Immunochemical Analysis of the Human Erythrocyte Rh Polypeptides

Avent, Neil D.; Liu, Wendy; Warner, Karen M.; Mawby, W. J.; Jones, Jeffrey W.; Ridgwell, K.; Tanner, Michael

doi:10.1074/jbc.271.24.14233

Cited by 60 publications

(41 citation statements)

References 30 publications

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“…6, both proteins migrated to approximately the same position on SDS-PAGE, although the calculated molecular mass of DD2 is approximately 7 kDa more than that of DD3. This may be due to the fact that Rh protein, which constituted mostly of DD2 and DD3, is known to migrate in SDS gel anomalously (23). On the other hand, although the expression of DD1 (80 kDa) was confirmed as shown in Figs.…”

Section: Discussionmentioning

confidence: 89%

Use of RhD Fusion Protein Expressed on K562 Cell Surface in the Study of Molecular Basis for D Antigenic Epitopes

Zhu¹,

Haller²,

Li³

et al. 1999

Journal of Biological Chemistry

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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.As one of the most important blood group systems involved in blood transfusion and hemolytic disease, the Rh antigens are present on the erythrocyte surface as a complex including Rh30 proteins (RhD and RhCE) and Rh50 glycoprotein (see recent reviews in Refs. 1 and 2). Hartel-Schenk and Agre (3) first demonstrated that the Rh polypeptides migrated through sucrose gradients as a complex with an apparent M r of 170,000. Based on proteolytic digestion and immunoblotting, Eyers et al. (4) proposed a model that consists of two of each Rh30 and Rh50 molecules, with their N-terminal portions forming the core of the complex. The genes encoding RhD and RhCE are located at chromosome 1p34-p36 (5), whereas RH50 gene is at chromosome 6p11-21 (6). These three genes share not only a similar exon-intron composition but also homology in their deduced amino acid sequences (92% identity between RhD and RhCE, and 36% identity between Rh30 and Rh50) (2). Therefore, it appears that all three of these molecules belong to a family of structurally related membrane proteins.The multiprotein complex provides the basis for enormously complicated D antigenic epitopes, which have been serologically classified into nine epitopes, epD1 to epD9 (7). Most of the information regarding the molecular basis for D epitopes has been derived from genetic analysis of RhD variants where part of the RhD gene is missing or replaced by the highly homologous RhCE sequence (2). Thus, D VI phenotype, characterized as lack of epD1, 2, 5, 6, 7, and 8, contains rearranged RHD gene where its exons 4 -6 are replaced with RHCE equivalents (8). In D IVa phenotype that lacks epD 1, 2, 3, and 9, there is a rearrangement of exon 3 and part of exon 7 of the RHD gene (9). These observations suggest that serologically defined D epitopes are organized into overlapping m...

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Section: Discussionmentioning

confidence: 89%

Use of RhD Fusion Protein Expressed on K562 Cell Surface in the Study of Molecular Basis for D Antigenic Epitopes

Zhu¹,

Haller²,

Li³

et al. 1999

Journal of Biological Chemistry

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“…As additional proof that permeability to CH 3 NH 2 ͞NH 3 is facilitated by a membrane protein, transport experiments were performed with ghosts prepared from protease-digested RBCs. Because bromelain cleaves RhAG and RhD, but not the RhCE protein (47), the experiments were carried out with RhD-negative cells. Ghosts from bromelain-digested RBCs exhibited a significant reduction of the alkalinization rate constant compared with the untreated control (Fig.…”

Section: Ch3nh2͞nh3 Transport Function Is Mediated By a Protein-depenmentioning

confidence: 99%

Human Rhesus-associated glycoprotein mediates facilitated transport of NH ₃ into red blood cells

Ripoche

Bertrand

Gane

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

149

154

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“…One region of the Rh D protein which has clear conformational differences to the Rh CcEe proteins has been localized to the predicted sixth external domain of the Rh D protein (Avent et al, 1992). The precise localization of a Rh D specific extracellular bromelain/papain cleavage site between amino acid residues 353 (Glycine) and 354 (Alanine) clearly indicates that these amino acids form part of a Rh D specific domain with different protease accessibility than the Rh CcEe proteins (Avent et al, 1996b).…”

Section: Summary the Discovery Of Rh Partial D Variant Red Cells By mentioning

confidence: 99%

Molecular basis of the D variant phenotypes DNU and D^II allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein

et al. 1997

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Summary. The discovery of Rh partial D variant red cells by discrepant reactions with different monoclonal anti-D hasdemonstrated the range of Rh D epitopes that have arisen due to alterations in Rh D protein structure. There are two current classification systems, one which uses a nine epitope model (epD1-epD9) whereas a more recent model proposes 30 different epitopes. We describe here the molecular basis of two D variants which lack epD4 and epD9 namely the DNU and D II phenotypes. These would have both been originally classified as D II phenotype individuals, but we have revealed subtle differences in the serological profile of these erythrocytes. Such a differential reactivity and determination of the molecular bases of these phenotypes allows us to predict critical amino acids for epD3, epD4 and epD9 expression. The DNU phenotype arises from a single point mutation in the RHD gene resulting in a single amino acid change (Gly353Arg). Sequence analysis of exon 7 of the RHD gene derived from the D II propositus indicates that there is a single point mutation in this exon resulting in a single amino acid change (Ala354Asp). It is likely that this point mutation gives rise to the D II phenotype. Both mutations result in the change to Rh D-specific residues. Our results indicate that the following amino acids are crucial for epD3a

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Immunochemical Analysis of the Human Erythrocyte Rh Polypeptides

Cited by 60 publications

References 30 publications

Use of RhD Fusion Protein Expressed on K562 Cell Surface in the Study of Molecular Basis for D Antigenic Epitopes

Use of RhD Fusion Protein Expressed on K562 Cell Surface in the Study of Molecular Basis for D Antigenic Epitopes

Human Rhesus-associated glycoprotein mediates facilitated transport of NH ₃ into red blood cells

Molecular basis of the D variant phenotypes DNU and D^II allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein

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Immunochemical Analysis of the Human Erythrocyte Rh Polypeptides

Cited by 60 publications

References 30 publications

Use of RhD Fusion Protein Expressed on K562 Cell Surface in the Study of Molecular Basis for D Antigenic Epitopes

Use of RhD Fusion Protein Expressed on K562 Cell Surface in the Study of Molecular Basis for D Antigenic Epitopes

Human Rhesus-associated glycoprotein mediates facilitated transport of NH 3 into red blood cells

Molecular basis of the D variant phenotypes DNU and DII allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein

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Human Rhesus-associated glycoprotein mediates facilitated transport of NH ₃ into red blood cells

Molecular basis of the D variant phenotypes DNU and D^II allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein