Cysteine 230 modulates tumor necrosis factor-related apoptosis-inducing ligand activity.
Biologically active tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein is known to form a homotrimer in solution. Unexpectedly, the recombinant active human TRAIL protein purified from bacteria produced two bands (a Mr 21,000 monomer derived from the disruption of the trimer in SDS gels and a Mr 42,000 dimer) on nonreducing SDS gels. The treatment of this TRAIL protein with DTT, a reducing agent, abolished formation of the Mr 42,000 band, suggesting that the Mr 42,000 band was the result of intermolecular disulfide bridge formation. Inspection of the amino acid sequence of human TRAIL protein identified a unique cysteine residue at position 230, and subsequent site-directed mutagenesis revealed that this amino acid residue is responsible for the appearance of the Mr 42,000 dimer. The binding analysis using the TRAIL protein and a TRAIL receptor (death receptor 5) revealed that both the dimer and the trimer bind to death receptor 5 with similar affinity. Interestingly, mutation of cysteine 230 to glycine completely abolished the apoptotic activity of TRAIL protein. The disruption of the dimer in the mixture of TRAIL dimer and trimer increased the apoptotic activity slightly, suggesting that the dimer has less apoptotic activity than the trimer. Therefore, our data indicate that cysteine 230 is not only required for TRAIL function but also modulates the apoptotic activity of TRAIL by forming an intermolecular disulfide bridge.
Seol
D W
DW
Department of Surgery, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA. seold+@pitt.edu
Billiar
T R
TR
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D003545:Cysteine / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
D051017:Apoptosis Regulatory Proteins; D004220:Disulfides; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D003545:Cysteine; D004229:Dithiothreitol; D005998:Glycine