Two Lipoproteins Extracted fromEscherichia coliK-12 LCD25 Lipopolysaccharide Are the Major Components Responsible for Toll-Like Receptor 2-Mediated Signaling

Abstract

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-α 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, IκB kinase β 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 NH2-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.

Lipopolysaccharide-Induced Leukocyte-Endothelial Cell Interactions: A Role for CD14 Versus Toll-Like Receptor 4 Within Microvessels

“…More recently, TLR4 Ϫ/Ϫ mice showed hyporesponsiveness to LPS to an extent similar to that of C3H/HeJ mice (19). Although TLR2 was initially also thought to function as an LPS receptor, this was likely a result of contaminant found in commercial LPS preparations (29,34). Current evidence argues against a major role for TLR2 in the physiological response to LPS (19,35,36).…”

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“…NF-B Reporter Assay-NF-B reporter assay was performed as previously described (17). Briefly, HEK293 or HeLa cells (1.5 ϫ 10 5 ) were plated in 12-well plates with Dulbecco's modified Eagle's medium/ 10% fetal calf serum.…”

The costimulatory immunogen LPS induces the B-Cell clones that infiltrate transplanted human kidneys

“…S2) that carries the Pam3Cys modification and a mutant of Lip12 that lacks the Braun modification ( Fig. 2A) (31). The VH169 antibody from the kidney again reacted strongly and selectively with an antigen present in the protein markers (marker lane), the E. coli cell extract (lane 3), and a band that did not stain with ponceau red that was present in both the wild type Lip12-Pam3cys and mutant Lip12 preparations (lanes 5 and 6) but not with yeast or Wil2 cell extracts or with BSA (lane 1, 2 and 4).…”