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Ultrastructure of a transmembrane glycoprotein, glycophorin A
TISSUE & CELL 1988 20 (2) 21Y-227 0 lY88 Longman Group UK Ltd
GARY E. WISE, LAWRENCE X. OAKFORD and JAMES
ULTRASTRUCTURE GLYCOPROTEIN,
K. DZANDU
OF A TRANSMEMBRANE GLYCOPHORIN A
Keyworda: Glycophorin A. rotary shadowing. catmmzcd ferrltm. electron m~crovxp). cyte memhranc
crbthro-
ABSTRACT. The major sialoglycoprotcin of the human red cell membrane, glycophorin A. wa isolated and examined by rotary shadowing and transmlssion electron microscopy. ‘Ihc glycophorin A molecule appeared as a cloud-like structure with a short. dense cow within a larpi: cloud. Mild acid hydrolysis in 0.05 M HISO,. 80°C for I hr reduced the ac ot the cloud significantly hut left the dense core intact indicatmg that the orlginal cloud reprcsentcd the sialylated oligosaccharide chains of glycophorin A wth the dense core being the polypeptide chain and its associated linkage proteins. Incubating glycophorin A with cationized fcrrnin (Cl-‘) revealed that the CF was bound only to the cloud. a finding that supports the view that the clotld is comprised of the sialylated ohgosaccharide chains of the glycophorm A molecule SDSpolyacrylamide gel electrophoresis revealed that our preparation of glycophorin A. a\ well a\ commeraal preparations, consisted of monomers, dimera and ohgomers of glycophorin A wth trace amounts of the minor glycophorins and lmkage proteins. Knowledge of the ultraatructulc of this important integral protein will enable one to design studies to dcterminc its functional role in the membrane.
Introduction Glycophorin A is a transmembrane glycoprotein of the red blood cell (RBC) that contains 15 O-linked oligosaccharide chains and 1 N-linked chain with the carbohydrate chains comprising about 60% (w/w) of the molecule (Thomas and Winzler, 1969; Tomita and Marchesi, 1975). Recentiy, it has been discovered that the lectin, wheat germ agglutinin (WGA), binds more to sickle RBCs that to normal RBCs, as well as having a more clustered distribution on the sickle cell surface (Wise et al., 1987). Because glycophorin A binds to a WGA-sepharose column (Bhavanandan and Katlic, 1979). it may be the molecule responsible for this difference in binding of WGA. This membrane surface difference between sickle and normal red ceils with respect to the conformation and distribution of glycophorin A is significant because in vitro, sickle RBCs adhere to endothelium more than normal RBCs (Hoover et al., 1979; Hebbel et al., 1980a), an Texas College of Osteopathic Medicine, Department of Anatomy. 3516 Camp Bowie Boulevard, Fort Worth. Texas 76107. Received I? January 1988. 219
adherence that may be the basis for vasoocclusive episodes that cause the painful crisis syndrome (Hebbel et al., 198Ob: Wise. 1984). In view of these findings it was decided to determine if isolated glycophorin A molecules could be visualized bv first rotarv shadowing the molecules with tantalum) tungsten followed by examination with the transmission electron microscope. As reported here, we were able to do so. as well as determine which portions of the visualized molecule represented the oligosaccharide and polypeptide chains. Materials and Methods isolation
of glycophorin
A
Glycophborin A was isolated and purified from red cell ghosts according to a modification of the method of Taraschi et ul. (1982) which was based on the butanol extraction procedure of Azuma etal. (1973) as described by Verpoorte (1975). The sialoglycoprotein fraction contained in the aqueous phase of the initial n-butanol extract was mixed with an equal volume of 50% phenol (v/v). After centrifugation, the phenol-poor aqueous
WISE ETAL.
phase was dialyzed extensively against 1-O mM Tris-HCl (pH 8-O) at 4°C for 3 days. The dialyzed solution was adjusted with 0.2 volume of 1 .O M Tris-HCI (pH 8-O) and then mixed with 2.1 volumes of 95% ethanol (-20°C). After incubation at 0-4’C for 2 hr, the precipitate formed was removed by centrifugation at 8000 g for 15 min. One volume of the clear supernatant fluid containing 60% ethanol was mixed with an equal volume of cold acetone (-20°C) and allowed to incubate at 4°C for 30 min. Glycophorin precipitates were harvested by centrifugation at 8000 g for 10 min and then dialyzed overnight against 1-O mM Tris-HCI (pH 8-O). Buffer salts were removed by dialysis against de-ionized distilled water. The extract was freeze-dried and stored in a desiccator at -20°C. The average yield of sialoglycoprotein was 22 mg powder per unit of packed red cells. As ascertained by periodic acid-Schiff (PAS) and the silver/Coomassie blue (Ag/CB) staining procedures (Dzandu et al., 1984), the preparation contained the entire array of the major and minor red cell membrane sialoglycoproteins. To obtain a pure isolate containing glycophorin A only, a sample of the lyophilized powder was dissolved in distilled water (5-10 mg/ml) and then chromatographed on Sepharose-6B (1 .Ox 60 cm) column (bed volume 39 ml, 12 ml/hr flow rate, and O-8 ml fraction volume). The elution buffer contained 1 .O mM Tris-HCl (pH 8.0). Column fractions were analyzed for protein by the method of Lowery et al. (1951); neuraminic acid was assayed by the method of Warren (1959) and gel electrophoretic profiles were ascertained using the method of Laemmhi (1970). Glycophorin A dimer eluted at a peak column volume of 13.6 ml. After dialysis overnight against deionized distilled water, the fractions containing glycophorin A exclusively were lyophilized and stored at -20°C until used.
