Biochhnica et Biophysica A cta, 490 (1977) 178-191

© Elsevier/North-Holland Biomedical Press BBA 37545 MAMMALIAN EPIDERMAL KERATIN ISOLATION A N D C H A R A C T E R I Z A T I O N OF T H E a - H E L I C A L PROTEINS F R O M N E W B O R N RAT

VIRGINIA B. CULBERTSON and IRWIN M. FREEDBERG Department o f Dermatology, Har yard Medical School and the Thorndike Research Laboratories o f the Harvard Medical School at the Beth Israel Hospital, Bostotl, Mass. (U.S.A.)

(Received June 22nd, 1976)

SUMMARY

Neutral buffer-insoluble proteins extracted from newborn rat epidermis with alkaline urea have been purified by chromatography on Sephadex G-150 columns run in the presence of sodium dodecyl sulfate. Two proteins with apparent molecular weights of 60 000 and 68 000, respectively have been isolated and characterized. Spectropolarimetric studies show both of them to be a-helical in contrast to the non-helical heavier and lighter species also solubilized with alkaline urea. The amino acid composition of the two proteins, their electrophoretic behavior and their immunological characteristics are essentially identical. Both proteins appear to be major constituents of rat epidermal tonofilaments.

INTRODUCTION Previous reports from our laboratory have focused upon the major phenotypic organelles of newborn rat epidermis, the keratohyaline granules and the tonofilaments. The keratohyaline granules have been solubilized with sodium deoxycholate, yielding a heterogeneous group of proteins [1 ] which have been purified and characterized [2]. The tonofilament proteins, which are insoluble in sodium deoxycholate, have been isolated following subsequent extraction with alkaline urea [3]. Since the proteins solubilized by alkaline urea were also heterogeneous, we undertook the studies reported in this communication. They were aimed at the definition of the fibrous proteins in the alkaline urea extract of newborn rat epidermis. EXPERIMENTAL TECHNIQUE Materials

Newborn rats (Sprague-Dawley strain) were obtained from Charles River Laboratories, Wilmington, Mass.; New Zealand white rabbits were purchased from the Gloucester Rabbitry, Gloucester, R.1.; phosphorylase a (rabbit muscle), bovine

179 serum albumin, ovalbumin, carbonic anhydrase (bovine erythrocytes) and cytochrome c (horse heart) from Sigma Chemical Co., St. Louis, Mo.; Sephadex G-150 and G-200, CM-Sephadex (C-50) and Blue Dextran 2000 from Pharmacia Fine Chemicals, Inc., Piscataway, N.J.; Bio-Gel A-50m, Bio-Lyte (ampholine) 4/6 and 6/8 and AG-1-X2 from Bio-Rad Laboratories, Richmond, Calif.; DEAE-cellulose and iodoacetamide from Eastman Organic Chemicals, Rochester, N.Y.; XM 300 Diaflo Ultrafilters from Amicon, Lexington, Mass.; Freund's complete and incomplete adjuvants from Difco Laboratories, Detroit, Mich.; guanidine. HCI, ultra pure, from Matheson, Coleman and Bell, East Rutherford, N.J. Reagent grade urea and Rexyn 1-300 were obtained from Fisher Scientific Co., Fair Lawn, N.J. All urea solutions were de-ionized by passage over a mixed anion/cation exchange resin (Rexyn 1-300) prior to use. All other chemicals were of reagent or analytical grade.

