Characterization of polypeptide chains of epidermal prekeratin and reconstituted filaments

160

Biochimica et Biophysica Acta, 668 (1981) 160--166

© Elsevier/North-Holland Biomedical Press

BBA 38636 CHARACTERIZATION OF POLYPEPTIDE CHAINS OF EPIDERMAL PREKERATIN AND RECONSTITUTED FILAMENTS

A. GEDEON MATOLTSY, MARGIT N. MATOLTSY and PATRICIA J. CLIFFEL Department o f Dermatology, Boston University School o f Medicine, 80 East Concord Street, Boston, MA 02118 (U.S.A.)

(Received October 7th, 1980) Key words: Kera tin; Prekera tin filam en t; Polypep tide composition; Molecular weigh t determination; (Bovine snout epidermis)

Summary Prekeratin was isolated from bovine snout epidermis with 0.1 M citric acid/ sodium citrate buffer, pH 2.6 (buffer A). Filaments, 6.0--9.0 nm wide, were produced by dialysis against low ionic strength buffer A or by dissociating prekeratin in 8 M urea solution followed by dialysis against 0.005 M Tris-HC1 buffer, pH 8.0. The polypeptide composition of both prekeratin and filaments was studied by four different SDS-polyacrylamide gel electrophoresis methods. The best resolution was obtained by Laemmli's technique in which both prekeratin and filaments were separated into three major and seven distinct minor bands of polypeptides. The major ones comprise approx. 70% of total polypeptides and their estimated molecular weights are 68 000, 54 000, and 50 000. The molecular weight of minor ones is in decreasing order 65 000, 63 000, 61 000, 58 000, 47 000, 44 000 and 42 000. It is proposed that the major polypeptides form the backbone structure of epidermal filaments and the minor polypeptides play a role in its stabilization.

Introduction Studies of bovine snout epidermis have shown that its lower viable layers are solubilized selectively by 0.1 M citric acid/sodium citrate buffer, pH 2.6, and a low-sulfur protein can be isolated from such extracts by serial precipitation in the pH range 4.5--7.0 [1]. The low-sulfur protein has been designated prekeratin and found to represent a structural unit of 6.0--9.0-nm filaments of epidermal cells. It was shown that prekeratin is a rod-like macromolecule approx. 100 nm long and 4.0 nm wide possessing three-chain units having a-helical and non-a-helical segments [2,3].

161

Although the polypeptide composition of prekeratin has been extensively studied by various sodium dodecyl sulfate polyacrylamide gel electrophoresis methods, the number of chains, their relative amounts and molecular weights have not been found to be in agreement. In early studies, Baden et al. [4] separated prekeratin into three bands of polypeptides ranging in molecular weight from 9 8 0 0 0 to 47000. Skerrow [5] found only two bands, a narrow and a 'wide' band, and estimated the molecular weight of the polypeptides 72 000 and 60 000, respectively. Since they appeared in molar ratios of 1 : 2 it was proposed that prekeratin consists of three polypeptide species. Subsequently, Lee et al. [6] separated prekeratin into four distinct groups of polypeptides with molecular weights ranging from 67 000 to 45 000, a range that is quite different from those found in earlier studies. In a later study, Steinert [7] repeated Skerrow's work and showed that the 'wide' band could be separated into five distinct components by an improved technique. His gels revealed three major and three minor bands of polypeptides and molecular weight estimates indicated a range of 58000--47 000 for the six polypeptide species. Recently, Skerrow [8] has reinvestigated the polypeptide composition of prekeratin by a modified method and found that the 'wide' band separates only into two distinct bands. The molecular weight of the three polypeptides was estimated 72 000, 60 000, and 57 000, respectively. In this study, SDS-polyacrylamide gel electrophoresis methods, such as those employed by previous investigators and also Laemmli's method were used for the study of the polypeptides comprising prekeratin and filaments produced from prekeratin samples. Both consisted of three major and seven minor polypeptide species, the latter were demonstrable as distinct components only by Laemmli's method. Materials and Methods Bovine snouts were obtained chilled from a local abbatoir within 6 h after slaughter of the animals. After washing in running tap water, the epidermis was removed in sheets 0.5 mm thick with a keratotome (Storz Instrument Co.) and cut into small pieces with scissors. Prekeratin was extracted from the minced epidermis with 0.1 M citric acid/sodium citrate buffer, pH 2.6 (buffer A) and purified by serial precipitation at pH 4.5--7.0 as described previously [2]. Subsequently, 10 ml of 0.1% prekeratin solution was dialyzed against 1 1 of buffer A, pH 2.6, for 17 h at 4°C and cleared by centrifugation at 260 000 × g for 2 h. Two different methods were used for the production of filaments: (1) 2 ml of 0.05% prekeratin solution was dialyzed against three changes of 300 ml 0.01 M, 0.005 M and 0.001 M buffer A for 40 h at 4°C. (2) 10 ml 0.05% prekeratin solution was precipitated at pH 4.5 and the precipitate sedimented by centrifugation at 3500 rev./min for 10 min. The sediment was redissolved in 10 ml of 0.05 M Tris-HC1 buffer, pH 8.0, containing 8 M urea and 0.025 M 2-mercaptoethanol and stirred for 1 h at 20°C to dissociate prekeratin into polypeptide chains. This solution was then dialyzed against three changes of 300 ml 0.005 M Tris-HC1 buffer, pH 8.0, containing 0.025 M 2-mercaptoethanol for 40 h at 4°C as described by Steinert and Gullino [9]. Samples taken from both preparations were negatively stained with 0.7% uranyl acetate on

