Mycoplasma fermentans lipoprotein M161Ag-induced cell activation is mediated by Toll-like receptor 2: role of N-terminal hydrophobic portion in its multiple functions.
M161Ag is a 43-kDa surface lipoprotein of Mycoplasma fermentans, serving as a potent cytokine inducer for monocytes/macrophages, maturing dendritic cells (DCs), and activating host complement on affected cells. It possesses a unique N-terminal lipo-amino acid, S:-diacylglyceryl cysteine. The 2-kDa macrophage-activating lipopeptide-2 (MALP-2), recently identified as a ligand for Toll-like receptor 2 (TLR2), is derived from M161Ag. In this study, we identified structural motifs sustaining the functions of M161Ag using wild-type and unlipidated rM161Ag with (SP(+)) or without signal peptides (SP(-)). Because the SP(+) rM161Ag formed dimers via 25Cys, we obtained a monomeric form by mutagenesis (SP(+)C25S). Only wild type accelerated maturation of human DCs as determined by the CD83/86 criteria, suggesting the importance of the N-terminal fatty acids for this function. Wild-type and the SP(+) form of monomer induced secretion of TNF-alpha and IL-12 p40 by human monocytes and DCs. Either lipid or signal peptide at the N-terminal portion of monomer was required for expression of this function. In contrast, murine macrophages produced TNF-alpha in response to wild type, but not to any recombinant form of M161Ag, suggesting the species-dependent response to rM161Ag. Wild-type and both monomeric and dimeric SP(+) forms possessed the ability to activate complement via the alternative pathway. Again, the hydrophobic portion was associated with this function. These results, together with the finding that macrophages from TLR2-deficient mice did not produce TNF-alpha in response to M161Ag, infer that the N-terminal hydrophobic structure of M161Ag is important for TLR2-mediated cell activation and complement activation.
Nishiguchi
M
M
Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka, Japan.
Matsumoto
M
M
Takao
T
T
Hoshino
M
M
Shimonishi
Y
Y
Tsuji
S
S
Begum
N A
NA
Takeuchi
O
O
Akira
S
S
Toyoshima
K
K
Seya
T
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003176:Complement C3 / Q000378:metabolism; D003170:Complement Pathway, Alternative / Q000276:immunology; D029721:Drosophila Proteins; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008262:Macrophage Activation / Q000276:immunology*; D017737:Macrophages, Peritoneal / Q000276:immunology; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016858:Mycoplasma fermentans / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology; D011956:Receptors, Cell Surface / Q000502:physiology*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D003176:Complement C3; D029721:Drosophila Proteins; D008074:Lipoproteins; C091221:M161 antigen; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors