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Red cell surface cysteine residue (285) of D polypeptide is not essential for D antigenicity
K Suyama1, R Lunn, J Goldstein
1Cell Biochemistry Laboratory, Lindsley F. Kimball Research Institute, New York Blood Center, New York, USA.
Background:
Only one surface cysteine residue (285) has been thought to be involved in D antigenicity, according to studies using lyophilized 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 antigenicity is assessed by using intact red cells treated with the reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and 2-(4'-maleimidylanilino)- naphthalene-6-sulfonic acid. Antigenicity was appraised by hemagglutination titers and immunoprecipitation using human anti-D, -c, and -E.
Results:
Treatment with N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid) at various concentrations (< or = 5 mM) or for various times (< or = 120 min) did not cause significant decrease in hemagglutination titers as compared to untreated intact red cells. Moreover, immunoprecipitation of Rh antigen-carrying peptides by human anti-D was not affected by prior treatment with N-ethylmaleimide or 2-(4'maleimidylanilino)-naphthalene-6-sulfonic acid. Efficacy of blockage of SH groups was demonstrated by inhibition of palmitic acid uptake by the Rh polypeptides for prior treatment with N-ethylmaleimide and by the presence of fluorescent Rh polypeptides for prior treatment with 2-(4'maleimidylanilino)-naphthalene-6-sulfonic acid.
Conclusion:
SH group involvement is not essential for D, c, or E antigenic expression in intact red cells.
Insights
Sulfhydryl groups are not essential for RhD, Rhc, and RhE antigen expression on intact red blood cells. Studies show that blocking these groups does not affect antigenicity, challenging previous assumptions.
Area of Science:
- Immunology
- Hematology
- Biochemistry
Background:
- Previous research suggested a single surface cysteine residue (285) is crucial for RhD antigenicity.
- Conflicting reports indicated a 17-kDa fragment lacking this cysteine is associated with D antigenicity.
Purpose of the Study:
- To investigate the role of sulfhydryl (SH) groups in the antigenic expression of RhD, Rhc, and RhE on intact red blood cells.
- To determine if SH group modification affects antigenicity as measured by hemagglutination and immunoprecipitation.
Main Methods:
- Intact red blood cells were treated with SH-modifying reagents: N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid.
- Antigenicity was assessed using hemagglutination titers and immunoprecipitation with specific human anti-D, anti-c, and anti-E antibodies.
- SH group blockage efficacy was confirmed by inhibition of palmitic acid uptake and fluorescence.
Main Results:
- Treatment with SH-modifying reagents did not significantly decrease hemagglutination titers for D, c, or E antigens.
- Immunoprecipitation of Rh antigen-carrying peptides by anti-D was unaffected by SH group modification.
- Successful blockage of SH groups was confirmed through functional assays.
Conclusions:
- Sulfhydryl group involvement is not essential for the antigenic expression of RhD, Rhc, and RhE on intact red blood cells.
- These findings challenge the established understanding of cysteine residue involvement in RhD antigenicity.