Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans

Mari Fujita, Takeshi Into, Motoaki Yasuda, Tsugumi Okusawa, Sumiko Hamahira, Yoshio Kuroki, Akiko Eto, Toshiki Nisizawa, Manabu Morita, Ken Ichiro Shibata

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Abstract

S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-κB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2 ΔS40-I64 (a TLR2 mutant with a deletion of the region of Ser40 to Ile64) failed to activate NF-κB in response to FSL-1. The deletion mutant TLR2ΔC30-S39 induced NF-κB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu178 to Ala (TLR2E178A), TLR2 E180A, TLR2E190A, and TLR2L132E induced NF-κB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2L107E, TLR2 L112E (a TLR2 point mutant with a substitution of Leu112 to Glu), and TLR2L115E failed to induce NF-κB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2L115E, TLR2 L112E, and TLR2ΔS40-164 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2E190A were. In addition, these mutants, except for TLR2E180A, functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser40-Ile64 and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.

Original languageEnglish
Pages (from-to)3675-3683
Number of pages9
JournalJournal of Immunology
Volume171
Issue number7
Publication statusPublished - Oct 1 2003
Externally publishedYes

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Lipopeptides
Toll-Like Receptor 2
Peptidoglycan
Leucine
Lipoproteins
Staphylococcus aureus
Mycoplasma salivarium
Kidney
human TLR2 protein

ASJC Scopus subject areas

  • Immunology

Cite this

Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans. / Fujita, Mari; Into, Takeshi; Yasuda, Motoaki; Okusawa, Tsugumi; Hamahira, Sumiko; Kuroki, Yoshio; Eto, Akiko; Nisizawa, Toshiki; Morita, Manabu; Shibata, Ken Ichiro.

In: Journal of Immunology, Vol. 171, No. 7, 01.10.2003, p. 3675-3683.

Research output: Contribution to journalArticle

Fujita, Mari ; Into, Takeshi ; Yasuda, Motoaki ; Okusawa, Tsugumi ; Hamahira, Sumiko ; Kuroki, Yoshio ; Eto, Akiko ; Nisizawa, Toshiki ; Morita, Manabu ; Shibata, Ken Ichiro. / Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans. In: Journal of Immunology. 2003 ; Vol. 171, No. 7. pp. 3675-3683.
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abstract = "S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-κB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2 ΔS40-I64 (a TLR2 mutant with a deletion of the region of Ser40 to Ile64) failed to activate NF-κB in response to FSL-1. The deletion mutant TLR2ΔC30-S39 induced NF-κB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu178 to Ala (TLR2E178A), TLR2 E180A, TLR2E190A, and TLR2L132E induced NF-κB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2L107E, TLR2 L112E (a TLR2 point mutant with a substitution of Leu112 to Glu), and TLR2L115E failed to induce NF-κB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2L115E, TLR2 L112E, and TLR2ΔS40-164 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2E190A were. In addition, these mutants, except for TLR2E180A, functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser40-Ile64 and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.",
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T1 - Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans

AU - Fujita, Mari

AU - Into, Takeshi

AU - Yasuda, Motoaki

AU - Okusawa, Tsugumi

AU - Hamahira, Sumiko

AU - Kuroki, Yoshio

AU - Eto, Akiko

AU - Nisizawa, Toshiki

AU - Morita, Manabu

AU - Shibata, Ken Ichiro

PY - 2003/10/1

Y1 - 2003/10/1

N2 - S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-κB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2 ΔS40-I64 (a TLR2 mutant with a deletion of the region of Ser40 to Ile64) failed to activate NF-κB in response to FSL-1. The deletion mutant TLR2ΔC30-S39 induced NF-κB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu178 to Ala (TLR2E178A), TLR2 E180A, TLR2E190A, and TLR2L132E induced NF-κB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2L107E, TLR2 L112E (a TLR2 point mutant with a substitution of Leu112 to Glu), and TLR2L115E failed to induce NF-κB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2L115E, TLR2 L112E, and TLR2ΔS40-164 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2E190A were. In addition, these mutants, except for TLR2E180A, functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser40-Ile64 and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.

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