162 carbon-coated holey grids and surveyed in the Philips 300 electron microscope. For electrophoresis samples were prepared as follows: Prekeratin was precipitated at pH 4.5 and the precipitate collected by centrifugation at 3500 rev./ min for 10 min. Filaments were pelletted by centrifugation at 260 000 × g for 2 h. Samples of prekeratin, filaments and molecular weight standards were heated for 5 min at 100°C in Laemmli's sample buffer [10] containing 1% SDS and 1% 2-mercaptoethanol. Electrophoresis was performed by Weber-Osborn's (10% gel), Neville's (7.5% gel), and modified Davis' (7.5% g e l ) m e t h o d as used by Skerrow [5], Lee et al. [6] and Steinert [7], respectively. Furthermore, electrophoresis was also performed by Laemmli's column and slab gel methods (9% gel) [10]. In each gel system, 10--50 ~l samples ofprekeratin and filaments were run simultaneously with molecular weight standards at 20°C. Electrophoresis was performed until the marker dye, bromophenol blue, was at approx. t cm distance from the b o t t o m of the gel. The dye position was marked with India ink after electrophoresis, the gels were stained with 0.25% Coommassie brilliant blue dissolved in 50% (v/v) methanol/10% (v/v) acetic acid, destained in 7.5% acetic acid/5% methanol and stored in 7.5% acetic acid. The mobility of stained bands was measured and plotted on semilogarithmic paper for calculation of molecular weights. The following proteins were obtained from Bio-Rad and used as molecular weight standards: phosphorylase b ( 9 4 0 0 0 ) ; bovine serum albumin ( 6 8 0 0 0 ) ; ovalbumin ( 4 3 0 0 0 ) and carbonic anhydrase (30 000). Densitometer tracing of each gel was done at a wavelength of 540 nm using a Zeiss PM 6 spectrophotometer fitted with a linear transport system. The contribution of the bands to the total stain was assessed by cutting o u t and weighing the area under the peaks. The values obtained were interpreted as relative amounts of polypeptides by assuming that the polypeptides of our samples have very similar color yields and that the intensity of stain is a linear function of the amount of protein present [11]. Results Prekeratin

Typical band patterns formed from denatured prekeratin in different polyacrylamide gel systems are demonstrated in Fig. 1. In Weber-Osborn's system, three major bands and two poorly defined minor bands of polypeptides are apparent as shown in column 1 PK. In the system of Neville and the modified system of Davis, the band patterns are almost identical consisting of three major, four minor and two poorly resolved minor bands of polypeptides as shown in columns 2 PK and 3 PK. In Laemmli's system, the polypeptides separate into ten distinct bands, including three major and seven minor bands as seen in column 4 PK. The relative amounts of major and minor polypeptide species separated in the different gel systems are given in Table I. The major polypeptides account for 85% of total polypeptides in Weber-Osborn's gel system which provides the lowest resolution. In Neville's and Davis' gel systems, 79% and 75% values are obtained, in Laemmli's gel system with the best resolution, the major polypeptides represent 70% of total polypeptides. The estimated molecular weights of polypeptides are given in Table II as

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Fig. 1. T y p i c a l b a n d p a t t e r n s f o r m e d in f o u r d i f f e r e n t S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s s y s t e m s are d e m o n s t r a t e d . PK, p r e k e r a t i n ; F, r e c o n s i t u t e d f i l a m e n t s .

determined in the various gel systems. The data show that the weights are close to 68 000, 54 000 and 50 000 obtained in Laemmli's gel system. The molecular weight of minor polypeptide species estimated by Laemmli's method is in decreasing order 65 000, 63 000, 61 000, 58 000, 47 000, 44 000 and 42 000 (Fig. 1, column 4 PK, bands 2--5 and 8--10). TABLE

