Abstract
Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response. Recent reports have identified accessory proteins that provide essential support to TLR function through ligand delivery and receptor trafficking. Herein, we introduce leucine-rich repeats (LRRs) and calponin homology containing 4 (Lrch4) as a novel TLR accessory protein. Lrch4 is a membrane protein with nine LRRs in its predicted ectodomain. It is widely expressed across murine tissues and has two expression variants that are both regulated by lipopolysaccharide (LPS). Predictive modeling indicates that Lrch4 LRRs conform to the horseshoe-shaped structure typical of LRRs in pathogen-recognition receptors and that the best structural match in the protein database is to the variable lymphocyte receptor of the jawless vertebrate hagfish. Silencing Lrch4 attenuates cytokine induction by LPS and multiple other TLR ligands and dampens the in vivo innate immune response. Lrch4 promotes proper docking of LPS in lipid raft membrane microdomains. We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response and a potential molecular target in inflammatory disease.
Highlights
Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response
We recently identified Lrch4 in a proteomic screen as a protein increased in lipid raft microdomains of macrophages upon LPS exposure, suggestive of a potential role in TLR4 signaling [13]
amino acid (AA) sequence-based prediction of conserved motifs (UniProt) in conjunction with transmembrane prediction (TMpred [15] and Philius [16]) indicates that Lrch4 has an ectodomain composed of a 19 AA N-terminal signal peptide followed by nine leucine-rich repeats (LRRs), a central disordered region, and a calponin homology (CH) motif; this is followed by a transmembrane domain (TMD) and a short cytoplasmic tail (Fig. 1A)
Summary
We recently identified Lrch in a proteomic screen as a protein increased in lipid raft microdomains of macrophages upon LPS exposure, suggestive of a potential role in TLR4 signaling [13]. Confirming a role for Lrch in regulation of TLR4 signaling, both Lrch knockdown lines induced significantly less TNF␣ than the Scr line in response to two doses of LPS (Fig. 2C). JNK phosphorylation was reduced at 15 min but not at 30 min post-LPS, consistent with a delay in its activation by LPS in Lrch4-silenced cells Taken together, these results indicate that Lrch regulates early signaling events in the proximal TLR4 pathway, with effects upon both MyD88 and TRIF arms, but with varying temporal effects on the MAPKs. Consistent with Lrch not exerting a global or indiscriminate effect upon TLR4 pathway output, we confirmed that neither silencing nor overexpression of Lrch altered cell surface display of TLR4 as assessed by flow cytometry (Fig. 5A and Fig. S3, A and B); nor did Lrch silencing or overexpression modify TLR4 gene expression (Fig. 5B). This finding suggests that Lrch plays a modulatory role in the innate immune response in vivo
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