The Human Kell Blood Group Binds the Erythroid 4.1R Protein: New Insights into the 4.1R-Dependent Red Cell Membrane Complex
Authors: Azouzi Slim , Collec Emmanuel , Mohandas Narla , An Xiuli , Colin Aronovicz Yves , Le Van Kim Caroline 
Published: October 2019

Abstract
Introduction: Protein 4.1R is a cytoskeletal adaptor protein that is responsible for the control of the mechanical stability of erythrocyte membranes, and for the proper anchoring of transmembrane proteins to the membrane skeletal network. Analysis of 4.1R-deficient human and murine erythrocytes revealed the complex array of membrane proteins that bind 4.1R and link these proteins to the spectrin-based skeletal network. 4.1R is composed of four functional domains: the N-terminal 30 kDa domain referred to as the FERM domain, the 16 kDa domain, the 10 kDa spectrin-actin binding domain, and the C-terminal 24 kDa domain. The Kell glycoprotein (93 kDa) is a type II single-span membrane protein which carry the Kell blood group system including the K1 (Kell) and K2 (cellano) antigens. Kell protein has endothelin-3 converting enzyme activity of type II membrane glycoproteins.
In this study we have analyzed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(-) HE) and performed detailed characterization of the interaction between 4.1R and Kell glycoprotein. Furthermore we also investigated the expression of membrane proteins exhibiting blood group antigens and the functional activities of AQP1, Band 3 and RhAG in the 4.1(-) HE erythrocyte membrane.
Results: Flow cytometry and western blot analyses revealed a severe reduction of Kell in the absence of 4.1R. In vitro pull down and co-immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R46 R motif in the juxta-membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(-) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO3-/Cl- exchange (band 3-dependent), but a normal water (AQP1-dependent) and ammonia (RhAG-dependent) transport in the absence of 4.1R.
Discussion: In this study, we provide evidence for a direct interaction between Kell and 4.1R and we propose an updated model for the 4.1R- multiprotein complex in human erythrocyte (Fig 1). The lobe A in the 4.1R FERM domain binds to protein transporters such as band 3, NHE1 and UT-B. Functional and structural experiments are required to confirm the presence of UT-B in this complex. The transmembrane proteins GPC, XK, Duffy and Kell bind to the lobe B and the binding site of p55 is located in lobe C.
The deficiency of blood group antigens carrying proteins in HS and HE erythrocytes can be explained by various molecular mechanisms including perturbed traffickingto the erythroblast membrane, aberrant protein sorting duringerythroblast enucleation, and selective loss during reticulocytemembrane remodelling. Establishing when and where these proteins associate during erythroid differentiation should provide mechanistic insights into membrane multi-protein complex formation in both normal and abnormal erythropoiesis.
Conclusion: The findings from the present study using 4.1(-) HE human erythrocytes have enabled us to obtain novel insights into the 4.1R complex organization.
Table 1. Antigen and protein expression of human erythrocytes.Specific antibody binding capacity, as determined by indirect immunofluorescence using QIFIKIT calibrated beads. *Mean of fluorescence intensity given in arbitrary units. Proteins Normal 4.1(-) HE GPC 54075 (± 1075) 4000 (± 500) Band 3 415000(±7000) 317000(±6000) Kell 4150 (±66) 1220 (±35) CD47 21300 (±1556) 21000 (± 2350) Rh 135200 (±283) 121000 (±113) RhAG 90450 (±2192) 71950 (±13150) GPA 357500 (±7072) 310500(±12500) DARC 2500 (±283) 1850 (±250) CD44 4150 (±71) 4100 (±200) UT-B* 13125 (± 1968) 2702 (±142) Lu/BCAM 1300 (±141) 1624 (±153) AQP1* 256 (±15) 202 (± 22) GLUT-1 10961 (± 239) 11542 (± 806)
Figure 1. Proposed model of the 4.1R-multiprotein complex in human erythrocyte. Figure 1. Proposed model of the 4.1R-multiprotein complex in human erythrocyte.

Disclosures
No relevant conflicts of interest to declare.