Chemical removal of N-acetylneuraminic acid from glycophorin
A sample of lyophilized glycophorin A was dissolved in 0.05 M H2SO4 and then heated at 80°C for 1 hr as previously described by Aminoff et al. (1976). Under the established conditions, hydrolytic release of N-acetylneuraminic acid was virtually complete as determined by chemical analysis of released neuraminic acid. After hydrolysis, the sample was either dialyzed overnight against deionized distilled water or chromatographed on a Sepharose 6B column as described to remove released sugar. SDS/polyacrylamide
gel electrophoresis
Electrophoresis was performed on isotropic 11% (w/v) acrylamide slab gels using the discontinuous buffer system of Laemmli (1970). Using the double-staining technique devised by Dzandu et al. (1984) gels were first stained with silver followed by staining with Coomassie blue R-250. Parallel gels were stained with Coomassie blue or periodic acid-Schiff reagent as described by Fairbanks et al. (1971). Rotary shadowing and transmission electron microscopy
The glycophorin A was prepared for shadowing using a modified technique of Mould et al. (1985) in which a solution of glycophorin A (1 mg/ml) was placed in double distilled HZ0 at pH 3.0. This was then diluted to 0.11 mg/ml and a 5 ~1 aliquot was sandwiched between two pieces of mica for 7 min. The resultant mica sandwich with the adsorbed glycoprotein was separated and dried in a vacuum evaporator overnight. It was then rotary shadowed in a Balzer’s BAF 400 freeze-fracture apparatus at -196°C in which 2 nm of tantalum/tungsten was deposited from an angle of 6”. The replica on
Fig. 1. Transmission electron micrograph of an isolated glycophorin A molecule that had been rotary shadowed in a Balzer’s BAF 400 freeze-fracture apparatus. Arrows mark the ‘cloud’ of oligosaccharide chains at one end of the molecule. The double arrows (++) denote the two polypeptide chains of a dimer, possibly spectrin that is attached to the molecule. ~300,ooO.
222
WISE ETAL.
mica was placed in a closed jar with glacial acetic acid for 1 hr. The loosened replica was floated on to double distilled H20 where it was picked up on a 300 mesh grid and examined with an Hitachi H-600 TEM. Cationized ferritin labeling
To determine where on the glycophorin A molecule cationized ferritin (CF) would bind, an aqueous solution of glycophorin A (1 mg/ml) was incubated with cationized ferritin (Miles-Yeda Ltd) at a dilution of 1:5000 for 30 min at room temperature. The glycophorin A/CF solution was then prepared for rotary shadowing and transmission electron microscopy as described in the previous section. Results
As seen in Fig. 1, glycophorin A appears as a cloud-like matrix with a double stranded chain attached to it. The length of the chain in this micrograph is 183 nm (as measured from the edge of the cloud to the end of the chain) and approximates the contour length of a spectrin tetramer (Elgsaeter et al., 1986). The
portion of the chain outside the cloud is usually absent (see Fig. 2). Within the cloud, the chain continues but not as a dimer. The mean area of the cloud is 25,208 nm2 (N=22, s.d.=+18,775) and ranges from 975 to 73,000 nm2. The commercial preparations and our isolated preparation of glycophorin A appear morphologically similar. The polypeptide composition of the glycophorin A preparation isolated in our laboratory (see Materials and Methods section) or glycophorin A purchased from Sigma Chemical Co. was examined by SDS-gel electrophoresis. As seen in Fig. 3 (lanes l-6), glycophorin A dimer (M,=SS,OOO) is clearly the major component present on gels stained with Coomassie blue (CB, lanes 1,2), silver/ Coomassie blue (Ag/CB, lanes 3,4), or periodic acid-Schiff reagent (PAS, lanes 5, 6). Monomeric and oligomeric forms of glycophorin A can also be seen on these gels (lanes 1, 3-6). Also present on the gels but not easily seen in these photographs were small amounts of Coomassie blue stained band 4.1 (M,=SO,OOO), band 4.2 (M,=78,000) and spectrin. Slight amounts of the minor sialoglycoproteins were also pre-
Fig. 2. Electron micrograph of an isolated glycophorin A molecule from a commercial preparation (Sigma Chemical Co.) in which spectrin is not attached. The oligosaccharide chains resemble the wings of a butterfly (arrows) and the body of the butterfly is presumably the unfolded polypeptide chain(s) of the glycophorin A. x 133,000. Fig. 3. SDS-polyacrylamide gel electrophoresis of glycophorin A isolated as described in Materials and Methods section (lanes 1,3,5) or obtained from a commercial source (lanes 2.4, 6). Gels were stained with Coomassie blue (CB, lanes 1,2), silver/Coomassie blue double stain (Ag/CB, lanes 3, 4) or periodic acid-Schiff reagent (PAS, lanes 5, 6). Forty micrograms of protein were applied to gels stained with either Coomassie blue or silver/Coomassie blue, whereas for PAS stained gels, aliquots containing 120 pg were used. Glycophorin A dimer (M,=SS,ooO) can be seen to be the major polypeptide band stained on these gels. In addition to the minor sialoglycoproteins seen best in the PAS stained gel (lanes 5, 6), Coomassie stained erythrocyte membrane polypeptide band 4.1 (M,=80,0@3) band 4.2 (M,=78,000) and spectin bands 1 and 2 were also clearly visible on the original gels. Clearly visible on these gels (lanes 3-6) were at least three high M, forms which may be oligomeric forms of glycophorin A. Fig. 4. Preparation of glycophorin A that had been subjected to acid hydrolysis. As can be seen in this lower power electron micrograph, the cloud of oligosaccharide chains are absent and all that remains are intact or fragmented polypeptide chains. Arrow denotes one heterodimer which may be a spectrin molecule. ~124,000. Fig. 5. Electron micrograph of a glycophorin A molecule that had been incubated with cationized ferritin (CF) before being rotary shadowed. Small arrows denote the CF molecules binding to the cloud of oligosaccharide chains and the large arrow marks what may be the unfolded polypeptide chain of the glycophorin A molecule. Note the absence of CF in the background surrounding the glycophorin A molecule. ~240,000.
