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ABSTRACT: Staphylococcal superantigen-like (SSL)5 family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1+2) as well as factor Xa in pull down analysis. Equilibrium dissociation constant between SSL10 and prothrombin was 1.36 x 10-7 M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla)-domain less coagulation factors and prothrombin from warfarin-treated mouse, suggesting that Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca2+ and factor Xa. SSL10 did not affect the protease activity of thrombin, but inhibited the generation of thrombin activity in recalcificated plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering activation of coagulation cascade via binding to Gla domain of coagulation factor, but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting Gla domain of coagulation factors.
Journal of Biological Chemistry 06/2013; · 4.77 Impact Factor
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ABSTRACT: Staphylococcal superantigen-like protein (SSL), a family of exotoxins composed of 14 SSLs, exhibits no superantigenic activity despite of its structural similarity with superantigens. Several SSLs have been revealed to bind to host immune molecules such as IgA, IgG, complement and cell surface molecules expressed on immune cells, but the physiological function of SSL family has not been fully identified. In this study we attempted to isolate host target proteins of SSLs from human breast milk using SSLs-conjugated Sepharose. SSL8-conjugated Sepharose specifically recovered tenascin C (TNC), a multimodular and multifunctional extracellular matrix protein. Pull down analysis using SSL8-conjugated Sepharose and recombinant truncated fragments of TNC revealed that SSL8 interacts with fibronectin (FN) type III repeats 1-5 of TNC. The interaction of TNC with immobilized FN was attenuated, the scratch wound closure by HaCaT human keratinocytes was delayed and theinhibitionofcellspreadingonFNbyTNCwasrecovered in the presence of SSL8. These findings suggest that SSL8 binds to TNC, thereby inhibits the TNC-FN interaction and motility of keratinocytes. The present study added a novel role of SSL family protein as an interrupting molecule against the function of extracellular matrix.
Biochemical and Biophysical Research Communications 02/2013; · 2.48 Impact Factor
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Go Kamoshida,
Ayaka Matsuda,
Kouji Katabami,
Takumi Kato,
Hiromi Mizuno,
Wakana Sekine,
Teruaki Oku,
Saotomo Itoh,
Makoto Tsuiji,
Yoshiyuki Hattori,
Yoshie Maitani,
Tsutomu Tsuji
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ABSTRACT: The α3β1 integrin is an adhesion receptor for extracellular matrix proteins, and plays crucial roles in cell motility, proliferation, and differentiation. The aberrant expression of this adhesion molecule on tumor cells is frequently associated with their malignant behaviors. We previously reported that the Ets transcription factor-binding consensus sequence at 133 bp upstream of the mouse α3 integrin gene is an important element for its expression in various tumor cell lines. In the present study, we attempted to identify a transcription factor bound to the Ets-consensus sequence and found that Ets-1 bound to this sequence in an electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and pull-down assay using a tandem repeat of the sequence as adsorbent. We next examined the role of Ets-1 in α3 integrin gene expression by a luciferase assay with a reporter plasmid containing the 5'-flanking region of the α3 integrin gene. Co-transfection of HEK293T cells with an Ets-1 expression construct and the reporter plasmid increased luciferase activity. By contrast, transfection of HT1080 cells (high α3 integrin expresser) with a dominant-negative mutant of Ets-1 decreased luciferase activity. Overexpression of Ets-1 in HepG2 hepatocellular carcinoma cells (low α3 integrin expresser) upregulated α3 integrin expression as assessed by immunoprecipitation. Finally, the induction of α3 integrin gene expression in HepG2 cells after transforming growth factor-β1 treatment was abrogated by the dominant-negative mutant of Ets-1. These results suggest that Ets-1 is involved in transcriptional activation of the α3 integrin gene through its binding to the Ets-consensus sequence at -133 bp. © 2012 The Authors Journal compilation © 2012 FEBS.
FEBS Journal 10/2012; · 3.79 Impact Factor
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ABSTRACT: Macrophages are a major population of immune cells, and those that infiltrate into tumor tissues and affect the malignant behavior of tumor cells are called tumor-associated macrophages (TAMs). We previously reported that human peripheral blood monocytes could be induced in vitro to differentiate into TAM-like cells by co-culture with tumor cells. In the present study, we characterized changes in the invasive phenotype of tumor cells after co-culture with monocytes, and found that MKN1 gastric carcinoma cells acquired higher invasive potential into Matrigel-reconstituted basement membranes, accompanied by enhanced production of matrix metalloproteinase (MMP)-9. The increased invasiveness was inhibited in the presence of an arginyl-glycyl-aspartic acid peptide, suggesting that the process is dependent on the integrin-extracellular matrix interaction. We also found that these cells secreted fibronectin into the culture medium and expressed α5 integrin on their surface at higher levels after the co-culture with monocytes for 5 days. The conditioned medium of monocytes also potentiated MKN1 cell invasion; however, the potentiation was lowered by the depletion of tumor necrosis factor (TNF)-α from the conditioned medium with an antibody-protein G-Sepharose conjugate. In addition, the treatment of MKN1 cells with TNF-α promoted invasion of these cells, as well as secretion of MMP-9 and fibronectin. These results suggest that TNF-α secreted from monocytes is, at least in part, involved in the changes in invasive phenotype of tumor cells during co-culture with monocytes.
