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Blood, Vol. 112, Issue 9, 3661-3670, November 1, 2008
Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response
Blood Shi et al.
112: 3661
Supplemental materials for: Shi et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 1.02 MB)
- Figure S1. SDS-PAGE and Western blot analysis of rTMD1 obtained from Pichia and mammalian protein expression systems and Western blot analysis of tagged rTMD1 and non-tagged rTMD1 (JPG, 81.4 KB)
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(A) Both Pichia and mammalian-expressed rTMD1 were purified by an affinity nickel-chelating column. The purified rTMD1 proteins were subjected to electrophoresis on 12.5% SDS-PAGE and visualized by silver staining (lane 1 and 2) and Western blotting with c-Myc antibody (Lane 3 and 4). (B) Mammalian expressed rTMD1 protein (1 mg) was cut by adding 5 ng enterokinase and incubated at 4°C for 16 hours. After incubation, the sample was applied to a nickel-chelating Sepharose column and the non-bound fraction containing the non-tagged rTMD1 was collected. The sample was further treated with immobilized soybean trypsin inhibitor-Sepharose gel to adsorb residual enterokinase. The samples before and after treatment with enterokinase were analyzed with SDS-PAGE and Western blot. rTMD1 with the glycosylation modification had a molecular mass of 35 kDa. The result demonstrated that a non-tagged rTMD1 with molecular mass 2 kDa less than the tagged rTMD1 reacted positively with anti-TMD1 polyclonal antibody (H-300, Santa Cruz Biotechnology, Santa Cruz, CA) but not with anti–c-Myc antibody (9E10, Santa Cruz Biotechnology).
- Figure S2. A polyclonal antibody against rTMD1 reverses the blocking effect of rTMD1 on the LPS-induced TNF-α release in vitro and in vivo (JPG, 193 KB)
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The rabbit polyclonal antibody against Pichia-expressed rTMD1 was prepared by a company (GlycoNex, Inc., Taiwan). rTMD1-immunized rabbit anti-serums were collected and further purified by protein G-Sepharose column (Amersham Pharmacia Biotech AB). (A) Characterization of the protein G-purified rabbit polyclonal anti-TMD1antibody (TMD1 Ab). Specificity of TMD1 Ab was examined by recognizing pichia-expressed rTMD1 with silver stain and Western blot (WB). (B) The human full-length or lectin-like domain-deleted TM was constructed in pEGFPN1 vector (BD Biosciences Clontech; Palo Alto, CA) and transfected into HEK293 cells. The cell lines stably expressed GFP, full-length TM+GFP, or lectin-like domain-deleted TM+GFP were subjected to Western blot analysis by TMD1 Ab. (C) The anti-inflammatory function of rTMD1 was neutralized by TMD1 Ab in vitro. Pichia-expressed rTMD1 and TMD1 Ab or control rabbit IgG were pre-incubated 30 minutes with LPS before adding to RAW 264.7 cells. After 6 hour incubation, culture media were collected for the measurement of TNF-α release. The anti-inflammatory effect of rTMD1 was specifically inhibited by TMD1Ab. ###P <0.001 compared with PBS group. ***P <0.001 compared with LPS group. (D) rTMD1 was co-incubated with TMD1 Ab for 30 minutes at 37°C and then i.v.-administrated before i.p. injection of LPS (20 mg/kg). Serum samples were collected 6 hours after administration of LPS for the assay of TNF-α release. Values are the mean ± SD (n=10). *P < 0.05 and ***P < 0.001 compared with the LPS-treated group. ###P < 0.001 compared with the PBS group. Similar results were obtained in two independent experiments.
- Figure S3. Surface plasmon resonance (SPR) analysis of LPS binding to rTMD1 (JPG, 52.6 KB)
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SPR analysis demonstrated the binding curve of LPS to rTMD1-coated BIAcore sensor chip. E. coli LPS O111:B4 was applied at 20, 40, 60, 80, and 100 µg/mL. The dissociation constants calculated from the binding curve were 8.24 × 10−6 mole/L, 1.16 × 10−6 mole/L, and 9.01 × 10−8 mole/L with prediction molecular masses of LPS at 10 kDa, 100 kDa, and 1000 kDa, respectively. An activated and blocked flow-cell without immobilized rTMD1 was used to evaluate nonspecific binding.