Methods Tissue preparation and extraction. 3-day-old rats (Sprague-Dawley) were killed by cervical dislocation and exsanguination. The skin was removed, rinsed in 0.9 NaCI (4 °C) and frozen at --20 °C overnight. The tissue was thawed and suspended in 10 mM Tris. HC1 buffer (pH 8.8) containing 1 mM magnesium acetate, 10 mM NaC1, 5 mM 2-mercaptoethanol, 250 mM sucrose and 0.03 mM sodium tetraphenylboron for 4 h after which sheets of epidermis could be separated from the dermis by gentle scraping [3]. All tissue preparation procedures and extractions were carried out at 4 °C. Soluble proteins, keratohyaline granules and other non-filamentous, intracellular organelles were extracted as previously described [1] in 50 mM Tris.HCl buffer (pH 8.8) containing 10 mM 2-mercaptoethanol and 3 mM sodium deoxycholate (sodium deoxycholate buffer). After 3-5 extractions had removed all sodium deoxycholate-soluble materials, the resulting insoluble pellets were homogenized and extracted overnight with 8 M urea/100 mM Tris.HC1 (pH 9.4) (alkaline urea) [3]. In some experiments 70 mM 2-mercaptoethanol was added to the alkaline urea. Following centrifugation (7000 × g for 15 min) the urea-solubilized proteins were recovered and the pellet was re-extracted three or four times until filaments could no longer be demonstrated by electron microscopy. The extracts were pooled, centrifuged at 270 000 × g for 120 min (Beckman-Spinco L2-65) and the resulting supernatant fractions were concentrated by ultrafiltration (Amicon 8 MC micro-ultrafiltration system, XM 300 membrane). Fibrous proteins were purified from the concentrated retenates. Histology and electron microscopy. For light microscopy, samples were fixed in 10% buffered formalin, routinely embedded, sectioned and stained with hematoxylin and eosin. For electron microscopy, intact tissues and pellets were fixed in either 2% OsO4, buffered with 100 mM sodium phosphate (pH 7.3), or 3 % glutaraldehyde buffered in I00 mM sodium cacodylate (pH 7.4). Glutaraldehyde-fixed samples were post-fixed in osmium and all samples were embedded in epon and sectioned on a Porter-Blum MT-2 ultratome (Ivan Serval, Inc.). Thick sections were stained with methylene blue and thin sections were stained with 1 9/00uranyl acetate, counter-stained with lead and examined in a Philips EM200 electron microscope. Electrophoresis. Sodium dodecyl sulfate gel electrophoresis was performed on 10~ polyacrylamide gels according to the technique of Weber and O3born [7];

180 15 mM 2-mercaptoethanol was added to the reservoir buffer solutions. Gels were stained with Coomassie Brillant Blue (0.4~ for 2 h or 0.1 ~ overnight) in 5 0 ~ methanol, 10~ acetic acid and destained in methanol/acetic acid. The protein standards used to determine molecular weights included phosphorylase a (94 000), bovine serum albumin (68 000), ovalbumin (45 000), carbonic anhydrase (29 000) and cytochrome c (12 400). Disc gel electrophoresis was performed on 7 . 2 ~ acrylamide gels using the pH 8.3 Tris/glycine buffer system described by Davis [8]. 5-8 M urea and 5 mM 2-mercaptoethanol were added to the gels and running buffer. Analytic immunoelectrophoresis was undertaken following initial resolution on polyacrylamide gels. The proteins migrated into 1.0 ~ ionagar (50 mM Tris. HC1, pH 8.0) which contained appropriately spaced antibody troughs and the resulting precipitin lines were stained by the procedure used for Ouchterlony plates (see below). Analytic isoelectric focusing gels (10 × 0.4 cm) were run using a modification of the procedure of Righetti and Drysdale [9]. Samples were dialyzed against 8 M urea containing 0.5 ~ ampholine (pH 6-8) and were incorporated into gels containing 6.5 M urea and 4.4~/o ampholine (pH 4-8). They were focused for 20-24 h and pH gradients were determined on sliced gels while sample gels were stained with Coomassie Brilliant Blue in CuSO4 [10]. Preparative isoelectric focusing of 6-7 mg protein samples was done at 4 °C in an LKB 8102 isofocusing column (440 ml) using the technique suggested by the manufacturer. The buffer reservoirs and the sucrose gradient contained 6 M urea and the gradient contained a 1.5 °/~ solution of pH 4-8 ampholines. Several gel filtration and ion-exchange column techniques were used. Sephadex G-200 (1.5 x 60 cm column) was prepared in 50 mM sodium citrate (pH 4.0) containing 7.5 M urea; Sephadex G-150 (two tandem 2.5 x 90 cm columns) was prepared in 100 mM Tris. HC1 buffer (pH 7.5) containing 0.5 ~o sodium dodecyl sulfate and 15 mM 2-mercaptoethanol; agaro~e A-50m (1 x 95 cm column) was prepared in 6 M guanidine. HC1 containing 50 mM Tris. HC1 (pH 9.0) and 15 mM 2-mercaptoethanol. Carboxymethyl-Sephadex C-50 in 50 mM sodium citrate (pH 4.0) containing 7.5 M urea was eluted with both salt and pH gradients and DEAE-cellulose chromatography was performed using the technique of Waugh et al. [1 l]. All columns were run at room temperature. Immunologic techniques. Antibodies to the fibrous epidermal proteins were raised in New Zealand white rabbits. 200-500 #g of immunogen suspended in 50 mM Tris" HCI (pH 8.8), 10 mM 2-mercaptoethanol was emulsified with Freund's complete adjuvant for primary injections. 50-100 #g of antigen in incomplete Freund's adjuvant was used for boosting injections. ;/-Globulins were concentrated by precipitation with 2.0 M (NH4)2SO4 and redissolved in 100 mM NaHCO3. Antigen-antibody reactions were identified by the Ouchterlony technique [12] (1 ~i ionagar in 50 mM Tris. HC1 buffer, pH 8.0) using antigens dissolved in alkaline urea. Ouchterlony plates were washed with physiological saline and stained with I ~ amido black in 7 ~o acetic acid. Spectropolarimetry. Optical rotatory dispersion (ORD) and circular dichroism (CD) were measured at 25 °C in 1 cm quartz cells with a Cary 6001 recording spectropolarimeter calibrated with d-10 camphor sulfonic acid. In preliminary studies samples for CD analysis were dissolved in alkaline urea and dialyzed against: 2.5 or

