|Detection of Human TLR4 by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line. PVDF Membrane was probed with 2 µg/mL of Mouse Anti-Human TLR4 Monoclonal Antibody (Catalog # MAB14782) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for TLR4 at approximately 120 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
TLR4 is a 100 kDa type I transmembrane glycoprotein that belongs to the mammalian Toll-Like Receptor family of pathogen pattern recognition molecules. MD-2, also known as ESOP-1, is a 25 kDa secreted protein that is required for TLR4-mediated responses to bacterial lipopolysaccharide (LPS) (1‑3). The human TLR4 cDNA encodes an 839 amino acid (aa) precursor that contains a 23 aa signal sequence, a 608 aa extracellular domain (ECD), a 21 aa transmembrane segment, and a 187 aa cytoplasmic domain. TLR4 contains 21 leucine rich repeats in its ECD and one cytoplasmic Toll/IL-1 receptor (TIR) domain (4). The ECD of human TLR4 shares approximately 25% aa sequence identity with other TLRs and 60%‑74% aa sequence identity with bovine, equine, feline, mouse, rat, and porcine TLR4. The human MD-2 cDNA encodes a 160 aa precursor with an 18 aa signal sequence (5). Human MD-2 shares 20% aa sequence identity with MD-1 and 62%‑64% aa sequence identity with bovine, mouse, and rat MD-2. MD-2 associates with TLR4 on monocytes, macrophages, dendritic cells, and B cells (5‑7). MD-2 expression is required for cell surface localization of TLR4 and for optimal LPS-induced TLR4 signaling (7, 8). MD-2 also forms soluble disulfide-linked homo-oligomers which can interact with TLR4 (6). Through a domain separate from its TLR4-binding domain, MD-2 extracts LPS from circulating CD14-LPS complexes and carries the LPS into a ternary complex with TLR4 (9‑11). The interaction of MD-2/LPS with TLR4 induces receptor oligomerization and the triggering of an inflammatory response (12). Increased levels of plasma MD-2 in septic shock patients sensitizes MD-2 non-expressing epithelial cells to LPS and promotes widespread tissue inflammation (13).