4.1R-deficient human red blood cells have altered phosphatidylserine exposure pathways and are deficient in CD44 and CD47 glycoproteins

Abstract

Background: Protein 4.1R is an important component of the red cell membrane skeleton. It imparts structural integrity and has transmembrane signaling roles by direct interactions with transmembrane proteins and other membrane skeletal components, notably p55 and calmodulin.

Design and methods: Spontaneous and ligation-induced phosphatidylserine exposure on erythrocytes from two patients with 4.1R deficiency were studied, using CD47 glycoprotein and glycophorin C as ligands. We also looked for protein abnormalities in the 4.1R-based multiprotein complex.

Results: Phosphatidylserine exposure was significantly increased in 4.1R-deficient erythrocytes obtained from the two different individuals when ligands to CD47 glycoprotein were bound. Spontaneous phosphatidylserine exposure was normal. 4.1R, glycophorin C and p55 were missing or sharply reduced. Furthermore there was an alteration or deficiency of CD47 glycoprotein and a lack of CD44 glycoprotein. Based on a recent study in 4.1R-deficient mice, we found that there are clear functional differences between interactions of human red cell 4.1R and its murine counterpart.

Conclusions: Glycophorin C is known to bind 4.1R, and we have defined previously that it also binds CD47. From our evidence, we suggest that 4.1R plays a role in the phosphatidylserine exposure signaling pathway that is of fundamental importance in red cell turnover. The linkage of CD44 to 4.1R may be relevant to this process.

Figure 1.

Flow cytometry of erythrocytes from 4.1R (−) patients A and B and controls following ligation with natural and synthetic CD47 and GPC ligands and resultant PS exposure, detected by annexin V binding. Erythrocytes were incubated with ligands as described in the Design and Methods section, and treated with annexin V-fluorescein isothiocyanate (FITC). Mean percentages of annexin V-FITC positive cells are shown with respect to each lig-and. Spontaneous PS exposure was not statistically different in patients A and B, despite the significant protein deficiencies, from that in normal controls. However, the exposure was relatively higher in patient B in all experiments. This might be a transport or preparation artifact. (A) Ligation of CD44 had no effect. Ligation of CD47 (monoclonal antibody BRIC 126) resulted in increased PS exposure with anti-CD47. Ligation of GPA had no effect. Ligation of GPC triggered less PS exposure than in the normal control. (B) Ligation with synthetic peptide CD47 ligand (4N1K) and control peptide (4NGG) produced a similar effect to that seen with monoclonal anti-CD47 ligands. Each experiment was repeated three times, and statistical analysis was performed using a paired Student’s t test. The statistical significance is indicated on the figure as follows *p<0.05, **p<0.01, ***p<0.0005.

Figure 2.

Sypro Ruby stained SDS-PAGE gels of red cell membrane from 4.1R (−) patients A and B, and from controls. The absence of 4.1R is clearly visible in samples from patients A and B and is labeled with an arrow.

Figures 3.

Western blotting of red cell membranes from 4.1R-deficient patients A and B and age- matched controls with various anti-red cell membrane and membrane skeletal antibodies. The following antibodies were used: anti-4.1R (polyclonal antibody); anti-p55 (rabbit polyclonal antibody); anti-GPA (murine monoclonal antibody BRIC 256); anti-GPC (BRIC 10); CD47 (BRIC 126); anti-band 3 (BRIC 155); CD44 (BRIC 235). Multiple lanes were loaded for anti-4.1R and anti-p55 blots. (A) Patient A. The most salient findings were the absence of 4.1R and of p55, the reduction in GPC, the absence of the high molecular weight isoforms of CD47, and the absence of CD44. (B) Patient B. The most salient findings were the absence of 4.1R and near total absence of p55, the reduction in GPC, the absence of CD47, and the near complete absence of CD44.