[21] Glycophorins: Isolation, orientation, and localization of specific domains

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[21] G l y c o p h o r i n s : Isolation, Orientation, a n d L o c a l i z a t i o n o f Specific D o m a i n s

By H E I N Z

F U R T H M A Y R a n d V I N C E N T T . MARCHESI

Glycophorins are relatively small and heavily glycosylated proteins that are found in plasma membranes of many, possibly all, cell types. This class of proteins was initially discovered and studied extensively in human erythrocyte membranes. 2 More recently, similar proteins have been isolated from pig 3 and horse erythrocytes, 4 from plasma membranes of murine T lymphocytes, 5 rat hepatoma cells, 6 or platelets. 7 From these studies it became clear, that in most species and at least in some cell types the glycophorins represent a family of related or similar sialoglycoproteins, which are difficult to separate. In human red cells three distinct molecules, termed glycophorin A, B, and C, 8 have been identified, and similar heterogeneity is observed for platelet membranes. Despite a considerable body of information on the primary structure of the protein and carbohydrates of human glycophorins, 9-13 in general the function of these proteins is unknown. Genetically determined changes in the amount or even lack of one or the other of the various proteins expressed at the cell surface of the mature erythrocyte appear to be of little biological consequence, 2 and mutational events that lead to structural alterations 14,15within the glycosylated domain are of interest mainly H. Furthmayr, Protides Biol. Fluids 29, 49 (1982). 2 H. Furthmayr, in "Biology of Complex Carbohydrates" (V. Ginsburg and P. Robbins, eds.), p. 123. Wiley, New York, 1981. 3 K. Honma, M. Tomita, and A. Hamada, J. Biochem. (Tokyo) 88, 1679 (1980). 4 j. Murayama, K. Takeshita, M. Tomita, and A. Hamada, J. Biochem. (Tokyo) 89, 1593 (1981). 5 W. R. A. Brown, A. N. Barcley, C. A. Sunderland, and A. F. Williams, Nature (London) 289, 456 (1981). 6 S. Nakajo, K. Nakaya, and Y. Nakamura, Biochim. Biophys. Acta 579, 88 (1979). 7 T. Okumura and G. A. Jamieson, J. Biol. Chem. 251, 5944 (1976). s H. Furthmayr, J. Supramol. Struct. 9, 79 (1978). 9 M. Tomita, H. Furthmayr, and V. T. Marchesi, Biochemistry 17, 4756 (1978). io H. Furthmayr, Nature (London) 271, 519 (1978). N W. Dahr, K. Beyreuther, E. Bause, and M. Kordowicz, Protides Biol. Fluids 29, 57 (1982). 12 H. Yoshima, H. Furthmayr, and A. Kobata, J. Biol. Chem. 255, 9713 (1980). ~3R. Prohaska, T. A. W. Koerner, I. M. Armitage, and H. Furthmayr, J. Biol. Chem. 256, 5781 (1981). ~4H. Furthmayr, M. N. Metaxas, and M. Metaxas-Biihler, Proc. Natl. Acad. Sci. U.S.A. 78, 631 (1981).

METHODS IN ENZYMOLOGY.VOL.96

Copyright© 1983by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181996-5

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because of effects on the expression of blood group antigens. Thus it remains unknown which genetic events compatible with life are required to affect red cell function severely in humans. In platelets, the expression of these proteins appears to be more tightly linked to the biological function of these particles, since different sialoglycoprotein profiles on polyacrylamide gels are correlated with a number of disorders. ~6 Yet, the molecular basis of deficient function is not understood. Various ideas have been put forward, suggesting that the cell surface domain of these proteins serves as receptors for viruses and other ligands, and that the cytoplasmic domain of these transmembrane protein provides attachment sites for cytoskeletal proteins.~7 These observations are compatible with the idea that these proteins mediate functions via the cytoskeleton.18 The function of these proteins could, however, be related to more general cellular requirements--to provide negatively charged molecules that may be required to prevent cell fusion. Although it is known, that glycophorins are expressed in early precursor cells during erythropoiesis, J9 we do not know whether the function of these membrane proteins in the early precursor cells is related to the postulated function in the mature red cells, which are much more readily accessible to study. In this chapter the isolation procedures and properties of intact glycophorins are described, as well as simplified procedures to obtain preparative amounts of the glycosylated domains of the three glycophorin molecules.

