Abstract
Surfactant protein-A (SP-A) and Toll-like receptor-4 (TLR4) proteins are recognized as pathogen-recognition receptors. An exaggerated activation of TLR4 induces inflammatory response, whereas SP-A protein down-regulates inflammation. We hypothesized that SP-A–TLR4 interaction may lead to inhibition of inflammation. In this study, we investigated interaction between native baboon lung SP-A and baboon and human TLR4-MD2 proteins by coimmunoprecipitation/immunoblotting and microwell-based methods. The interaction between SP-A and TLR4-MD2 proteins was then analyzed using a bioinformatics approach. In the in silico model of SP-A–TLR4–MD2 complex, we identified potential binding regions and amino acids at the interface of SP-A-TLR4. Using this information, we synthesized a library of human SP-A-derived peptides that contained interacting amino acids. Next, we tested whether the TLR4-interacting SP-A peptides would suppress inflammatory cytokines. The peptides were screened for any changes in the tumor necrosis factor-α (TNF-α) response against lipopolysaccharide (LPS) stimuli in the mouse JAWS II dendritic cell line. Different approaches used in this study suggested binding between SP-A and TLR4-MD2 proteins. In cells pretreated with peptides, three of seven peptides increased TNF-α production against LPS. However, two of these peptides (SPA4: GDFRYSDGTPVNYTNWYRGE and SPA5: YVGLTEGPSPGDFRYSDFTP) decreased the TNF-α production in LPS-challenged JAWS II dendritic cells; SPA4 peptide showed more pronounced inhibitory effect than SPA5 peptide. In conclusion, we identify a human SP-A-derived peptide (SPA4 peptide) that interacts with TLR4-MD2 protein and inhibits the LPS-stimulated release of TNF-α in JAWS II dendritic cells.
Footnotes
This work was supported by the National Institutes of Health National Center for Research Resources [Grant P40-RR012317]; the Presbyterian Health Foundation [Grant PHF1539]; and the College of Pharmacy Seed Grant Program, University of Oklahoma Health Science Center, Oklahoma City [Grant PSXA1].
The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the U.S. Army or of the U.S. Department of Defense.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.173765.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- PPRR
- pathogen-pattern recognition receptor
- SP
- surfactant protein
- TLR4
- Toll-like receptor-4
- IP-SP-A
- immunoprecipitated SP-A
- IP-TLR4
- immunoprecipitated TLR4
- IB-SP-A
- immunoblotted SP-A
- IB-TLR4
- immunoblotted TLR4
- LPS
- lipopolysaccharide
- DTT
- dithiothreitol
- PAGE
- polyacrylamide gel electrophoresis
- CRD
- carbohydrate recognition domain
- TNF-α
- tumor necrosis factor-α
- HEK
- human embryonic kidney
- TBST
- Tris-buffered saline containing 0.4% Tween 20
- LAL
- limulus amebocyte lysate
- BSA
- bovine serum albumin
- PDB
- Protein Data Bank
- GRAMM
- Global Range Molecular Matching.
- Received August 6, 2010.
- Accepted December 8, 2010.
- U.S. Government work not protected by U.S. copyright
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