181 5 mM Tris. HCI (pH 9.0); 40 mM urea containing 10 mM Tris. HC1 (pH 9.0); 40 mM urea; 30mM KF containing 1 mM Tris.HC1 (pH 9.3); 1.6mM Tris.HC1 buffer (pH 9.0) containing 0.5 mM sodium deoxycholate; 0.1 ~o dimethyl sulfoxide; 1 ~o sodium dodecyl sulfate and 2.5 mM citric acid (pH 3.0). For comparisons of relative helicity, CD samples were dissolved in alkaline urea or in 6 M guanidine. HC1 containing 5 mM Tris. HC1 (pH 9.0) and dialyzed for 48 h against 2.5 or 5 mM Tris. HC1 (pH 9.0). Some preparations of whole urea extract were reduced and carboxymethylated [13] prior to CD analysis. ORD samples were dissolved in alkaline urea and dialyzed against 2.5 mM Tris-HC1 (pH 9.0) or 4 M urea containing 2.5 mM Tris. HC1 (pH 9.0). All samples were filtered (0.45/~m Millipore filter) prior to analysis and protein analyses were performed immediately following spectropolarimetric studies. Chemical analyses and procedures. Protein concentrations were determined in several ways. For samples without mercaptoethanol the technique of Lowry et al. [14] was used. The methods of Bramhall et al. [15], and of Radola [16] were applied to samples containing 2-mercaptoethanol or guanidine. HC1. Bovine serum albumin was used as a standard for all procedures. Chromatographic columns run in the absence of guaninidine were monitored by measurement of absorbance at 280 nm. Amino acid composition was determined with a Beckman model 121 amino acid analyzer following 22 h hydrolysis in 6 M HC1 in vacuo at 110 °C. Cysteic acid content was determined on performic acid-oxidized samples [18]. Sodium dodecyl sulfate was removed from some samples analyzed by CD using a modification of the procedure of Weber and Kuter [19]. Samples were dialyzed overnight at room temperature against 6 M urea containing 50 mM Tris.HC1 (pH 7.8) and 10 mM 2-mercaptoethanol and subsequently against AG-1-X2, an anion exchange resin, equilibrated in the same buffer. RESULTS

Tissue preparation and extraction The epidermis of 3-day-old rats consists of a stratum corneum, several layers of cells containing large keratohyaline granules, spinous cells without keratohyaline granules and a basal cell layer. Tonofilaments, the morphologic entity corresponding to the a-helical keratinous proteins, can be identified in all layers. These studies were aimed at the isolation and characterization of the proteins which comprise these tonofdaments. When newborn rat skin is frozen, thawed and soaked in tetraphenylboron, the epidermis can be separated from the dermis and all epidermal cell layers are represented in the preparation obtained (Fig. 1). Extraction of this tissue with sodium deoxycholate buffer solubilizes protein species which are predominantly of low molecular weight (less than 20 000) (Fig. 2a). Previous studies [1] have shown that the filamentous proteins are not solubilized by this procedure. Subsequent extraction with alkaline urea solubilizes polypeptides of predominantly larger size which are derived from the tonofilaments [3]. The results are the same whether or not mercaptoethanol is included in the extraction medium. As is shown in Fig. 2a, the alkaline urea extract contains polypeptides of 100 000 daltons and over, a predominant group of 48 000-68 000 dalton species and a third group of lower molecular weight. We have

182

Fig. 1. Epidermis from 3-day-old rat which has been separated from dermis following exposure to sodium tetraphenylboron. Basal cells (B), spinous cells (S), granular cells (G) and stratum corneum (SC) are present. (1/~m epon-embedded specimen stained with methylene blue x 400). designated these groups A, B and C, respectively. Our current studies have focused on the resolution and characterization of the major proteins of the B group, two species of 68 000 and 60 000 molecular weight. Most of the C group species were removed and the higher molecular weight species (A and B) concentrated by ultrafiltration with an XM 300 membrane (Fig. 2b).