Isolation Procedures

Preparation of the Crude Glycophorin Fraction A crude sialoglycoprotein fraction can be readily prepared by the lithium diiodosalicylate (LIS)/phenol extraction procedure originally described by Marchesi and Andrews. 2° The methods outlined here include modifications and are currently used in the laboratory.

15 O. O. Blumenfeld, A. M. Adamani, and K. V. Puglia, Proc. Natl. Acad. Sci. U.S.A. 78, 747 (1981). 16 A. T. Nurden, D. Dupuis, D. Pidard, T. Kunicki, and J. P. Caen, Ann. N.Y. Acad. Sci. 370, 72 (1981). t7 T. J. Mueller and M. Morrison, Prog. Clin. Biol. Res. 56, 95 (1981). 18 R. G. Painter and M. Ginsberg, J. Cell Biol. 92, 565 (1982). 19 p. D. Yurchenco and H. Furthmayr, J. Supramol. Struet. 13, 255 (1980). 20 V. T. Marchesi and E. P. Andrews, Science 174, 1247 (1971).

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Reagents

Lithium 3,5-diiodosalicylate (DIS) is prepared from twice recrystallized 2-hydroxy-3,5-diiodosalicylate (Eastman Kodak No. 2166, Mr 389.91). Three hundred grams of DIS are dissolved in 4 liters of methanol at 38 °, the solution is filtered through two layers of Whatman No. 1 filter paper on a B/]chner funnel under suction, and the solution is kept at 4 ° (light protected) for 1-2 days. Crystals are collected on a B~chner funnel and dried in vacuo (yield approximately 50%). These steps are repeated once more. The mother liquor from this second crystallization step can be stored and used in the first step for future preparation. LIS is prepared by dissolving DIS in water to an end concentration of 0.6 M by adding dropwise a concentrated solution of lithium hydroxide (LiOH • HzO, Mr 41.96) to a final concentration of 0.6 M or to neutrality. The solution can be warmed to 45 °. After filtration of the almost clear solution through two layers of Whatman No. 1 filter paper, the solution is kept for 1-2 days at 4 ° (light protected), and crystals are collected and dried as above (end product 3 x crystallized LIS). Phenol (Mallinckrodt, loose crystals). LIS/Phenol Extraction

1. Freshly drawn or outdated human blood is washed free of plasma by centrifugation and resuspension in phosphate-buffered saline. Hemoglobin-free ghosts are then prepared by lysis of the packed red cells in 20fold excess (v/v) of 5 mM sodium phosphate, pH 8.0, containing 0.3 mM phenylmethylsulfonyl fluoride (PMSF) and stirring at ice temperature for 10 min. The red cell ghosts are pelleted by centrifugation at approximately 30,000 g for 30 min and are then washed repeatedly. The last wash should be done with 25 m M Tris-HC1, pH 8.0, and the pellet is then lyophilized. During the washing procedure and centrifugation of the membranes a small darker pellet is formed, which is carefully removed by suctioning with a Pasteur pipette. 2. Three grams of lyophilized membranes (white to pink) are dispersed by brief homogenization with a homogenizer (Tekman Co., Cincinnati, Ohio) in 100 ml of 0.3 M LIS (12 g/100 ml) in 50 mM Tris-HCl, pH 8.0. After stirring for 10 min at room temperature (light protected), 200 ml of cold distilled water is added and the solution is stirred for an additional 10 min on ice. 3. Undissolved material is removed by centrifugation for 1 hr at 40,000-50,000 g in a refrigerated centrifuge.

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4. Fifty percent aqueous phenol is prepared by mixing 150 g of phenol and distilled water at room temperature to a final volume of 300 ml in a closed vessel. The solution is stored at 4 ° until use. 5. Equal volumes of supernatant from step 3 and 50% phenol are mixed, and the mixture is stirred at room temperature for 20 min (light protected). After centrifugation at 4000 rpm for 1 hr, in a refrigerated centrifuge, two phases are formed; the upper (water) phases are collected by suction into a sidearm flask (occasionally this phase is cloudy, but will become clear as the solution warms up). The interphase and phenol phase are discarded. 6. The water phase is extensively dialyzed in the cold against distilled water and is then lyophilized. After lyophilization the material is suspended in excess ice-cold ethanol by stirring for 30-60 min to extract excess LIS; the protein precipitate is recovered by centrifugation at 4000 rpm. The ethanol extraction is repeated once more. The final precipitate is dissolved in distilled water, then dialyzed against distilled water and lyophilized. The solution should be clear at this step and a precipitate indicates contamination with other membrane proteins. Removal of the precipitate by centrifugation before lyophilization is then required, and this may result in some loss of sialoglycoprotein. Excess amount of precipitate at this step indicates problems with the procedure and usually results in considerably lower yields of material still contaminated by other proteins. The approximate yield of ethanol extracted protein is 30 mg/unit of blood.

