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Glycobiology Advance Access originally published online on June 29, 2007
Glycobiology 2007 17(9):1015-1028; doi:10.1093/glycob/cwm071
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

C-Mannosylated peptides derived from the thrombospondin type 1 repeat enhance lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells

Eiji Muroi2,3,4,5,, Shino Manabe5,6,, Midori Ikezaki3,5,, Yoshishige Urata3, Shinichi Sato4, Takahito Kondo3, Yukishige Ito5,6, and Yoshito Ihara1,2,3,5,7,

3 Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute
4 Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan
5 CREST, JST Kawaguchi 332-1102, Kawaguchi, Japan
6 RIKEN, Saitama 351-0198, Japan
7 Department of Biochemistry, Wakayama Medical University, Wakayama 641-8509, Japan

1 To whom correspondence should be addressed: Tel/Fax: +81-73-441-0628; e-mail: y-ihara{at}wakayama-med.ac.jp

Received on May 18, 2007; revised on June 20, 2007; accepted on June 24, 2007


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Funding
 Conflict of interest statement
 References
 
C-Mannosylation is a unique type of glycosylation occuring at the first Trp (W) in the WXXW motif, which is found in the thrombospondin type 1 repeat (TSR) of proteins. However, the biological function of C-mannosylation is not fully understood. In this study, we investigated the effect of C-mannosylated TSR-derived peptides on lipopolysaccharide (LPS)-induced signaling in macrophage-like RAW264.7 cells. The cells were stimulated with LPS in the presence or absence of chemically synthesized peptides with or without C-mannose (e.g., (C-Man)-Trp-Ser-Pro-Trp [C-Man-WSPW], C-Man-W, WSPW, etc.), then the effects of the peptides on cellular viability and signaling were examined. We found a cytotoxic effect in the cells treated with LPS and C-Man-WSPW, but not in the cells solely treated with LPS or C-Man-WSPW. We also found that production of tumor necrosis factor-{alpha} (TNF-{alpha}) was upregulated more in response to LPS plus C-Man-WSPW, than in response to LPS plus WSPW or LPS alone. Among the LPS-induced signaling pathways that induce production of TNF-{alpha}, the activation of c-Jun N-terminal kinase (JNK) was greatly enhanced by LPS and C-Man-WSPW, and the production of TNF-{alpha} was suppressed by an inhibitor for JNK. Together, these results demonstrate a novel function of the C-mannosylated TSR-derived peptide motif, to promote LPS-induced JNK signaling, and this leads to an enhancement of cytotoxicity via the upregulation of TNF-{alpha} production.

Key words: C-mannosylation / lipopolysaccharide / macrophage / thrombospondin


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Funding
 Conflict of interest statement
 References
 
C-Mannosylation is unique in that an {alpha}-mannose is directly bound to the indole C2 carbon atom of a Trp (W) residue through a C-C bond to produce C-mannosyl Trp (C-Man-W) (Hofsteenge et al. 1994Go). C-Mannosylation occurs at the first Trp in the consensus amino acid sequence Trp-X-X-Trp (WXXW) in proteins (Furmanek and Hofsteenge 2000Go). The WXXW motif is found in the thrombospondin type 1 repeat (TSR), which is predicted to be a functional peptide module in various integral proteins such as thrombospondin-1 (TSP-1) (Tucker 2004Go). The motif is known to bind with heparin, heparan sulfate proteoglycans, and collagen, suggesting a functional significance in cell–cell interaction and/or cellular signaling. There are a number of examples of C-mannosylated proteins including ribonuclease 2 (Hofsteenge et al. 1994Go), Interleukin-12 (Doucey et al. 1999Go), complements (C6, C7, C8a, C8b, and C9) (Hofsteenge et al. 1999Go), properdin (Hartmann and Hofsteenge 2000Go), thrombospondin (Hofsteenge et al. 2001Go; De Peredo et al. 2002Go), F-spondin (Furmanek and Hofsteenge 2000Go), the erythropoietin receptor (Furmanek et al. 2003Go), mucins (MUC5AC and MUC5B) (Perez-Vilar et al. 2004Go). C-Mannosylation is thought to be carried out by a specific unidentified mannosyltransferase located in the microsomes. This suggests that C-mannosylation is involved in conventional glycosylation through the secretory pathway (Doucey et al. 1998Go).

Glycosylation has been revealed to have functional relevance to various cellular events including cell development, growth, differentiation, and death (Varki 1993Go; Dennis et al. 1999Go; Haltiwanger and Lowe 2004Go). However, the biological significance of C-mannosylation has yet to be fully investigated because a suitable methodology has not been established. In general, it is difficult to control the formation of glycoforms in cultured cells, which hampers progress in glycobiology and is a major obstacle to research into glycoprotein functions. One approach to conducting research in glycobiology is to generate well-defined synthetic glycopeptides or small glycoproteins using chemical or chemoenzymatic synthesis (Buskas et al. 2006Go). Since the discovery of C-mannosylated glycoproteins, several ways to obtain C2-{alpha}-D-C-Man-L-Trp building blocks have been reported (Manabe and Ito 1999Go; Nishikawa et al. 2001Go). Furthermore, Manabe et al. (2003)Go reported a route that differs from conventional approaches to C-glycosylation (Beau and Gallagher 1997Go). Recently, using a synthetic C-Man-W, we prepared a specific antibody against C-Man-W, then found that C-mannosylated proteins were expressed in mouse macrophage-like RAW264.7 cells, and the expression was upregulated under hyperglycemic conditions (Ihara et al. 2005Go).

To know whether C-mannosylation plays a functional role in macrophages, we focused on lipopolysaccharide (LPS)-induced signaling in macrophages, and investigated the effect of chemically synthesized C-mannosylated TSR-derived peptides (e.g., (C-Man)-Trp-Ser-Pro-Trp [C-Man-WSPW] [Figure 1]) on the signaling pathway in RAW264.7 cells. Here we report that C-mannosylated TSR-derived peptides modulate lipopolysaccharide (LPS)-induced signaling including the pathways involving mitogen activating protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK), and enhance the cytotoxicity of LPS.


Figure 1
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  		Fig. 1. The structure of C-mannosyl tryptophan in the TSR-derived peptide.

