Int. J. Eiochem. Vol. 24, No. II, pp. 170~1709, 1992 Rioted in Great Britain. All rights reserved

0020-711X/92 SS.00 + 0.00 Copyright 0 1992 Pergamon Press Ltd

RAT ERYTHROCYTE GLYCOPHORINS CAN BE ISOLATED BY THE LITHIUM DIIODOSALICYLATE METHOD USED FOR OTHER GLYCOPHORINS ANGEL HERRAEZ, Josh C. DIEZ and Jos6 LUQUE Departamento

de Bioquimica y Biologia Molecular, Universidad de Alcald, 28871 Alcalb de Henares, Madrid, Spain [Tel. +34-l-885 45 28; Fax +34-l-885 45 441 (Received 22 May 1992)

Attstraet-1. The lithium diiodosalicylate/phenol method, widely employed for the isolation of membrane sialoglycoproteins (glycophorins) from mammalian erythrccytes, was applied for the first time to the purification of homologous glycoproteins from rat erythrocyte membranes. 2. The resulting preparations showed to be composed of four components, fractionated on SDS-PAGE. All four were positive for periodic acid-Schiff’s reagent stain, the two largest of them being major. 3. Isolated rat glycophorins accounted for 60% of the ghost sialic acid and 1.5% of their protein. The presence of O-acetyl groups was confirmed in one-third of the sialic acid residues. 4. The molecular masses of the four glycophorin components were determined by a method which takes into account the anomalous mobility of glycoproteins on SDS-electrophoresis. Estimated values thus obtained for the actual molecular masses were 74, 32, 25 and 17 kDa.

INTRODUCTION

Glycophorins are the major sialoglycoproteins on mammalian erythrocyte membrane. On human erythrocytes this family of glycoproteins is responsible for a major part of the negative charge of the cell surface; furthermore, they bear determinants for blood groups and for the interaction with viruses, bacteria, lectins and another proteins both in membrane and the submembranous cytoskeletal network (Marchesi et al., 1976; Bizot, 1990; Anderson and Lovrien, 1984; Bhavanandan and Katlic, 1979; Paul and Lee, 1987). Homologous sialoglycoproteins have been found in erythrocytes from various animal species (Krotkiewski, 1988), though no functional role has yet been conclusively ascribed to them. These sialoglycoproteins are restricted to the erythroid cell lineage, and changes in them have been described in association with differentiation, maturation and aging of the cell (Fukuda and Fukuda, 1981; Loken et al., 1987; Skutelsky and Farquhar, 1976). A study in depth of the involvement of such sialoglycoproteins in these processes requires, first of all, their obtention in purified form. The method most widely used for the isolation of human glycophorins is that using lithium diiodosalicylate (LIS) solubilization and phenol extraction (Marchesi and Andrews, 1971; Marchesi, 1972). It has also been successfully applied to erythrocytes of other species, such as rabbit (Honma et al., 1982), dog (Murayama ef al., 1983), pig (Kawashima et al., 1982) cow (Murayama et al., 1982) horse (Murayama et al., 1981), mouse (Sarris and Palade,

1982) and monkey (Murayama et al., 1989) (see Krotkievski (1988) for a review). This is a well-established method which has allowed the comparison of glycophorins from different species, but in rat no successful attempt of isolation by this method has been reported. In this work we carried out the isolation of rat erythrocyte glycophorins by the LIS extraction method, as a prerequisite for the further study of their possible implication as markers of differentiation and aging. MATERIAL AND METHODS Preparation

of ghosts

Blood from male Wistar rats (Rotter noroegicus Berk var. alba, 150-200g weight) was drawn into cold 320 mosM citrate/NaCl buffer (PH 7.4) (40 mM sodium citrate/80 mM NaCl adjusted to pH 7.4 with 160 mM citric acid/80 mM NaCI). Erythrocytes were sedimented at 500g for 10 min at 4°C and plasma and buffy coat removed. The cell pellet was washed three times with 320mosM phosphate buffer t&H 7.4) (16OmM NaH,PO, adjusted to pH 7.4 with 107 mM Na,HPG,). Erythrocyte ghosts were prepared by hypotonic lysis in 30-40 volumes of 20mosM phosphate buffer @H 7.4) as described (Fairbanks et al., 1971). Human ghosts were prepared similarly to rat ghosts, from blood from healthy donors. Purification of glycophorins

Lyophilized ghosts were resuspended in 0.3 M LIS (lithium diiodosalicylate, Sigma)/O.OSM Tris (PH 7.5) at 25 mg protein/ml. This suspension was stirred for 15 min at room temperature, 2 volumes of cold water added, and

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ANGEL HERR& et al.