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3
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5
4
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W 85-O-
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67.0.
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- 66.2
.+W*
- 45-2
- 21-5 - 14.4
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PAS
ULTRASTRUCTURE
OF GLYCOPHORIN
A
sent but accounted for less than 2% of the total PAS stainable polypeptides seen (Fig. 3 lanes 5, 6). Acid hydrolysis greatly reduces the size of the cloud (Fig. 4). The small dense structures remaining are probably the non-hydrolyzed protein portions of the glycophorin A, any of the accompanying proteins that may be linked and possibly any remaining nonhydrolyzed oligosaccharide chains. Incubating glycophorin A with CF reveals that the CF binds only to the cloud as seen in Fig. 5. The CF does not bind to the chain portion of the molecule, nor does it appear to bind to the denser center of the cloud. Discussion
In interpreting the images of glycophorin A following rotary shadowing and transmission electron microscopy, one can conclude that the clouds visualized represent oligosaccharide chains. This inference is confirmed by subjecting glycophorin A to acid hydrolysis to remove the sialic acid residues and observing a significant reduction in the size of the cloud. In addition, incubating glycophorin A with cationized ferritin results in CF binding only to the cloud (Fig. 5). Because CF is known to bind to surface anionic sites in the RBC (primarily sialic acid), the observed CF binding reported in this study strongly suggests that the cloud represents sialylated oligosaccharide chains of the glycophorin molecule. Although CF could bind to phosphatidyl serine, it is unlikely that the clouds represent complexes of CF/phosphatidyl serine because the isolated glycophorin was subjected to exhaustive extractions with organic solvents during the procedure isolation purification (see Materials and Methods). The short, dense portion of each cloud probably represents the polypeptide chain of glycophorin A, 131 amino acids in the monomeric form (e.g. see Tomita et al., 1978), and band 4.1, the protein that links glycophorin A to spectrin (Anderson and Lovrien, 1984; Anderson and Marchesi, 1985). Occasionally, a double stranded chain is seen attached to the cloud (Fig. 1). The chain’s mean length of 171 nm (s.d.?35.8, N=13) is similar to the contour length of a spectrin tetramer (e.g. see ElgsaeteretaE., 1986). The fact that there are slight amounts of spectrin
225
and band 4.1 in the isolated glycophorin A preparations as determined by SDS-PAGE (see Fig. 3) also supports the above conclusions. Moreover, acid hydrolysis of the glycophorin A preparation results in removal of the cloud with only dense structures remaining (Fig. 4). As determined by SDS-PAGE (results not shown), the dense bodies which are resistant to acid hydrolysis were comprised of the protein portion of glycophorin A, with the remaining non-hydrolyzed oligosaccharide chains and band 4.1. In addition, an occasional tetramer comparable in size to a spectrin tetramer is observed in the acid hydrolyzed preparation. Whether or not glycophorin A exists in the intact membrane as monomers, oligomers or both is not known. It is known, however, that the oligosaccharide chains of the molecule are exposed on the external surface of the RBC (Gahmberg and Hakomori, 1973; Steck and Dawson, 1974; Gahmberg, 1976), at least to the extent that 90% of the cell’s total sialic acid on the surface can be removed by neuraminidase treatment of the intact cell (Eylar et al., 1962; Dzandu et al., 1984). Thus, variation in the size of the clouds may be explained in part by the fact that the preparations contain various oligomers, as well as the monomeric forms, of glycophorin A. Assuming that the smallest cloud was a monomer, 975 nm2 in area, and that there are 500,000 monomers of glycophorin A in a red cell (Steck, 1974) then the monomers would occupy an area of 487 pm’. The surface area of the red cell is approximately 163 ,um* (Eylar et al., 1962) which means that if only monomers existed in the membrane in a distended state they would cover the entire surface three times. However, the molecules may not be extended insitu to the degree they are in vitro when they are subjected to a pH of 3.0 and dried on mica. Another minor contribution to the possible variation in cloud size is the fact that small amounts of the minor sialoglycoproteins (less than 2% of the total PAS stainable polypeptides) were present. Regardless, it is interesting to note that all the molecules observed in our preparations had a similar morphology; i.e. a cloud with a short, dense portion within it. Thus, it is probable that all the glycophorins have a similar ultrastructure. Estimates based on dividing the mean area of the cloud (approximately 25,000 nm2), by the
226
WISE ET AL.
minimum size of the monomer (i.e. 975 nm2), suggest that 25 molecules may be present in the average cloud. Although the normal function of glycophorin A in the RBC membrane is unknown, the ability to visualize the overall morphology of the glycophorin molecule as shown in this study, coupled with the available biochemical data on the various structural domains will aid in determining the probable function of this molecule. The majority of the surface anionic sites of the red cell membrane appear to be due to sialic acid (N-acetylneuraminic acid) which is present in the glycophorin A molecules because removal of sialic acid for RBCs by neuraminidase reduces the electrophoretic mobility of RBCs by at least 95% (Eylar et al., 1962). It is likely that the net negative charge on the surface of RBCs due to sialic acid prevents their adherence to each other. However, there is a mutation in which RBCs lack glycophorin A and yet the red cells appear to function normally (Bachi et al., 1977; Gahmberg et al., 1978). Moreover, these red cells have normal profiles of intramembranous particles (IMPS) within the membrane in terms of size and number indicating that these particles are not composed of glycophorin A (Bachi et al., 1977).