 

   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Funding
 Conflict of interest statement
 References
 
C-Mannosylated TSR-derived peptides enhance LPS-induced cell damage in RAW264.7 cells
C-Mannosylated TSR-derived peptides and derivatives were chemically synthesized as described in Materials and methods. Cells were cultured for 48 h with different concentrations of LPS in the presence or absence of C-Man-WSPW or WSPW (10 µM). The WSPW peptides, in which the first W has the potential to be C-mannosylated, are derived from TSR2 of human TSP-1, and the first W corresponds to Trp423 in the amino acid sequence (Lawler and Hynes 1986Go). Cell viability was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as described in the methods (Figure 2A). Cell viability was little influenced by LPS between 0.1 and 5 µg/mL. In the presence of C-Man-WSPW (10 µM), viability was apparently reduced by LPS, especially at 1 µg/mL. However, no enhancement of LPS-dependent cell damage was observed in the presence of WSPW (10 µM). To further investigate the effect of C-Man-WSPW on LPS-induced cell damage, damaged cells were examined microscopically after staining with SYTOX Green, a noncell-permeable dye used to stain the nucleus (Figure 2B). Cells were cultured for 48 h with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). After the treatment with LPS, there was a slight enlargement of the cell body with or without C-Man-WSPW or WSPW. The cells stained with SYTOX Green were slightly observed in the cases with LPS plus WSPW or LPS alone. However, SYTOX Green-stained nuclei were observed more in the cells treated with LPS plus C-Man-WSPW, indicating that the plasma membrane was damaged in these cells. Together, these results suggest that C-Man-WSPW specifically induces damage in the cells treated with LPS.


Figure 2
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  		Fig. 2. C-Mannosylated TSR-derived peptides enhance the cytotoxicity of LPS in RAW264.7 cells. (A) RAW264.7 cells were cultured for 48 h in medium containing one of the several different concentrations of LPS in the presence or absence of C-Man-WSPW or WSPW (10 µM). Cell viability was examined by MTT assay, as described in Materials and methods. Each value represents the mean ± SD of four independent experiments. (B) Cells were treated as in A, then stained with SYTOX Green (1 µM) for 10 min. After fixation, signals were visualized by fluorescence microscopy with excitation at 485 nm and emission at 520 nm. Over 200 cells were counted to determine the proportion of SYTOX Green-positive cells (%). The values represent the means ± SD of three independent experiments. (C) Cells were cultured for 48 h with LPS (1 µg/mL) in the presence or absence of various concentrations of C-Man-WSPW (0.5–10 µM). Cell viability was examined by MTT assay, as described. (D) Cells were cultured for 48 h with or without LPS (1 µg/mL) in the presence or absence of mannose, C-Man-W, C-mannosylated peptides with different structures (C-Man-WS, C-Man-WSP, C-Man-WSPW, C-Man-WSPWS, C-Man-WSG, and C-Man-WSK), or WSPW (10 µM), and then cell viability was examined by MTT assay. Each value represents the mean ± SD of three to five independent experiments.

 
Next, to see the dose-responsive effect of C-Man-WSPW on LPS-induced cell damage, cells were cultured for 48 h with LPS (1 µg/mL) in the presence of various concentrations of C-Man-WSPW (0.5–10 µM). Cell viability was examined as described above. The results showed that C-Man-WSPW enhanced the LPS-induced cell damage in a dose-dependent manner (Figure 2C). To further investigate the structural requirement of C-mannosylated peptides for enhancement of the damage due to LPS, cells were cultured for 48 h with or without LPS (1 µg/mL) in the presence or absence of mannose, C-Man-W, C-mannosylated peptides with different structures (e.g., C-Man-WS, C-Man-WSP, C-Man-WSPW, C-Man-WSPWS, C-Man-WSG, and C-Man-WSK) or WSPW (10 µM), then cell viability was examined. As shown in Figure 2D, C-mannosylated peptides apparently enhanced the LPS-induced cell damage, especially in the cases of C-Man-WSP, C-Man-WSPW, and C-Man-WSPWS. C-Man-WSG and C-Man-WSK also moderately enhanced LPS-induced cell damage. On the other hand, C-Man-WS, C-Man-W, and mannose had a minor effect compared with other C-mannosylated peptides, and nonglycosylated WSPW had little effect as well. Furthermore, without LPS, cell viability was not reduced by mannose, C-Man-W, C-Man-WSPW, or WSPW (10 µM). These results indicate that C-mannosylated peptides, which contain tripeptides such as WSP, effectively enhance LPS-induced damage in the cells.

Effect of C-mannosylated TSR-derived peptides on the binding of LPS to the cells
To investigate whether C-Man-WSPW influences the binding of LPS to RAW264.7 cells, the cells were incubated at 37°C for 20 min with FITC-conjugated LPS (LPS-FITC) (1 µg/mL) in the presence or absence of WSPW or C-Man-WSPW (10 µM) as described in the methods. After a wash with PBS, the binding of LPS-FITC to the cells was analyzed by flow cytometry (Figure 3A). The results showed that LPS-FITC was similarly bound to the cells in the presence of C-Man-WSPW or WSPW, indicating that C-Man-WSPW or WSPW did not directly influence the binding of LPS to the cells. Next, to determine the amount of WSPW or C-Man-WSPW bound to RAW264.7 cells, the cells were incubated with biotin, biotin-labeled WSPWC (WSPWC-biotin) or C-Man-WSPWC (C-Man-WSPWC-biotin) (10 µM), and then the molecules bound to the cells were detected by flow cytometry after incubation with FITC-conjugated avidin (Figure 3B). The results showed that C-Man-WSPWC-biotin was bound to the cells, although the binding was weak. On the other hand, biotin and WSPWC-biotin showed less of a background signal. To further examine the interaction between C-mannosylated peptides and target proteins in the cell, the cells were incubated with biotin, WSPWC-biotin, or C-Man-WSPWC-biotin (10 µM), and then the proteins bound to the biotin conjugates were detected, as described in the methods, by blot analysis using peroxidase-conjugated avidin after treatment with or without dithiobis[succinimidylpropionate] (DSP), a membrane-permeable crosslinker. As shown in Figure 3C, various proteins were bound to C-Man-WSPWC-biotin to a greater extent than those to WSPWC-biotin, under the conditions with DSP (arrows). The binding was not apparent under the conditions without DSP, although some non-specific binding of peroxidase-conjugated avidin to proteins was detected (arrow heads). These results suggest a specific but weak interaction between C-Man-WSPWC-biotin and the proteins.


Figure 3
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  		Fig. 3. Effect of C-Man-WSPW or WSPW on the binding of LPS to RAW264.7 cells. (A) Cells were incubated at 37°C for 20 min in medium containing LPS-FITC (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). After a wash with PBS, the binding of LPS-FITC to the cells was analyzed by flow cytometry, as described in Materials and methods. (B) Cells were incubated at 37°C for 20 min in PBS with biotin, WSPWC-biotin, or C-Man-WSPWC-biotin (10 µM). After a wash with PBS, the cells were blocked with 3% BSA in PBS, incubated with FITC-conjugated avidin for 15 min, and washed with PBS containing 1% BSA. After another wash, the binding of biotin conjugates to cells was analyzed by flow cytometry. (C) Cells were incubated with the biotin conjugates as described in B, and treated for 10 min with or without 1 mM DSP. After quenching with 1M Tris–HCl (pH 7.2), cells were washed with PBS, and subsequently harvested. The cells were lysed and protein samples were electrophoresed on 7.5% SDS-polyacrylamide gels under nonreducing conditions and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in TBS and then incubated at 4°C for 10 min with the peroxidase-conjugated avidin in TBS containing 0.1% Tween 20. After a wash with TBS containing 0.1% Tween 20, the blots were developed using an ECL chemiluminescence detection kit.

