Two Lipoproteins Extracted from Escherichia coli K-12 LCD25 Lipopolysaccharide Are the Major Components Responsible for Toll-Like Receptor 2-Mediated Signaling

• Published in: The Journal of immunology (1950) Vol. 168; no. 8; pp. 4012 - 4017
• Main Authors: Lee, Hyun-Ku, Lee, Jongdae, Tobias, Peter S
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 15-04-2002
• Subjects: Amino Acid Sequence; Amino Acid Substitution - genetics; Animals; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - physiology; Cell Line; Cloning, Molecular; Drosophila Proteins; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - physiology; Humans; I-kappa B Kinase; JNK Mitogen-Activated Protein Kinases; Lip12 protein; Lip19 protein; Lipopolysaccharides - isolation & purification; Lipopolysaccharides - pharmacology; Lipoproteins - biosynthesis; Lipoproteins - genetics; Lipoproteins - isolation & purification; Lipoproteins - physiology; Membrane Glycoproteins - physiology; Mice; Mitogen-Activated Protein Kinases - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; NF-kappa B - genetics; NF-kappa B - metabolism; Protein Transport - genetics; Protein-Serine-Threonine Kinases - biosynthesis; Receptors, Cell Surface - physiology; Signal Transduction - genetics; Signal Transduction - physiology; TLR2 protein; Toll-Like Receptor 2; Toll-Like Receptors; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha - biosynthesis
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.168.8.4012

Toll-like receptor 2 (TLR2)-mediated cell activation induced by commercial preparations of LPS was recently shown to arise from impurities whose identities are not known. In this work, we determined the molecules responsible for TLR2-mediated cell activation in LPS derived from Escherichia coli K-12 strain LCD25. When LCD25 LPS was phenol extracted, two proteins capable of TLR2-mediated cell activation were purified and identified as E. coli lipoproteins. We cloned, expressed, and purified these two lipoproteins, Lip19 and Lip12. Lip19 or Lip12 activated TNF-alpha production from RAW264.7 and THP-1 cells in a TLR2-dependent manner. However, neither Lip19 nor Lip12 activated HUVECs, which lack endogenous TLR2. Additionally, IkappaB kinase beta and c-Jun N-terminal kinase 1 activation in THP-1 cells induced by Lip19 or Lip12 was observed. TLR2 activation by Lip19 and Lip12 in HEK293 cells was blocked by inhibitory anti-TLR2 mAbs. The unlipidated mutants, Lip19-C19S and Lip12-C21S, in which the NH(2)-terminal cysteine was substituted by serine, lost their ability to activate TLR2-transfected HEK 293 cells. Taken together, these results demonstrate that two lipoproteins constitute the major contaminants responsible for TLR2-mediated cell activation in E. coli LCD25 LPS.