- Figure S4. Interaction of LPS to endogenous and overexpressed membrane-bound TM (JPG, 53.6 KB)
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(A) Binding of LPS and endogenous TM on THP-1 cells. Biotinylated ultrapure E. coli O111:B4 LPS (5 µg/mL; InvivoGen, San Diego, CA) was incubated with THP-1 for 30 minutes at 37°C. After the incubation, cells were washed with ice-cold PBS and the cell lysates were harvested and incubated with streptavidin agarose resin (Pierce ,Rockford, IL) at 4°C. After 1 hour incubation, the cell lysate interacted streptavidin agarose resin was washed three times with PBS containing 0.05% Tween-20. Samples were analyzed by SDS-PAGE and Western blotting by anti-TM antibody (clone D3, Santa Cruz Biochemicals). (B) LPS interacts with TM through lectin-like domain in HEK293 stable cell line. TMUL (lectin-like domain truncated TM) and TMG (full length TM) were constructed into pEGFP-N1 vector (BD Bioscience Clontech, CA) and transfected to HEK293 cell line for stably overexpression. biotinylated ultrapure E. coli O111:B4 LPS (5 µg/mL) was incubated with the cell lines at 37°C. After 30 minutes incubation, cells were washed with ice-cold PBS and the cell lysates were harvested and incubated with streptavidin agarose resin at 4°C as described above. Samples were analyzed by SDS-PAGE and Western blotting by anti-GFP antibody (Zymed).
- Figure S5. Binding of HMGB1 with rTMD1 and no competing effect of Ley on HMGB1-rTMD1 binding (JPG, 30.0 KB)
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rTMD1 (50 µg/mL) in 100 µL of bicarbonate buffer (pH 9.6) were coated onto wells of high-binding microtiter plate. The nonspecific binding was blocked with a binding buffer (20 mM Tris-pH 7.4, 150 mM NaCl, and 5 mM CaCl2) containing 50 mg/mL BSA at 37°C for 2 hours. Various concentrations of HMGB1 (1690-HM, R&D Systems) without or with Ley (20 µg/mL) in binding buffer containing 1 mg/mL BSA were added into rTMD1-coated wells at 37°C for 2 hours. The monoclonal anti-human HMGB1antibody (MAB1690, R&D Systems) in binding buffer containing 1 mg/mL BSA was added to wells and incubated for 2 hours. The wells were followed by incubation with goat anti-mouse horseradish peroxidase-conjugated IgG at 37°C for 2 hours. The peroxidase reaction was performed using 3,3′,5,5′-tetramethylbenzidine as a substrate and was stopped by 2N H2SO4. The products were detected by measuring absorbance at 450 nm.
- Figure S6. Sedimentation velocity analytical ultracentrifugation of rTMD1 (JPG, 33.5 KB)
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Sedimentation velocity experiments were performed with a Beckman model XL-A analytical ultracentrifuge for pichia-expressed rTMD1. rTMD1 and reference solutions were loaded into double-sector centerpieces and mounted in a Beckman An-50 Ti rotor. The experiments were performed at 20°C with a rotor speed of 42,000 rpm. Absorbance of the sample at 280 nm was monitored in a continuous mode. Multiple scans at different time points were globally fitted to a monomer-dimer rapid self-association model by using the Sednterp program (accessed on Jan. 23, 2004). The partial specific volume used for rTMD1 and calculated from amino acid composition, was 0.68. All samples were visually checked for clarity after ultracentrifugation, and no indication of precipitation was observed. The red line shows that rTMD1 consists of monomer (M, 35 kDa) and dimer (D, 70 kDa). The blue line shows that rTMD1 predominantly consists of dimer (D) in solution containing 5 mM CaCl2.
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