 
Taken together, these results indicate that C-Man-WSPWC was predominantly bound to some target proteins of RAW264.7 cells, although it did not directly influence the binding of LPS to the cells.

C-Mannosylated TSR-derived peptides enhance LPS-induced production of TNF-{alpha} in the cells
LPS is cytotoxic due to the TNF-{alpha} or nitric oxide produced by the macrophages it stimulates (Guha and Mackman 2001Go). Thus, we focused on the effect of C-mannosylated TSR-derived peptides on the LPS-induced production of TNF-{alpha} and cell signaling. To investigate whether the C-mannosylated peptides influence the LPS-induced production of TNF-{alpha}, RAW264.7 cells were treated with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM), then the amount of TNF-{alpha} secreted from the cells was estimated by enzyme-linked immunoabsorbent assay (ELISA). As shown in Figure 4A, secretion of TNF-{alpha} increased in the culture medium following the treatments. In the case of LPS plus C-Man-WSPW, the secretion was especially enhanced at 1 and 2 h after the treatment, compared with the conditions with LPS plus WSPW or LPS alone. The transcriptional expression of TNF-{alpha} was also examined by reverse transcription-polymerase chain reaction (RT-PCR) in the cells treated with LPS in the presence or absence of C-Man-WSPW or WSPW. The cells were treated for 1 and 2 h with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM), then total RNA was extracted and subjected to RT-PCR. As shown in Figure 4B, the transcriptional level of TNF-{alpha} was enhanced more with LPS plus C-Man-WSPW than with LPS plus WSPW or LPS alone. Next, to investigate the structural requirement of C-mannosylated peptides for the enhancement of the LPS-induced production of TNF-{alpha}, cells were cultured for 2 h with LPS (1 µg/mL) in the presence or absence of mannose, C-Man-W, C-mannosylated peptides with different structures (e.g., C-Man-WS, C-Man-WSP, C-Man-WSPW, and C-Man-WSPWS), or WSPW (10 µM), then the amount of TNF-{alpha} secreted was measured by ELISA as described. As shown in Figure 4C, C-mannosylated peptides, such as C-Man-WSP, C-Man-WSPW, and C-Man-WSPWS, apparently enhanced the LPS-induced production of TNF-{alpha}, compared with LPS alone. C-Man-WSG and C-Man-WSK also similarly enhanced the LPS-induced production of TNF-{alpha} (data not shown). On the other hand, mannose, C-Man-W, and C-Man-WS had a minor effect compared with other C-mannosylated peptides, and nonglycosylated WSPW had very little effect. Furthermore, without LPS, the production of TNF-{alpha} was not triggered by mannose, C-Man-W, C-Man-WSPW or WSPW (10 µM). These results indicate that C-mannosylated peptides, which contain tripeptides such as WSP, effectively enhance the LPS-induced production of TNF-{alpha} in RAW264.7 cells. Collectively, they indicate that LPS-induced expression of TNF-{alpha} is enhanced with C-mannosylated peptides such as C-Man-WSPW.


Figure 4
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  		Fig. 4. C-Mannosylated TSR-derived peptides enhance the LPS-induced production of TNF-{alpha} in RAW264.7 cells. (A) Cells were plated in 96-well plates (10,000 cells/well), and cultured overnight. After being washed, the cells were stimulated for the periods indicated with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). After the incubation, the culture medium was collected, and the concentration of TNF-{alpha} in the sample was measured using a mouse TNF-{alpha} ELISA kit as described in Materials and methods. Each value represents the mean ± SD of four independent experiments. *P < 0.05 vs. value for the cells treated with LPS or WSPW plus LPS. (B) Cells were stimulated for the periods indicated with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). Total RNA was prepared from the cells, and a RT-PCR analysis was performed to evaluate the transcriptional expression of TNF-{alpha}. The expression of ß-actin was also shown as a control. The band intensity was estimated densitometrically, and the relative value of TNF-{alpha} to ß-actin represents the mean ± SE of three independent experiments (*P < 0.05 vs. value at same time point (1 h) for the cells treated with LPS or WSPW plus LPS). (C) Cells were cultured for 2 h with or without LPS (1 µg/mL) in the presence or absence of mannose, C-Man-W, C-mannosylated peptides with different structures (C-Man-WS, C-Man-WSP, C-Man-WSPW, and C-Man-WSPWS), or WSPW (10 µM), and then the TNF-{alpha} concentration in the medium was evaluated using a mouse TNF-{alpha} ELISA kit. Each value represents the mean ± SD of three independent experiments (*P < 0.05 vs. value for the cells treated with LPS alone).

 
C-Mannosylated TSR-derived peptides enhance LPS-induced activation of JNK in the cells
LPS induces a variety of cellular signaling events, including the activation of NF-{kappa}B and activation of the members of the MAPK family (Guha and Mackman 2001Go). The LPS signaling cascade leading to the production of TNF-{alpha} is controlled at the levels of both transcription of the TNF-{alpha} gene and translation of the mRNA. Transcriptional control of the gene is mediated primarily through the NF-{kappa}B signaling pathway (Shakhov et al. 1990Go), and translation of the mRNA is regulated through the JNK pathway (Swantek et al. 1997Go). To investigate the effect of C-Man-WSPW on NF-{kappa}B signaling with LPS, the transcriptional activity of NF-{kappa}B was examined by conducting a luciferase reporter assay for NF-{kappa}B as described in the methods. After transfection with the lufciferase vector for NF-{kappa}B, the cells were treated with LPS (1 µg/mL) in the presence or absence of WSPW or C-Man-WSPW (10 µM), and reporter activity was measured (Figure 5A). The results showed that LPS-induced activation of NF-{kappa}B was not influenced by C-Man-WSPW or WSPW. To further investigate the NF-{kappa}B signaling with LPS, degradation of inhibitor of {kappa}B{alpha} (I{kappa}B{alpha}) was also examined by immunoblot analysis using specific antibodies, because degradation of I{kappa}B{alpha} occurred during the activation of NF-{kappa}B (Guha and Mackman 2001Go). The results showed that there was no difference in the LPS-induced degradation of I{kappa}B{alpha} in the cells treated with or without C-Man-WSPW or WSPW (Figure 5B).


Figure 5
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  		Fig. 5. Effect of C-Man-WSPW or WSPW on the NF-{kappa}B signaling in RAW264.7 cells treated with LPS. (A) After 24 h of transfection with the lufciferase vector for NF-{kappa}B, the cells were incubated at 37°C for the periods indicated in medium containing LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM), and the reporter activity was measured as described in the methods. (B) Cells were incubated at 37°C for the periods indicated in medium containing LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). Degradation of I{kappa}B{alpha} was examined with samples of cell lysate by immunoblot analysis using specific antibodies as described in Materials and methods.

