Innate immunity: Bacterial cell‐wall muramyl peptide targets the conserved transcription factor YB‐1

Abstract

Edited by Renee Tsolis Keywords:Innate immunity Muramyl peptide Glucosaminyl-muramyl dipeptide Y-box protein 1 a b s t r a c tThe bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-jB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-jB2 cleavage, transport of activated NF-jB2 p52 to the nucleus, and upregulation of NF-jB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling.

Muramyl peptides activate innate immunity conjointly via YB1 and NOD2

“…YB1 Abs were as described previously. 36 The specificity of both Abs was confirmed by detection of bands of the expected molecular mass on Western blotting and by their abrogation in gene-specific knockdown cells ( Figure S1 in the Supplementary Data Online).…”

“…Specific antisense RNA coding sequences 5'-GGGTCC TCCACGCAATTAC-3' (for YB1) 36 and 5'-TTGATC TCGCTGCGGTGAA-3' (for NOD2) were introduced downstream of a Pol III promoter in plasmid pSIREN-RetroQ-TetH (Clontech Laboratories Inc); mouse WEHI-3 monocytes were stably transfected with the expression plasmids using selection in 250 mg/ml hygromycin.…”

“…Encoded polypeptides were isolated following bacterial disruption (model 300VT Ultrasonic Homogenizer) and Ni chelate chromatography (Ni-NTA agarose, Quiagen 30210) according to suppliers' recommendations and as described previously for YB1. 36 To measure affinity for YB1 purified proteins (100 nM) were applied to immunoplates, washed (PBST), and different concentrations of YB1 were added as described in the Figure 1 legend. After incubation (1h, 37 C) and washing the plates were incubated with anti-YB1 Abs, washed, and developed using anti-mouse HRP conjugate.…”

“…Values were normalized against maximal stimulation; the maximal level of induction (+GMDP +YB1) was not significantly different in control versus NOD2 antisense transfected cells. The right-hand panel is provided only for comparison and is adapted from Laman et al 36 No addition 25 100 150 Cells were pretreated with GMDP (1 mg/ml) or YB1 (1 mg/ml); after cell disruption in the presence of 1% Triton X-100 cell extracts were resolved by sucrose gradient centrifugation. Aliquots were immobilized on membranes and probed with peroxidase-labeled anti-YB1 or anti-NOD2 as indicated, and developed colorimetrically.…”

“…35 More recently, we confirmed specific binding of GMDP to YB1, subcellular colocalization of YB1 and GMDP, and demonstrated that YB1 knockdown impaired the biological response to GMDP. 36 Independent lines of evidence thus suggest that both NOD2 and YB1 may act as receptor targets for muramyl peptides, and we therefore addressed the possibility of overlap between YB1 and NOD2 in the innate immune response of mammalian cells to GMDP. We report here that YB1 interacts with NOD2 in the cellular response to muramyl peptide.…”