CELL-SURFACE CYSTEINE RESIDUE-(285) OF D-POLYPEPTIDE IS NOT ESSENTIALFOR D-ANTIGENICITY

Background: Only one surface cysteine residue (285) has been thought t o be involved in D antigenicity, according to studies using lyophilize d or nonlyophilized red cell membranes. However, it has been reported that a 17-kDa chymotryptic fragment containing the N-terminus but not this cysteine residue is associated with D antigenicity. Study Design and Methods: The role of the sulfhydryl (SH) group in D, c, and E anti genicity Is assessed by using intact red cells treated with the reagen ts N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and 2-(4'-ma leimidylanilino)-naphthalene-6-sulfonic acid. Antigenicity was apprais ed by hemagglutination titers and immunoprecipitation using human anti -D, -c, and -E. Results: Treatment with N-ethylmaleimide or 5,5'-dithi obis(2-nitrobenzoic acid) at various concentrations (less than or equa l to 5 mM) or for various times (less than or equal to 120 min) did no t cause significant decrease in hemagglutination titers as compared to untreated intact red cells. Moreover, immunoprecipitation of Rh antig en-carrying peptides by human anti-D was not affected by prior treatme nt with N-ethylmaleimide or 2-(4'maleimidylanilino)-naphthalene-6-sulf onic acid. Efficacy of blockage of SH groups was demonstrated by inhib ition of palmitic acid uptake by the Rh polypeptides for prior treatme nt with N-ethylmaleimide and by the presence of fluorescent Rh polypep tides for prior treatment with 2-(4'maleimidylanilino)-naphthalene-6-s ulfonic acid. Conclusion: SH group involvement is not essential for D, c, or E antigenic expression in intact red cells.