 
To investigate how C-Man-WSPW enhances the LPS-induced production of TNF-{alpha}, the activation status of several MAPKs was examined in the cells treated with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). The cells were treated with the reagents for given periods, then the phosphorylation status of MAPKs, such as extracellular signal-regulated kinase (ERK) 1/2, p38-MAPK, and JNK, was examined by immunoblot analysis using specific antibodies recognizing the phosphorylated form of each kinase. As shown in Figure 6A, B, and C, phosphorylation was induced in all tested MAPKs by the treatment with LPS, indicating that LPS upregulated MAPK signaling, and was consistent with previous findings related to LPS-induced MAPK signaling (Guha and Mackman 2001Go). The results also showed that the LPS-induced phosphorylation of MAPKs was little influenced by WSPW peptides. On the other hand, the LPS-induced phosphorylation was apparently enhanced by C-Man-WSPW especially in the case of JNK, although slight enhancement was observed in the cases of ERK1/2 and p38-MAPK. The phosphorylation of MAPKs was not induced in the cells solely treated with WSPW or C-Man-WSPW (data not shown), indicating that LPS was required to upregulate all of the MAPK signaling. To confirm the enhanced activation of JNK in the cells treated with LPS plus C-Man-WSPW, the activity of JNK was examined using GST-c-Jun fusion protein as a substrate in samples prepared from cells treated for 15 min with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). As shown in Figure 6D, phosphorylation of GST-c-Jun was upregulated more in the cells treated with LPS plus C-Man-WSPW, than those treated with LPS plus WSPW or LPS alone. This indicates that JNK activity is specifically enhanced by LPS plus C-Man-WSPW. Collectively, these results indicate that LPS-induced JNK signaling was upregulated by C-Man-WSPW, but not by WSPW, despite that LPS-induced NF{kappa}B signaling was little influenced by C-Man-WSPW or WSPW.


Figure 6
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  		Fig. 6. Effect of C-Man-WSPW or WSPW on the MAPK signaling in RAW264.7 cells treated with LPS. A, B, C, and cells were treated with LPS, LPS plus WSPW, or LPS plus C-Man-WSPW, as described in Figure 5B. The phosphorylation status of MAPKs, such as ERK1/2 (Thr-202/Tyr-204) (A), p38-MAPK (Thr-180/Tyr-182) (B), and JNK (Thr-183/Tyr-185) (C), was examined with samples of cell lysate by immunoblot analysis using specific antibodies as described in Materials and methods. (D) The activity of JNK was examined, as described in the methods using GST-c-Jun as a substrate in the samples prepared from cells treated at 37°C for 15 min with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM).

 
JNK signaling pathway is involved in enhancement of LPS-induced upregulation of TNF-{alpha} by C-Man-WSPW
To investigate whether the signaling pathways of JNK and/or NF-{kappa}B are involved in the LPS-induced production of TNF-{alpha}, levels of TNF-{alpha} were examined in cells treated with or without LPS, LPS plus WSPW, and LPS plus C-Man-WSPW in the presence or absence of specific signaling inhibitors. SP600125 (a JNK Inhibitor) and a NF-{kappa}B activation inhibitor were used to inhibit the activities of JNK and NF-{kappa}B, respectively. In Figure 7A, the cells were pretreated for 10 min with the JNK inhibitor (10 µM), then treated for 15 min with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). The activation status of JNK was examined in the cell lysates by immunoblot analysis using the anti-phospho-JNK antibody as described. The results showed that the phosphorylation of JNK was significantly suppressed by the JNK inhibitor in all cases tested. In Figure 7B, after 24 h of transfection with the luciferase vector for NF-{kappa}B, the cells were pretreated for 10 min with the NF-{kappa}B activation inhibitor (10 µM), then treated for 90 min with LPS (1 µg/ mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). The activation status of NF-{kappa}B was examined in the cell lysates by luciferase reporter assay as described. The results showed that the level of transcriptional activity for NF-{kappa}B was similarly enhanced by LPS with or without WSPW or C-Man-WSPW, but was suppressed by the inhibitor to approximately 60% of each level of NF-{kappa}B activity without the inhibitors. The cells were pretreated for 10 min with or without the JNK inhibitor and/or NF-{kappa}B activation inhibitor, and treated for 2 h with LPS, LPS plus WSPW, or LPS plus C-Man-WSPW (Figure 7C). Then, the amount of TNF-{alpha} released into the medium was estimated by an ELISA for TNF-{alpha}. The results showed that LPS-induced production of TNF-{alpha} was suppressed by the inhibitors for JNK and/or NF-{kappa}B with or without WSPW or C-Man-WSPW. Although the suppression was enhanced more with the JNK inhibitor plus NF-{kappa}B activation inhibitor, the production of TNF-{alpha} was not completely suppressed to the level of the untreated background (see Figure 4A).


Figure 7
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  		Fig. 7. JNK and NF-{kappa}B contribute to the LPS-induced production of TNF-{alpha} in RAW264.7 cells in the presence or absence of C-Man-WSPW or WSPW. (A) Cells were pretreated at 37°C for 10 min with the JNK inhibitor (SP600125, 10 µM), then treated for 15 min with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). The activation status of JNK was examined in the cell lysates by immunoblot analysis using the antiphospho-JNK (Thr183/Tyr-185) antibody as described in Materials and methods. (B) After 24 h of transfection with the luciferase vector for NF-{kappa}B, the cells were pretreated at 37°C for 10 min with a NF-{kappa}B activation inhibitor (10 µM), then treated for 90 min with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). The activation status of NF-{kappa}B was examined in the cell lysates by luciferase reporter assay as described in the methods. (C) Cells were pretreated at 37°C for 10 min with or without JNK inhibitor and/or the NF-{kappa}B activation inhibitor, and treated for 2 h with LPS (1 µg/mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM), as described above. Then, amount of TNF-{alpha} released into the medium was estimated by ELISA for TNF-{alpha}.

 
Taken together, these results demonstrate that both the JNK and NF-{kappa}B pathways are important to the production of TNF-{alpha} induced in all cases with LPS, LPS plus WSPW, and LPS plus C-Man-WSPW, suggesting that enhanced JNK signaling in the cells treated with LPS plus C-Man-WSPW may be part of the signaling pathway responsible for upregulating the production of TNF-{alpha}.

