ABCB6 is dispensable for erythropoiesis and specifies the new blood group system Langereis

Abstract

The human ATP-binding cassette (ABC) transporter ABCB6 has been described as a mitochondrial porphyrin transporter essential for heme biosynthesis, but it is also suspected to contribute to anticancer drug resistance, as do other ABC transporters located at the plasma membrane. We identified ABCB6 as the genetic basis of the Lan blood group antigen expressed on red blood cells but also at the plasma membrane of hepatocellular carcinoma (HCC) cells, and we established that ABCB6 encodes a new blood group system (Langereis, Lan). Targeted sequencing of ABCB6 in 12 unrelated individuals of the Lan(-) blood type identified 10 different ABCB6 null mutations. This is the first report of deficient alleles of this human ABC transporter gene. Of note, Lan(-) (ABCB6(-/-)) individuals do not suffer any clinical consequences, although their deficiency in ABCB6 may place them at risk when determining drug dosage.

Figure 1. Characterization of the Lan blood group antigen expression on RBCs with the monoclonal antibody OSK43

(a) Specificity of the monoclonal antibody OSK43 as shown by flow cytometry analysis of Lan+ and Lan− RBCs. OSK43 shows a strict Lan specificity so that the histogram profiles of Lan− RBCs labeled with and without OSK43 were superposable. (b) Immunofluorescence analysis of Lan+ and Lan− RBCs co-labeled with the human monoclonal OSK43 (green) and a mouse monoclonal anti-glycophorin A, the most abundant glycoprotein on RBCs (red). Scale bars represent 5 µm. (c) Lan antigen expression on RBCs from 39 random blood donors from France (ABO type O) as measured by flow cytometry with OSK43 as in (a). The geometric means of fluorescence intensity (in arbitrary units of the flow cytometer) are shown as a box-and-whisker diagram: lower and upper boundaries of boxes represent the 25th and 75th percentiles, horizontal lines the medians, whiskers the minimum and maximum non-atypical values, and circles the atypical values. (d) Lan antigen expression on RBCs from adult or cord blood as measured by flow cytometry with OSK43 as in (a). Bars represent the means of fluorescence intensity, and error bars the s.d. (n=4); **P < 0.01 by Mann-Whitney U test.

Figure 2. Identification of ABCB6 as the carrier of the Lan blood group antigen

(a) Purification of the Lan antigen from RBCs by immunoprecipitation with OSK43. Lysates (lane 1: 1/300^th, lane 2: 1/900^th and lane 3: 1/2,700^th) were prepared from membranes of Lan+ RBCs labeled with or without OSK43, and corresponding immune complexes (lanes 4 and 5) were analyzed by polyacrylamide gel electrophoresis and silver staining. Identity and position of heavy and light chains of OSK43 are indicated. (b) ABCB6 peptides identified by mass spectrometry from the band of approximately 80 kDa that was immunoprecipitated by OSK43 from RBCs. In total, 27 tryptic peptides of ABCB6 were identified (green); the predicted trypsin cleavage sites are underlined. (c) ABCB6 is present in the plasma membrane of RBCs from Lan+ but not Lan− individuals as shown by western blot analysis. RBC membranes were prepared from 2 Lan+ (lanes 3 and 6) and 6 Lan− (lanes 1, 2, 4, 5, 7 and 8) individuals and analyzed by western blot with a rabbit polyclonal anti-ABCB6 (top panel). Of note, ABCB6 runs as monomeric and multimeric forms even under reducing conditions (arrows, see Supplementary Figure 6 for details). The western blot membrane was reprobed with a mouse monoclonal anti-ABCG2, another ABC transporter able to transport porphyrin (bottom panel; only monomeric form of ABCG2 is shown, see Supplementary Figure 6). (d) Exogenous expression of ABCB6 in K-562 cells results in cell surface expression of the Lan antigen. Live cells of representative K-562 clones stably transfected with an expression construct of ABCB6 cDNA (green profile) or the corresponding empty vector (red profile) were analyzed by flow cytometry with OSK43. (e) Diagram showing the positions of ABCB6 null mutations identified in this study (see Table 1 for details). Green boxes represent exons and broken lines introns. Red diamonds at the top represent the number of unrelated Lan− subjects found with each mutation.

Figure 3. Expression of the Lan antigen on human cancer cell lines

Cell surface expression of the Lan antigen was analyzed on native HepG2 (a), A-498 (b), A-431 (c), MOLT-4 (d), HeLa (e) and HuH-7 (f) cells by flow cytometry with OSK43 (green profiles) or T27S (a non relevant human monoclonal antibody, grey profiles).