Matriptase Research Articles

Post-ischemic protection of hepatocyte growth factor requires the type II transmembrane serine protease matriptase-A reciprocal regulation of the two for neuroprotection in stroke brain.

In a rat middle cerebral artery occlusion (MACo) model of ischemic stroke, intracerebroventricular administration of human recombinant hepatocyte growth factor (HGF) mitigated motor impairment and cortical infarction. Recombinant HGF reduced MCAo-induced TNFα and IL1β expression, and alleviated perilesional reactivation of microglia and astrocyte. All of the aforementioned beneficial effects of HGF were antagonized by an inhibitor to the type II transmembrane serine protease matriptase (MTP). MCAo upregulated MTP mRNA and protein in the lesioned cortex. MTP protein, not the mRNA, was increased further by recombinant HGF but reduced when MTP inhibitor (MTPi) was added to the treatment. Changes of the endogenous active HGF by MCAo, HGF or MTPi paralleled with the changes of MTP protein under the same conditions whilst neither HGF mRNA nor the total endogenous HGF protein were altered. These data showed that the therapeutic effects of HGF in stroke brain is attributed to its proteolytic activation and that MTP is a main protease of the event. MCAo enhanced MTP mRNA and thus protein expression; the initial use of the recombinant active HGF stabilized MCAo-induced MTP protein and subsequent activation of endogenous latent HGF which in turn stabilized further MTP protein. A reciprocal regulation between MTP and HGF appears to be present where MTP promotes HGF activation and the active HGF prevents MTP protein turnover. This study, for the first time, shows that MTP can participate in neural protection in stroke brain through activation of HGF. The cycles of HGF-MTP regulation achieved preservation of the neurological activity.

Abstract 550: Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity

While immune checkpoint blockade leads to potent anti-tumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in multi-organ inflammation including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA-4 antibody (anti-CTLA-4 DVD) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA-4 binding domain. Upon reaching the tumor, the outer domain is cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA-4 blockade. Anti-CTLA-4 DVD significantly reduced multi-organ immune toxicity by preserving tissue-resident regulatory T cells (Tregs) in Rag 1-/- mice that received naive donor CD4+ T cells from wild-type C57BL/6j mice. Moreover, anti-CTLA-4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2 bearing C57BL/6j mice. Tregs depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA-4 without the FcγR binding portion (anti-CTLA-4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrated a new approach to anti-CTLA-4 blockade that depletes tumor-infiltrating, but not tissue-resident Tregs, preserving anti-tumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA-4 DVD provides an avenue to uncouple anti-tumor efficacy from immunotherapy-induced toxicities. Citation Format: Steven C. Pai, Donald M. Simons, Xiaoqing Lu, Wendy Ritacco, Michael Evans, Gillian Kingsbury, Lawrence Fong. Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 550.

Abstract 550: Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity

Abstract While immune checkpoint blockade leads to potent anti-tumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in multi-organ inflammation including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA-4 antibody (anti-CTLA-4 DVD) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA-4 binding domain. Upon reaching the tumor, the outer domain is cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA-4 blockade. Anti-CTLA-4 DVD significantly reduced multi-organ immune toxicity by preserving tissue-resident regulatory T cells (Tregs) in Rag 1-/- mice that received naïve donor CD4+ T cells from wild-type C57BL/6j mice. Moreover, anti-CTLA-4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2 bearing C57BL/6j mice. Tregs depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA-4 without the FcγR binding portion (anti-CTLA-4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrated a new approach to anti-CTLA-4 blockade that depletes tumor-infiltrating, but not tissue-resident Tregs, preserving anti-tumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA-4 DVD provides an avenue to uncouple anti-tumor efficacy from immunotherapy-induced toxicities. Citation Format: Steven C. Pai, Donald M. Simons, Xiaoqing Lu, Wendy Ritacco, Michael Evans, Gillian Kingsbury, Lawrence Fong. Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 550.

Mitochondrial localization of St14-encoding transmembrane serine protease is involved in neural stem/progenitor cell bioenergetics through binding to F0F1-ATP synthase complex.