C-Mannosylated TSR-derived peptides enhance LPS-induced phosphorylation of TAK1 in RAW264.7 cells
In terms of signaling pathways for LPS-induced TNF-{alpha} production, we found that the activation of JNK was specifically upregulated by LPS in the presence of C-Man-WSPW. To further investigate how C-Man-WSPW enhances LPS-induced activation of JNK, we focused on the upstream signaling molecules, such as interleukin-1 receptor-associated kinase 1 (IRAK1) and transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1). IRAK1 is one of the upstream serine/threonine kinases activated by LPS through Toll-like receptor 4 (TLR4) (Janssens and Beyaert 2003Go). The activation of IRAK1 occurs with a concomitant self-phosphorylation of serine or threonine (Kollewe et al. 2004Go). In Figure 8A, cells were treated with LPS (1 µg/ mL) in the presence or absence of C-Man-WSPW or WSPW (10 µM). The activation status of IRAK1 was examined in the cell lysates by immunoblot analysis using the anti-phospho-IRAK1 (Thr-209) antibody, as described. The results showed that the pattern of phosphorylation of IRAK1 did not differ with LPS, LPS plus WSPW, or LPS plus C-Man-WSPW, suggesting that the activation of IRAK1 was little influenced by WSPW or C-Man-WSPW. On the other hand, TAK1, a MAPK kinase kinase (MAPKKK) activated by LPS through TLR4 signaling, is also involved in the LPS-induced signaling pathways to activate NF-{kappa}B and MAPKs, such as JNK (Irie et al. 2000Go; Chen et al. 2002Go; Silverman et al. 2003Go) (Figure 8B). The activation of TAK1 occurs with a concomitant autophosphorylation of serine or threonine (Kishimoto et al. 2000Go; Singhirunnusorn et al. 2005Go). The activation status of TAK1 was also examined in the cell lysates by immunoblot analysis using the anti-phospho-TAK1 (Thr-187) antibody, as described in Materials and methods. The results showed that the phosphorylation of TAK1 was significantly enhanced by LPS plus C-Man-WSPW, but not by LPS plus WSPW or LPS alone (Figure 8B).


Figure 8
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  		Fig. 8. LPS-induced phosphorylation of TAK1 is highly upregulated in RAW264.7 cells treated with C-Man-WSPW. (A) Cells were treated with LPS, LPS plus WSPW, or LPS plus C-Man-WSPW, as described in Figure 5B. The activation status of IRAK1 was examined in the cell lysates by immunoblot analysis using the antiphospho-IRAK1 (Thr-209) antibody as described in the methods. (B) Cells were treated with LPS, LPS plus WSPW, or LPS plus C-Man-WSPW, as described above. The activation status of TAK1 was also examined in the cell lysates by immunoblot analysis using the antiphospho-TAK1 (Thr-187) antibody, as described in Materials and methods.

 
Taken together, C-Man-WSPW or WSPW did not influence the LPS-induced activation of IRAK1, a pivotal LPS-induced kinase located upstream of TAK1, which was consistent with the finding that the quantity of LPS-FITC bound to the cells was not affected by C-Man-WSPW or WSPW. However, C-Man-WSPW specifically upregulated LPS-induced activation of TAK1, a pivotal MAPKKK for LPS-induced activation of MAPK pathways. This was consistent with enhanced LPS-induced activation of MAPKs such as JNK by C-Man-WSPW, resulting in increased production of TNF-{alpha} and further LPS-induced cell damage.


   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Funding
 Conflict of interest statement
 References
 
In the present study, to know how C-mannosylation is involved in the functions of macrophages, we chemically synthesized C-mannosylated peptides such as C-Man-WSPW derived from the TSR motif of human TSP-1, and investigated their effect on LPS-induced signaling in macrophage-like RAW264.7 cells.

LPS, a component of the outer cell membrane of gram- negative bacteria, is a strong stimulator of macrophage/ monocytes and shows a clearly inflammatory effect. It is also known that LPS is toxic to several types of cells, such as macrophages and endothelial cells, and that LPS is involved in the pathology of sepsis and septic shock (Akira 2001Go; Bannerman and Goldblum 2003Go). In this study, we found that C-mannosylated peptides enhanced LPS-induced damage in RAW264.7 cells, especially in the cases of C-Man-WSP, C-Man-WSPW, and C-Man-WSPWS. However, C-Man-WS, C-Man-W, and mannose had a minor effect compared with other C-mannosylated peptides, and nonglycosylated WSPW also had little effect. These results strongly suggest that C-mannosylated peptides, which contain tripeptides such as WSP, effectively enhance LPS-induced damage, and also that C-mannosylation confers a specific function on WSPW peptides which is commonly seen in the TSR motif of cellular proteins. However, it is not clear yet whether these C-mannosylated peptides can be naturally generated in the cell. In addition, to test functional importance of Pro in C-Man-WSP, we prepared C-Man-WSG and C-Man-WSK, in which Pro was replaced by Gly and Lys, respectively, and examined the effect of the C-mannosylated tripeptides on LPS-induced cell damage. As shown in Figure 2D, both C-Man-WSG and C-Man-WSK moderately enhanced LPS-induced cell damage, suggesting that the effect seems not necessarily dependent on Pro in the specific sequence of WSP.

A variety of C-mannosylated proteins have been identified, and some of these proteins, such as TSP, F-spondin, Interleukin-12, complements, and mucins, mainly function in the extracellular spaces (Furmanek and Hofsteenge 2000Go). Recently, it has been reported that TSP-1 and 2 are cleaved by ADAMTS1 (A Disintegrin And Metalloprotease with ThromboSpondin 1), resulting in the production of functional antiangiogenic peptides (Lee et al. 2006Go). Furthermore, C-Man-W was detected in human serum or urine from patients of renal diseases (Takahira et al. 2001Go), suggesting that C-Man-W could be a novel biomarker for renal dysfunction. These results suggest that some of the functional C-mannosylated peptides might be generated in vivo by proteolytic degradation of C-mannosylated proteins. However, it is not clear yet what endogenous C-mannosylated peptides or proteins enhance LPS-induced signaling in macrophages. Thus, further investigation is required to identify such endogenous proteins bearing the C-mannosylated peptides in vivo.

In this study, we showed that the enhanced LPS-induced signaling with C-Man-WSPW was not simply due to the enhanced binding of LPS to cells treated with LPS and C-Man-WSPW, suggesting that the cellular binding of LPS was not directly affected by C-Man-WSPW. On the other hand, we found that the binding of C-Man-WSPWC-biotin to cells was greater than that of biotin or WSPWC-biotin on flow cytometry. In addition, under the conditions with DSP, we found that C-Man-WSPWC-biotin was predominantly bound to some specific proteins of RAW264.7 cells, compared with the lower binding of biotin or WSPWC-biotin. Together, these results strongly suggest that C-Man-WSPW peptides exert an enhancing effect on LPS-induced cytotoxicity by their binding to some specific target proteins in cells, but not by affecting LPS binding to the cells.

