Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4.
MD-2, a eukaryotic accessory protein, is an essential component for the molecular pattern recognition of bacterial endotoxins. MD-2 interacts with lipid A of endotoxins [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] to activate human toll-like receptor (TLR) 4. The structure of lipid A influences the subsequent activation of human TLR4 and the immune response, but the basis for the discrimination of lipid A structures is unclear. A recombinant human MD-2 (rMD-2) protein was produced in the Pichia pastoris yeast expression system. Human embryonic kidney (HEK293) cells were transfected with human TLR4 and were stimulated with highly purified LOS (0.56 pmol) from Neisseria meningitidis or LPS from other structurally defined bacterial endotoxins in the presence or absence of human rMD-2. Human rMD-2 restored, in a dose-dependent manner, interleukin (IL-8) responsiveness to LOS or LPS in TLR4-transfected HEK293 cells. The interaction of endotoxin with human rMD-2 was then assessed by enzyme-linked immunosorbent assays. Wild-type meningococcal LOS (Wt m LOS) bound human rMD-2, and binding was inhibited by an anti-MD-2 antibody to MD-2 dose-dependently (P < 0.005). Wt m LOS or meningococcal KDO(2)-lipid A had the highest binding affinity for human rMD-2; unglycosylated meningococcal lipid A produced by meningococci with defects in the 3-deoxy-d-manno-2-octulosonic acid (KDO) biosynthesis pathway did not appear to bind human rMD-2 (P < 0.005). The affinity of meningococcal LOS with a penta-acylated lipid A for human rMD-2 was significantly less than that for hexa-acylated LOS (P < 0.05). The hierarchy in the binding affinity of different lipid A structures for human rMD-2 was directly correlated with differences in TLR4 pathway activation and cytokine production by human macrophages.
Zimmer
Shanta M
SM
Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA. szimmer@emory.edu
Zughaier
Susu M
SM
Tzeng
Yih-Ling
YL
Stephens
David S
DS
D001665:Binding Sites; D002236:Carbohydrate Conformation; D002478:Cells, Cultured; D004731:Endotoxins / Q000378:metabolism; D006801:Humans; D008050:Lipid A / Q000737:chemistry* / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000737:chemistry*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014162:Transfection
D004731:Endotoxins; C495377:LY96 protein, human; D008050:Lipid A; D051218:Lymphocyte Antigen 96; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4