Regardless of its normal function, there are indications that glycophorin A may play a role in the pathophysiology of erythrocytes. For example, we have recently discovered that glycophorin A has a different distribution and probably different conformation in sickle RBCs than in normal RBCs as determined by the binding of wheat germ agglutinin (WGA) (Wise et al., 1987). Because sickle RBCs adhere more to endothelium in vitro than do normal RBCs (Hoover et al., 1979; Hebbel et al., 1980a), the difference in adhesion may be due to the different distribution and/or conformation of glycophorin A molecules in the sickle cell membrane. Thus, future studies will focus on the binding of WGA to isolated glycophorin A molecules as well as determining if modification of the glycophorin A by proteolysis will prevent the binding of sickle cells to endothelium. Acknowledgements
This work was supported in part by a grant from the Texas College of Osteopathic Medicine to G.E.W. The authors would like to thank Carolyn Bannon for typing the manuscript and Stan Clason for preparation of the photographs.
References Aminoff, D., Bell, W. C., F&on, I. and Ingebrigtsen, N. 1976. Effect of sialidase on the viability of erythrocyte in circulation. Am. J. HemafoL, 1, 419-432. Anderson, R. A. and Lovrien, R. E. 1984. Glycophorin is linked by band 4.1 protein to the human erythrocyte membrane skeleton. Nature, Land., 307, 655460. Anderson, R. A. and Marchesi, V. T. 1985. Regulation of the association of membrane skeleton protein 4.1 with glycophorin by a phosphoinositide. Nature, Land., 318, 295298. Azuma, J. I., Janada, M. and Onodera, K. 1973. Effect of glycophorin on lipid polymorphism. A 31P-NMR study. Biochim. Biophys. Acta, 685, 153-159. Bachi, T., Whiting, K., Tanner, M. J. A., Metaxes, M. N. and Anstee, D. J. 1977. Freeze-fracture electron microscopy of human erythrocyte lacking the major membrane sialoglycoprotein. Biochim. Biophys. Acta, 464, 635-639. Bhavanandan, V. P. and Kattic, A. W. 1979. The interaction of wheat agglutinin with sialoglycoproteins. The role of sialic acid. J. BioL Chem., 254, 4000-4008. Danon, D., Goldstein, L., Marikovsky, Y. and Skutelsky, E. 1972. Use of cationized ferritin as a label of negative charges on cell surfaces. J. Urrastrucr. Rex, 38, 500-510. Dzandu, J. K., Deh, M. E., Barr&t, D. L. and Wise, G. E. 1984. Detection of erythrocyte membrane proteins, sialoglycoproteins and lipids, in the same polyacrylamide gel using a novel staining technique. Proc. nad. Acnd. Sci. U.S.A., 81, 173S1737. Elgasaeter, A., Stokke, B. T., Mikkelsen, A. and Branton, D. 1986. The mole&r basis of erythrocyte shape. Science, 234, 1217-1233. Eylar, E. H., Madoff, M. A., Brody, D. V. and Oncley, J. L. 1962. The contribution of sialic acid to the surface charge of the erythrocyte. J. Biol. Chew., 237, 1992-2000.
ULTRASTRUCTURE Fairbanks.
OF GLYCOPHORIN
G., Steck, T. L. and Wallach,
erythrocyte membrane. Gahmberg, C. G., Tauren,
Biochembrry, G., Virtanen,
227
A
D. F. H. 1971. Electrophoretic 10, 26006-2617. I. and Wartiovaora,
analysisof
J. 1978. Distribution
the majorpolypeptideof of glycophorm
the human
on the surface of
freeze-fracture study of normal and En(a-)erythrocytes. J. Supmmolec. Struct., 8, 337-347. Gahmberg, C. G. 1976. External labelling of human erythrocyte glycoproteins. J. Biol. Chem , 251. 511&515 Gahmberg, C. G. and Hakomori, S. 1973. External labelling of cell surface glactose and galactosamine in glycolipid and glycoprotein of human erythrocytes. J. Biol. Chem., 248, 4311-4317. Hebbel, R. P., Yamada, O., Moldow, C., Jacob. H. S., White, J. G. and Eaton, J. W. 198Oa. Abnormal adherence of sickle erythrocytes to cultured vascular endothelium. J. Clin. Invest., 65, 154-160 Hebbel, R. P., Boogaerts, M. A. B., Eaton, J. W. and Steinberg, M. H. 1980b. Erythrocyte adherence toendothelium In sickle cell anemia: a possible determinant of disease severity. New En@. J. Med., 302, 992-995. Hoover, R.. Rubin. R., Wise, G., and Warren, R. 1979. Adhesion of normal and sickle erythrocytes to endothehal monolayer cultures. Blood. 54, 872-876. L.aemmh, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage 7,. l’uturr. Land., 227, 68&685. Lowry. 0. H., Rosebrough, N. .I., Farr, A. L. and Randall, R. J 1951. Protein measurement with the F&n phenol reagent. 1. Biol. Chent.. 193, 265-275. Mould. A P., Holmes. D. F., Kadler. K. E. and Chapman, J. A. 1985. Mica sandwch techmque for prcparmg macromolecules for rotary shadowing. J. Uftractruct. Rev., 91, 6676. Steck, T. L. 1974. The organization of proteins in the human red blood cell membrane (a review). 1. Cell Rio/. 62. l-19 Steck. T. L. and Dawson, G. 1974. Topographical distribution of complex carbohydrates in the erythrocyte membrane. 