The inflammatory effects of LPS are mediated through several secreted factors, such as TNF-{alpha} (Guha and Mackman 2001Go) and nitric oxide (Nathan 1992Go). We also found that C-Man-WSPW apparently enhanced the production of TNF-{alpha} in cells treated with LPS, compared to those treated with LPS plus WSPW or LPS alone. LPS starts the signal transduction by engaging itself with cell surface TLR4 via sequential binding with LPS-binding protein and CD14 (Guha and Mackman 2001Go). LPS has been shown to initiate multiple intracellular signaling events, such as the activation of NF-{kappa}B, and three distinct MAPKs (i.e., ERK1/2, p38-MAPK, and JNK), and these pathways are involved in the LPS-induced upregulation of TNF-{alpha} production. In this study, we found that JNK was significantly activated in the cells treated with LPS plus C-Man-WSPW, compared with the cells treated with LPS plus WSPW or LPS alone, although other kinases such as ERK1/2 and p38-MAPK were also slightly upregulated in the activation by C-Man-WSPW plus LPS. On the other hand, the LPS-induced activation of NF-{kappa}B was little influenced by C-Man-WSPW or WSPW. Wilson et al. (1999)Go reported that TSP-1 or TSP-1-derived peptides containing a WSXW motif enhanced CD3-induced activation of MAPK signaling including JNK signaling in T lymphocytes. In addition, Jimenez et al. (2001)Go reported that TSP-1 inhibits angiogenesis in corneal neovascularization via the activation of JNK signaling. However, it is not clear how the WXXW motif in the TSR is involved in the signaling to JNK.

The proximal signaling molecules involved in the LPS-induced activation of MAPKs have not been fully identified. LPS is thought to activate ERK1/2 through the Ras/Raf-1/MAPKK pathway, p38-MAPK through the activation of MAPK/ERK kinase (MEK)-3, and JNK through the MEKK-1/MEK4 pathway (Swantek et al. 1997Go; van der Bruggen et al. 1999Go). In this study, we focused on the upstream signaling of JNK, and examined the effect of C-Man-WSPW on the activation status of some upstream kinases induced by LPS. IRAK1 is one of the upstream kinases associated with the LPS-specific receptor, TLR-4, and is phosphorylated by LPS to be activated (Li et al. 2000Go). Unexpectedly, LPS-induced phosphorylation of IRAK1 was not influenced by C-Man-WSPW or WSPW, which was consistent with the finding that both C-Man-WSPW and WSPW did not influence the binding of LPS to cells. However, these results suggested that C-Man-WSPW indirectly acts on some intermediate in the LPS-induced signaling pathways to JNK. Next, we examined TAK1, a MAPKKK mainly involved in LPS-induced JNK signaling (Chen et al. 2002Go; Silverman et al. 2003Go), and found that LPS-induced phosphorylation of TAK1 was apparently enhanced by C-Man-WSPW but not by WSPW. This suggests that C-Man-WSPW influenced LPS-induced signaling to activate JNK at the level of TAK1 activity, although how the phosphorylation of TAK1 was upregulated by C-Man-WSPW is not known. Although TAK1 is located downstream of the pathway of LPS/TLR4/IRAK1/TNF-{alpha} receptor-associated factor (TRAF6) that activates JNK, an alternative pathway for the activation of TAK1 has been identified in cells expressing latent membrane protein 1 (LMP1) derived from Epstein-Barr virus (Wan et al. 2004Go). LMP1 does not require myeloid differentiation factor 88 (MYD88), IRAK1, and IRAK4 to engage TRAF6, and selectively utilize TRAF6, TAK1/TAK1-binding protein, and JNK kinases 1 and 2 to activate JNK. Very recently, Uemura et al. (2006)Go demonstrated that TAK1 is involved in the LMP1 complex and is essential for activation of JNK but not of NF-{kappa}B. This suggests that the LPS signaling pathway can be influenced by other modulators, which are not directly involved in the authentic LPS/TLR-4/TRAF6/IRAK/TAK1 pathways, implying that C-mannosylated TSR-derived peptides may act as another modulator of LPS-induced MAPK signaling by mimicking the functions of modulators such as LMP1, although the precise mechanism for the signal transduction by C-Man-WSPW is yet to be clarified.

In macrophages/monocytes, there is a variety of mannose recognizing proteins included in the mannose receptor family (East and Isacke 2002Go; McGreal et al. 2005Go). The mannose receptor family is a subgroup of the C-type lectin superfamily and comprises four members; the mannose receptor (MR), the M-type phospholipase A2 receptor, DEC-205 (CD205), and Endo-180. Among the members, MR and Endo-180 have the capacity to bind carbohydrates such as mannose, fucose, N-acetylglucosamine, etc., in a Ca2+-dependent manner via their carbohydrate recognition domains. On the other hand, among the C-type lectin receptors, Dectin-1, which recognizes ß-linked glucans from fungi, is known to work in collaboration with TLR2 to facilitate inflammatory responses (Brown et al. 2003Go; Gantner et al. 2003Go). This also suggests a possible regulation of TLR-related signaling by C-mannosylated peptides via some co-lateral regulatory pathways. Hence, another question raised is whether the C-mannosylated peptides also modulate other receptor signaling pathways in macrophages (i.e., TLR2, TLR6, TLR9, interleukin-1 receptor, and TNF-{alpha} receptor), compared with the LPS/TLR4 pathway. Nishikawa et al. (2004)Go reported that C-Man-W is not recognized in vitro by conventional mannose-binding lectins such as concanavaline A and mannose-binding lectin-C, a lectin abundant in mouse serum. However, it is not clear yet what specific molecule binds to C-Man-W and C-mannosylated peptides. Further investigation to clarify the specific receptors recognizing the motif-containing C-mannosylated peptides is required.

In conclusion, C-mannosylated TSR-derived peptides have an enhancing effect on the cytotoxicity of LPS by increasing the production of TNF-{alpha} through enhanced activation of JNK. This study demonstrates a novel regulatory function of the C-mannosylated TSR-derived peptide motif in the innate immunity of macrophages, and also suggests unrevealed functions of C-mannosylated proteins or peptides in macrophages.


   Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Funding
 Conflict of interest statement
 References
 
Materials
Antibodies against ERK1/2, phospho-ERK1/2 (Thr-202/Tyr-204), p38-MAPK, phospho-p38MAPK (Thr-180/Tyr-182), JNK, phospho-JNK (Thr-183/Tyr-185), TAK1, phospho-TAK1 (Thr-187), IRAK1, phospho-IRAK1 (Thr-209), and I{kappa}B{alpha} were purchased from Cell Signaling Technology (Beverly, MA). Peroxidase-conjugated secondary antibodies against IgG of rabbit and mouse were from Dako (Glostrup, Denmark). LPS from Escherichia coli O55:B5 and fluorescein isothiocyanate (FITC)-conjugated LPS (LPS-FITC) were obtained from Sigma-Aldrich (St. Louis, MO). Nuclear factor-{kappa}B (NF-{kappa}B) activation inhibitor [i.e., 6-amino-4-(4-phenoxypheny- lethylamino)quinazoline] was from Calbiochem. DSP was from Pierce Biotechnology (Rockland, IL). The other reagents used in the study were all of high grade, from Sigma-Aldrich or Wako pure chemicals (Osaka, Japan).

