The role of disulfide bonds in the assembly and function of MD-2.
MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDSPAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-kappaB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.
Mullen
Gregory E D
GE
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1360, USA.
Kennedy
Margaret N
MN
Visintin
Alberto
A
Mazzoni
Alessandra
A
Leifer
Cynthia A
CA
Davies
David R
DR
Segal
David M
DM
D019943:Amino Acid Substitution; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine; D004220:Disulfides / Q000032:analysis / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney; D051218:Lymphocyte Antigen 96; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine
D000954:Antigens, Surface; D004220:Disulfides; C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D011994:Recombinant Proteins; D012694:Serine; D003545:Cysteine