Alternative titles; symbols
HGNC Approved Gene Symbol: LY96
Cytogenetic location: 8q21.11 Genomic coordinates (GRCh38): 8:73,991,392-74,099,857 (from NCBI)
Leucine-rich repeat (LRR) molecules are found in diverse organisms and mediate innate defenses against pathogens. By searching an EST database for sequences homologous to MD1 (605241), a protein associated with the LRR-bearing molecule RP105 (LY64; 602226), Shimazu et al. (1999) identified MD2. The predicted 160-amino acid MD2 protein, which shares 23% amino acid identity with MD1, contains a 16-amino acid signal peptide and 2 N-glycosylation sites. Northern blot analysis detected a 0.7-kb MD1 transcript in all human cell lines and mouse tissues tested.
By RT-PCR of human THP-1 monocytic cells and human dermal microvascular endothelial cells, Gray et al. (2010) cloned a short variant of MD2 that they termed MD2s. MD2s lacks exon 2 and encodes a 130-amino acid protein. Like full-length MD2, MD2s was glycosylated and secreted. A variant equivalent to MD2s appeared to be absent in mouse.
Gray et al. (2010) determined that the MD2 gene contains 5 exons and spans approximately 38 kb.
Scott (2000) mapped the MD2 gene to chromosome 8 based on sequence similarity between the MD2 sequence (GenBank AB018549) and the chromosome 8 clone RP11-3506 (GenBank AC009672).
Using flow cytometric and confocal microscopy analyses, Shimazu et al. (1999) found that MD2 did not interact with LY64, but it did appear on cells expressing Toll-like receptor-4 (TLR4; 603030). Toll-like receptors, which contain extracellular LRRs and have intracellular regions homologous to the intracellular signaling domain of the interleukin-1 receptor (IL1R; 147810), activate nuclear factor kappa-B (NFKB; see 164011). TLR4 is involved in the response to lipopolysaccharide (LPS) from gram-negative bacteria. Functional analysis showed that expression of MD2 enhanced TLR4-dependent activation of NFKB. Furthermore, cells expressing both MD2 and TLR4, but not TLR4 alone, responded to LPS.
Trompette et al. (2009) noted that the main allergen of the house dust mite, Der p 2, has structural homology with MD2. Using human and mouse cells, they found that the mite allergen also exhibited functional homology with MD2, in that it facilitated signaling through direct interactions with the TLR4 complex and reconstituted LPS-driven TLR4 signaling in the absence of MD2. Airway sensitization and challenge with the mite allergen led to experimental allergic asthma, as shown by eosinophil numbers in bronchoalveolar lavage fluid, IgE production, and histopathologic analysis, in wildtype and Md2-deficient mice, but not in Tlr4-deficient mice. Trompette et al. (2009) proposed that Der p 2 and other allergens belonging to the MD2-like lipid-binding family have intrinsic adjuvant activity together with their accompanying lipid cargo and may thus account for the phenomenon of allergenicity.
Gray et al. (2010) showed that the human MD2s isoform interacted with LPS and TLR4 but failed to mediate LPS-induced NFKB activation and IL8 (146930) production. MD2s was upregulated by IFNG (147570), IL6 (147620), and TLR4 stimulation and negatively regulated LPS-mediated TLR4 signaling. MD2s competitively inhibited binding of full-length MD2 to TLR4. Gray et al. (2010) proposed that MD2s may regulate TLR4 activation and have therapeutic potential to treat disease characterized by an excessive immune response to LPS.
Crystal Structure
Endotoxic lipopolysaccharide (LPS) with potent immunostimulatory activity is recognized by the receptor complex of MD2 and TLR4. Ohto et al. (2007) reported the crystal structure of human MD2 and its complex with the antiendotoxic tetra-acylated lipid A core of LPS 2.0- and 2.2-angstrom resolutions, respectively. MD2 shows a deep hydrophobic cavity sandwiched by 2 beta sheets, in which 4 acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. Ohto et al. (2007) concluded that these structures suggested that MD2 plays a principal role in endotoxin recognition and provided a basis for antiseptic drug development.