I

PERCENTAGE OF RELATIVE AND FILAMENTS (F)

AMOUNTS

OF

POLYPEPTIDES

COMPRISING

PREKERATIN

Major p o l y p e p t i d e s

Weber and Osborn

Neville

PK

F

PK

F

PK

F

PK

F

Band 1 Band 6 Band 7

30 32 23

25 31 26

29 31 19

22 34 23

24 29 19

21 29 22

22 25 23

21 26 22

Total of minor polypeptides

15

18

21

22

28

28

30

31

Davis

(PK)

Laemmli

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T A B L E II M O L E C U L A R W E I G H T O F M A J O R P O L Y P E P T I D E S C O M P R I S I N G P R E K E R A T I N (PK) A N D F I L A M E N T S (F) Major polypeptides

Weber and Osborn

Neville

Davis

Laemmli

PK

F

PK

F

PK

F

PK

F

Band 1 Band 6 Band 7

67 0 0 0 57 0 0 0 53 0 0 0

67 0 0 0 57 0 0 0 54000

66 0 0 0 53 0 0 0 50000

66 0 0 0 54 0 0 0 50000

67 0 0 0 54 0 0 0 51 0 0 0

66 0 0 0 54 0 0 0 51 0 0 0

68 0 0 0 54 0 0 0 50 0 0 0

68 0 0 0 54 0 0 0 50 0 0 0

Filaments Samples of prekeratin dialyzed against buffer A of various low ionic strengths were surveyed in the electron microscope. It was found that reassembly of macromolecules into filaments took place in 0.005 M buffer A. The filaments are relatively short and 6.0--9.0 nm wide. They have sticky surfaces and

Fig. 2. E l e c t r o n m i c r o g r a p h of f i l a m e n t s f o r m e d f r o m p r e k e r a t i n m a c r o m o l e c u l e s in 0 . 0 0 5 M b u f f e r A, p H 2.6. F, single f i l a m e n t ; A, a g g r e g a t e o f f i l a m e n t s . Bar: 1 ~ m . M a g n i f i c a t i o n : X46 0 0 0 . Fig. 3. E l e c t r o n m i c r o g r a p h of f i l a m e n t s r e a s s e m b l e d f r o m p o l y p e p t i d e c h a i n s of p r e k e r a t i n in Tris-HC! b u f f e r , p H 8.0. Bar: 1 pM. M a g n i f i c a t i o n : X46 000.

165 readily clump together to form fibrous aggregates such as those shown in Fig. 2. In prekeratin samples which were first dissociated into polypeptide chains in urea solution and subsequently dialyzed against low ionic strength Tris-HC1 buffer, relatively long and 6.0--9.0 nm wide filaments were formed (Fig. 3) similar to those produced from a polypeptide mixture extracted from epidermal tissue with urea/mercaptoethanol by Steinert and Gullino [9]. Since these filaments resemble more closely in situ filaments, they were pelletted and used for SDS-polyacrylamide gel electrophoresis studies. Typical band patterns formed from polypeptide chains of filaments in the different gel systems are demonstrated in Fig. 1, columns 1 F, 2 F, 3 F, and 4 F. It can be seen that they axe very similar or identical to those formed from polypeptides of prekeratin. The relative amount of major and minor polypeptide species is variable as can be seen in Table I. The estimated molecular weight of major polypeptides is close or identical to that found for major polypeptides of prekeratin as shown in Table II. Discussion

In this work, four different SDS-polyacrylamide gel electrophoresis systems were used to study the polypeptide species of prekeratin. When the results are compared, it is apparent that Laemmli's system provides the best resolution. The polypeptides are separated into ten distinct bands. Bands 1, 6, and 7 represent three major polypeptide species and bands 2--5 and 8--10 seven minor ones (Fig. 1, PK 4). The other gel systems are useful for the demonstration of the major polypeptide species but inadequate for the identification of all minor species. For instance, minor species 2--5 are well resolved in the system of Neville and the modified system of Davis, while 8--10 are not well separated. Weber-Osborn's m e t h o d is the least satifactory for separation of minor polypeptides. The data shown in Table II indicate that there are only minor differences in molecular weight of the major polypeptides of prekeratin estimated in four different gel systems. Significant differences are found, however, when previously estimated molecular weights in identical gel systems axe compared with our data as shown in Table III. It can be seen that Skerrow [8] generally obtained higher values for major polypeptides by using Weber-Osborn's system. Lee et al. [6] values for the two major polypeptides, 6 and 7, are lower as estimated in Neville's system and the value of one major polypeptide, 1, is lower as