1. Biol. Chem., 249, 2135-2142. Taraschi. T.. DeKruijff. B., Verkleij, A. and Van Echteld, C. J. A. 1982. Heterogeneity of a human erythrocytc membrane glycoprotein. J. Biochem.. 73, 1127-1134. Thomas. D. B. and Win&r, R. J. 1969. Structural studies on human erythrocyte glycoproteins. J Rio/. Chern.. 244, 5943-5946. Tomita. M and Marchesi, V. T. 1975. Amino-acid sequence and oligosaccharide attachment sites of human erythrocytc glycophorin. Proc. natl. Acad. Sci. U.S.A., 72, 29642968. Tomlta. M., Furthmayr, H. and Marchesi, V. T. 1978. Primary structure of human erythrocyte glycophorm A Iwlatmn and characrerization of peptides and complete amino acid sequence. Biochemistry, 17, 47564770. Vcrpoorte. J A. 1975. Purification and characterization of glycoprotein from human crythrocyte membrane\ ini. J Biochem., 6, 855-862. Warren. L. 1959. The thiobarbituric acid assay of sialic acids. J. Bio[. Chem., 234, 1971-1975 Wise, G. E. 1984 Identification and function of transmembrane glycoproteins-the red cell model ?‘rssue & (‘r[/. 16, 665-676. Ww. G. E., Oakford, L. X. and Cantu-Crouch, D. B. 1987. A comparison of wheat germ agglutinin binding between normal and sickle red blood cells. Cell Es. Res., 248, 267-273.
GARY E. WISE, LAWRENCE X. OAKFORD and JAMES
ULTRASTRUCTURE GLYCOPROTEIN,
K. DZANDU
OF A TRANSMEMBRANE GLYCOPHORIN A
Keyworda: Glycophorin A. rotary shadowing. catmmzcd ferrltm. electron m~crovxp). cyte memhranc
crbthro-
ABSTRACT. The major sialoglycoprotcin of the human red cell membrane, glycophorin A. wa isolated and examined by rotary shadowing and transmlssion electron microscopy. ‘Ihc glycophorin A molecule appeared as a cloud-like structure with a short. dense cow within a larpi: cloud. Mild acid hydrolysis in 0.05 M HISO,. 80°C for I hr reduced the ac ot the cloud significantly hut left the dense core intact indicatmg that the orlginal cloud reprcsentcd the sialylated oligosaccharide chains of glycophorin A wth the dense core being the polypeptide chain and its associated linkage proteins. Incubating glycophorin A with cationized fcrrnin (Cl-‘) revealed that the CF was bound only to the cloud. a finding that supports the view that the clotld is comprised of the sialylated ohgosaccharide chains of the glycophorm A molecule SDSpolyacrylamide gel electrophoresis revealed that our preparation of glycophorin A. a\ well a\ commeraal preparations, consisted of monomers, dimera and ohgomers of glycophorin A wth trace amounts of the minor glycophorins and lmkage proteins. Knowledge of the ultraatructulc of this important integral protein will enable one to design studies to dcterminc its functional role in the membrane.
Introduction Glycophorin A is a transmembrane glycoprotein of the red blood cell (RBC) that contains 15 O-linked oligosaccharide chains and 1 N-linked chain with the carbohydrate chains comprising about 60% (w/w) of the molecule (Thomas and Winzler, 1969; Tomita and Marchesi, 1975). Recentiy, it has been discovered that the lectin, wheat germ agglutinin (WGA), binds more to sickle RBCs that to normal RBCs, as well as having a more clustered distribution on the sickle cell surface (Wise et al., 1987). Because glycophorin A binds to a WGA-sepharose column (Bhavanandan and Katlic, 1979). it may be the molecule responsible for this difference in binding of WGA. This membrane surface difference between sickle and normal red ceils with respect to the conformation and distribution of glycophorin A is significant because in vitro, sickle RBCs adhere to endothelium more than normal RBCs (Hoover et al., 1979; Hebbel et al., 1980a), an Texas College of Osteopathic Medicine, Department of Anatomy. 3516 Camp Bowie Boulevard, Fort Worth. Texas 76107. Received I? January 1988. 219
adherence that may be the basis for vasoocclusive episodes that cause the painful crisis syndrome (Hebbel et al., 198Ob: Wise. 1984). In view of these findings it was decided to determine if isolated glycophorin A molecules could be visualized bv first rotarv shadowing the molecules with tantalum) tungsten followed by examination with the transmission electron microscope. As reported here, we were able to do so. as well as determine which portions of the visualized molecule represented the oligosaccharide and polypeptide chains. Materials and Methods isolation
of glycophorin
A
Glycophborin A was isolated and purified from red cell ghosts according to a modification of the method of Taraschi et ul. (1982) which was based on the butanol extraction procedure of Azuma etal. (1973) as described by Verpoorte (1975). The sialoglycoprotein fraction contained in the aqueous phase of the initial n-butanol extract was mixed with an equal volume of 50% phenol (v/v). After centrifugation, the phenol-poor aqueous
WISE ETAL.