Park et al. (2009) determined the crystal structure of the TLR4-MD2-LPS complex. LPS binding induced the formation of an m-shaped receptor multimer composed of 2 copies of the TLR4-MD2-LPS complex arranged symmetrically. LPS interacts with a large hydrophobic pocket in MD2 and directly bridges the 2 components of the multimer. Five of the 6 lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD2, forming a hydrophobic interaction with the conserved phenylalanines of TLR4. The F126 loop of MD2 undergoes localized structural change and supports this core hydrophobic interface by making hydrophobic interactions with TLR4. Comparison with the structures of tetra-acylated antagonists bound to MD2 indicated that 2 other lipid chains in LPS displace the phosphorylated glucosamine backbone of approximately 5 angstroms towards the solvent area. This structural shift allows phosphate groups of LPS to contribute to receptor multimerization by forming ionic interactions with a cluster of positively charged residues in TLR4 and MD2.
Nagai et al. (2002) generated Md2-deficient mice lacking the 37 amino acids encoded by exon 1. RT-PCR and flow cytometric analyses demonstrated expression of Tlr4 mRNA, but the Md2-Tlr4 complex on the cell surface was absent. Md2 -/- B cells, macrophages, and dendritic cells responded to CpG, anti-RP105, and proteoglycan, but not to LPS. The response to CpG indicated that Tlr9 (605474) was intact, and the response to proteoglycan indicated that Tlr2 (603028) was intact. In vivo, Md2 null mice, like Tlr4 null mice, failed to produce the acute-phase reactant, serum amyloid A. Md2-deficient mice all survived LPS challenge and did not produce inflammatory cytokines. However, the mutant mice showed enhanced susceptibility to Salmonella typhimurium, probably due to a defect in LPS sensing. Confocal microscopy demonstrated that mouse embryonic fibroblasts from Md2 -/- mice only expressed Tlr4 in the Golgi apparatus and not on the cell surface. Nagai et al. (2002) concluded that MD2 is essential for the correct intracellular distribution, cell surface expression, and LPS recognition of TLR4. They proposed that MD2 may be a target for neutralizing the toxic effects of endotoxin.
Gray, P., Michelsen, K. S., Sirois, C. M., Lowe, E., Shimada, K., Crother, T. R., Chen, S., Brikos, C., Bulut, Y., Latz, E., Underhill, D., Arditi, M. Identification of a novel human MD-2 splice variant that negatively regulates lipopolysaccharide-induced TLR4 signaling. J. Immun. 184: 6359-6366, 2010. [PubMed: 20435923] [Full Text: https://doi.org/10.4049/jimmunol.0903543]
Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y., Akira, S., Kitamura, T., Kosugi, A., Kimoto, M., Miyake, K. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nature Immun. 3: 667-672, 2002. [PubMed: 12055629] [Full Text: https://doi.org/10.1038/ni809]
Ohto, U., Fukase, K., Miyake, K., Satow, Y. Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa. Science 316: 1632-1634, 2007. [PubMed: 17569869] [Full Text: https://doi.org/10.1126/science.1139111]
Park, B. S., Song, D. H., Kim, H. M., Choi, B.-S., Lee, H., Lee, J.-O. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 458: 1191-1195, 2009. [PubMed: 19252480] [Full Text: https://doi.org/10.1038/nature07830]
Scott, A. F. Personal Communication. Baltimore, Md. 8/30/2000.
Shimazu, R., Akashi, S., Ogata, H., Nagai, Y., Fukudome, K., Miyake, K., Kimoto, M. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189: 1777-1782, 1999. [PubMed: 10359581] [Full Text: https://doi.org/10.1084/jem.189.11.1777]
Trompette, A., Divanovic, S., Visintin, A., Blanchard, C., Hegde, R. S., Madan, R., Thorne, P. S., Wills-Karp, M., Gioannini, T. L., Weiss, J. P., Karp, C. L. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature 457: 585-588, 2009. [PubMed: 19060881] [Full Text: https://doi.org/10.1038/nature07548]