Clinical and Experimental Metastasis 09/2012; · 3.52 Impact Factor
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ABSTRACT: Background/Aim: P-selectin is a carbohydrate-recognizing cell adhesion molecule expressed on activated platelets and endothelial cells. It plays a crucial role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. Cell adhesion mediated by P-selectin induces leukocyte activation, such as the generation of reactive oxygen species and the expression of blood coagulation factors. We assessed how P-selectin-mediated cell adhesion affects cytokine secretion from monocytes. Methods: Human peripheral blood monocytes were cultured in a plate that had been coated with P-selectin purified from human platelets, and cytokines released in the culture supernatant from monocytes were determined by ELISA. Results: The secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-12 and macrophage inflammatory protein-1β increased 3- to 10-fold in response to P-selectin compared with unstimulated monocytes. We next examined the effects of cytokine treatment of monocytes on their susceptibility to P-selectin. The secretion of TNF-α from monocytes in response to P-selectin was increased when monocytes were preincubated with granulocyte/macrophage colony-stimulating factor, monocyte chemotactic protein-1 or interferon-γ (IFN-γ); IFN-γ was the most effective in potentiating TNF-α secretion from monocytes. Conclusion: These results suggest that the interaction of monocytes with P-selectin plays an important role not only in their trafficking but also in the regulation of cytokine production by these cells.
International Archives of Allergy and Immunology 09/2012; 160(2):152-160. · 2.40 Impact Factor
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ABSTRACT: Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins sharing structural similarity with superantigens, but no superantigenic activity. Corresponding host target proteins or receptors against a portion of SSLs in the family have been identified. In this study, we show that SSL3 specifically binds to Toll-like receptor 2 (TLR2) and inhibits the stimulation of macrophages by TLR2 ligands. An approximately 100-kDa protein was recovered by using recombinant His-tagged SSL3-conjugated Sepharose from the lysate of porcine spleen, and the protein was identified as porcine TLR2 by peptide mass fingerprinting analysis. The SSL3-conjugated Sepharose recovered human and mouse TLR2 but not TLR4 from human neutrophils and mouse macrophage RAW 264.7 cells, as well as a recombinant TLR2 extracellular domain chimera protein. The production levels of interleukin 12 (IL-12) from mouse macrophages treated with heat-killed Staphylococcus aureus and of tumor necrosis factor alpha (TNF-α) from RAW 264.7 cells induced by peptidoglycan or lipopeptide TLR2 ligands were strongly suppressed in the presence of SSL3. The mutation of consensus sialic acid-containing glycan-binding residues in SSL3 did not abrogate the binding ability to TLR2 or inhibitory activity on TLR2, indicating that the interaction of SSL3 with TLR2 was independent of the sialic acid-containing glycan-binding residues. These findings demonstrate that SSL3 is able to bind the extracellular domain of TLR2 and interfere with TLR2 function. The present study provides a novel mechanism of SSL3 in immune evasion of S. aureus via interfering with its recognition by innate immune cells.
Infection and immunity 06/2012; 80(8):2816-25. · 4.21 Impact Factor
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ABSTRACT: Macrophages that infiltrate tumor tissues, or tumor-associated macrophages (TAMs), affect the malignant behaviors of tumor cells. In this study, we attempted to induce monocytes to differentiate into TAM-like cells producing matrix metalloproteinases (MMPs) by co-culture with tumor cells. When human monocytes were co-cultured for 3-7 days with tumor cell lines, monocytes differentiated to produce MMP-9, accompanied by morphological changes. The in vitro cell invasion of MKN1 human gastric carcinoma cells into Matrigel membranes was promoted in the presence of differentiated monocytes, and the enhancement of cell invasion by differentiated monocytes was correlated with their MMP-9 productivity. The addition of an RGD (Arg-Gly-Asp) peptide to the culture significantly inhibited monocyte differentiation. The MMP-9 production from monocytes was diminished by the depletion of fibronectin from the conditioned media with gelatin-Sepharose, and potentiated by culturing them in fibronectin-coated plates. These results suggest that cell adhesion to the extracellular matrix plays a crucial role in monocyte differentiation into TAM-like cells.
Cancer letters 11/2011; 315(2):145-52. · 4.86 Impact Factor
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ABSTRACT: Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [K(D)] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (K(i) = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking.
Infection and immunity 07/2010; 78(7):3298-305. · 4.21 Impact Factor
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ABSTRACT: Staphylococcal superantigen-like (SSL) proteins are a family of exoproteins that share structural similarity with staphylococcal superantigens but exhibit no superantigenic activity. It was previously reported that two members (SSL5 and SSL7) bound to serum components and cell adhesion molecules involved in host immune response; however, the other family members have not been functionally characterized. In this study, we attempted to isolate SSL10-binding proteins from human serum and found that recombinant His-tagged SSL10 bound two major polypeptides of approximately 50 and approximately 25 kDa after affinity purification and SDS-polyacrylamide gel electrophoresis. These polypeptides were identified as heavy and light chains of human IgG by peptide mass fingerprinting analysis. The specific interaction between recombinant SSL10 and human IgG was confirmed by far Western blot analysis using immobilized SSL10 and pull-down analysis using SSL10-conjugated Sepharose. Surface plasmon resonance analysis revealed that the dissociation equilibrium constant for the interaction between human IgG and recombinant SSL10 was estimated to be 220 nM. We also found that recombinant SSL10 inhibited the binding of complement component C1q to IgG-Sepharose and hemolysis of IgG-sensitized sheep erythrocytes via the classical complement activation pathway. These results suggest that SSL10 may play a role in the evasion of Staphylococcus aureus from the host immune system via interfering complement activation.
Molecular Immunology 11/2009; 47(4):932-8. · 2.90 Impact Factor
