Use of RhD fusion protein expressed on K562 cell surface in the study of molecular basis for D antigenic epitopes

Alex Zhu, Stephanie Haller, Hua Li, Asok Chaudhuri, Antoine Blancher, Kimita Suyama

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323-331(A) and 350-354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes.

Original languageEnglish (US)
Pages (from-to)5731-5737
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number9
DOIs
StatePublished - Feb 26 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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