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Immunochemical analysis of the human erythrocyte Rh polypeptides
1International Blood Group Reference Laboratory, Southmead Rd., Southmead, Bristol BS10 5ND, United Kingdom.
The Journal of Biological Chemistry
|June 14, 1996
Summary
This study reveals new protease cleavage sites on Rh D proteins and Rh glycoprotein, clarifying their membrane organization. Findings support that Rh Cc and Ee antigens are on a single polypeptide chain, not truncated ones.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- The Rh blood group system is crucial for transfusion compatibility.
- Understanding Rh protein structure and organization in the erythrocyte membrane is vital.
Purpose of the Study:
- To investigate the topography and organization of Rh polypeptides and Rh glycoprotein in the human erythrocyte membrane.
- To identify and characterize exofacial protease cleavage sites on Rh proteins.
Main Methods:
- Rabbit polyclonal antisera against synthetic peptides of Rh polypeptides and glycoprotein.
- Protease digestion (bromelain, trypsin, subtilisin) of erythrocyte membranes.
- Immunoprecipitation assays.
Main Results:
- Identified previously unrecognized exofacial protease sites on Rh CcEe, D proteins, and Rh glycoprotein.
- Rh D protein has two bromelain cleavage sites in external domains 1 and 6 (residues 353-354 in domain 6).
- Rh glycoprotein has two bromelain cleavage sites flanking its N-glycosylation site (residues 39-40).
- Bromelain treatment deglycosylated the Rh glycoprotein.
- Immunoprecipitation confirmed reactivity with antisera against the fourth external loop and C-terminal domain of Rh proteins.
Conclusions:
- The Rh C/c antigens are expressed on a single polypeptide chain, refuting the alternate splicing hypothesis.
- Rh Cc and Ee antigens are co-expressed on the same polypeptide.
- New insights into Rh protein topography and membrane organization were provided.