Chemical synthesis of C-mannosylated peptides and derivatives
C2-{alpha}-D-C-Mannosylpyranosyl-L-tryptophan (C-Man-W) and C-mannosylated TSR-derived peptides (C-Man-WS, C-Man-WSP, C-Man-WSG, C-Man-WSK, C-Man-WSPW, and C-Man-WSPWS) were synthesized essentially, as described previously (Manabe and Ito 1999Go; Manabe et al. 2003Go). WSPW peptides, in which the first W has the potential to be C-mannosylated, are derived from TSR2 of human TSP-1, and the first W corresponds to Trp423 in the amino acid sequence (Lawler and Hynes 1986Go; NCBI Accession number NP_003237 [GenBank] ) (Figure 1). Silica gel 60N (spherical, neutral, Kanto Chemical Co., Inc, Tokyo) was used for flash column (40–100 µM) and open column (100–200 µM) chromatography. SephadexTM LH-20 (GE Healthcare Biosciences, Buckinghamshire, UK) was used for size-exclusion chromatography. Silica gel 60 F254 (E. Merck, Whitehouse Station, NJ) was used for analytical thin-layer chromatography. MALDI-TOF MS spectra were measured with a Shimadzu AXIMA-CFR using 2,5-dihydrobenzoic acid or {alpha}-cyano-4-hydroxycinnamic acid as a matrix. 1H-NMR (nuclear magnetic resonance) spectra were recorded at ambient temperature (23–24°C) in CDCl3, CD3OD or DMOS using JEOL EX 400 MHz spectrometer. A Waters C18 SepPack Cartridge was used for reverse phase column chromatography (Waters, Milford, MA). Boc amino acids were purchased from Kokusan Laboratory Chemicals, TCI, or NOVA BioChem. The biotin derivative was purchased from Pierce Biotechnology. Other chemicals were purchased from Sigma-Aldrich, TCI, or Kanto chemicals.

Synthesis of C-Man-WSPW
Boc-Ser-Pro-Trp-OMe and other peptides were prepared by the conventional solution-phase Boc peptide synthesis protocol. For example, Boc-Pro-Trp-OMe (767 mg, 1.85 mmol) was dissolved in CH2Cl2 (20 mL), and 4.0 M HCl (20 mL, dioxane solution) was added. After the mixture had been stirred at room temperature for 2–5 h, the solvent was evaporated. Then, dioxane was added and removed in vacuo several times to remove HCl. The mixture was dissolved in CH2Cl2, then diisopropylethylamine (i-Pr2NEt) (0.48 mL, 2.78 mmol), Boc-Ser-OH (570 mg, 2.78 mmol), and 1-hydroxybenzotriazole (HOBt) (375 mg, 2.78 mmol) were added. Subsequently, water soluble carbodiimide (WSCDI, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl) (530 mg, 2.78 mmol) was added at 0°C. After the mixture had been stirred overnight, the solution was diluted with CHCl3 and an aqueous 10% citric acid solution. The aqueous layer was extracted with CHCl3. The combined layers were washed with brine and dried over Na2SO4. After removal of the solvent in vacuo, the residue was purified by silica gel column chromatography (CHCl3:MeOH 9:1–4:1) to give the product.

The Boc group of Boc-Ser-Pro-Trp-OMe (235 mg, 0.468 mmol) was removed, as described above. Then, the residue was dissolved in dimethylformamide (DMF) (1 mL), and i-Pr2NEt (0.13 ml, 0.729 mmol), Fmoc-(C-Man)-Trp (229.5 mg, 0.39 mmol), and (7-azabenzotriazole-1-yloxy)tri- pyrrolidinophosphonium hexafluorophosphate (193 mg, 0.508 mmol) were added at 0°C (Miller et al. 2003Go). After the mixture had been stirred overnight, the residue was directly purified by size-exclusion chromatography [LH 20 (MeOH as eluent)] and subsequent silica gel column chromatography (CHCl3:MeOH 9:1–4:1) to give Fmoc-(C-Man)-Trp-Ser-Pro-Trp-OMe. MS m/z 995 (M++Na). Fmoc-(C-Man)-Trp-Ser-Pro-Trp-OMe (297 mg 0.201 mmol) was dissolved in MeOH (3 mL) and aqueous 10% NaOH (3 mL) was added. After 1 h, the mixture was purified with a SepPak cartridge (H2O-H2O:MeOH 4:1) to give the product. MS m/z 759 (M++Na). (C-Man)-Trp-Ser-Pro and (C-Man)-Trp-Ser were synthesized similarly. (C-Man)-Trp-Ser-Pro; MS m/z 596 (M++Na). (C-Man)-Trp-Ser; MS m/z 476 (M++Na).

C-Man-WSPWC-biotin
To a solution of Fmoc-(C-Man)-Trp-Ser-Pro-Trp-Cys(StBu)-OMe (55.6 mg, 0.0478 mmol) in MeOH (1 mL), H2O (0.5 mL), and DMF (0.5 mL), tributylphosphine (PBu3) (23 mL, 0.0919 mmol) was added and the mixture was stirred at room temperature for 1 h under a N2 atmosphere. After the solvent was removed in vacuo, the residue was dissolved in DMF (0.5 mL). To the solution, (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (27 mg, 0.050 mmol) and i-Pr2NEt (10 µL, 1.78 mmol) were added and the mixture was stirred overnight. The mixture was purified using LH 20 (MeOH as eluent), then silica gel column chromatography (CHCl3:MeOH 9:1–4:1) to give the product. MS m/z 1512. Then Fmoc and methyl ester were removed and purified as described above. MS m/z 1276. The purity of C-Man-W containing peptides was determined by 400 MHz 1H-NMR. The purity of other peptides (i.e., non-C-Man-W containing peptides) was determined by both 1H-NMR and HPLC (Itertsil ODS column, water:CH3CN containing 0.1% TFA). The purity of all peptides was more than 95%.

Cell lines and culture
RAW264.7 cells, a clonal line of mouse macrophage-like cells, were obtained from American Type Culture Collection (TIB-71). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 20% fetal calf serum (FCS) under a humidified atmosphere of 95% air and 5% CO2 at 37°C.

Cell injury assay
The viability of cultured cells was evaluated using MTT, as described (Ihara et al. 2006Go). The cells (1000–2000) were placed in 100 µL of medium per well in 96-well plates, and cultured overnight. After treatment with LPS for 48 h in the presence or absence of C-Man-W, C-mannosylated peptides, and peptides, 10 µL of 0.5% MTT solution was added, and the cells were incubated at 37°C for 4 h. The reaction was stopped by adding 100 µL of lysis buffer A (10% SDS and 0.1 M HCl), and then cell viability was evaluated by measuring the absorbance at 570 nm using a microplate reader. Dead cells were detected by fluorescent DNA staining using SYTOX Green (Invitrogen) (Cheung et al. 2000Go). Cultured cells were stained with SYTOX Green (1 µM) for 10 min, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.2) for 30 min under darkness, and visualized by fluorescence microscopy with excitation at 485 nm and emission at 520 nm using a Carl Zeiss LSM5 microscope (Carl Zeiss, Jena, Germany), and analyzed by PASCAL analytical software.