phase was dialyzed extensively against 1-O mM Tris-HCl (pH 8-O) at 4°C for 3 days. The dialyzed solution was adjusted with 0.2 volume of 1 .O M Tris-HCI (pH 8-O) and then mixed with 2.1 volumes of 95% ethanol (-20°C). After incubation at 0-4’C for 2 hr, the precipitate formed was removed by centrifugation at 8000 g for 15 min. One volume of the clear supernatant fluid containing 60% ethanol was mixed with an equal volume of cold acetone (-20°C) and allowed to incubate at 4°C for 30 min. Glycophorin precipitates were harvested by centrifugation at 8000 g for 10 min and then dialyzed overnight against 1-O mM Tris-HCI (pH 8-O). Buffer salts were removed by dialysis against de-ionized distilled water. The extract was freeze-dried and stored in a desiccator at -20°C. The average yield of sialoglycoprotein was 22 mg powder per unit of packed red cells. As ascertained by periodic acid-Schiff (PAS) and the silver/Coomassie blue (Ag/CB) staining procedures (Dzandu et al., 1984), the preparation contained the entire array of the major and minor red cell membrane sialoglycoproteins. To obtain a pure isolate containing glycophorin A only, a sample of the lyophilized powder was dissolved in distilled water (5-10 mg/ml) and then chromatographed on Sepharose-6B (1 .Ox 60 cm) column (bed volume 39 ml, 12 ml/hr flow rate, and O-8 ml fraction volume). The elution buffer contained 1 .O mM Tris-HCl (pH 8.0). Column fractions were analyzed for protein by the method of Lowery et al. (1951); neuraminic acid was assayed by the method of Warren (1959) and gel electrophoretic profiles were ascertained using the method of Laemmhi (1970). Glycophorin A dimer eluted at a peak column volume of 13.6 ml. After dialysis overnight against deionized distilled water, the fractions containing glycophorin A exclusively were lyophilized and stored at -20°C until used.
Chemical removal of N-acetylneuraminic acid from glycophorin
A sample of lyophilized glycophorin A was dissolved in 0.05 M H2SO4 and then heated at 80°C for 1 hr as previously described by Aminoff et al. (1976). Under the established conditions, hydrolytic release of N-acetylneuraminic acid was virtually complete as determined by chemical analysis of released neuraminic acid. After hydrolysis, the sample was either dialyzed overnight against deionized distilled water or chromatographed on a Sepharose 6B column as described to remove released sugar. SDS/polyacrylamide
gel electrophoresis
Electrophoresis was performed on isotropic 11% (w/v) acrylamide slab gels using the discontinuous buffer system of Laemmli (1970). Using the double-staining technique devised by Dzandu et al. (1984) gels were first stained with silver followed by staining with Coomassie blue R-250. Parallel gels were stained with Coomassie blue or periodic acid-Schiff reagent as described by Fairbanks et al. (1971). Rotary shadowing and transmission electron microscopy
The glycophorin A was prepared for shadowing using a modified technique of Mould et al. (1985) in which a solution of glycophorin A (1 mg/ml) was placed in double distilled HZ0 at pH 3.0. This was then diluted to 0.11 mg/ml and a 5 ~1 aliquot was sandwiched between two pieces of mica for 7 min. The resultant mica sandwich with the adsorbed glycoprotein was separated and dried in a vacuum evaporator overnight. It was then rotary shadowed in a Balzer’s BAF 400 freeze-fracture apparatus at -196°C in which 2 nm of tantalum/tungsten was deposited from an angle of 6”. The replica on
Fig. 1. Transmission electron micrograph of an isolated glycophorin A molecule that had been rotary shadowed in a Balzer’s BAF 400 freeze-fracture apparatus. Arrows mark the ‘cloud’ of oligosaccharide chains at one end of the molecule. The double arrows (++) denote the two polypeptide chains of a dimer, possibly spectrin that is attached to the molecule. ~300,ooO.
222
WISE ETAL.
