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Biochimiea et Biophysica Acta, 579 (1979) 241--245 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 31275

SELECTIVE EXTRACTION OF DESMOSOMAL PROTEINS BY LOW IONIC STRENGTH MEDIA

CHRISTINE J. SKERROW

University of Glasgow, Department of Dermatology, Anderson College Building, 56 Dumbarton Road, Glasgow G11 6NU (U. K.) (Received March 2nd, 1979)

Key words: Desmosome; Epidermis; Membrane protein

Summary Desmosomes, isolated using an acidic buffer, have been subjected to extraction at low ionic strength. This treatment removes more than 35% of their protein in the form of two polypeptide chains of molecular weight 210 000 and 230 000, but the desmosomes show only subtle changes in ultrastructure. It is concluded that the use of low ionic strength media for desmosome isolation yields residual structures specifically depleted in high molecular weight proteins.

Desmosomes have been isolated from cows nose epidermis by the selective solubilizing action of citric acid/sodium citrate buffer, pH 2.6 [1]. They contain over 75% of protein, which can be resolved into approximately 24 chains by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The action of citrate buffer dissolves the tonofilaments attached to the desmosomes in situ, and only 6% of the desmosomai protein is accounted for by chains whose mobilities correspond with those of tonofilament proteins [2]. Citrate buffer-isolated desmosomes thus consist almost entirely of desmosomal membrane and membrane associated proteins [2]. Two chains (bands 1 and 2) are present in approximately equimolar amounts and have unusually high molecular weights (210 000; 230 000) corresponding with those of an immunologicaUy heterogeneous group of fibrous proteins associated with the cytoplasmic side of plasma membranes [ 3, 4]. The most studied member of the group, spectrin, supports and strengthens the bilayer of the erythrocyte ghost, and also exerts transmembrane control on cell surface glycoproteins [ 5, 6]. In other cell types, high molecular weight membrane-associated proteins are generally considered to form part of the

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cytoskeleton [4]. Desmosomes, which are components of an epidermal cytoskeleton designed to transmit tension and resist mechanical deformation, contain a far higher proportion (35%) of high molecular weight chains than any other membrane preparation so far reported [2, 3]. Filaments with diameters (4--5 nm) similar to those of spectrin-like proteins have been observed in freeze-fractured desmosomai plaques [7]. High molecular weight proteins such as spectrin can be selectively eluted from membranes by extraction with media of low ionic strength, free from divalent cations [4, 8]. In order to obtain further data on the relationship of desmosome bands 1 and 2 to these proteins, isolated desmosomes were extracted at low ionic strength. This communication describes the effects of such treatment on desmosomal structure and protein composition. Desmosomes were isolated from cows nose epidermis as previously described [1]. The low ionic strength treatment was based on that of Tillack et al. [8], with the entire procedure carried out at 4°C. 2 mg (wet weight) of freshly prepared desmosomes were suspended in 20 ml of low ionic strength medium: deionized distilled water containing 1 mM disodium ethylenediaminetetraacetic acid (EDTA) and 5 mM 2-mercaptoethanol, pH 7.8. The suspension was diai~ysed against low ionic strength medium, with continuous stirring for 48 h, during which the solution was changed three times. It was then centrifuged at 100 000 X g for 2 h. The pellet was resuspended in 20 ml of deionized distilled water, the suspension stirred overnight and then recentrifuged for 2 h at 100 000 X g. The final pellet was suspended in 1 ml denaturing buffer, and processed for electrophoresis as previously described [2]. Complete denaturation of desmosomes, in c o m m o n with other membrane preparations, requires the inclusion of EDTA and 2-mercaptoethanol in both denaturing and electrophoresis buffers, and prolonged boiling in SDS. Omission of these essential precautions leads to loss of resolution and reduction in the relative amounts of bands 1, 2, 3 and 4 seen on the gels [2]. The molecular weights and relative abundance of the major desmosomal protein chains were determined as before [2]. To act as a control, a further 2 mg portion of desmosomes was subjected to the same procedure, using calcium and magnesium free phosphate buffered saline in place of deionized distilled water. After both treatments, an 0.5 ml aliquot of the final suspension in water was centrifuged separately and the pellet processed for electron microscopy using standard procedures. Figs. l a and l b are electron micrographs of desmosomes subjected to the control procedure and the low ionic strength treatment respectively. Control desmosomes {Fig. la) show no significant differences from untreated desmosomes [1]. The midlines are visible and the desmosomal plaques are divided into t w o zones: a dense layer immediately adjacent to the plasma membrane and a filamentous zone extending from the dense layer. In previous publications [1,2] evidence has been presented that this filamentous zone is not the remnants of tonofilament bundles but represents a fraying out of plaque material, probably as a result of the solubilization of attached tonofilaments in citrate buffer. After low ionic strength treatment (Fig. l b ) desmosomes remain grossly intact, although the preparation contains more membrane vesicles than the

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Fig. 1. (a) Isolated desmosomes subjected to the control procedure. Midlines (m) are visible. The plaque consists of a dense layer (d) and an outer filamentous zone (f). Magnification X 33 700. (b) Desmosomes extracted at low ionic strength. The dense layer is reduced in electron density and in width, being absent in places (arrows). Plasma membranes (p) have greatly enhanced contrast. Magnification X 67 500.