Isolation of Glycophorin A The various glycoproteins contained in the crude glycophorin fraction can be separated by analytical polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate and staining of the gels with the periodic acid-Schiff's reagent. Depending on the gel system used, different protein patterns are obtained. A minimum number of four bands are seen on gels prepared according to Fairbanks, 21,22 and eight to ten bands are observed on gels prepared according to Laemmli. 8,23 The major glycoprotein, glycophorin A, can be separated from the other sialoglycopeptides by gel filtration in the presence of detergents. We 2~ H. Furthmayr, M. Tomita, and V. T. Marchesi, Biochem. Biophys. Res. Commun. 65, 113 (1975). ~ G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry 10, 2606 (1971). 23 U. K. Laemmli, Nature (London) 227, 680 (1970).

272

[21]

B I O G E N E S IAND S ASSEMBLY OF MEMBRANE PROTEINS

a

0

°

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80 120 Fraction Number

150

Ft~. 1. Elution profile of human erythrocyte membrane glycophorins on agarose A-1.5 in the presence of the detergent Ammonyx-Lo. A maximum load of 100 mg of a crude sialoglycoprotein fraction is placed onto an 85 × 5 cm column. - - , Absorbance 230 nm; ---, fluorescence of tryptophan (excitation, 290 nm; emission, 355 nm); LIS: position at which excess lithium diiodosalicylate will elute. Phenol red is used as marker dye. Peak a contains nearly pure glycophorin A; peaks b and c contain mixtures of several glycophorins.

have determined that the Zwitterionic detergent Ammonyx-Lo (Onyx Chem. Co., Jersey City, New Jersey; stock solution 30%) is ideally suited for preparative separation, since column effluents can be monitored in the far UV range and the detergent does not interfere with measurements of protein, sialic acid, or fluorescence. An agarose A-1.5 (Bio-Rad Laboratories Richmond, California) column (90 × 5 cm) is equilibrated with a buffer containing 0.1% Ammonyx-Lo, 25 mM NaC1, 5 mM sodium phosphate, pH 8.0, and 0.01% NaN3. The size of this column allows one to separate 100 mg of lyophilized crude glycophorin into three distinct peaks (Fig. 1). The first, and largest, peak contains almost pure glycophorin A, and the second and third peaks contain mixtures of two minor glycoproteins, glycophorins B and C (in addition to variable amounts of glycophorin A). 8 Figure 2 shows the complex patterns observed on SDSpolyacrylamide gels. It has become clear from a number of studies that some of the bands represent oligomeric species of the glycophorins that either require certain conditions for dissociation 2,24or cannot be disaggregated once they have been formed. 8 The proteins from appropriately pooled fractions can be separated from salt and most of the detergent by extensive dialysis against distilled water. After lyophilization, residual detergent and lipid that is still contained in the material can be extracted by chloroform-methanol (2 : l, v/v). The proteins will still be soluble in aqueous solutions (see below). 24 H. Furthmayr and V. T. Marchesi,

Biochemistry

15, 1137 (1976).

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GLYCOPHORINS: ISOLATION OF SPECIFIC DOMAINS

273

Q

T

"a

c"

ff

Fro. 2. Sodium dodecyl sulfate (SDS-polyacrylamide gel) electrophoresis of glycophorins separated by gel filtration. T: crude glycophorin fraction; a, b, c: protein pools from Fig. 1. Arrows and arrowheads indicate positions in the gel at which specific glycophorins are found: ~glycophorin A, ~' glycophorin B, A glycophorin C.

