Aggregates of human erythrocyte membrane sialoglycoproteins in the presence of deoxycholate and dodecyl sulfate

347

Biochimica et Biophysiea Acta, 532 (1978) 347--353 © Elsevier/North-Holland Biomedical Press

BBA 37841

AGGREGATESOFHUMANERYTHROCYTE MEMBRANE SIALOGLYCOPROTEINSINTHEPRESENCEOFDEOXYCHOLATE AND DODECYLSULFATE

LARS LILJAS * The Membrane Group, Institute of Biochemistry, Biomedical Center, University of Uppsala, Box 576, S-751 23 Uppsala, (Sweden)

(Received September 6th, 1977)

Summary Gel electrophoresis in the presence of deoxycholate of human erythrocyte membranes solubilized with deoxycholate resolves four glycoprotein zones. Electrophoresis in dodecyl sulfate in a second dimension reveals several components, three of which migrate in the region of PAS-2. One of the zones in deoxycholate gel electrophoresis contains component PAS-3, and this giycoprotein seems to exist as a monomer in deoxycholate, but aggregates partially upon addition of dodecyl sulfate. The major sialoglycoprotein migrates as a diffuse zone in deoxycholate gel electrophoresis, indicating association and dissociation during the electrophoresis. The use of deoxycholate followed by dodecyl sulfate in two-dimentional electrophoresis gives high resolution of membrane proteins and can be used for detection of complexes in one of the detergents.

Introduction

Much work has been devoted to elucidatingthe interrelationshipbetween,the erythrocyte membrane sialoglycoproteincomponents obtained in dodecyl sulfate gel electrophoresis after stainingwith the periodic acid-Schiffprocedure. In earlierstudies three bands, calledPAS-I, PAS-2 and PAS-3, were mainly discussed. Of these,PAS-1 has been shown to be a dimer of PAS-2, with the equilibrium between monomer and dimer in dodecyl sulfate being dependent on several parameters [i--4]. However, the band PAS-2 has recently been shown to be heterogeneous [5--7]. * Present address: The Wallenberg Laboratory, University of Uppsala, Box 562, S-751 2 2 U p p s a l a , Sweden. A b b r e v i a t i o n : SDS, s o d i u m d o d e c y l sulfate.

348 In the present investigation a two-dimensional electrophoretic technique was used to resolve the glycoprotein components of the erythrocyte membrane. The membranes were solubilized with the mild ionic detergent sodium deoxycholate, and run in gel electrophoresis in the presence of this detergent in the first direction and in dodecyl sulfate in the second direction. This technique proved to be very useful for demonstrating interrelationships among certain membrane glycoprotein components. Materials and Methods Human erythrocyte membranes were prepared, and part of the water-soluble protein was extracted at low ionic strength as described in ref. 8. The glycoprotein pattern was the same as for unextracted membranes. The washed membranes were suspended in 20 mM glycine-NaOH buffer (pH 9.8) containing 20 mM sodium deoxycholate (Merck) to a final concentration of 3 g protein/1. After 0.5 h at 4°C the mixture was centrifuged. Polyacrylamide gel electrophoresis was performed in 3-mm-thick gel slabs, 90 mm in length and in polyacrylamide gradient gels (PAA 4/30, Pharmacia Fine Chemicals, Uppsala). The gradient gels were pre-run for at least 2 h at 75 V in the electrophoresis buffer to introduce dodecyl sulfate into the gel [4]. Buffer and gel compositions are given in the legends to the figures. The gels were stained first with periodic acid-Schiff and then with Coomassie Brilliant Blue essentially according to Fairbanks et al. [9]. Gels were scanned at 560 nm for the periodic acid-Schiff stain in a scanner built at the institute (Hjert~n, S., to be published). Results and Discussion Fig. 1 shows the high resolution obtained in two-dimensional gel electrophoresis in the presence of deoxycholate in the first direction and dodecyl sulfate in the second. The periodic acid-Schiff pattern in deoxycholate gel electrophoresis shows only four zones, here called A, B, C and D (Fig. la). Zone A, which normally has a diffuse front and a sharp back edge, corresponds to the bands PAS-1, PAS-2 and one band migrating slower than PAS-1 in dodecyl sulfate, zone B to PAS-2a, zone C to PAS-3b, and zone D to bands PAS-la, PAS-2b and PAS-3 and one band migrating slower than PAS-la. (The denotations PASla, PAS-2b, PAS-2a and PAS-3b are extensions of the nomenclature used by Fairbanks et al. [9] .) The resolution in the region of PAS-2 is much higher when a gradient gel or a gel with high polyacrylamide concentration is used [ 5,7 ] than when a normal gel is used in dodecyl sulfate electrophoresis. Most proteins are separated mainly according to their molecular weight in dodecyl sulfate gel electrophoresis. The major erythrocyte membrane sialoglycoprotein binds greater amounts of dodecyl sulfate than normal water-soluble proteins [10] and has a high carbohydrate content. Therefore molecular weight estimations from gel electrophoresis give too high a value for this protein (and possibly also for the other erythrocyte membrane sialoglycoproteins), especially when a gel of low polyacrylamide concentration is used [11]. In the polyacrylamide gradient gel used in the present work the migration distance of the dimer of the major sialoglycoprotein (PAS-1) seems to correspond reasonably