244 controls. The midline is not visible: instead, the desmosomal interspace is occupied by material with randomly variable electron density. The demarcation of the plaque into dense and filamentous zones is still seen, and the appearance and extent of the filamentous zone is unchanged. However, the dense layer is reduced in electron denSity and in width, and is absent in places. As a consequence of these changes, both leaflets of the plasma membrane show greatly enhanced contrast. Compared with these subtle changes in ultrastructure, the effect of low ionic strength treatment on the protein composition of desmosomes is much more striking (Fig. 2, Table I). Bands 1 and 2, representing a third of the desmosomal protein, are completely removed. The identification of the remaining peak close to band 2 with band 2a (Mr = 2 0 5 000) was confirmed by coelectrophoresis. Bands 3 and 4 are correspondingly increased in relative abundance, b u t band 5 (Mr = 90 000) is slightly decreased, indicating that a small amount of this c o m p o n e n t has also been extracted. All other minor bands show the expected slight increase in amount. The values for the control procedure were very similar to those obtained from untreated desmosomes [2]. The complete and selective extraction of the protein chains with molecular weights 210 000 and 230 000 from desmosomes using low ionic strength media provides further evidence that these desmosomal components are related to the high molecular weight membrane-associated proteins found in other membrane preparations. The partially extracted appearance of the plaque of the treated desmosomes suggests that the high molecular chains are present in the desmosomal plaque b u t not its sole constituent. More specifically, the persistence of the outer filamentous zone and its unchanged appearance even in regions at which the dense layer is lacking indicates that these chains are present,in the dense region of the plaque, closest to the plasma

22 a

3

5

Fig. 2. D e n a i t o m e t e r traces o f 5% S D S - p o l y a c r y l a m i d e gels, s t a i n e d w i t h C o o m a s s i e Blue and s c a n n e d at 540 n m . A: c o n t r o l d e s m o s o r n e s , B: d e s r n o s o m e s e x t r a c t e d at l o w i o n i c strength.

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TABLE I M O L E C U L A R WEIGHTS A N D A B U N D A N C E O F M A J O R D E S M O S O M A L P R O T E I N CHAINS Band

1 2 2a 3 4 5

Molecular weight

230 210 205 140 120 90

000 000 000 000 b 000 b 000

% o f stain Control

Extracted desmosomes

18.0 19.8 a -9.1 13.8 13.0

0 0 7.2 11.1 16.0 10.4

a v a l u e includes b o t h b a n d s 2 a n d 2a, w h i c h are i n c o m p l e t e l y resolved. b B a n d s 3 a n d 4 are p e r i o d i c a c i d - S c h i f f positive, h e n c e these values are a p p a r e n t m o l e c u l a r weights only.

membrane. This is in agreement with the invariably cytoplasmic location of other high molecular weight membrane-associated proteins [4]. Recently, desmosomes have been prepared from cows nose epidermis by a method involving prolonged exposure to alkaline media (pH 9, 10 or 11) of low ionic strength, and the use of sonication to remove attached tonGfilaments [9]. The protein composition of the pure desmosome fraction (fraction D) is strikingly different from that citrate buffer-isolated junctions. SDS-polyacrylamide gels are dominated by protein chains with molecular weights of 68 000 and below, ascribed to tonofilament proteins. The nontonofilament proteins, with molecular weights of 90 000 and above, which comprise 75% of the protein of citrate buffer-isolated desmosomes (Fig. 2, Table I) are present only as very minor components. No explanation is given for these marked differences in gel patterns between junctions isolated by the two methods. Ultrastructurally, low ionic strength-isolated desmosomes resemble low ionic strength extracted citrate buffer-isolated desmosomes (Fig. lb), in that their midlines are frequently indistinct and their plasma membranes show unusually high contrast. The results described above indicate that the use of a preparation method involving low ionic strength media, even at near neutral pH and in the absence of sonication, would result in extraction of much of the high molecular weight desmosomal protein and would also account for the ultrastructural differences observed. The residual structure remains morphologically identifiable as a desmosome, but in contrast to citrate buffer-isolated desmosomes, has a low content of specifically desmosomal non-tonofilament proteins. This work was supported by a grant from the Medical Research Council. References 1 2 3 4 5 6 7 8 9

Skemcow, C.J. a n d M a t o l t s y , A.G. ( 1 9 7 4 ) J. Cell Biol. 63, 5 1 5 - - 5 2 3 Sker~ow, C.J. a n d M a t o l t s y , A.G. ( 1 9 7 4 ) J. Cell Biol. 63, 5 2 4 - - 5 3 0 Hiller, G. a n d Weber, K. ( 1 9 7 7 ) N a t u r e 2 6 6 , 1 8 1 - - 1 8 3 H i t c h c o c k , S.G. ( 1 9 7 7 ) J. Cell Biol. 74, 1 - - 1 5 J u l i a n o , R.L. ( 1 9 7 3 ) B i o c h i m . Biophys. A c t a 3 0 0 , 3 4 1 - - 3 7 8 Elgsaetar, A., S h o t t o n , D.M. a n d B r a n t o n , D. ( 1 9 7 6 ) Biochim. B i o p h y s . A c t a 4 2 6 , 1 0 1 - - 1 2 2 M c N u t t , N.S. ( 1 9 7 0 ) A m . J. Cardiol. 25, 1 6 9 - - 1 8 3 TillaclL T.W., Marchesi, S.L., Marchesi, V.T. a n d Steers, D.E. ( 1 9 7 0 ) Biochim. B i o p h y s . A c t a 2 0 0 , 125--131 D r o c h m a n s , P., F r e u d e n s t e i n , C,, Wanson, J.-C., L a u r e n t , L., K e e n a n , T.W., Stadler, J. L e l o u p , R. a n d F r a n k e , W.W. ( 1 9 7 8 ) J. Cell Biol. 79, 4 2 7 - - 4 4 3

Selective extraction of desmosomal proteins by low ionic strength media.

241 Biochimiea et Biophysica Acta, 579 (1979) 241--245 © Elsevier/North-Holland Biomedical Press BBA Report BBA 31275 SELECTIVE EXTRACTION OF DESMO...
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