Knockdown of the suppression of tumorigenicity 14-encoding type II transmembrane serine protease matriptase (MTP) in neural stem/progenitor (NS/P) cells impairs cell mobility, response to chemo-attractants, and neurovascular niche interaction. In the present study, we showed by Western blot that a portion of MTP can be detected in the mitochondrial fraction of mouse NS/P cells by immunostaining that it is co-stained with the mitochondrial dye MitoTracker (Thermo Fisher Scientific, Waltham, MA, USA) inside the cells. Co-immunoprecipitation showed that MTP is bound to the β subunit of mitochondrial F0F1-ATP synthase complex (ATP-β). Cyto-immunofluorescence staining and an in situ proximity ligation assay further confirmed a physical interaction between MTP and ATP-β. This interaction relied on the presence of both Cls/Clr urchin embryonic growth factor, bone morphogenic protein 1 and low-density lipoprotein receptor motifs of MTP. We found that NS/P cell mitochondrial membrane potential is impaired by MTP knockdown, and ATP synthesis and oxygen consumption rate are significantly reduced in MTP-knockdown NS/P cells. Among the oxidative phosphorylation functions, the greatest effect of MTP knockdown is the reduction by over 50% in the mitochondrial energy reserve capacity. This made MTP-knockdown NS/P cells unable to overcome hydrogen peroxide stress, which leads to cessation of cell growth. This work identifies 2 previously unknown functions for MTP: first as a binding protein in the mitochondrial F1F0-ATP synthase complex and second as a regulatory mechanism of mitochondrial bioenergetics. Mitochondrial MTP may serve a protective function for NS/P cells in response to stress.-Fang, J.-D., Tung, H.-H., Lee, S.-L. Mitochondrial localization of St14-encoding transmembrane serine protease is involved in neural stem/progenitor cell bioenergetics through binding to F0F1-ATP synthase complex.

Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.

LB966 Cleavage of EpCAM and TROP2 by matriptase destabilizes claudin-1 and claudin-7 in keratinocytes

LB966 Cleavage of EpCAM and TROP2 by matriptase destabilizes claudin-1 and claudin-7 in keratinocytes

Physical Binding of Endothelial MCAM and Neural Transmembrane Protease Matriptase\u2014Novel Cell Adhesion in Neural Stem cell Vascular Niche

Brain neural stem cells and transit amplifying cells in the subventricular zone (SVZ) of the lateral ventricles are in direct contact with the microvascular endothelium. The mechanisms/molecules of direct cell contact in the SVZ neurovascular niche are not fully understood. We previously showed that neural stem/progenitor (NS/P) cells induce brain endothelial signaling in direct cell contact through matriptase (MTP) on NS/P cell surface. In the present study, using pull-down and LC-MS/MS, we identified melanoma cell adhesion molecule (MCAM) the brain endothelial molecule that interacts with MTP. MCAM physically binds to the CUB domains of MTP and induces a chain of brain endothelial signaling including p38MAPK activation, GSK3β inactivation and subsequently β-catenin activation; none of these signaling events occurred when either MTP or MCAM is deleted. MTP-MCAM binding and induction of endothelial signaling were all sensitive to cholera toxin. Together, we identified key molecules that may represent a mechanism in neural stem cell vascular niche regulation.

Progress in iron metabolism research.

Iron is essential for various cellular processes, but an excess of iron may cause organ damage through the production of reactive oxygen species. Therefore, the amount of iron in the body must be strictly controlled. The central regulator of systemic iron homeostasis is hepcidin, which is primarily produced in the liver. Various molecules, including HFE, transferrin receptor 2 (TFR2), and hemojuvelin (HJV), are involved in sensing systemic iron status. Hepatocytes produce hepcidin in response to excess iron and inflammatory stimuli (e.g., interleukin-6), whereas hepcidin expression is downregulated by hypoxia, anemia, and erythropoietic activity. In mice, erythroferrone, secreted from erythroblasts, suppresses hepcidin expression. Hepcidin downregulates the protein expression of ferroportin, the only iron exporter in mammalian cells, and thereby downregulates iron absorption from intestine and iron release from macrophages. Mutations in the genes HFE, TFR2, HJV, HAMP (encoding hepcidin), and SLC40A1 (encoding ferroportin) cause hereditary hemochromatosis, whereas mutations in TMPRSS6 (which encodes matriptase 2) cause iron-refractory iron deficiency anemia through the upregulation of hepcidin expression. In chronic anemias, such as β-thalassemia, myelodysplastic syndromes, and aplastic anemia, repeated red blood cell transfusion can cause systemic iron overload. Iron chelation therapy improves the prognosis of patients with such conditions.

Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia.

Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

Molecular Cloning and Heterologous Expression in Pichia pastoris of X-Prolyl-dipeptidyl Aminopeptidase from Basidiomycete Ustilago maydis

Dipeptidyl aminopeptidases are enzymes involved in the posttranslational control of bioactive peptides. Here we identified the gene dapUm in Ustilago maydis by homology with other fungal dipeptidyl aminopeptidases. Analysis of the dapUm-deduced amino acid sequence indicated that it encodes for membrane-type serine protease with a characteristic prolyl oligopeptidase catalytic motif triad: Ser, Asp, His. In order to overexpress the DapUm, the gene encoding for it was cloned and transformed into Pichia. Using this system, we observed a ∼ 125-kDa recombinant protein with an optimal enzymatic activity at pH 6.0 and at 40 °C for the Ala-Pro-p-nitroanilide substrate and an experimental pH of 6.9. U. maydis DapUm was specifically inhibited by phenylmethylsulfonyl fluoride and Pefabloc, confirming the presence of a serine residue in the active site. To our knowledge, this study is the first report on the cloning and expression of a DPP IV dipeptidyl aminopeptidase from a basidiomycete organism. Moreover, the use of recombinant DapUm will allow us to further study and characterize this enzyme, in addition to testing chemical compounds for pharmaceutical purposes.

Imbalanced Matriptase Pericellular Proteolysis Contributes to the Pathogenesis of Malignant B-Cell Lymphomas

Membrane-associated serine protease matriptase is widely expressed by epithelial/carcinoma cells in which its proteolytic activity is tightly controlled by the Kunitz-type protease inhibitor, hepatocyte growth factor activator inhibitor (HAI-1). We demonstrate that, although matriptase is not expressed in lymphoid hyperplasia, roughly half of the non-Hodgkin B-cell lymphomas analyzed express significant amounts of matriptase. Furthermore, a significant proportion of these tumors express matriptase in the absence of HAI-1. Aggressive Burkitt lymphoma was more likely than indolent follicular lymphoma to express matriptase alone (86% versus 36%). In the absence of significant HAI-1 expression, the lymphoma cells activate and shed active matriptase when the cells are stimulated with mildly acidic buffer or the hypoxia-mimicking agent, CoCl2. The shed active matriptase can initiate pericellular proteolytic cascades by activating urokinase-type plasminogen activator on the cell surface of monocytes, and it can activate prohepatocyte growth factor. In addition, matriptase knockdown suppressed proliferation and colony-forming ability of neoplastic B cells in culture and growth as tumor xenografts in mice. Furthermore, exogenous expression of HAI-1 significantly suppressed proliferation of neoplastic B cells. These studies suggest that dysregulated pericellular proteolysis as a result of unregulated matriptase expression with limited HAI-1 may contribute to the pathological characteristics of several human B-cell lymphomas through modulation of the tumor microenvironment and enhanced tumor growth.

Expression and significance of matriptase in ovarian cancer cells with diverse metastatic potential

To study the expression and significance of matriptase in different metastatic potential of human ovarian cancer cells. High-metastatic human ovarian cancer cell HO8910PM and ovarian cancer cell HO8910 were collected.The ability of metastatic of the former was stronger than that of the latter. Compared the ability of invasion and migration in HO8910PM and HO8910 by scratch assay and by millicell chamber artificial reconstituted basement membrane invasion assay. Detected the matriptase mRNA and protein expression levels in HO8910PM and HO8910 through reverse transcription(RT)-PCR and immunocytochemistry methods. The 24 hours' migration distance (347 ± 8) µm of HO8910PM cells were significantly higher than that in HO8910 group (154 ± 10) µm (P < 0.01);The number of HO8910PM cells that penetrated the matrigel after 24 hours' incubation were significantly higher than that in HO8910 group (90.7 ± 2.1 vs 63.3 ± 1.5,P < 0.01). The expression of matriptase mRNA in HO8910PM cells was higher than that in HO8910 group (0.72 ± 0.03 vs 0.38 ± 0.04,P < 0.01). The migration was positively correlated with the matriptase mRNA expression levels (r = 0.992, P < 0.01); and the invasiveness was also positively correlated with the matriptase mRNA expression levels (r = 0.973, P < 0.01). As far protein level,the expression of matriptase protein in HO8910PM cells was higher than that in HO8910 group (15.6 ± 0.8 vs 7.6 ± 1.3,P < 0.01). The migration was positively correlated with matriptase protein expression levels (r = 0.971, P < 0.01); And the invasiveness was also positively correlated with the matriptase protein expression levels (r = 0.958, P < 0.01). The relationship between the expression levels of matriptase and the metastatic of ovarian cancer cells may be correlative. The function of matriptase in ovarian cancer cells metastatic mechanism still need to be confirmed.