Fractionation by charge and size separation Several techniques were used to fractionate the solubilized proteins. Preparative isoelectric focusing of the whole urea extract produced an absorbance profile with a major peak at pH 5.8 and a shoulder and minor peak at pH 5.6 and p H 5.5, respectively. When these were subsequently analyzed by sodium dodecyl sulfate electrophoresis, the p H 5.8 fraction consisted primarily of the 68 000 dalton species, the p H 5.6 fraction contained primarily the 60 000 dalton species and the pH 5.5 fraction contained the 60 000 dalton species plus an admixture of larger and smaller proteins. All three fractions contained a relatively small amount of protein of approx. 120 000 daltons.

183

Fig. 2. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate. (a) Electrophoretic profile of proteins extracted by sodium deoxycholate buffer (D) and alkaline urea (U). (b) Electrophoretic profile of alkaline urea extract before and after ultrafiltration over an XM 300 membrane: starting material (U), retentate (a) and filtrate (b). The filtrate was concentrated approx. 20-fold prior to electrophoresis. Two ion-exchange procedures (Methods) were used to resolve the B group proteins. The 68 000 and 60 000 dalton species could be separated on either DEAEcellulose or CM-Sephadex but the amounts recovered were low and we did not use this technique for preparative purposes. A successful preparative method was developed based upon size separation although the relative insolubility of the proteins under study necessitated the use of denaturing solvents. We initially used Sephadex G-200 in 50 mM sodium citrate buffer (pH 4.0) [20] containing 7.5 M urea to separate the rat proteins. When the alkaline urea extract was dialyzed against this urea/citrate buffer, a precipitate formed which could be removed by centrifugation (7000 × g for 10 min). This precipitate contained the 68 000 dalton protein with minor contamination by heavier material. Following chromatography on Sephadex G-200, the urea/citrate-soluble fraction yielded a single absorbance peak which contained the 60 000 dalton protein and moderate amounts of both higher and lower molecular weight materials (data not shown). Agarose columns (A-50m) developed in 6 M guanidine at alkaline pH and Sephadex G-150 eluted with a buffer containing 0.5 ~ sodium dodecyl sulfate improved the fractionation. The results obtained using these two techniques were similar. Chromatography on Sephadex G-150 with sodium dodecyl sulfate was more efficient and this technique was used routinely. When the alkaline urea-soluble

184

Fig. 3. (a) Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of allcaline urea extract proteins fractionated on Sephadex G-150 (2.5 × 180 cm) with 0.1 M Tris. HCI (pH 7,5) containing 0.5 ~ sodium dodecyl sulfate and 15 mM 2-mercaptoethanol. 3.1-ml fractions were collected. Gel U represents the starting material and gels a-g represent selected fractions between tubes 74 and 100. The reactions of the fractionated species with an antibody (ab) to whole urea extract of newborn rat epidermis (ag) are indicated diagrammatically below each gel. (b) Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the alkaline urea extract (U) and of the 68 000 (a) and 60 000 (b) dalton species isolated from this extract by recycling molecular sieve chromatography on Sephadex G-150 run as in a. e p i d e r m a l p r o t e i n s were r u n on such a column, a single p e a k o f a b s o r b a n c e was a p p a r e n t in the m o l e c u l a r weight range between 220 000 a n d 35 000 daltons. The smaller C g r o u p p o l y p e p t i d e s eluted subsequently. W h e n the p e a k fractions were a n a l y z e d by electrophoresis in the presence o f s o d i u m dodecyl sulfate, it was a p p a r e n t (Fig. 3a) t h a t significant f r a c t i o n a t i o n h a d been achieved. R e p e a t e d c h r o m a t o g r a p h y in this same system resulted in the p r e p a r a t i o n s o f 60 000 a n d 68 000 d a l t o n proteins s h o w n in Fig. 3b. These p r e p a r a t i o n s have been used for subsequent characterization.