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further stirred at 4°C for another 10 min. From then on, all steps were conducted at 4°C. After centrifuging at 45,OOOg for 90 min, the supematant was mixed with an equal volume of freshly made 50% (w/v) aqueous phenol and stirred for 15 min. Aqueous phase was separated by centrifugation at 4000g for 60 min, dialyzed against distilled water for 2436 hr and finally lyophilized. The resulting material was suspended in cold absolute ethanol and mixed for l-2 hr at 4°C. The suspension was centrifuged at 16,ooOg for 30 min and the pellet similarly washed another 3 times with ethanol. The final pellet was dissolved in water, dialyzed overnight and clarified by centrifugation at lO,OOOgfor 30 min. The resulting supematant constituted the purified preparation of glycophorins and was stored frozen. For the analysis of the purification process, samples were separated at three different stages: (a) the initial ghosts before lyophili~tion, (b) the aqueous phase subsequent to phenol extraction, after being dialyzed and (c) the final purified preparation. As a control for the process and for comparison studies, glycophorins were also isolated from human erythrocytes ~ploying the same method. Analylical determinations

Protein was determined by the Lowry method (Lowry ef al., 1951) using crystalline bovine serum albumin (Merck} as standard. Sialic acid was measured using the thiobarbituric acid method (Warren, 1959), after release from the sialoglycoproteins by incubation at 80°C in 50mM H,SO, for 60min. For the determination to include 0-acetylsialic acids, protein samples were first treated for 45 min in ice-water bath with 0.1 M NaOH and then neutralized and hydrolyzed in 50 mM H,SO, as above.

11% polyacrylamide slab gels were used for the analysis of samples, in the presence of SDS, using the discontinuous buffer system of Laemmli (1970). Proteins were detected with Coomassie blue (Fairbanks er al., 1971) and glycoproteins using the periodic acid-ehiff’s reagent stain (Zacharius et al., 1969). For the determination of molecular mass of glycophorins, rod gels of several different concentrations of acrylamide were used instead. Two sets of standards were used: first, a mixture of /3_galactosidase (116 kDa), phosphorylase b (97.4 kDa), serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), p-lactoglobulin (18.4 kDa) and lysozyme

(14.3 kDa) (Sigma), revealed by Coomassie blue staining, and second, ~-macroglobulin (180 kDa), ~-galactosid~

(116 kDa), fructose-6-phosphate kinase (84 kDa), pyruvate kinase (58 kDa), fumarase (48.5 kDa), lactate dehydrogenase (36.5 kDa) and triose-phosphate isomerase (26.6 kDa)

(Sigma prestained markers). RESULTS AND DISCUSSION

The LIS/phenol method of Marchesi and coworkers (1971, 1972) was applied to the isolation of glycophorins from rat erythrocyte ghosts. The progress of purification was checked through quantitation of protein and sialic acid contents in three different stages along the process (Table 1). After solubilization of the ghosts with LIS and subsequent phenol partition, an aqueous extract was obtained which contained 60% of the ghosts sialic acid with only 7% of the protein, showing a preferential retention of sialoglycoproteins along the isolation procedure. After the final ethanol washes, 1.5% of the protein was recovered, still containing 32% of the membrane sialic acid. The sialic~protein ratio, used as an index of the enrichment in sialoglycoproteins achieved, consequently increased by 1l-fold from the initial erythrocyte ghosts to the post-phenolic aqueous phase, and a further nearly 2-fold to the final glycophorins preparation (a total of 19-fold with respect to ghosts). Similar values were obtained in a control isolation of human glycophorins (see Table 1). Hence, the method seems readily applicable to rat glycophorins. It is known that, unlike human erythrocytes, murine and rat red cells contain 0-acetylated sialic acids and that these are not reactive on the usual assay for sialic acids unless the O-acetyl groups are previously removed by alkaline hydrolysis (Sarris and Palade, 1979; Reuter et al., 1980). To account for this, sialic acids were determined both with and without previous treatment of the samples with sodium hydroxide. The ratio of total sialic acid (N-acetylneuraminic + O,N-diacetylneuraminic) to non-0 acetylated sialic acid (i.e. N-acetylneuraminic acid only) was found to be 1.5 for both rat erythrocyte ghosts, the aqueous phase obtained from them and purified rat glycophorins. This means that one-third of all sialic acid residues are O-acetylated in rat sialogly~oproteins. On the contrary, and in accordance

Table I. Determination of protein and sialic acid alona the isolation arocedure

Rat Human

Ghosts Aqueous phase Glycophorins Ghosts Aqueous phase Glycophorins

Sialic acid yield (% w/w)

Protein yield (% w/w)

