We thank Dr William C. Mentzer for providing the sample from patient W.D. and ... ROBERT M. JOHNSON. Depamnent of ... Greenquist A, Schrier. SL, Lande W ...
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1992 80: 1624-1626
Hereditary stomatocytosis and Rh-deficient patients exhibit distinct molecular defects [letter] P Bailly, JP Cartron, D Wang and RM Johnson
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HEREDITARY STOMATOCYTOSIS AND Rh-DEFICIENT PATIENTS EXHIBIT DISTINCT MOLECULAR DEFECTS To the Editor: Hereditary stomatocytosis is a rare chronic hemolytic anemia due to defective cell volume regulation. Affected red blood cells (RBCs) exhibit an increased Na+ and K+ permeability, normal Na, K-ATPase activity, and increased water content of undetermined aetiology, but an integral membrane protein migrating to the band 7 region on sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) was found to be clearly decreased.' Further investigations of RBC membrane proteins from several patients by
two-dimensional gel electrophoresis indicated that the band 7 region on one-dimensional gels could be resolved into four distinct proteins (2 integral and 2 peripheral), among which only the basic integral membrane protein called 7.2b was completely a b ~ e n t . ~ - ~ Protein 7.2b is a recently purified 31-Kd protein that is phosphorylated and ~almitylated.~ During these investigations it was noted that protein 7.2b and the blood group Rh polypeptides6 exhibited common features, including solubility properties, molecular weight (Mr), and palmitylation.s Also, the absence or severe reduction of Rh antigens from RBCs of Rh-deficient individuals
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1625
A
Anti-protein 7.2b
B
Anti-Rh proteins
CRh
MPC1
CRh
MPC8
Fig 1. lmmunostrining anr)yrb of Rh and 7.2b protoinr on human RBC mombranos from Rh varknta and horeditly rtomrtocytorh Miviehub. Memhne proteins from the Rh varknta and the heredltay atomatocytosh patlent (W.D.) were prepared and ~ I I byOWestam ~ Mot arutpb aa pmviwaly described,@ using a detection system of goat antimbbk IgG conjugatedto phosphatase alkaline. (A) Immunortaining with antipratein 7.2b antiserum (no. R1107,1:5,000 dilution). (E) Immunortaining with two polyclonal anti-Rh proteins, MPCl (1:2,000 dilution) and MPCE (1:4,000 dilution), raised against the N-tor (res. 34-46)and C-tor (res. 409917) of Rh proteins, respectively. A n o m indicae the migration positionsof the 7.2b and Rh proteins.
(Rh..u and R h d ) is associatedwith a chronic hemolyticanemia of varying severity characterized by stomatocytosis. reduced osmotic fragility, and increased cation permeability.’ Although Rh antigens have been found normally expressed in one patient with hereditary stomatocytosis,” it is not known whether the Rh polypeptides are normal in these cells and, conversely, whether protein 7.2b is present or absent in Rhdeficient erythrocytes. In the present study, we report on the characterization of Rh proteins and protein 7.2b in erythrocytes from Rhdeficient and hereditary stomatocytosis patients using specific polyclonal antisera raised against these proteins. Accordingly, RBC membrane proteins from individualsof known common and rare Rh phenotypes, including those from Rhdeficient patients (Rh..ll and R h d ) of both the regulatory and amorphic types were prepared, separated by SDS-PAGE. transferred to nitrocellulose sheets, and analyzed by immunoblotting with the antiprotein 7.2b antiserum: as shown in Fig 1A. Membranes from a hereditary stomatocytosis patient (W.D.)8 were simultaneously investigated as control. We found that protein 7.2b (apparent Mr, 31,000) was normally present in all membranes from the Rh variants, including those of 4 unrelated Rh-deficient cells, whereas this protein was. as expected, undetectable in membrane from patient W.D. In parallel experiments, we found that the Rh proteins (apparent Mr, 31,000) detected with the polyclonal antisera MPCl and MPC8 (specific for the N-ter and C-ter domains of the Rh proteins, respectively9)could be identified on RBCs from RhD-positive and RhD-negative individuals, as well as those from patient W.D.. but were absent, as expected, from
Rh-deficient samples (Fig 19). Together, these results indicate that, although RBCs from hereditary stomatocytosis and Rhdeficient patients exhibit similar features, these syndromes are associated with the molecular defect of distinct integral membrane proteins. This finding is in agreement with the known heterogeneity of disorders associated with stomatocytosis of peripheral blood c e l l ~ , but ~ ~ how J ~ such defects are correlated with morphologic and functional abnormalities needs further investigations. ACKNOWLEDGMENT
We thank Dr William C. Mentzer for providing the sample from patient W.D. and Martine Huet for technical assistance. Support from the Institut National de la Santt et de la Recherche Medicale. the Caisse Nationale d’Assurances Maladie des Travailleun Salaries, and the NATO organization (Grant No. 0556/88) is gratefully acknowledged. PASCAL BAILLY JEAN-PIERRE CARTRON Unite INSERM U76 Instirut National de TtunsjkionSanguine Paris, Fmnce DING WANG ROBERT M. JOHNSON Depamnent of Biochemistry Wayne Srare University Detroit, MI
REFERENCES 1. Mentzer WC, Lam GKH. Lubin BH. Greenquist A, Schrier association with an integral membrane protein deficiency. Br J SL, Lande W Membrane effects of hidoesters in hereditary Haematol72:452,1989 stomatocytosis.J Supramol Struct 9275,1978 4. Mode L, Pothier 9, Alloisio N, Fto C, Gamy R, Bast M, 2. Lande WM. Thiemann PVW. Mentzer W C Missing band 7 Delaunay J: Reduction of membrane band 7 and activation of membrane protein in two patients with high Na, low K erythrovolume stimulated (K+.CI-)-cotransport in a case of congenital cytes. J Clin Invest 701273,1982 stomatwosis. Br Haematol 1989 3. Eber SW, bride WM, la-i TA, Mentzer WC, Hehn p, Wiley JS. Schriiter W Hereditary stomatocytosis: Consistent 5. Wang D, Mentzer WC, Cameron T. Johnson RM: Purifica71:1419
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tion of band 7.2b, a 31-kDa integral phosphoprotein absent in hereditary stomatocytosis.J Biol Chem 26617826,1991 6. Agre P, Cartron J P Molecular biology of the Rh antigens. Blood 7831,1991 7. Nash R, Shojania AM: Hematological aspect of Rh deficiency syndrome: A case report and a review of the literature. Am J Hematol24:267,1987 8. Mentzer WC, Smith WB,Goldstone J, Shohet SB: Hereditary stomatocytosis:Membrane and metabolism studies. Blood 46:659, 1975
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9. Perez-Perez C, Taliano V, Mouro I, Huet M, Salat-Marti A, Martinez A, Rouger P, Cartron JP: Spanish Rh,,ll family caused by a silent Rh gene. Hematological, serological and biochemical studies. Am J Hematol (in press) 10. Miller DR, Rickles FR, Lichtman MA, La Celle PL, Bates J, Weed R A new variant of hereditary hemolytic anemia with stomatocytosis and erythrocyte cation abnormality. Blood 38:184,1971 11. Parker JC, Berkowitz L R Physiologically instructive genetic variants involving the human red cell membrane. Physiol Rev 63:261,1983