349

MS-

I

la

=i b " ~ 2a

Fig. I . T w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s o f e r y t h r o c y t e m e m b r a n e p r o t e i n s solubilized w i t h d e o x y c h o late. T h e s u p e r n a t a n t a f t e r d e o x y e h o l a t e ( D O C ) s o l u b i l i z a t i o n w a s r u n in a gel w i t h t h e c o m p o s i t i o n T = 10% (4% in t h e u p p e r 1 e m ) , C = 2% [ 1 9 ] . T h e b u f f e r w a s 0.1 M ( 0 . 0 2 M in t h e u p p e r 1 c m ) glycineN a O H , p H 9 . 8 / 0 . 0 1 M d e o x y c h o l a t e . T h e e l e c t r o p h o r e s i s w a s r u n f o r 2 h a t 1 5 0 V. A gel strip c o r r e s p o n d i n g t o one well w a s c u t o u t a n d i m m e r s e d in a s o l u t i o n o f 0.1 M d o d e c y l s u l f a t e f o r 1 5 m i n . T h e strip w a s t h e n p l a c e d o n t o p o f a P h a r m a c i a g r a d i e n t gel ( p r e r u n w i t h d o d e c y l s u l f a t e b u f f e r ) a n d t h e seco n d e l e c t r o p h o r e s i s w a s r u n at 6 0 V f o r 16 h in 0 . 0 5 M g l y c i n e - N a O H , p H 9 . 8 / 4 m M d o d e c y l sulfate. Patt e r n s o b t a i n e d in t h e first d i m e n s i o n a n d in o n e - d i m e n s i o n a l d o d e c y l s u l f a t e e l e c t r o p h o r e s l s u n d e r t h e s a m e c o n d i t i o n s are i n s e r t e d f o r c o m p a r i s o n . D e n o t a t i o n s are essentially a c c o r d i n g t o F a i r b a n k s e t al. [ 9 ] . I n b t h e gel w a s s t a i n e d w i t h C o o m a s s i e Brilliant Blue a f t e r t h e p e r i o d i c a c i d - S c h i f f staining.

well to the molecular weight for this protein as determined by analytical ultra° centrifugation [10] or calculated from the amino acid and carbohydrate composition [ 12]. In the presence of deoxycholate above the critical micellar concentration membrane proteins bind this detergent in substantial amounts [ 13]. The bound charged detergent molecules tend to some extent to decrease the difference in surface charge density between different proteins, and they will separate

350 mainly according to their size in a gel of sufficiently high polyacrylamide concentration as in the case with dodecyl sulfate gel electrophoresis. In two-dimensional gel electrophoresis in deoxycholate/dodecyl sulfate, membrane proteins should therefore fall roughly on a "diagonal". This is true for the majority of the deoxycholate-solubilized proteins of the e r y t h r o c y t e membrane (Fig. lb). A hypothetical c o m p o n e n t that exists as a (slowly migrating) aggregate in deoxycholate but is split into (faster migrating) subunits in dodecyl sulfate will give spots below this "diagonal". One example of this is c o m p o n e n t 6 (Fig. lb), which in this gel is represented by a line. Component 6 is identical to the subunit of glyceraldehyde-3-phosphate dehydrogenase. This enzyme is present as a tetramer bound to specific sites in the e r y t h r o c y t e membrane [14], and the pattern in Fig. l b indicates that a tetramer of the enzyme is continually split to monomers during the electrophoresis in the presence of deoxycholate. If the sample is heated to 100°C in deoxycholate before the run in the first dimension, c o m p o n e n t 6 is found on the diagonal. Considerable material has either not entered the gel or migrated very slowly in the deoxycholate run, indicating the existence of large aggregates (Fig. lb). Components PAS-la and PAS-2b migrate much slower in dodecyl sulfate relative to the components on the "diagonal" than could be expected from their rapid migration in deoxycholate. These components, in addition to comp o n e n t PAS-3, all seem to be derived from the same zone in deoxycholate gel electrophoresis, regardless of the polyacrylamide concentration and distance migrated in the first dimension run. It is therefore reasonable to assume that zone D contains only one component, from which PAS-3, PAS-2b and PAS-la are derived. Since PAS-3 falls roughly on the "diagonal" (Fig. lb), the zone D in deoxycholate gel electrophoresis seems to represent the migration of the m o n o m e r of the PAS-3 glycoprotein. PAS-la and PAS-2b seem to be aggregates of c o m p o n e n t PAS-3 formed from the m o n o m e r before the second dimension run. The view that these three components obtained in dodecyl sulfate gel electrophoresis might represent different levels of aggregation of one glycoprotein is also supported by the work of Dahr et al. [5,15]. Since the bands PAS-la and PAS-2b are also obtained in dodecyl sulfate gel electrophoresis of e r y t h r o c y t e membranes, they are probably not artefacts introduced by the deoxycholate. A weak spot, possibly corresponding to a higher aggregate of PAS-3, can be seen in Fig. la, but is not present in all experiments. Zone A in deoxycholate gel electrophoresis gives rise to the two components PAS-1 and PAS-2 in dodecyl sulfate (Fig. la). These components seem to represent the m o n o m e r and dimer of the major sialoglycoprotein [1]. (The material between PAS-1 and PAS-2 in Fig. l a probably represents dimers formed from monomers during the electrophoresis, cf. Fig. 2.) The sharp back edge of zone A in deoxycholate gel electrophoresis can be interpreted as representing a dimer of the major sialoglycoprotein, whereas the diffuse front may represent the monomer. The profile of the zone indicates that the dimer dissociates into the faster migrating m o n o m e r during the electrophoresis in deoxycholate. The profile of the zone is dependent on the glycoprotein concentration in the sample in such a way that at high concentration most of the material migrates at the back edge (not shown here). Upon addition of dodecyl sulfate, an equilibrium is rapidly attained, and the entire zone A gives rise to both PAS-1 and

351

PAS-2. The different aggregates obtained in the both detergents are summarized in Table I. As has been shown earlier for the components PAS-1 and PAS-2 [1], the relative amounts of the SDS components PAS-la, PAS-2b and PAS-3 in gel electrophoresis can be changed by, for example, heating of samples in the presence of dodecyl sulfate (Fig. 2). Fig. 2 shows a scan of the SDS gel electrophoresis pattern of a sample heated to 100°C in the presence of dodecyl sulfate compared to a normal sample. Peak PAS-1 decreases drastically, whereas peaks PAS-2 and PAS-3 increase in the heated sample. In view of the results from the two-dimensional gel electrophoresis, the changes in staining intensities can be explained by dissociation of the dimer PAS-1 into PAS-2 and possibly also of PAS-2b into PAS-3. The diffuse background between PAS-1 and PAS-2 is probably due to a partial reaggregation of the monomer to dimer during the electrophoresis. The amount of PAS-2b has probably diminished in the heated sample (cf. the insert of a photograph of the corresponding gel, but since the background intensity has increased, the peak value is about the same. When two~limensional gel electrophoresis as in Fig. 1 is performed with a gel strip from deoxycholate electrophoresis that has been heated to 100°C in dodecyl sulfate solution for 10 rain, the same change in relative staining intensities can be observed, indicating a partial dissociation of PAS-1 to PAS-2 and of PAS-2b to PAS-3. The exceptional behaviour of the monomeric and dimeric forms of the major sialoglycoprotein in the presence of dodecyl sulfate has been thoroughly discussed [1--3,8,16] but not satisfactorily explained. At room temperature the monomer and dimer seem to be relatively stable (e.g. there is normally only little trailing of the monomer zone in dodecyl sulfate gel electrophoresis), but upon heating the ratio between monomer and dimer is changed, and the original value is not immediately regained upon cooling. The equilibrium ratio between monomer and dimer depends in addition to temperature on, for example, the glycoprotein concentration during the solubilization with SDS [16], the presence of other agents [2,4,16] and treatment or modification of the molecule [3,17]. Grant and Hjert~n [4] have shown that the ratio between monomer and dimer of the major sialoglycoprotein in dodecyl sulfate gel electrophoresis is greatly affected by non-ionic detergents. Supernatants after soluTABLE I A G G R E G A T E S O F H U M A N E R Y T H R O C Y T E M E M B R A N E S I A L O G L Y C O P R O T E I N S IN T H E P R E S E N C E O F D E O X Y C H O L A T E A N D D O D E C Y L S U L F A T E AS S U G G E S T E D BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

