The effect of cysteine modification and proteinases on the major antigens (D, C, c, E and e) of the Rh blood group system

We have confirmed and extended previous observations showing that the (Rh) D antigen of erythrocyte membranes is destroyed by various reagents that modify cysteine (Cys) residues (Res.) and by trypsin as well as chymotrypsin, using thirty examples of monoclonal or polyclonal anti-D in heamglutination inhibition assays. We have also shown that most C, c, E, e and BS58 epitopes are inactivated or weakened by most Cys reagents and by these proteinases, using monoclonal and polyclonal antibodies. Inactivation by 5,5-dithiobis-(2-nitrobenzoic acid) was always fully reversible after subsequent dithioerythritol treatment. The essential Cys Res. appear to be buried in the membrane in view of the inability of some reagents to inactivate (iodoacetamide, iodoacetic acid) or reactivate (reduced glutathione) the antigens. Data obtained with N-ethylmaleimide indicate that inactivation of the C and c antigens is, at least in part, attributable to (a) Cys Res. that is (are) different from that (those) involved in the E and e antigens. Data obtained with the Cys reagents and the proteinases suggest that more than one peptide loop of the Rh proteins is involved in the major Rh antigens.