Isolation of Glycophorin B and Glycophorin C The two minor glycophorins could not be separated by column chromatography. However, larger amounts of intact glycophorins B and C can be isolated by elution from gels after preparative SDS-gel electrophoresis. 8 The proteins contained in the minor peaks in Fig. 1 are labeled with dansyl chloride or fluorescamine in the presence of SDS and mixed with unlabeled material at a ratio of 1 : 9 before electrophoretic separation. Ten milligrams of protein are dissolved in 1 ml of 0.02 M sodium phosphate, pH 8.0, 1% SDS; 0.5 ml of freshly prepared dansyl chloride at a concentration of 0.5% in acetone is added. After incubation at 37 ° for 30 min, acetone is added and the precipitate is collected by centrifugation. The

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[21]

precipitate is dissolved in 20 mM Tris-HCl, pH 6.8, 4 M urea, 6% SDS at a concentration of 10 mg/ml, and aliquots are mixed with unlabeled protein dissolved in the same buffer. After incubation at 37° for 30 min and 2 rain at 100°, 20 mg per 2 ml are loaded onto a slab gel (17 x 15 x 0.5 cm) prepared according to Laemmli, 23 and electrophoresis is done for 12 hr at 50 mA or until pyronine Y, a dye included into the sample before loading, has moved 9-10 cm into the gel bed. The labeled protein bands are visualized under UV light, and the fluorescent gel strips are cut with razor blades. After homogenization of the gel with a Teflon pestle the elution buffer, containing 0.05% SDS, 50 mM sodium bicarbonate, and 0.02% sodium azide, is added. The protein is extracted into excess buffer at 37 ° by shaking overnight. The extract is dialyzed against water and then lyophilized. Since it was found to be difficult to remove excess glycine and acrylamide by dialysis, the lyophilized material is subjected to column chromatography on LKB Ultrogel AcA-54 in 0.05% SDS, 5 mM sodium phosphate, pH 8.0 (column dimensions 2.5 × 90 cm). The protein-containing fractions are pooled, dialyzed against distilled water and lyophilized. To remove residual SDS, the lyophilized material is extracted twice with ethanol, then solubilized in distilled water and lyophilized.

Preparation of the Glycosylated (Cell Surface) Domains of Glycophorins The glycosylated domains of glycophorins A and C can be obtained in larger amounts by a rather simple procedure, s To 200 ml of packed and carefully washed (isotonic phosphate-buffered saline, pH 7.2) red blood cells, 400 ml of 0.02 M sodium phosphate, pH 8.0, 0.2 M NaCI is added. Then 30 mg of Tos-PheChzCl-treated trypsin (Worthington) are added and the suspension is gently shaken at 37° for 2 hr. The cells are removed by centrifugation at 3000 g for 20 min and washed once with phosphatebuffered saline; both supernatants are combined, mixed with an equal volume of 50% phenol (see above). After stirring for 20 min at ice temperature, the solution is centrifuged in a Sorvall RC-3 centrifuge using a swinging-bucket rotor for 1 hr at 5000 rpm. The upper (water) phase is extensively dialyzed against water at 4° and then lyophilized. The glycopeptide material is then extracted twice with chloroform-methanol (2 : 1, v/v) at 4° and the peptides are lyophilized again. Separation of the glycopeptides is done by gel filtration on a column (2.5 x 150 cm) of LKB Ultrogel AcA-54 equilibrated with 100 mM sodium acetate, pH 6.8, containing 0.02% sodium azide (Fig. 3). Two peptide peaks are separated, which contain sialic acid. The first peak contains tryptophan, indicating

[2 1]

GLYCOPHORINS: ISOLATION OF SPECIFIC DOMAINS

275

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FIG. 3. Gel filtration of cell surface glycopeptides obtained by tryptic digestion of intact human erythrocytes. Peptides recovered from the water phase after phenolic partitioning of a tryptic digest of intact cells are separated by gel filtration on AcA-54 (panel A). Two major sialic acid-containing peaks are obtained ( ) absorbance 230 nm), the first of which yields fluorescence indicative of tryptophan (excitation, 290 nm; emission, 355 nm; ---). CT1, AT1, and AT2 indicate the major glycopeptides derived from glycophorins A and C. 8,9 Panel b: Separation of peptides ATI and CT1 by DEAE-cellulose chromatography. The peak indicated as CTI yields tryptophan fluorescence (not shown).

