(CANCER RESEARCH 49. 2626-2632, May 15, 1989] Early Expression of Glycophorin C during Normal and Leukemic Human Erythroid Differentiation1 Jean-Luc Villeval, Caroline Le Van Kim, Ali Bettaieb, Najet Debili, Yves Colin, Bouchra El Maliki, Dominique Blanchard, William Vainchenker,2 and Jean-Pierre Cartron Unite Inserm U 91, Hôpital Henri Mondar, 94010 Créíeil[J-L.K, A. B., N. D., W. V.], and UnitéInserm U 76, Institut National de Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015 Paris [C. L. V. K., Y. C., B. E. M., D. B., J-P. C.J, France ABSTRACT red cell membrane. They carry the MN and Ss blood group antigens, respectively, and have extensive structural homolo- Glycophorins C and D (GPC and GPD) are two erythrocyte glycopro- teins which originate from the same gene but differ in their NHj-terminal gies, suggesting that they have derived by duplication of a common ancestral gene. GPC and GPD are minor components residues. The cell surface expression of these glycoproteins during normal and erythroid differentiation has been investigated with monoclonal and of the red cell membrane carrying the blood group Gerbich polyclonal antibodies and has been compared to the expression of gly- determinants. Several lines of evidence indicate that these GPs cophorin A (GPA), the major sialoglycoprotein of human red cells. Using play a critical role in regulating membrane deformability and glycosylation-independent antibodies (monoclonal or polyclonal), GPC or mechanical stability of red cells (2) by interacting via protein GPD was detected in erythroid and nonerythroid cell lineages. However, 4.1 with the membrane skeleton (3). Recently, it was suggested a glycosylation-dependent monoclonal antibody (MR4-130) detected an that GPC and GPD are encoded by the same gene (4) and that epitope on GPC which appears to be erythroid specific, suggesting that GPD might be a shortened form of GPC (5). Indeed, these two lineage specificity of this glycoprotein is related to some carbohydrate GPs differ in their NH2-terminal region (4) but are immunolog structures. During normal erythroid differentiation, GPC was expressed ically (6) and structurally (5) indistinguishable in their COOH- early at the level of erythroid progenitors (part of erythroid burst-forming unit and erythroid colony-forming unit) as detected with a glycosylation- terminal domain. independent monoclonal antibody (APO 3), whereas GPA is only present Since the red cell membrane glycophorins are now well during terminal erythroid differentiation. The MR4-130 epitope was not characterized, they represent useful markers to follow the se coordinately expressed on the cell surface with the GPC molecule in the quential steps of erythroid differentiation, and GPA has been erythroid differentiation, since it was detected at the level of the more already used for this purpose (7). GPA is synthesized during mature erythroid colony-forming unit slightly later than the GPC poly- terminal erythroid differentiation but is detected slightly earlier peptide. with PoAbs which recognize peptidic epitopes than with MoAbs In four erythroleukemic patients, blast cells blocked at discrete stages which react with carbohydrate-dependent determinants (8). of the erythroid differentiation were also investigated with antibodies and However, even with a PoAb, GPA is not detected on the surface complementary DNA probes for GPA and GPC. GPA was immunologi- of BFU-E and CFU-E (9). Only preliminary data, however, cally detected in three of four cases, and its cell surface expression was have been obtained concerning the expression of GPC during correlated with the amount of specific mRNA in the cells, as seen by Northern blot analysis. GPC was immunologically detected on the blast normal (10) and leukemic differentiation (11). In the present cells of all four patients. However, in two cases including one with positive study, we have compared the expression of GPA and GPC expression of GPA, the MR4-130 epitope was absent from the GPC during normal and leukemic erythroid differentiation using molecule. By Northern blot analysis, we found that the GPC/GPD mRNA specific antibodies, and we have investigated their biosynthesis was present at a high level in all four patient samples. Western blot by Northern blot analysis of their mRNA in four patients analysis of GPC and GPD in two of these patients revealed that these suffering from erythroleukemia. mRNAs were mostly translated into the GPD molecule, suggesting that these glycoproteins might be differently processed in certain cases of erythroleukemia. MATERIALS AND METHODS Samples. Normal bone marrow and blood samples obtained from INTRODUCTION normal donors after informed consent were collected on heparin. In