MD-2, an LPS-binding protein is essential for the recognition of LPS by TLR4. MD-2 belongs to the ML superfamily of lipid-binding proteins. The tertiary structure of mite allergen protein Der p 2 was identified as having the protein fold most compatible with the sequence of MD-2. Comparison of MD-2 and Der p 2 reveals that they have many common biochemical characteristics: they are both rich in β-structure and they are both very stable proteins as they both unfold only above 90°C. In Der p 2, six cysteine residues form three disulfide bridges. We determined one free cysteine residue per recombinant biologically active MD-2 molecule, supporting similar disulfide topology with three disulfides bridges as in Der p 2. MD-2 binds LPS with high affinity; however, only weak binding of LPS was detected with Der p 2. Comparison of electrostatic potentials of the structural model of MD-2 and Der p 2 indicates a region of high positive potential on MD-2 and its absence in Der p 2, which may be the reason for its weak binding of LPS. We suggest that Der p 2 and its homologues probably do not have a role in response to Gram-negative bacteria in insects and that MD-2 family members with their specific role in innate immunity probably evolved from an ML ancestor only in higher vertebrates.
Janeway Jr CA , Medzhitov R.Innate immune recognition. Annu Rev Immunol2002; 20: 197—216.
2.
Poltorak A., He X., Smirnova I. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science1998; 282: 2085—2088.
3.
Qureshi ST, Lariviere L., Leveque G. et al. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med1999; 189: 615—625.
4.
Shimazu R., Akashi S., Ogata H. et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med1999; 189: 1777—1782.
5.
Nagai T., Ibata K., Park ES, Kubota M., Mikoshiba K., Miyawaki A.A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol2002; 20: 87—90.
6.
Re F., Strominger JLMonomeric recombinant MD-2 binds Toll-like receptor 4 tightly and confers lipopolysaccharide responsiveness. J Biol Chem2002; 277: 23427—23432.
7.
Viriyakosol S. , Tobias PS, Kitchens RL, Kirkland TNMD-2 binds to bacterial lipopolysaccharide . J Biol Chem2001; 276: 38044—38051.
8.
Akashi S., Saitoh S., Wakabayashi Y. et al. Lipopolysaccharide interaction with cell surface Toll-like receptor 4—MD-2: higher affinity than that with MD-2 or CD14. J Exp Med2003; 198: 1035—1042.
9.
Schromm AB, Lien E., Henneke P. et al. Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling. J Exp Med2001; 194: 79—88.
10.
Re F., Strominger JLSeparate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness. J Immunol2003; 171: 5272—5276.
11.
Mancek M., Pristovsek P., Jerala R.Identification of LPS-binding peptide fragment of MD-2, a Toll-receptor accessory protein. Biochem Biophys Res Commun2002; 292: 880—885.
12.
Visintin A. , Latz E., Monks BG, Espevik T., Golenbock DTLysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction . J Biol Chem2003; 278: 48313—48320.
13.
Kawasaki K. , Nogawa H., Nishijima M.Identification of mouse MD-2 residues important for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 antibodies, and for conferring LPS and Taxol responsiveness on mouse TLR4 by alanine-scanning mutagenesis. J Immunol2003; 170: 413—420.
14.
Inohara N., Nunez G.ML — a conserved domain involved in innate immunity and lipid metabolism . Trends Biochem Sci2002; 27: 219—221.
15.
Derewenda U. , Li J., Derewenda Z. et al. The crystal structure of a major dust mite allergen Der p 2, and its biological implications. J Mol Biol2002; 318: 189—197.
16.
Wright CS, Li SC, Rastinejad F.Crystal structure of human GM2-activator protein with a novel beta-cup topology . J Mol Biol2000; 304: 411—422.
17.
Wright CS, Zhao Q., Rastinejad F.Structural analysis of lipid complexes of GM2-activator protein. J Mol Biol2003; 331: 951—964.
18.
Friedland N. , Liou HL, Lobel P., Stock AMStructure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease. Proc Natl Acad Sci USA2003; 100: 2512—2517.
19.
Heymann PW, Chapman MD, Aalberse RC, Fox JW, Platts-Mills TAAntigenic and structural analysis of group II allergens (Der f II and Der p II) from house dust mites (Dermatophagoides spp). J Allergy Clin Immunol1989; 83: 1055—1067.
20.
Mueller GA, Benjamin DC, Rule GSTertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies. Biochemistry1998 ; 37: 12707—12714.
21.
Gruber A., Mancek M., Wagner H., Kirschning CJ, Jerala R.Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition. J Biol Chem2004; 279: 28475—28482.
22.
Altschul SF , Madden TL, Schaffer AA et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res1997; 25: 3389—3402.
23.
Edgar RCMUSCLE: a multiple sequence alignment method with reduced time and space complexity . BMC Bioinformatics2004; 5: 113—131.
24.
Sali A., Potterton L., Yuan F., van Vlijmen H., Karplus M.Evaluation of comparative protein modeling by MODELLER. Proteins1995; 23: 318—326.
25.
Kennedy MN, Mullen GE, Leifer CA et al. A complex of soluble MD-2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4. J Biol Chem2004; 279: 34698—34704.
26.
Cain G., Elderfield AJ, Green R. et al. The effect of dry heat on mite, cat, and dog allergens. Allergy1998; 53: 1213—1215.
27.
Mullen GE, Kennedy MN, Visintin A. et al. The role of disulfide bonds in the assembly and function of MD-2. Proc Natl Acad Sci USA2003; 100: 3919—3924.
28.
Gioannini TL , Teghanemt A., Zhang D. et al. Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations. Proc Natl Acad Sci USA2004 ; 101: 4186—4191.
29.
Ichikawa S. , Hatanaka H., Yuuki T. et al. Solution structure of Der f 2, the major mite allergen for atopic diseases. J Biol Chem1998; 273: 356—360.
30.
Thomas WR, Chua KYThe major mite allergen Der p 2 — a secretion of the male mite reproductive tract? Clin Exp Allergy1995; 25: 667—669.
31.
Spellman PT , Rubin GMEvidence for large domains of similarly expressed genes in the Drosophila genome. J Biol2002 ; 1: 5—12.
32.
Guex N., Peitsch MCSWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis1997; 18: 2714—2723.
