Journal of Biological Chemistry
Volume 274, Issue 4, 22 January 1999, Pages 2416-2423
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MEMBRANES AND BIOENERGETICS
Redox Control of Exofacial Protein Thiols/Disulfides by Protein Disulfide Isomerase*

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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.

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*

This work was supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia, and the New South Wales Cancer Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.