Presence of closely spaced protein thiols on the surface of mammalian cells.
It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the gamma-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active.
Donoghue
N
N
Centre for Thrombosis and Vascular Research, School of Pathology, University of New South Wales, Sydney, Australia.
Yam
P T
PT
Jiang
X M
XM
Hogg
P J
PJ
D000818:Animals; D001152:Arsenicals / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000737:chemistry / Q000166:cytology; D005354:Fibrosarcoma / Q000737:chemistry / Q000473:pathology; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008322:Mammals; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
C426645:4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide; D001152:Arsenicals; D007202:Indicators and Reagents; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; C029341:oxophenylarsine; D013879:Thioredoxins; D001710:Biotin; D019704:Protein Disulfide-Isomerases; D005978:Glutathione