Glycobiology Advance Access published online on June 2, 2007
Glycobiology, doi:10.1093/glycob/cwm057
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Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4
1 Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, USA 30322
2 Laboratories of Microbial Pathogenesis, Atlanta Veterans' Affairs Medical Center, Atlanta, Georgia, USA 30033
3 Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA 30322
Corresponding author: Shanta M. Zimmer, MD Atlanta VAMC, Research 5A161, 1670 Clairmont Rd., Atlanta, GA 30033. Phone: (404) 3231-6111 x7570. Fax: (404) 329-2210 Email: szimmer{at}emory.edu
Received on February 15, 2007; revised on May 16, 2007; accepted on May 17, 2007
The eukaryotic accessory protein MD-2 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 was produced in the Pichia pastoris yeast expression system (rMD-2). Human embryonic kidney (HEK293) cells were transfected with human TLR4 and were simulated with highly purified LOS (0.56 pmoles) 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, IL-8 responsiveness to LOS or LPS in TLR4 transfected HEK293 cells. The interaction of endotoxin with human rMD-2 was then assessed by ELISAs. Wild-type meningococcal LOS (Wt m LOS) bound human rMD-2 and binding was inhibited by a monoclonal antibody to MD-2 (p<0.005) in a dose-dependent manner. Wt m LOS or meningococcal KDO2-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-mannooctulosonic 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 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.
Key words: endotoxin / lipid A / MD-2 / toll-like receptor 4
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