the presence of the glycosylated peptide CT 1 derived from glycophorin C. Fractions are pooled, concentrated by evaporation, and desalted on a BioGel P2 column equilibrated with 0.05 N acetic acid. After lyophilization, the fractions are chromatographed on a DEAE-cellulose (Whatman DE-52) column (15 × 1.5 cm) equilibrated with 50 mM sodium formate, pH 6.1, and eluted with a linear salt gradient from 0 to 0.3 M sodium chloride over a total volume of 400 ml. 9 This procedure separates peptides CT1 from AT1 (the larger amino-terminal glycopeptide derived from glycophorin A) as indicated in Fig. 3B. Trypsin does not release the glycosylated fragment of glycophorin B from intact erythrocytes. 8 Thus, an alternative procedure is proposed to obtain this fragment. Glycophorins B and C are contained in peaks b and c after gel filtration of the crude glycophorin fraction (Figs. I and 2) in addition to traces of glycophorin A and possibly other glycoproteins. After trypsin digestion of peak b or peak c material in 50 mM Tris-HC1,

276

BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS

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Fro. 4. Separation of tryptic fragments of glycophorin mixtures. (a) After tryptic digestion of protein pools b and c, peptides are separated by gel filtration on AcA-54. Positions in the chromatogram at which specific sialoglycopeptides elute are indicated by ATI, AT2, BT1, and CTI. (b) These peptides are further purified by DEAE-cellulose ion-exchange chromatography as in Fig. 3. , Absorbance at 230 nm; .... , sialic acid at 549 nm; ---, fluorescence (excitation 290 nm; emission, 355 nm).

p H 8.0, at an e n z y m e - t o - s u b s t r a t e ratio of 1 : 30 for 20 hr at 37 °, the digest is brought to p H 4.5 b y dropwise addition of 1 N HCI. The precipitate is r e m o v e d b y centrifugation, and the peptides in the soluble fraction are separated by gel filtration on AcA-54 as described a b o v e (Fig. 4a, b). The peptides in the first two p e a k s are r e c h r o m a t o g r a p h e d by ion-exchange c h r o m a t o g r a p h y on D E A E - c e l l u l o s e equilibrated with 50 m M sodium formate, p H 6.1, as a b o v e to purify C T I and BT1, the sialoglycopeptide fragments of glycophorins B and C. Purity Glycophorin A is heterogeneous with regard to c a r b o h y d r a t e composition and amino acid sequence. The latter is related to the M N blood group antigens, m,25 and a h o m o g e n e o u s product can be obtained by using t y p e d blood of individuals h o m o z y g o u s for these antigens. F o r corrections of 2s K. Wasniowska, Z. Drzeniek, and E. Lisowska, Biochem. Biophys. Res. Commun. 76, 385 (1977).

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277

the amino acid sequence proposed earlier 9 in positions 11 and 17, it is referred to Dahr et al. 26 Heterogeneity of the oligosaccharides attached to threonine or serine has been inferred in the structural studies 9 and has been documented for the asparagine-linked oligosaccharide. 27 Similar heterogeneity in amino acid sequence exists for glycophorin B. Depending on the Ss-blood type in position 29 a methionine or threonine is present. 26 All of the glycophorins contain a hydrophobic region, but structural studies have been done only on glycophorin A. 28 Glycophorin preparations contain various contaminants that interact with this region tenaciously and are difficult to remove unless detergents such as SDS are used. Invariably, phospholipids, phosphatidylinositol, 29 heme, and possibly glycolipids can be detected in the preparations obtained by procedures as outlined above. In addition, it is difficult to exclude the possibility that minute amounts of other glycoproteins are present as contaminants. Chemical and Physicochemical Properties Glycophorins may be associated to form dimeric structures in the membrane. This is suggested on the basis of data on the protein in solution. When erythrocyte membranes or the isolated glycophorin A preparation are dissolved in sodium dodecyl sulfate-containing solutions and the proteins are analyzed by polyacrylamide gel electrophoresis, glycophorin A migrates as a homodimer that is in equilibrium with the monomeric form. The equilibrium is sensitive to ionic strength, concentration, temperature, SDS concentration, and other parameters. The site of interaction resides within the hydrophobic domain of the molecule. 24,3°,31 Circular dichroism 32 and proton nuclear magnetic resonance studies 33 suggest differences in the conformation of the three domains of glycophorin A - - t h e extracellular glycosylated, the intramembranous hydrophobic, and the cytoplasmic carboxy-terminal peptide regions. The hydrophobic domain assumes an t~-helical conformation that is stable in a wide range of 26 W. Dahr, K. Beyreuther, H. Steinbach, W. Gielen, and J. Kriiger, Hoppe-Seyler's Z. Physiol. Chem. 361, 895 (1980). 27 H. Yoshima, H. Furthrnayr, and A. Kobata, J. Biol. Chem. 255, 9713 (1980). H. Furthmayr, R. E. Galardy, M. Tomita, and V. T. Marchesi. Arch. Biochem. Biophys. 185, 21 (1978). 29 I. M. Armitage, D. L. Shapiro, H. Furthmayr, and V. T. Marchesi, Biochemistry 16, 1317 (1977). 30 M. Silverberg, H. Furthmayr, and V. T. Marchesi, Biochemistry 15, 1448 (1976). 3~ M. Silverberg and V. T. Marchesi, J. Biol. Chem. 253, 95 (1978). 32 T. H. Schulte and V. T. Marchesi, Biochemistry 18, 275 (1979). 33 j. A. Cramer, V. T. Marchesi, and I. M. Armitage, Biochim. Biophys. Acta 595, 235 (1980).