LPS-FITC binding assay
Cells were incubated at 37°C for 20 min in medium containing LPS-FITC (1 µg/mL) in the presence or absence of WSPW or C-Man-WSPW (10 µM). After a wash with PBS, the binding of LPS-FITC to the cells was analyzed using a Cytomics FC500 Flow Cytometry System (Beckman Coulter, Fullerton, CA).

Estimation of C-Man-WSPWC-biotin binding
Cells were incubated at 37°C for 20 min in PBS with biotin, WSPWC-biotin, or C-Man-WSPWC-biotin (10 µM). After a wash with PBS, the cells were blocked with 3% bovine serum albumin (BSA) in PBS, incubated with FITC-conjugated Nutr-avidin (Pierce Biotechnology) for 15 min, and washed with PBS containing 1% BSA. After another wash, the binding of biotin conjugates to the cells was analyzed using a Cytomics FC500 Flow Cytometry System. Alternatively, after incubation with the biotin conjugates as described above, the cells were treated for 10 min with or without 1 mM DSP, a membrane permeable cross linker. After quenching with 1 M Tris–HCl (pH7.2), the cells were washed with PBS, and harvested by using a scraper. The cells were lysed in lysis buffer B (20 mM Tris–HCl [pH 7.2], 130 mM NaCl, and 1% NP-40 including protease inhibitors [20 µM APMSF, 50 µM pepstatin, and 50 µM leupeptin]). Protein samples were electrophoresed on 7.5% SDS-polyacrylamide gels under non-reducing conditions and then transferred to a nitrocellulose membrane as described before (Ihara et al. 2005Go). The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS; 10 mM Tris–HCl [pH 7.5] and 150 mM NaCl) and then incubated at 4°C for 10 min with the peroxidase-conjugated Nutr-avidin (Pierce Biotechnology) in TBS containing 0.1% Tween 20. After a wash with TBS containing 0.1% Tween 20, the blots were developed using an ECL chemiluminescence detection kit (GE Healthcare Biosciences) according to the manufacturer's instructions.

Immunoblot analysis
Cultured cells were harvested and lysed in lysis buffer B, as described above. Protein samples were electrophoresed on 7.5–10% SDS-polyacrylamide gels under reducing conditions and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in TBS and then incubated at 4°C overnight with the primary antibody in TBS containing 0.1% Tween 20. The blots were coupled with the peroxidase-conjugated secondary antibodies, washed, and then developed using an ECL chemiluminescence detection kit.

JNK activity assay
JNK activity was assayed using a JNK assay kit (Cell Signaling Technology) according to the manufacturer's protocol with glutathione S-transferase-c-Jun fusion protein (GST-c-Jun) as a substrate. Phosphorylation of GST-c-Jun was assessed by immunoblot analysis using specific antibody.

TNF-{alpha} assay
Cells were plated in 96-well plates (10,000 cells/well), and cultured overnight. The medium was renewed. Then, cells were stimulated with LPS (1 µg/mL) in the presence or absence of C-Man-W, C-mannosylated peptides, and peptides (10 µM). After the incubation, the culture medium was collected, and the concentration of TNF-{alpha} in the sample was evaluated using a mouse TNF-{alpha} ELISA kit according to the manufacturer's instructions (BioSource International, Camarillo, CA). All experiments were done in quadruplicate.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from cultured cells using standard methods and was reverse transcribed using a One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals, Shiga, Japan) with avian myoblastosis virus-derived reverse transcriptase XL according to the manufacturer's instructions. PCR was run for 19 cycles of 95°C for 0.5 min/65°C for 0.5 min/72°C for 1.5 min. Primer sequences were as follows: For mouse TNF-{alpha} (GenBank accession number NM_013693 [GenBank] ) (Fransen et al. 1985Go), forward: 5'-TCT CAG CCT CTT CTC ATT CC-3'; reverse: 5'-GTC CCA GCA TCT TGT GTT TC-3'; for mouse ß-actin (BC063166 [GenBank] ), forward: 5'-GAG CTA TGA GCT GCC TGA CG-3'; reverse: 5'-AGC ATT TGC GGT GCA CGA GG-3'.

Luciferase activity assay
The PathDetect pNF{kappa}B-Luc Cis-Reporter Plasmid, a luciferase reporter construct for the NF{kappa}B gene promoter, was obtained from Stratagene (Tokyo, Japan). The vector was introduced into cells by using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. After 24 h of transfection, cells were treated with LPS (1 µg/mL) in the presence or absence of WSPW or C-Man-WSPW (10 µM), or left untreated for the periods indicated in the text. Then luciferase activity was assayed with cellular extracts by using a Dual-Luciferase Reporter Assay System (Promega), as described before (Yasuoka et al. 2004Go).

Statistical analysis
Statistical analysis was performed using Student's t-test or ANOVA (StatView software). Significance was set at P < 0.05.


   Funding
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Funding
Conflict of interest statement
 References
 
This work was supported in part by Grants-in-Aid from the President's Discretionary Fund of Nagasaki University, Japan, the Ministry of Education, Science, Sports, Culture, and Technology of Japan, and fellowship from the Tsukushi Foundation (E.M.).


   Conflict of interest statement
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Funding
 Conflict of interest statement
References
 
None declared.


   Acknowledgements
 
We are grateful to Akiko Emura for technical assistance.


   Footnotes
 
2 These authors contributed equally to this work. Back


   Abbreviations
 
DMEM, Dulbecco's modified Eagle's medium; DMF, dimethylformamide; DSP, dithiobis[succinimidylpropionate]; ELISA, enzyme-linked immunoabsorbent assay; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione S-transferase; I{kappa}B{alpha}, inhibitor of {kappa}B{alpha}; i-Pr2NEt, diisopropylethylamine; IRAK1, interleukin-1 receptor-associated kinase 1; JNK, c-Jun N-terminal kinase; LMP1, latent membrane protein 1; LPS, lipopolysaccharide; MAPK, mitogen activating protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-{kappa}B, nuclear factor-{kappa}B; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; TAK1, TGF-ß-activated kinase 1; TBS, Tris-buffered saline; TGF-ß, transforming growth factor ß; TLR, Toll-like receptor; TNF-{alpha}, tumor necrosis factor-{alpha}; TRAF6, TNF-{alpha} receptor-associated factor 6; TSP, thrombospondin; TSR, thrombospondin type 1 repeat


   References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Funding
 Conflict of interest statement
 References
 
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