1. Miltenberger blood group antigen type III (Mi
Miltenberger blood group antigen type III (Mi.III) enhances the expression of band 3
by Kate Hsu, Naiwen Chi, Marjan Gucek, Jennifer E. Van Eyk, Robert N. Cole, Marie Lin, and D. Brian Foster
Blood
Volume 114(9):
August 27, 2009
©2009 by American Society of Hematology

2. The expression levels of GPB and Gp. Mur in Mi
The expression levels of GPB and Gp.Mur in Mi.III+ RBCs were complementary.
The expression levels of GPB and Gp.Mur in Mi.III+ RBCs were complementary. (A) Mi.III-specific Gp.Mur probably evolved from homologous gene recombination between GYPA and GYPB, and shows a unique glycophorin B-A-B structure and the antigenic Mur (marked as checkered). The protein sequences of full-length Gp.Mur and GPB, and GPA lacking a cytoplasmic domain, were aligned by the CLUSTALW program. (B) Glycophorin immunoblot of ghost lysates from 6 Mi.III+ and 6 non-Mi.III (control) samples (30 mg/lane). Solubilized ghosts were resolved on 10% SDS–polyacrylamide gel electrophoresis. (Left) GPA immunoblot by E4 antibody. (Right) Immunoblot against GAB by E3 antibody. Homozygous Mi.III samples are marked +/+.
Kate Hsu et al. Blood 2009;114:
©2009 by American Society of Hematology

3. iTRAQ™ validation by immunoblot.
iTRAQ™ validation by immunoblot. Equal quantities of ghost lysates were pooled from 6 to 8 donors per group (control vs Mi.III) for IP, and one-tenth of the pulldown (vol/vol) was loaded onto 4% to 12% SDS-polyacrylamide gel electrophoresis for immunoblot comparison. (A) Immunoblots confirmed that glucose transporter type I, ANK1, EPB42, α spectrin, and β spectrin were not quantitatively different in AE1 immunoprecipitates from both groups. The pulldown experiments were repeated 2 to 8 times, and IP was also confirmed using another anti-AE1, BRIC170 (data not shown). (B) Both anti-4.1R and anti-Rh antibodies pulled down GPA, GPB, Gp.Mur, and AE1, indicating that 4.1R and Rh polypeptides were part of the AE1-based complexes. (C) Rh-associated glycoprotein was coimmunoprecipitated with AE1. AE1 IP was repeated and confirmed with both anti-AE1 (AE12-M and BRIC170). (D) AQP1 was more substantially associated with AE1 on Mi.III+ membrane. (Left) AQP1 immunoblot of the pooled ghost lysates showed similar expression levels for both Mi.III and the control. (Right) Both anti-AE1 antibodies coimmunoprecipitated more AQP1 from Mi.III. The coimmunoprecipitation experiment has been repeated and confirmed 7 times.
Kate Hsu et al. Blood 2009;114:
©2009 by American Society of Hematology

4. AE1 expressed more in Mi. III+ RBCs
AE1 expressed more in Mi.III+ RBCs. (A top panel) An outline for the iTRAQ™-based quantitative proteomic method.
AE1 expressed more in Mi.III+ RBCs. (A top panel) An outline for the iTRAQ™-based quantitative proteomic method. To compare the composition of AE1-based complexes in Mi.III versus the control, RBC samples were collected from 6 Mi.III+ and 6 control donors, and each subjected to immunoprecipitation and then deglycosylation. The deglycosylated samples were independently digested with trypsin, and then combined for labeling with 1 of the 4 iTRAQ™ reagents. Two pools of 3 control samples and 2 pools of 3 Mi.III+ samples were randomly formed from the 6 donors from each group. The iTRAQ™-labeled peptides from all 4 pools were mixed, fractionated by strong cation chromatography, and analyzed by liquid chromatography–MS/MS. (Bottom panel) A representative fragmentation spectrum on iTRAQ™-labeled AE1. A high-scoring spectrum with overlapping b- and y-series fragment ions assigned to the peptide ADFLEQPVLGFVR (99% confidence) from AE1. (Inset) Expansion of x-axis demonstrates the abundance (area under the curve) of the isobaric tags at 114, 115, 116, and 117 Da. (B) A representative immunoprecipitation experiment using AE12-M antibody. Equal protein quantities of ghost lysates (m/m) from 6 to 8 donors per group (control vs Mi.III) were pooled for immunoprecipitation. One-tenth of the immunoprecipitate (vol/vol) was loaded for immunoblot comparison. The IP experiment has been repeated 7 times and confirmed by another anti-AE1, BRIC170 (data not shown). (Left) Silver stain of the AE1 immunoprecipitates from Mi.III and the control groups. (Right) Immunoblots for AE1, GPA, and GAB (E3). More AE1 was expressed by Mi.III+ cells, whereas the levels of GPA and GPB/Gp.Mur were not significantly different between the 2 groups. (C) DIDS labeling of AE1 was significantly higher in Mi.III+ than the control erythrocytes. Fresh erythrocytes from 3 donors per group were labeled with DIDS. The background fluorescence intensities from the unlabeled erythrocytes were subtracted. Data are expressed as mean ± SE; *P < .01.
Kate Hsu et al. Blood 2009;114:
©2009 by American Society of Hematology