Human erythrocytes carry at least four main species of sial- addition, the blast cells from 4 patients presenting an early erythroid oglycoproteins known as GPA,3 GPB, GPC, and GPD, which leukemia were analyzed; 3 of these 4 cases have been previously de scribed (11). have been extensively investigated and have been used as models Isolation of the Cells. Cells from blood and normal marrows were of integral membrane proteins (for review, see Ref. l). These separated by a Ficoll-metrizoate gradient centrifugation (Lymphoprep; GPs have a high content in sialic acid which contributes to the Nyegaard, Norway). Adherent and nonadherent cells were separated by negative charge of red cells. Moreover, they carry blood group a 2-h incubation. T-cells were separated from other mononuclear cells determinants and receptors for viruses, bacteria, and parasites. by rosette formation with 2-amino-ethylisothiouronium bromide- GPA and GPB are major and well-known constituents of the treated sheep red blood cells (12). Granulocytes were isolated by sedi mentation on dextran followed by a Ficoll-metrizoate gradient centrif Received 10/17/88; revised 1/27/89; accepted 2/9/89. ugation. The remaining red cells in the granulocyte cell pellet were The costs of publication of this article were defrayed in part by the payment removed by lysis with ammonium chloride (13). Platelets were obtained of page charges. This article must therefore be hereby marked advertisement in from platelet-rich plasma. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was partly supported by grants from la Ligue Nationale sur le Antibodies. The antiserum directed against GPC and GPD (L 857) Cancer, from ARC (Association pour la Recherche sur le Cancer) and from the was produced by immunization of rabbits with purified GPD.4 Mono GEFLUG. clonal antibodies anti-GPC, MR4-130, and APO 3 were obtained from 2To whom requests for reprints should be addressed. 3The abbreviations used are: GPA. glycophorin A; GPB, glycophorin B; GPC, P. Rouger (14) and W. Dahr (15), respectively. Two previously de glycophorin C; GPD, glycophorin D; GP, glycoprotein; MoAb. monoclonal scribed antibodies against GPA, a murine MoAb (LICR-LON-R18) antibody; PoAb, polyclonal antibody; CA 1, carbonic anhydrase 1; BFU-E, erythroid burst-forming unit: CFU-E, erythroid colony-forming unit; CFU-GM, 4 B. El Maliki, D. Blanchard, W. Dahr, K. Beyreuther, and J-P. Carton. granulocyte-macrophage colony-forming unit; CFU-MK, megakaryocyte colony- Structural homology between the glycophorins C and D of the human erythrocyte forming unit. membrane, submitted for publication. 2626 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. EXPRESSION OF GLYCOPHORIN C DURING ERYTHROID DIFFERENTIATION (16,17) and a rabbit PoAb ( 18) (generous gifts of Dr. P. A. W. Edwards tion. The monoclonal antibody APO 3 reacted with a trypsin- and Dr. C. G. Gahmberg, respectively), have also been used. A murine sensitive but neuraminidase-resistant determinant located in MoAb against myeloid cells (80H5) (19) and several rabbit PoAbs the NH2-terminal region of GPC (15). Western blot studies against myeloperoxidase (20). CA 1 (21), hemoglobin (22), and von carried out with red cell membrane preparations further indi Willebrand factor (Dakopatts. Copenhagen, Denmark) have been used cated that APO 3 reacted with GPC but not with GPD (Fig. in double staining experiments. 1). It may be deduced from these data that MR4-130 and APO Fluorescent Labeling. Indirect immunofluorescence for murine 3 are both directed against GPC without cross-reactivity for MoAbs was performed on unfixed cells with an appropriate dilution of the antibody (1/50 to 1/500 depending upon the antibody). Binding GPD. However, the MR4-130 epitope was glycosylation de was revealed by incubation with a goat anti-mouse IgG Fab'2 fragment pendent, whereas the APO 3 epitope was not. The rabbit conjugated to fluorescein 500-fold diluted (Bioart, Meudon, France). antibody L 857 was obtained by immunization with a purified Cells were either spread on a slide and examined with a Zeiss micro GPD preparation.4 The immune serum was absorbed on intact scope (Oberkochen, Federal Republic of Germany) equipped with flu erythrocytes in order to select glycosylation-independent anti orescence epiillumination or analyzed by flow cytometry. Rabbit anti- bodies directed against the intracellular or intramembranous serum against either GPA or GPC was applied on cytospinned cells after 1-min fixation by methanol; their binding was revealed by a goat domain of the minor glycophorins. As expected, the absorbed anti-rabbit IgG Fab'2 fragment conjugated to rhodamine (Cappel, Coch- antibody reacted with both GPC and GPD in Western blot analysis