Two lipoproteins extracted from Escherichia coli K-12 LCD25 lipopolysaccharide are the major components responsible for Toll-like receptor 2-mediated signaling.
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.
Lee
Hyun-Ku
HK
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Lee
Jongdae
J
Tobias
Peter S
PS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D051550:I-kappa B Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D008070:Lipopolysaccharides / Q000302:isolation & purification* / Q000494:pharmacology; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000502:physiology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis; D021381:Protein Transport / Q000235:genetics; D011956:Receptors, Cell Surface / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D001426:Bacterial Proteins; D029721:Drosophila Proteins; D008070:Lipopolysaccharides; D008074:Lipoproteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases