Disease relevance of LY96

• MD-2 enables Toll-like receptor 2 (TLR2)-mediated responses to lipopolysaccharide and enhances TLR2-mediated responses to Gram-positive and Gram-negative bacteria and their cell wall components [1].
• Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules, including CD14, toll-like receptor (TLR) 4, and MD2 [2].
• MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex [3].
• The aim of this study was to characterize the expression and subcellular distribution of intestinal epithelial MD-2 and to delineate potential functional interactions with trypsin and then alteration in inflammatory bowel disease (IBD) [4].
• Although MD-2 protein expression was minimal in primary IEC of normal colonic or ileal mucosa, expression was significantly increased in IEC from patients with active IBD colitis, but not in ileal areas from patients with severe Crohn's disease [4].

High impact information on LY96

• Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype [5].
• Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it [5].
• The nonresponder phenotype of this mutant was reversed by transfection with MD-2 [5].
• Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange [5].
• Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling [5].

Chemical compound and disease context of LY96

• However, purified LOS from diverse strains of Neisseria (both N. meningitidis and N. gonorrhoeae) caused widely variant levels of IL-8 promoter induction in cells expressing MD2 that correlated with the production of TNF from THP-1 cells [6].

Biological context of LY96

• These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn(26) and Asn(114), and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS [7].
• Mutation of either one of two potential glycosylation sites (Asn(26) and Asn(114)) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn(26) and Asn(114) [7].
• Additional transfection with MD-2 did not affect stimulation of these cells by LTA [8].
• Here we propose two possible mechanisms for LPS recognition and signalling that take into account both the structural information available for TLR4 and MD-2, and the determinants of endotoxicity, namely, the acylation and phosphorylation patterns of LPS [9].
• Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2 [10].

Anatomical context of LY96

• Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane [11].
• Incubation of Chang epithelial cells with interferon-gamma (IFNgamma), but not TNF-alpha, stimulated MD2 mRNA expression and restored LPS responsiveness [12].
• When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum/cis Golgi with different-sized oligomers of MD-2, and excess MD-2 is secreted into the medium [13].
• An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors [13].
• A lipopolysaccharide-deficient mutant of Neisseria meningitidis elicits attenuated cytokine release by human macrophages and signals via toll-like receptor (TLR) 2 but not via TLR4/MD2 [14].

Associations of LY96 with chemical compounds

• N-linked glycosylations at Asn(26) and Asn(114) of human MD-2 are required for toll-like receptor 4-mediated activation of NF-kappaB by lipopolysaccharide [7].
• All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable [15].
• IFN-gamma regulates expression of the critical TLR4 co-receptor MD-2 through the Janus tyrosine kinase-STAT pathway [16].
• LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2 [17].
• AN ID of vecuronium prolonged the DUR25 of MD1 and MD2 of ORG 9487 (P < 0.05) [18].

Physical interactions of LY96

• Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling [19].
• Biochemical and visual studies shown here demonstrate that the role of CD14 in signal transduction is to enhance LPS binding to MD-2, although its expression is not essential for cellular activation [20].
• In order to mediate cellular response to lipopolysaccharide (LPS), Toll-like receptor (TLR) 4 must interact with MD-2, a secreted protein [21].
• Soluble G56R MD-2 showed markedly reduced LPS binding that was to a large degree rescued by TLR4 coexpression or presence of TLR4 ectodomain [22].

Regulatory relationships of LY96

• Little is known, however, as to whether MD-2 directly regulates LPS recognition by TLR4 [23].
• IFN-gamma positively regulated MD-2 promoter activity in IEC [16].
• An endotoxin-mutant (F126A) MD-2 complex that does not activate cells expressing TLR4 alone potently activates cells expressing mCD14, MD-2, and TLR4 by transferring endotoxin to mCD14, which then transfers endotoxin to endogenous wild-type MD-2.TLR4 [24].

Other interactions of LY96

• MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14 [19].
• In addition, when Chang cell cultures were supplemented with soluble MD2, LPS was able to stimulate IL-6 production [12].
• While LPS does not require endocytosis to functionally associate with the membrane expressed TLR4/MD2 complex, internalization and endosomal maturation is conditional for CpG-DNA to activate TLR9 [25].
• Lipopolysaccharide induces CD25-positive, IL-10-producing lymphocytes without secretion of proinflammatory cytokines in the human colon: low MD-2 mRNA expression in colonic macrophages [26].
• The cells that had been cotransfected with TLR4(Delta E24-P34) and MD-2 did not induce NF-kappa B activation in response to LPS [27].

Analytical, diagnostic and therapeutic context of LY96

• Through site-directed mutagenesis, more than two intermolecular disulfide bonds were found to stabilize the MD-2 multimer [21].
• However, HIEC expressed TLR4 and MD2 resulting in a fully functional signalling complex as demonstrated by RT-PCR, western blot, and immunofluorescence analyses [28].
• In contrast, other cell types of the normal human cornea, conjunctiva, retina, and sclera did not express TLR4/MD-2 protein in vivo as detectable by immunohistochemistry [29].
• Inhalation of LPS by healthy human volunteers resulted in enhanced alveolar macrophage expression of mRNAs encoding TLRs 1, 2, 7, 8 and CD14, and reduced expression of mRNAs encoding TLR4 and lymphocyte antigen 96 [30].
• Additionally, the expression of TLR1-6, MD2 and CD14 was examined by RT-PCR, and the CD14 expression was determined by flow cytometry analysis [31].

References