mica was placed in a closed jar with glacial acetic acid for 1 hr. The loosened replica was floated on to double distilled H20 where it was picked up on a 300 mesh grid and examined with an Hitachi H-600 TEM. Cationized ferritin labeling
To determine where on the glycophorin A molecule cationized ferritin (CF) would bind, an aqueous solution of glycophorin A (1 mg/ml) was incubated with cationized ferritin (Miles-Yeda Ltd) at a dilution of 1:5000 for 30 min at room temperature. The glycophorin A/CF solution was then prepared for rotary shadowing and transmission electron microscopy as described in the previous section. Results
As seen in Fig. 1, glycophorin A appears as a cloud-like matrix with a double stranded chain attached to it. The length of the chain in this micrograph is 183 nm (as measured from the edge of the cloud to the end of the chain) and approximates the contour length of a spectrin tetramer (Elgsaeter et al., 1986). The
portion of the chain outside the cloud is usually absent (see Fig. 2). Within the cloud, the chain continues but not as a dimer. The mean area of the cloud is 25,208 nm2 (N=22, s.d.=+18,775) and ranges from 975 to 73,000 nm2. The commercial preparations and our isolated preparation of glycophorin A appear morphologically similar. The polypeptide composition of the glycophorin A preparation isolated in our laboratory (see Materials and Methods section) or glycophorin A purchased from Sigma Chemical Co. was examined by SDS-gel electrophoresis. As seen in Fig. 3 (lanes l-6), glycophorin A dimer (M,=SS,OOO) is clearly the major component present on gels stained with Coomassie blue (CB, lanes 1,2), silver/ Coomassie blue (Ag/CB, lanes 3,4), or periodic acid-Schiff reagent (PAS, lanes 5, 6). Monomeric and oligomeric forms of glycophorin A can also be seen on these gels (lanes 1, 3-6). Also present on the gels but not easily seen in these photographs were small amounts of Coomassie blue stained band 4.1 (M,=SO,OOO), band 4.2 (M,=78,000) and spectrin. Slight amounts of the minor sialoglycoproteins were also pre-
Fig. 2. Electron micrograph of an isolated glycophorin A molecule from a commercial preparation (Sigma Chemical Co.) in which spectrin is not attached. The oligosaccharide chains resemble the wings of a butterfly (arrows) and the body of the butterfly is presumably the unfolded polypeptide chain(s) of the glycophorin A. x 133,000. Fig. 3. SDS-polyacrylamide gel electrophoresis of glycophorin A isolated as described in Materials and Methods section (lanes 1,3,5) or obtained from a commercial source (lanes 2.4, 6). Gels were stained with Coomassie blue (CB, lanes 1,2), silver/Coomassie blue double stain (Ag/CB, lanes 3, 4) or periodic acid-Schiff reagent (PAS, lanes 5, 6). Forty micrograms of protein were applied to gels stained with either Coomassie blue or silver/Coomassie blue, whereas for PAS stained gels, aliquots containing 120 pg were used. Glycophorin A dimer (M,=SS,ooO) can be seen to be the major polypeptide band stained on these gels. In addition to the minor sialoglycoproteins seen best in the PAS stained gel (lanes 5, 6), Coomassie stained erythrocyte membrane polypeptide band 4.1 (M,=80,0@3) band 4.2 (M,=78,000) and spectin bands 1 and 2 were also clearly visible on the original gels. Clearly visible on these gels (lanes 3-6) were at least three high M, forms which may be oligomeric forms of glycophorin A. Fig. 4. Preparation of glycophorin A that had been subjected to acid hydrolysis. As can be seen in this lower power electron micrograph, the cloud of oligosaccharide chains are absent and all that remains are intact or fragmented polypeptide chains. Arrow denotes one heterodimer which may be a spectrin molecule. ~124,000. Fig. 5. Electron micrograph of a glycophorin A molecule that had been incubated with cationized ferritin (CF) before being rotary shadowed. Small arrows denote the CF molecules binding to the cloud of oligosaccharide chains and the large arrow marks what may be the unfolded polypeptide chain of the glycophorin A molecule. Note the absence of CF in the background surrounding the glycophorin A molecule. ~240,000.
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ULTRASTRUCTURE
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sent but accounted for less than 2% of the total PAS stainable polypeptides seen (Fig. 3 lanes 5, 6). Acid hydrolysis greatly reduces the size of the cloud (Fig. 4). The small dense structures remaining are probably the non-hydrolyzed protein portions of the glycophorin A, any of the accompanying proteins that may be linked and possibly any remaining nonhydrolyzed oligosaccharide chains. Incubating glycophorin A with CF reveals that the CF binds only to the cloud as seen in Fig. 5. The CF does not bind to the chain portion of the molecule, nor does it appear to bind to the denser center of the cloud. Discussion
In interpreting the images of glycophorin A following rotary shadowing and transmission electron microscopy, one can conclude that the clouds visualized represent oligosaccharide chains. This inference is confirmed by subjecting glycophorin A to acid hydrolysis to remove the sialic acid residues and observing a significant reduction in the size of the cloud. In addition, incubating glycophorin A with cationized ferritin results in CF binding only to the cloud (Fig. 5). Because CF is known to bind to surface anionic sites in the RBC (primarily sialic acid), the observed CF binding reported in this study strongly suggests that the cloud represents sialylated oligosaccharide chains of the glycophorin molecule. Although CF could bind to phosphatidyl serine, it is unlikely that the clouds represent complexes of CF/phosphatidyl serine because the isolated glycophorin was subjected to exhaustive extractions with organic solvents during the procedure isolation purification (see Materials and Methods). The short, dense portion of each cloud probably represents the polypeptide chain of glycophorin A, 131 amino acids in the monomeric form (e.g. see Tomita et al., 1978), and band 4.1, the protein that links glycophorin A to spectrin (Anderson and Lovrien, 1984; Anderson and Marchesi, 1985). Occasionally, a double stranded chain is seen attached to the cloud (Fig. 1). The chain’s mean length of 171 nm (s.d.?35.8, N=13) is similar to the contour length of a spectrin tetramer (e.g. see ElgsaeteretaE., 1986). The fact that there are slight amounts of spectrin
225
and band 4.1 in the isolated glycophorin A preparations as determined by SDS-PAGE (see Fig. 3) also supports the above conclusions. Moreover, acid hydrolysis of the glycophorin A preparation results in removal of the cloud with only dense structures remaining (Fig. 4). As determined by SDS-PAGE (results not shown), the dense bodies which are resistant to acid hydrolysis were comprised of the protein portion of glycophorin A, with the remaining non-hydrolyzed oligosaccharide chains and band 4.1. In addition, an occasional tetramer comparable in size to a spectrin tetramer is observed in the acid hydrolyzed preparation. Whether or not glycophorin A exists in the intact membrane as monomers, oligomers or both is not known. It is known, however, that the oligosaccharide chains of the molecule are exposed on the external surface of the RBC (Gahmberg and Hakomori, 1973; Steck and Dawson, 1974; Gahmberg, 1976), at least to the extent that 90% of the cell’s total sialic acid on the surface can be removed by neuraminidase treatment of the intact cell (Eylar et al., 1962; Dzandu et al., 1984). Thus, variation in the size of the clouds may be explained in part by the fact that the preparations contain various oligomers, as well as the monomeric forms, of glycophorin A. Assuming that the smallest cloud was a monomer, 975 nm2 in area, and that there are 500,000 monomers of glycophorin A in a red cell (Steck, 1974) then the monomers would occupy an area of 487 pm’. The surface area of the red cell is approximately 163 ,um* (Eylar et al., 1962) which means that if only monomers existed in the membrane in a distended state they would cover the entire surface three times. However, the molecules may not be extended insitu to the degree they are in vitro when they are subjected to a pH of 3.0 and dried on mica. Another minor contribution to the possible variation in cloud size is the fact that small amounts of the minor sialoglycoproteins (less than 2% of the total PAS stainable polypeptides) were present. Regardless, it is interesting to note that all the molecules observed in our preparations had a similar morphology; i.e. a cloud with a short, dense portion within it. Thus, it is probable that all the glycophorins have a similar ultrastructure. Estimates based on dividing the mean area of the cloud (approximately 25,000 nm2), by the
226
WISE ET AL.