100 60 32 100 58 35

100 I 1.5 100 6 1.8

Sialic to protein ratio (% a/w) (relative) 1.8 20 35 2.3 18 41

I 11 19 1 8 18

Samples were taken at three stages of the process: (I) the washed ghosts; (2) the aqueous phase after phenol partition, once dialyzed; (3) the final preparation (purified glycophorins). Yields are referred to ghosts, as starting material. Sialic acid dete~ination does not include O-acetyisialic (yet this is only relevant for absolute siahc-to-protein ratios in rat samples). Enrichment is shown by the rightmost column, taken as purification index. Values are the average from two purification experiments, with at least duplicate determinations in each,

Rat glycophorins

isolated

with LlS

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-

SGP-

-

SGP-

II

-

SGP-

III

-

SGP-

JY

u

fr.

I

Fig. 1. SDS-PAGE of purified preparation of rat erythrocyte glycophorins. Periodic acid-Schiff’s reagent (PAS) staining was used for the detection. Denomination of the bands (SGP-I to -IV) is shown, as well as the position of the front (fr.).

ANGELHERR~EZef al.

1708

I

10 .V

0.25

relative

0.50

0.75

mobility

I

5

I

I

10 %

I

I

15

acrylamide

Fig. 2. Estimation of molecular masses for the four rat glycophorins bands. (Left panel) Calibration curves, obtained from the mobilities of marker proteins in gels of 5, 7.5, 10, 12.5 and 15% acrylamide. Mobility is relative to the tracking dye. (Right panel) Apparent molecular masses for the four glycophorin components. Purified glycophorins were run on separate gels of each acrylamide concentration and the mobility of the four bands interpolated in the standard curve for that concentration. Apparent molecular mass values so obtained are plotted versus acrylamide concentration, in order to estimate the actual masses by extrapolation to high acrylamide concentration. Missing points correspond to the situations in which the band moves too close to the front for a reliable estimation of its mass on the calibration curve.

with previous reports, the determination on human samples rendered the same values whether basic hydrolysis was performed or not, showing the absence of O-acetylated sialic acids in this species. When the purified preparation of glycophorins from rat erythrocytes was analyzed on SDS-PAGE, four bands could be appreciated using PAS staining (Fig. 1). They stained poorly with Coomassie blue, as is usual for sialylated glycoproteins (Segrest and Jackson, 1972). The bands are here designated SGP-I to SGP-IV, in order of increasing electrophoretic mobility. Their apparent relative molecular masses (in 11% gels) were around 74,39,34 and 24. Both the pattern and masses are similar to those found by Edge and Weber (1981) on their sialogIycoprotein preparations obtained with a different method of isolation. The two major bands, namely SGP-I and SGP-II (Fig. l), varied in relative intensity in different preparations. It could be possible that, similarly to what is found for human glycophorins (Furthmayr and Marchesi, 1976), both bands correspond to dime, and monomer; aggregation of amphiphilic transmembrane glycoproteins is often observed even in the presence of SDS. On the other hand, SGP-III band was always weaker, though still more apparent than SGP-IV, which showed a tendency to fade.

The mobility of glycoproteins in SDS electrophoresis is anomalously low with respect to non-glycosylated proteins, due to the different properties and conformation of the carbohydrate moiety (Segrest et al., 1971). To account for this, a special approach has been devised (Segrest and Jackson, 1972) to estimate the actual molecular mass of sialoglycoproteins. This consists on the determination of the apparent molecular mass in gels of several increasing concentrations of acrylamide and the extrapolation to high acrylamide concentrations. Hence, both the purified glycophorins and protein standards were electrophoresed in 5, 7.5, 10, 12.5 and 15% acrylamide gels, in the presence of SDS. Calibration curves are shown on the left of Fig. 2, and the apparent molecular masses estimated for the four bands on the right. These showed, as expected, a decrease in apparent size with increasing acrylamide concentration. Moreover, the anomaly in mobility, indicated by the slope of the lines, is stronger for the fastest-moving bands. This could arise, among other factors, from a differential carbohydrate content in the various glycophorin components, since a direct correlation has been previously found between the percentage of carbohydrate on a glycoprotein and the degree of anomaly in electrophoretic mobility (Segrest and Jackson, 1972). Extrapolation to high

Rat glycophorins isolated with LIS

acrylamide concentration rendered estimated molecular masses of 74 kDa for SGP-I, 32 kDa for SGP-II, 25 kDa for SGP-III and 17 kDa for SGP-IV. Acknowledgemenrs-This work was supported by Grants BI088-649 from C.I.C.Y.T., 87/1479 and 88/1034 from

F.I.S.S., and 90/A3 from the University of Alcali de Henares. A.H. was the recipient of a fellowship from the Spanish Ministry of Education and Science.

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