Crystal Structures of Matriptase in Complex with Its Inhibitor Hepatocyte Growth Factor Activator Inhibitor-1

Matriptase, a type II trans-membrane serine protease of the S1 trypsin-like family, is expressed on the surface of nearly all normal human epithelium and found in biological fluid-like human milk. Matriptase overexpression has been implicated in tumor progression in certain epithelium-derived cancer cells. Matriptase is tightly regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). It has been demonstrated that the Kunitz domain I (KD1) but not Kunitz domain II (KD2) of HAI-1 is responsible for the inhibitory activity of HAI-1 against matriptase. To investigate the molecular basis of inhibition of matriptase by HAI-1, we solved several crystal structures of matriptase serine protease domain in complex with the fragments of HAI-1. Based on these structures, we found that the binding of KD1 was different from previously predicted binding mode. The P3 arginine residue occupies the S3 specificity pocket of matriptase, but not the S4 pocket as in the cases of hepatocyte growth factor activator·HAI-1 KD1 and matriptase·sunflower trypsin inhibitor-1 complexes. The long 60-loop of matriptase makes direct contact with HAI-1 but remains flexible even in the complexes, and its apex does not bind with KD1 tightly. The interactions between this unique 60-loop and KD1 may provide an opportunity to increase the specificity and inhibitory activity of KD1 for matriptase. Furthermore, comparison between KD1 and a homology model of HAI-1 KD2 rationalizes the structural basis of why KD1 but not KD2 is responsible for the inhibitory activity of HAI-1 against matriptase.

Neural Transmembrane Protease and Endothelial Gs Protein Activation in Cell Contact-dependent Signaling between Neural Stem/Progenitor Cells and Brain Endothelial Cells

Vasculature is an important component of the neural stem cell niche in brain. It regulates neural stem/progenitor (NS/P) cell self-renewal, differentiation, and migration. In the neurogenic niches of adult brain, NS/P cells lie close to blood vessels, and proliferating NS/P cells frequently contact the vasculature. In the present study we showed that NS/P cells in co-culture with brain endothelial (bEND) cells activated endothelial G proteins and p38 mitogen-activated protein kinase (p38 MAPK) and stimulated cytokine/chemokine expression. These NS/P cell-induced endothelial responses took place during NS/P cell and bEND cell direct contact and were critically dependent on the expression of the type II transmembrane serine protease matriptase (MTP) by NS/P cells, because knocking down of MTP in NS/P cells impaired and re-expression of MTP restored their ability to induce endothelial cytokine/chemokine expression, p38 MAPK, or G protein activation. Cholera toxin blocked NS/P cell-induced endothelial responses, suggesting that the endothelial G protein activated by NS/P MTP is in the G(s) subfamily. The addition of p38 MAPK inhibitor impaired NS/P cell-induced endothelial cytokine/chemokine expression. The known G protein-coupled receptor substrate of MTP, protease-activated receptor 2, was not involved in this system. These results revealed a novel signaling pathway in neural stem cell vascular niches that is mediated by neural MTP and endothelial G(s) protein signaling at the cell-cell interface. This is the first report of direct cell-cell signaling between NS/P and bEND cells.

Inhibiting Alternative Pathway Complement Activation by Targeting the Factor D Exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.

A Reverse Binding Motif That Contributes to Specific Protease Inhibition by Antibodies