5. Mi.III+ RBCs exhibited higher Cl−/HCO3− exchange capacity upon HCO3− stimulation.
Mi.III+ RBCs exhibited higher Cl−/HCO3− exchange capacity upon HCO3− stimulation. (A) Intracellular Cl− was labeled by fluorescent dye SPQ, and the intracellular Cl− concentration ([Cl−]out) was measured by the degree of SPQ quenching inside erythrocytes. When the extracellular bicarbonate ([HCO3−]out) was 5 mM, [Cl−]in increased little with respect to [Cl−]out. [Cl−]in was similar between the control and Mi.III+ erythrocytes. In the milieu of 15 mM HCO3−, Mi.III+ erythrocytes contained more Cl− and showed higher Cl− permeability than the control cells. Data are expressed as mean ± SE; *P < .05 at 90 mM [Cl−]out. The numbers of donors tested were indicated next to sample labels. (B) The concentrations of intracellular bicarbonate ([HCO3−]in) were predicted according to Donnan equilibrium. In the milieu of 15 mM HCO3−, Mi.III+ erythrocytes also contained more HCO3− and showed higher HCO3− permeability.
Kate Hsu et al. Blood 2009;114:
©2009 by American Society of Hematology

6. The pHi-buffering capacities of Mi.III+ erythrocytes were superior.
The pHi-buffering capacities of Mi.III+ erythrocytes were superior. Fresh RBCs were loaded with fluorescent pH indicator SNARF-1, and its intracellular pH measurement at pHout 7.5 was measured by flow cytometry. In the absence of extracellular bicarbonate, the control cells became more acidified than Mi.III. Depletion of extracellular Cl− maximized HCO3− loading for both Mi.III+ and the control cells, and diminished their pHi differences. The number of donors tested was indicated next to each bar. Data are expressed as mean ± SE; *P < .05.
Kate Hsu et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Mi.III+ erythrocytes were more resistant to osmotic stress.
Mi.III+ erythrocytes were more resistant to osmotic stress. Osmotic fragility tests showed that Mi.III+ erythrocytes began and completed hemolysis at more hypotonic concentrations than the control cells. *P < .05. Data are expressed as mean ± SE. The number of donors tested was indicated next to each bar.
Kate Hsu et al. Blood 2009;114:
©2009 by American Society of Hematology

8. Gp.Mur exhibited similar functionality as GPA in promoting AE1 expression.
Gp.Mur exhibited similar functionality as GPA in promoting AE1 expression. (A) Both GPA and Gp.Mur promoted AE1 biosynthesis. AE1 was subcloned in pCIG, a bicistronic construct containing the reporter gene green fluorescent protein. pCIG-AE1 was expressed alone, or together with GPA or Gp.Mur, in HEK293 cells. The transiently transfected cells were fixed, permeabilized, and stained with anti-AE1, BRIC71. The degrees of BRIC71 staining in the fixed and permeabilized cells reflected the relative expression levels of AE1 on intracellular and plasma membranes. (B) Both GPA and Gp.Mur promoted similar degrees of AE1 surface expression. Intact HEK293 cells were directly stained with BRIC71 after scraped off from culture plates. (Top) A representative BRIC71 histogram from flow cytometry. (Bottom) The intensities of BRIC71 staining for different coexpression groups were compared with that for the singly expressed AE1 (set at 1). Data were averaged from 8 to 9 independent experiments (as indicated next to each bar), and expressed as mean ± SE. P > .05 was deemed not significant (n.s.).
Kate Hsu et al. Blood 2009;114:
©2009 by American Society of Hematology