278

BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS •

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GLYCOPHORINS; ISOLATION OF SPECIFIC DOMAINS

279

FIG. 6. Frozen thin sections of intact human red blood cells incubated with ferritinwheat germ agglutination conjugates (a) or with specific ferritin-antibody conjugates (b). x264,000. Reproduced from Cotmore e t al. 36

solvents, including SDS, guanidine, urea, but is disrupted by incubation at 100° for 3 min or in trifluoroacetic acid. The proteins can aggregate to even larger molecular weight forms. 8 In aqueous buffers particles are formed that appear to consist of 5 or 6 dimeric structures or 10-12 molecules consistent with a molecular weight of about 400,000.1 As shown in Fig. 5, glycophorin A in preparations obtained after metal shadow-casting with carbon-platinum gives starlike structures. These particles apparently are formed by association within the hydrophobic domain. The hydrophilic glycosylated portions appear to extend from the center of the particle. These images suggest that the glycosylated domains of glycophorin A may be fairly rigid rods, although it is not clear that the technique of metal shadow-casting visualizes the entire structure with sufficient detail.

280

BIOGENESIS AND ASSEMBLY OF MEMBRANE PROTEINS

[2 l]

Orientation G l y c o p h o r i n A is a t r a n s m e m b r a n e p r o t e i n , a n d t h e l i n e a r a r r a n g e m e n t of the three domains--extracellular, intramembranous, and cyto-

p l a s m i c - i s evident not only from studies on the primary structure, but also from experiments performed with cells. The strongly negative charge of sialic acid was utilized to label cells with colloidal iron. Sialic acid can be removed from intact cells with neuraminidase, and the oligosaccharides on glycophorin can interact with a variety of lectins (Fig. 6). Similarly, blood group antigens of the MNSs system are recognized by specific antibodies on intact cells. 2 The intramembranous domain can be labeled with chemical probes that partition into the hydrophobic environment of the lipid bilayer. 34 Finally, the cytoplasmic segment has been shown to be phosphorylated in the mature red cell, albeit to a minor extent, suggesting that it can act as a substrate for intracellular and possibly membrane-bound phosphokinases. 35 The location of the cytoplasmic domain of glycophorin A at the inner surface of the membrane has been demonstrated ultrastructurally by labeling with antibodies to a specific antigenic determinant of ultrathin frozen sections of intact c e l l s 36 as shown in Fig. 6. There is no information available on the mode of insertion or transmembrane nature of glycophorins B and C. It is interesting to note that red cells of a small number of individuals were discovered, by virtue of serological abnormalities during blood group testing or in population studies, to lack one or the other of the glycophorins.l.10 These genetically variant red cells appear to be structurally and functionally normal. It remains to be seen whether these genetic defects are expressed only in the mature red cell and not also in erythroid precursor cells.

34 D. W. Goldman, J. S. Pober, G. White, and H. Bayly, Nature (London) 280, 841 (1982). 35 D, L. Shapiro and V. T. Marchesi, J. Biol. Chem. 252, 508 (1977). 36 S. F. Cotmore, H. Furthmayr, and V. T. Marchesi, J. Mol. Struct. 153, 539 (1977).