The type II transmembrane serine protease family consists of 18 closely related serine proteases that are implicated in multiple functions. To identify selective, inhibitory antibodies against one particular type II transmembrane serine protease, matriptase [MT-SP1 (membrane-type serine protease 1)], a phage display library was created with a natural repertoire of Fabs [fragment antigen binding (Fab)] from human naïve B cells. Fab A11 was identified with a 720 pM inhibition constant and high specificity for matriptase over other trypsin-fold serine proteases. A Trichoderma reesei system expressed A11 with a yield of ∼ 200 mg/L. The crystal structure of A11 in complex with matriptase has been determined and compared to the crystal structure of another antibody inhibitor (S4) in complex with matriptase. Previously discovered from a synthetic single-chain variable fragment library, S4 is also a highly selective and potent matriptase inhibitor. The crystal structures of the A11/matriptase and S4/matriptase complexes were solved to 2.1 Å and 1.5 Å, respectively. Although these antibodies, discovered from separate libraries, interact differently with the protease surface loops for their specificity, the structures reveal a similar novel mechanism of protease inhibition. Through the insertion of the H3 variable loop in a reverse orientation at the substrate-binding pocket, these antibodies bury a large surface area for potent inhibition and avoid proteolytic inactivation. This discovery highlights the critical role that the antibody scaffold plays in positioning loops to bind and inhibit protease function in a highly selective manner. Additionally, Fab A11 is a fully human antibody that specifically inhibits matriptase over other closely related proteases, suggesting that this approach could be useful for clinical applications.

Expression and significance of matriptase and HAI-1 in prostate cancer

Objective To investigate the expression and significance of Matriptase and HAI-1 protein in prostate cancer (CaP). Methods Specimens of 46 prostate cancers,20 benign prostate hyperplasias (BPH),10 high-grade intraepithelial neoplasias (PIN),and 10 normal prostates (NP) were used. Expressions of Matriptase and HAI-1 proteins in specimens were detected by SP of immunohistochemistry. The results were analyzed in relation to the clinicopathological data. Results The protein levels of Matriptase in CaP tissues were significantly higher than PIN tissues(Z＝-2.150,P＝0.032),and the expression of matriptase in CaP and PIN was higher than that in BPH and NP (Z＝-3.270,P＝0.001;Z＝-2.817,P＝0.005). No statistically significant difference was observed between BPH and NP group (Z＝-0.895,P＝0.325). A progressive increase in the protein levels of Matriptase was observed with increasing tumor grade (rs＝0.583,P＜0.01) and clinical stages（rs＝0.611,P＜0.01）in CaP specimens. The protein levels of HAI-1 in BPH and NP tissues were significantly higher than CaP and PIN tissues（Z＝-3.277,-3.315,P＜0.01）,the levels of HAI-1 in PIN were higher than CaP (Z＝-2.310,P＝0.020). No statistically significant difference was found between BPH and NP (Z＝-0.872,P＝0.330). A progressive decrease in the protein levels of HAI-1 was observed with increasing tumor grades（rs＝-0.634,P＜0.01） and clinical stages（rs＝-0.521,P＜0.01）. The expressions of Matriptase and HAI-1 in CaP tissues showed negative correlations（rs＝-0.712,-0.560,-0.465,respectively,P＜0.01）. Conclusions The abnormal expressions of Matriptase and HAI-1 proteins may be important events during the progression of CaP in humans. Matriptase and HAI-1 Protein may be used as parameters for assessing the malignancy and prognosis of CaP. Key words: Serine endopeptidases/ME; Serine proteinase inhibitors/ME; Prostatic neoplasms/ME

Endogenous Expression of Matriptase in Neural Progenitor Cells Promotes Cell Migration and Neuron Differentiation

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.

Analysis of an engineered plasma kallikrein inhibitor and its effect on contact activation

Engineering of protein-protein interactions is used to enhance the affinity or specificity of proteins, such as antibodies or protease inhibitors, for their targets. However, fully diversifying all residues in a protein-protein interface is often unfeasible. Therefore, we limited our phage library for the serine protease inhibitor ecotin by restricting it to only tetranomial diversity and then targeted all 20 amino acid residues involved in protein recognition. This resulted in a high-affinity and highly specific plasma kallikrein inhibitor, ecotin-Pkal. To validate this approach we dissected the energetic contributions of each wild type (wt) or mutated surface loop to the binding of either plasma kallikrein (PKal) or membrane-type serine protease 1. The analysis demonstrated that a mutation in one loop has opposing effects depending on the sequence of surrounding loops. This finding stresses the cooperative nature of loop-loop interactions and justifies targeting multiple loops with a limited diversity. In contrast to ecotin wt, the specific loop combination of ecotin-Pkal discriminates the subtle structural differences between the active enzymes, PKal and Factor XIIa, and their respective zymogen forms. We used ecotin-Pkal to specifically inhibit contact activation of human plasma at the level mediated by plasma kallikrein.