minimum size of the monomer (i.e. 975 nm2), suggest that 25 molecules may be present in the average cloud. Although the normal function of glycophorin A in the RBC membrane is unknown, the ability to visualize the overall morphology of the glycophorin molecule as shown in this study, coupled with the available biochemical data on the various structural domains will aid in determining the probable function of this molecule. The majority of the surface anionic sites of the red cell membrane appear to be due to sialic acid (N-acetylneuraminic acid) which is present in the glycophorin A molecules because removal of sialic acid for RBCs by neuraminidase reduces the electrophoretic mobility of RBCs by at least 95% (Eylar et al., 1962). It is likely that the net negative charge on the surface of RBCs due to sialic acid prevents their adherence to each other. However, there is a mutation in which RBCs lack glycophorin A and yet the red cells appear to function normally (Bachi et al., 1977; Gahmberg et al., 1978). Moreover, these red cells have normal profiles of intramembranous particles (IMPS) within the membrane in terms of size and number indicating that these particles are not composed of glycophorin A (Bachi et al., 1977).
Regardless of its normal function, there are indications that glycophorin A may play a role in the pathophysiology of erythrocytes. For example, we have recently discovered that glycophorin A has a different distribution and probably different conformation in sickle RBCs than in normal RBCs as determined by the binding of wheat germ agglutinin (WGA) (Wise et al., 1987). Because sickle RBCs adhere more to endothelium in vitro than do normal RBCs (Hoover et al., 1979; Hebbel et al., 1980a), the difference in adhesion may be due to the different distribution and/or conformation of glycophorin A molecules in the sickle cell membrane. Thus, future studies will focus on the binding of WGA to isolated glycophorin A molecules as well as determining if modification of the glycophorin A by proteolysis will prevent the binding of sickle cells to endothelium. Acknowledgements
This work was supported in part by a grant from the Texas College of Osteopathic Medicine to G.E.W. The authors would like to thank Carolyn Bannon for typing the manuscript and Stan Clason for preparation of the photographs.
References Aminoff, D., Bell, W. C., F&on, I. and Ingebrigtsen, N. 1976. Effect of sialidase on the viability of erythrocyte in circulation. Am. J. HemafoL, 1, 419-432. Anderson, R. A. and Lovrien, R. E. 1984. Glycophorin is linked by band 4.1 protein to the human erythrocyte membrane skeleton. Nature, Land., 307, 655460. Anderson, R. A. and Marchesi, V. T. 1985. Regulation of the association of membrane skeleton protein 4.1 with glycophorin by a phosphoinositide. Nature, Land., 318, 295298. Azuma, J. I., Janada, M. and Onodera, K. 1973. Effect of glycophorin on lipid polymorphism. A 31P-NMR study. Biochim. Biophys. Acta, 685, 153-159. Bachi, T., Whiting, K., Tanner, M. J. A., Metaxes, M. N. and Anstee, D. J. 1977. Freeze-fracture electron microscopy of human erythrocyte lacking the major membrane sialoglycoprotein. Biochim. Biophys. Acta, 464, 635-639. Bhavanandan, V. P. and Kattic, A. W. 1979. The interaction of wheat agglutinin with sialoglycoproteins. The role of sialic acid. J. BioL Chem., 254, 4000-4008. Danon, D., Goldstein, L., Marikovsky, Y. and Skutelsky, E. 1972. Use of cationized ferritin as a label of negative charges on cell surfaces. J. Urrastrucr. Rex, 38, 500-510. Dzandu, J. K., Deh, M. E., Barr&t, D. L. and Wise, G. E. 1984. Detection of erythrocyte membrane proteins, sialoglycoproteins and lipids, in the same polyacrylamide gel using a novel staining technique. Proc. nad. Acnd. Sci. U.S.A., 81, 173S1737. Elgasaeter, A., Stokke, B. T., Mikkelsen, A. and Branton, D. 1986. The mole&r basis of erythrocyte shape. Science, 234, 1217-1233. Eylar, E. H., Madoff, M. A., Brody, D. V. and Oncley, J. L. 1962. The contribution of sialic acid to the surface charge of the erythrocyte. J. Biol. Chew., 237, 1992-2000.
ULTRASTRUCTURE Fairbanks.
OF GLYCOPHORIN
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