Uncein May Be a Potential Target for Sulfur Mustard Alkylation

Severe blister formation in theepidermal and dermal junction is one of the characteristic cutaneous lesions caused by bis-2-chloroethyl sulfide (sulfur mustard, HD), a bifunctional alkylating agent. Uncein is a newly discovered anchoring filament-associated antigen that has been shown to be undetectable in all forms of junctional epidermolysis bullosa, therefore suggesting its role in maintaining the integrity of the epidermal-dermal basement membrane zone. To test uncein as a potential target for HD alkylation in HD-induced vesication, uncein was biosynthetically labeled with 35 S-methionine and purified by immunoprecipitation with a 19-DEJ-1 monoclonal antibody from the medium of normal human keratinocyte (NHEK) cultures. The 3 5 S-labeled uncein was incubated with 50 muL of 10.0 mg/mL of HD in ethanol (ETOH) or ETOH, which served as the control in 50 mM Tris-Cl buffer (pH 7.4) for 2 h. In addition, 10.0 mg/mL of HD was incubated with uncein pretreated with 3 mM sodium thiosulfate, an HD scavenger. Uncein treated with HD was fractionated by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Uncein with no HD showed the typical profile of three subunit bands at 165, 130, and 100 kD. HD treatment reduced the intensity of uncein bands. Further, new higher molecular weight bands appeared at the top of the gel, and at the positions of 145 kD as well as 135 kD after HD treatment, suggesting that HD might cross-link uncein. In addition, sodium thiosulfate prevented the appearance of HD-crosslinked bands. Finally, monolayers of NHEK grown on cover slips were treated with 10.0 mg/mL of HD in ETOH or ETOH alone as control. Then, uncein was stained with a 19-DEJ-1 antibody and examined under immunofluorescence microscopy. In the control, uncein staining was localized intracellularly and was absent from the intercellular regions. Although the staining pattern did not change, the intensity of uncein staining was reduced after HD treatment, indicating that HD chemically modified uncein antigenic epitopes. These results suggest that HD alkylation of uncein may be ultimately responsible for weakening the epidermal-dermal junction, thereby contributing this chemical-induced subepidermal blister formation.