ookinete

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.     

Miropin,a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia,Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

All prokaryotic genes encoding putative serpins identified to date are found in environmental and commensal microorganisms, and only very few prokaryotic serpins have been investigated from a mechanistic standpoint. Herein, we characterized a novel serpin (miropin) from the human pathogen Tannerella forsythia, a bacterium implicated in initiation and progression of human periodontitis. In contrast to other serpins, miropin efficiently inhibited a broad range of proteases (neutrophil and pancreatic elastases, cathepsin G, subtilisin, and trypsin) with a stoichiometry of inhibition of around 3 and second-order association rate constants that ranged from 2.7 × 10⁴ (cathepsin G) to 7.1 × 10⁵ m⁻¹s⁻¹ (subtilisin). Inhibition was associated with the formation of complexes that were stable during SDS-PAGE. The unusually broad specificity of miropin for target proteases is achieved through different active sites within the reactive center loop upstream of the P1-P1′ site, which was predicted from an alignment of the primary structure of miropin with those of well studied human and prokaryotic serpins. Thus, miropin is unique among inhibitory serpins, and it has apparently evolved the ability to inhibit a multitude of proteases at the expense of a high stoichiometry of inhibition and a low association rate constant. These characteristics suggest that miropin arose as an adaptation to the highly proteolytic environment of subgingival plaque, which is exposed continually to an array of host proteases in the inflammatory exudate. In such an environment, miropin may function as an important virulence factor by protecting bacterium from the destructive activity of neutrophil serine proteases. Alternatively, it may act as a housekeeping protein that regulates the activity of endogenous T. forsythia serine proteases.     

The X-ray Crystal Structure of Mannose-binding Lectin-associated Serine Proteinase-3 Reveals the Structural Basis for Enzyme Inactivity Associated with the Carnevale,Mingarelli, Malpuech,and Michels (3MC) Syndrome

The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-Å structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.