Analysis of the purified fibrous epidermal proteins Immunological studies. A n t i b o d i e s were raised in rabbits against b o t h the u n f r a c t i o n a t e d alkaline u r e a extract a n d the purified 60 000 d a l t o n species. Yields o f the 68 000 d a l t o n species were t o o low to p e r m i t its use as an irnmunogen. The a n t i b o d y to the u n f r a c t i o n a t e d alkaline urea extract has been tested using the O u c h t e r l o n y m e t h o d with e p i d e r m a l a n d n o n - e p i d e r m a l antigens. A m a j o r line o f identity was p r e s e n t when the a n t i b o d y was tested against a p p r o p r i a t e concent r a t i o n s o f s o d i u m d e o x y c h o l a t e a n d u r e a extracts o f n e w b o r n rat epidermis a l t h o u g h the extent o f the r e a c t i o n was m u c h less with the s o d i u m d e o x y c h o l a t e t h a n the urea

185 extracts. Two additional minor lines were apparent with the unfractionated urea extract. When alkaline urea extracts of other rat, guinea pig or human tissues were studied, precipitin reactions were seen with human and guinea pig epidermis, guinea pig hair and hair roots and with rat hair shaft extract. None of the other rat or guinea pig tissues analyzed (liver, kidney, muscle, serum, or testis) gave any reaction. No reactions were seen with alkaline urea extracts of human exocervix, with an extract of a tonofilament-producing squamous cell carcinoma of human cervical origin (ME 180), with rat muscle actin or with the major protein of newborn rat keratohyalin. The antibody to the unfractionated urea extract was also used to analyze the partially purified fractions obtained by gel filtration. A single, major reaction of identity was apparent with the A, B and C group proteins. A second line, appearing close to the antigen well, was unique to fractions containing A group proteins and a third precipitin line close to the antibody well was demonstrable in most samples containing C group proteins and B group species of less than 60 000 daltons (Fig. 3a). Immunoelectrophoretic studies confirmed the fact that a common antigenic determinant exists among the proteins of the A, B and C groups. In addition a second antigen-antibody reaction, specific for the C group, was apparent, although we were unable to identify by immunoelectrophoresis the specific precipitin line unique to the A group proteins which was noted on the Ouchterlony plates. The latter observation probably is due to the low concentration of A group proteins in the unfractionated samples. Analysis of the antibody raised to the purified 60 000 dalton species revealed a reaction of identity between the 60 000 and 68 000 dalton polypeptides.

Spectropolarimetric analyses ORD spectra of the unfractionated fibrous epidermal proteins in the far ultraviolet range revealed a pattern typical of an a-helix-containing protein with a major negative Cotton effect at 233 nm. Moffit-Yang plots [22] of the ORD data from 600

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WAV(nm) ELENGTH

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Fig. 4. CD spectra of deoxycholate and alkaline urea-soluble proteins of newborn rat epidermis run in the presence of 2.5 mM Tris.HC1 buffer (pH 9.0).

186 TABLEI a-HELIX CONTENT OF ALKALINE UREA-SOLUBLE PROTEINS FROM NEWBORN RAT EPIDERMIS The data are calculated from optical rotatory dispersion and circular dichroism measurements. Proteins were dissolved in 2.5 mM Tris.HC1 buffer (pH 9.0) except where noted. Parameter used in calculation

Observed value

b0 (20 212) b0 (2o = 200) [m']233

338 516 6 990 5 100 13 000

[m']233" [0']2os *

cz-Helix content o/ (/o)

Reference

53 40 20 31

24 24 25

In 4 M urea, 2.5 mM Tris. HC1 (pH 9.0).

to 300 n m in 2.5 m M Tris. HC1 were used to calculate the relative helicity of the proteins. These plots only linearized at a 20 wavelength between 180 a n d 200 n m which is lower t h a n the u s u a l 20 of 212 nm. I n Fig. 4 are c o m p a r e d the far ultraviolet C D spectra of the proteins extracted f r o m n e w b o r n rat epidermis with s o d i u m deoxycholate buffer a n d with alkaline urea. The C D s p e c t r u m of the s o d i u m deoxycholate-soluble material exhibits m a j o r cont r i b u t i o n s f r o m r a n d o m coil a n d fl-structures. Consistent with the O R D data n o t e d above, the C D p a t t e r n of the u n f r a c t i o n a t e d alkaline urea extract indicates a signi-

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