Abstract
Toll-like receptor 2 (TLR2) and CD14 function as pattern recognition receptors for bacterial peptidoglycan (PGN). TLRs and CD14 possess repeats of the leucine-rich motif. To address the role of the extracellular domain of TLR2 in PGN signaling, we constructed CD14/TLR2 chimeras, in which residues 1-356 or 1-323 of CD14 were substituted for the extracellular domain of TLR2, and five deletion mutants of TLR2, in which the progressively longer regions of extracellular TLR2 regions were deleted. PGN induced NF-kappaB activation in HEK293 cells expressing TLR2 but not in cells expressing CD14/TLR2 chimeras. The cells transfected with a deletion mutant TLR2(DeltaCys30-Ile64) as well as TLR2(DeltaCys30-Asp160) and TLR2(DeltaCys30-Asp305) failed to respond to PGN, indicating the importance of the TLR2 region Cys(30)-Ile(64). Although TLR2(DeltaCys30-Ser39) conferred cell responsiveness to PGN, the cells expressing TLR2(DeltaSer40-Ile64) failed to induce NF-kappaB activation. In addition, NF-kappaB activity elicited by PGN was significantly attenuated in the presence of synthetic peptide corresponding to the TLR2 region Ser(40)-Ile(64). From these results, we conclude that; 1) CD14 cannot functionally replace the extracellular domain of TLR2 in PGN signaling; 2) the TLR2 region Cys(30)-Ser(39) is not required for PGN recognition; 3) the TLR2 region containing Ser(40)-Ile(64) is critical for PGN recognition.
Highlights
Gram-positive bacteria as well as Gram-negative bacteria cause life-threatening infection
To determine whether CD14, TLR2, and the CD14/TLR2 chimeras were expressed in HEK293 cells after transient transfection, mRNA and protein expressions were analyzed by using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting
Cotransfection of Membrane-bound CD14 (mCD14) with TLR2⌬Cys30-Ile64 or TLR2⌬Ser40-Ile64 failed to confer cellular responsiveness to PGN (Fig. 8B). These results demonstrate that the TLR2 region of Cys30-Ser39 is not required for PGN recognition and that the TLR2 region containing Ser40-Ile64 is critical for bestowal of cell responsiveness to PGN
Summary
Gram-positive bacteria as well as Gram-negative bacteria cause life-threatening infection. The cells transfected with a deletion mutant TLR2⌬Cys30-Ile64 as well as TLR2⌬Cys30-Asp160 and TLR2⌬Cys30-Asp305 failed to respond to PGN, indicating the importance of the TLR2 region Cys30-Ile64.
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