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 patent cytokine inducer for monocytes/macrophages, maturing dendritic ce lls (DCs), and activating host complement on affected cells, It possesses a unique N-terminal lipo-amino acid, S-diacylglyceryl cysteine. The 2-kDa ma crophage-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 (SPC) or without signal peptides (SP-). Because the SP+ rM161Ag formed dimers via 25Cys, we obtained a monomeric fo rm by mutagenesis (SP(+)C25S). Only wild type accelerated maturation of hum an DCs as determined by the CDS3/86 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 an d DCs. Either lipid or signal peptide at the N-terminal portion of monomer was required for expression of this function, In contrast, murine macraphag es produced TNF-alpha in response to mild type, but not to any recombinant form of M161Ag suggesting the species-dependent response to rM161Ag. Wild-t ype and both monomeric and dimeric SP+ forms possessed the ability to activ ate 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 res ponse to M161Ag, infer that the N-terminal hydrophobic structure of M161Ag is important for TLR2-mediated cell activation and complement activation. T he Journal of Immunology, 2001, 166: 2610-2616.