Expression Levels of the ABCG2 Multidrug Transporter in Human Erythrocytes Correspond to Pharmacologically Relevant Genetic Variations

Abstract

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.

Polymorphisms of the Multidrug Pump ABCG2: A Systematic Review of Their Effect on Protein Expression, Function, and Drug Pharmacokinetics

“…Saison et al (2012) also suggested that the R236X polymorphism (706 C.T) may be the basis of the Jr(a2) blood type in Romani Gypsy diaspora communities of Southwestern Europe, and a homozygous R246X (736 C.T) variant has been found, resulting in Jr(a2) erythrocytes in two Caucasian individuals. Heterozygous R246X was shown to lower ABCG2 expression approximately 50% in vivo during flow cytometry analysis of erythrocytes (Kasza et al, 2012). Along with these studies, it is predicted that the E334X variant also produces the Jr(a2) blood group (Zelinski et al, 2012).…”

“…The use of blood samples could be a welltolerated method of analyzing ABCG2 levels since it is noninvasive and quantifiable with good anti-ABCG2 antibodies. This method has been used to describe significant decreases in protein expression in heterozygous Q141K and R236X carriers (Kasza et al, 2012). This proved the method could be successful in showing the varying tissue levels of ABCG2 for differing genotypes.…”

Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene

“…According to our previous experience, strongly reduced RBC expression levels of the ABCG2 [7] and the ABCB6 [8] membrane proteins were connected to specific mutations and/or polymorphisms in the coding genes. Therefore, we analyzed the DNA extracts of the blood samples of the volunteers with low RBCPMCA4b expression by Sanger sequencing of the ATP2B4 gene, coding for this protein.…”

“…RBC membrane protein determinations were carried out according to our recently developed method [6][7][8][9]. In brief, we fixed and permeabilized the RBC membranes by using 1% formaldehyde solution, resulting in RBC "ghosts", and then equal number of cells were incubated with the PMCA-specific primary antibody (JA3, mouse monoclonal antibody, RRID: AB 628160) followed by a secondary, Alexa Fluor 488 labeled goat anti-mouse (H + L) antibody (Thermo Fisher/Life Technologies), in 96 well plates.…”

Cell Surface Membrane Proteins as Personalized Biomarkers: Where we Stand and Where we are Headed

“…As published recently, we have developed a simple, rapid, reliable flow cytometrybased diag nostic assay for the quantitative determination of the membrane protein, ABCG2, in the RBC membrane [82]. By using specific antibodies, we could detect ABCG2 specifically and sensitively in RBCs.…”