Redox control of exofacial protein thiols/disulfides by protein disulfide isomerase.
Protein disulfide isomerase (PDI) facilitates proper folding and disulfide bonding of nascent proteins in the endoplasmic reticulum and is secreted by cells and associates with the cell surface. We examined the consequence of over- or underexpression of PDI in HT1080 fibrosarcoma cells for the redox state of cell-surface protein thiols/disulfides. Overexpression of PDI resulted in 3.6-4. 2-fold enhanced secretion of PDI and 1.5-1.7-fold increase in surface-bound PDI. Antisense-mediated underexpression of PDI caused 38-53% decreased secretion and 10-33% decrease in surface-bound PDI. Using 5,5'-dithio-bis(2-nitrobenzoic acid) to measure surface protein thiols, a 41-50% increase in surface thiols was observed in PDI-overexpressing cells, whereas a 29-33% decrease was observed in underexpressing cells. Surface thiol content was strongly correlated with cellular (r = 0.998) and secreted (r = 0.969) PDI levels. The pattern of exofacial protein thiols was examined by labeling with the membrane-impermeable thiol reactive compound, 3-(N-maleimidylpropionyl)biocytin. Fourteen identifiable proteins on HT1080 cells were labeled with 3-(N-maleimidylpropionyl)biocytin. The intensity of labeling of 11 proteins was increased with overexpression of PDI, whereas the intensity of labeling of 3 of the 11 proteins was clearly decreased with underexpression of PDI. These findings indicated that secreted PDI was controlling the redox state of existing exofacial protein thiols or reactive disulfide bonds.
Jiang
X M
XM
Centre for Thrombosis and Vascular Research, School of Pathology, University of New South Wales, Sydney, New South Wales 2052, Australia.
Fitzgerald
M
M
Grant
C M
CM
Hogg
P J
PJ
D001483:Base Sequence; D002455:Cell Division; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004220:Disulfides; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D013438:Sulfhydryl Compounds; D019704:Protein Disulfide-Isomerases