TABLE III M O L E C U L A R W E I G H T OF M A J O R P O L Y P E P T I D E S OF P R E K E R A T I N E S T I M A T E D IN T H I S A N D PREVIOUS STUDIES Major polyp eptides

This s t u d y (Laemrnli's method)

Skerrow [S]

Lee et al. [6]

Steinert [7]

Band 1 Band 6 Band 7

68 0 0 0 54 0 0 0 50 0 0 0

72 0 0 0 60 0 0 0 57 0 0 0

67 0 0 0 47 0 0 0 45 0 0 0

58 0 0 0 54 0 0 0 48 0 0 0

166

determined by Steinert [7] with a modified system of Davis. Presumably, these differences are related to factors, such as modifications of the preparative procedure and storage of prekeratin, different denaturation methods of samples, the use of different molecular weight standards and different methods used for the estimation of molecular weights. It is n o t e w o r t h y that the filaments reconstituted from dissociated prekeratin consist of the same polypeptide species found in undissociated prekeratin. This indicates that all polypeptide species have participated in filament formation and that there are no contaminant proteins present in prekeratin samples. It is also remarkable that three major polypeptides comprise approx. 70% of both prekeratin and filament samples. They are present in nearly equal proportions, i.e., in ratios 1.00 : 1.14 : 1.05 in prekeratin and 1.00 : 1.24 : 1.05 in filaments as determined by Laemmli's method. Concerning these data, it is of -interest to recall that by the use of the modified method of Davis, Steinert [7] found that 87% of prekeratin consists of three major polypeptide species appearing in ratios 1.67 : 1.00 : 1.48. These major polypeptides most probably form the backbone structure of in situ epidermal filaments and are deposited in three~tranded units as originally proposed by Skerrow et al. [ 3 ] . The location of minor polypeptides is not known. Presently, one can only make assumptions a b o u t their site by relating them to similar polypeptides found in other ~class proteins. For instance, several minor polypeptides were found in purified samples of myosin. One of these was named C-protein and considered to act like a clamp to prevent disruption of the structure of myosin [12]. Thus, it is possible that minor polypeptides originate from spacer proteins and their role is to stabilize the backbone structure of epidermal filaments. Acknowledgements The authors wish to thank Drs. Loretta Lee and Peter Steinert for providing detailed instructions for the preparation of Neville's gel system and the modified gel system of Davis as used in their laboratories. This work was supported by Grant AM 05924 from the National Institute of Arthritis, Metabolism and Digestive Diseases U.S. Public Health Service. References 1 Matoltsy, A.G. (1964) Nature 201, 1130--1131 2 M a t o l t s y , A . G . ( 1 9 6 5 ) in B i o l o g y o f t h e S k i n a n d H a i r G r o w t h ( L y n e , A . G . a n d S h o r t , B . F . , eds.), p p . 291--305, Angus and Robertson, Sydney 3 S k e r r o w , D., M a t o l t s y , A . G . a n d M a t o l t s y , M.N. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 4 8 2 0 - - 4 8 2 6 4 B a d e n , H . P . , G o l d s m i t h , L . A . a n d F l e m i n g , B. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 7 , 3 0 3 - - 3 1 1 5 S k e r r o w , D. ( 1 9 7 4 ) B i o c h e m . B i o p h y s . R e s . C o m m u n . 5 9 , 1 3 1 1 - - 1 3 1 6 6 L e e , L . D . , F l e m i n g , B.C., W a i t k u s , R . F . a n d B a d e n , H.P. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 1 2 , 8 2 - - 8 9 7 S t e i n e r t , P.M. ( 1 9 7 5 ) B i o c h e m . J. 1 4 9 , 3 9 - - 4 8 8 S k e r ~ o w , D. ( 1 9 7 7 ) B i o c h i m . B l o p h y s . A c t a 4 9 4 , 4 4 7 - - 4 5 1 9 S t e i n e r t , P.M. a n d G u l l i n o . M.I. ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 7 0 , 2 2 1 - - 2 2 7 10 L a e m m l i , U . K . ( 1 9 7 0 ) N a t u r e 2 2 2 , 6 8 0 - - - 6 8 5 11 F a z e k a s de G r o t h , S., W e b s t e r , R . G . a n d D a t y n e r , A. ( 1 9 6 3 ) B i o c h i m . B i o p h y s . A c t a 71, 3 7 7 - - 3 9 1 12 O f f e r , G. ( 1 9 7 3 ) C o l d S p r i n g H a r b o r S y r u p . Q u a n t . Biol. 3 7 , 8 7 - - 9 5