Component

D e n o t a t i o n and aggregation level in Deoxycholate

D o d e e y l sulfate

Major s i a l o g l y e o p r o t e i n

A (dimer, m o n o m e r )

PAS-2a PAS-3b PAS-3

B (monomer) C (monomer) D (monomer)

PAS-1 ( d i m e r ) PAS-2 ( m o n o m e r ) PAS-2a ( m o n o m e r ) PAS-3b ( m o n o m e r ) P A S - l a (trlmer) PAS-2b (dimer) PAS-3 ( m o n o m e r )

352 0.8

0.6

I;


0.2

I J 4.

2

6

t t

PAS-I

I=, 2b

¢m

'tt

2 2a 3 6 3

Fig; 2. E f f e c t o f h e a t i n g in d o d e c y l s u l f a t e o n t h e g l y c o p r o t e i n p a t t e r n of d e o x y c h o l a t e - s o l u b i l i z e d m e m b r a n e p r o t e i n s . S c a n n i n g o f p e r i o d i c a c i d - S c h i f f - s t a l n e d d o d e c y l s u l f a t e g r a d i e n t gels. T o t h e d e o x y c h o l a t e solubilized s a m p l e d o d e c y l sulfate solution w a s a d d e d t o a final c o n c e n t r a t i o n o f 0.1 M d o d e c y l sulfate, 0 . 0 2 M d i t h i o t h r e i t o l , 0 . 0 1 M g l y c i n e - N a O H , p H 9 . 8 , 0 . 5 m M E D T A a n d 2% sucrose. One portion o f t h e s a m p l e was h e a t e d t o 1 0 0 ° C f o r 10 rain a n d c o o l e d t o r o o m t e m p e r a t u r e b e f o r e a p p l i c a t i o n t o t h e grad i e n t s gels. T h e e l e c t r o p h o r e s i s was r u n as in Fig. 1 a n d t h e gel s t a i n e d w i t h p e r i o d i c a c i d - S c h i f f a n d scanned. - - , normal sample; ...... , h e a t e d s a m p l e . A p h o t o g r a p h o f t h e c o r r e s p o n d i n g gel is i n s e r t e d : a, n o r m a l s a m p l e ; b, h e a t e d s a m p l e .

bilization of membranes with, for example, Triton X-100 gives almost only PAS-2 upon analysis in dodecyl sulfate, indicating that these mild detergents dissociate this protein more efficiently than does dodecyl sulfate. Potempa and Garvin [18] have recently reported that at a moderately high ionic strength (0.28), bands PAS-1 and PAS-2 disappear in dodecyl sulfate gel electrophoresis in favour of a band of intermediate migration velocity. If the equilibrium between monomer and dimer in dodecyl sulfate is attained quickly, a single zone with a migration velocity between those of PAS-1 and PAS-2 would be expected in dodecyl sulfate gel electrophoresis. Potempa and Garvin's results may therefore be interpreted as indicating that the equilibrium is attained more rapidly at high than at low ionic strength. The results presented here indicate that deoxycholate dissociates the erythrocyte membrane sialoglycoproteins more efficiently than does dodecyl sulfate, and that aggregates can even be formed from dissociated components in deoxycholate upon addition of dodecyl sulfate. This is probably due to differences in the associations between the glycoproteins and the two types of detergent molecules. Preliminary two-dimensional electrophoretic experiments indicate that addition of dodecyl sulfate to these components dissociated by nonionic detergents can also cause aggregation. Two-dimensional gel electrophoresis with the use of deoxycholate in the first direction and dodecyl sulfate in the second gives high'resolution, and might be a valuable tool for the demonstration of possible protein complexes in the presence of deoxycholate

353

as well as, in this case, aggregation of components upon addition of dodecyl sulfate. Acknowledgements I thank Professor Stellan Hjert~n and Drs. Per Lundahl and Magnus Malmqvist for suggestions. This work has been supported by Hierta-Retzius Stipendiefond, the Foundation Lars Hiertas Minne, L~gmanska kulturfonden, the yon Kantzow Foundation and the National Science Research Council. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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