Biochimica et Biophysica Acta, 491 (1977) 193-204 © Elsevier/North-Holland Biomedical Press BBA 37598
PURIFICATION AND CHARACTERIZATION OF A BASIC PROTEIN FROM THE STRATUM CORNEUM OF M A M M A L I A N EPIDERMIS
BEVERLY A. DALE
Department of Periodontics, SM-44, School of Dentistry, University of Washington, Seattle, Wash. 98195 (U.S.A.) (Received August 19th, 1976)
SUMMARY
A basic protein has been isolated and purified from the stratum corneum of newborn rat epidermis. This protein is referred to as stratum corneum basic protein. It was purified by ion-exchange chromatography on DE-52 and CM-52 cellulose. The protein has a molecular weight of 50 000 on sodium dodecyl sulfate-polyacrylamide gels. It is composed of one polypeptide chain and contains no detectable carbohydrate. The protein has an isoelectric point in the range o f p H 9-10, but decomposes during isoelectric focusing giving rise to a polypeptide of less than 10 000 daltons. Amino acid analysis reveals high quantities of glutamic acid, glycine, serine, arginine and relatively high levels of histidine, with these five amino acids composing 74 ~ of the total residues. The amino acid analysis is very similar to histidinecontaining keratohyalin proteins isolated from the granular layer of epidermis by several investigators. The stratum corneum basic protein differs from fibrous proteins isolated from the same cell layer with respect to net charge, amino acid composition, and molecular weight. The protein does not react with antibody to the fibrous protein. The basic protein has properties which are consistent with its possible function as a stratum corneum interfilamentous matrix protein.
INTRODUCTION
The stratum corneum, or keratinized layer, of mammalian epidermis is composed of flattened cells devoid of organelles but filled with filamentous material embedded in a matrix [1]. The filamentous proteins, referred to as a-keratin, have been biochemically characterized in newborn rat, human, and bovine snout and hoof epidermis [2-6]. In the newborn rat these proteins are composed of polypeptide subunits of approx. 61 000 and 66 000 daltons as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis [4]. A prominent additional component with approximate molecular weight 50 000 was also identified in the sodium dodecyl sulfate gels of the crude extract of the stratum corneum [4, 7]. The quantity of this component suggested that it serves an important role in the stratum corneum, possibly as an interfilamentous matrix protein. The protein was previously shown to have different
194 solubility properties and sulfhydryl content than the fibrous proteins, but was extracted in approximately equal quantities as the fibrous proteins [4]. This component, referred to as stratum corneum basic protein, is the subject of the present study. MATERIALS AND METHODS
Isolation of stratum corneum. The dorsal skin from 1-2-day-old SpragueDawley, Berkeley strain rats was used in these studies. The subcutaneous fat was removed by gentle wiping, then the skins were placed in cold 0.15 M NaCI. Skins were incubated 10 min in cold 0.24 M NH4C1 (pH 9.5) with stirring 30 s at the beginning and end of the incubation period as described by Sibrack et al. [8], after which the epidermis could be separated from the dermis with tissue forceps. The epidermal tissue from about 50 rats was pooled, finely minced, and stirred 45 min in 75 ml 1 sodium dodecyl sulfate with 0.05 M sodium phosphate (pH 7.1) to remove the lower cell layers. Intact stratum corneum was removed by centrifugation at 12 000 × g for 10 min and stored frozen until enough material was accumulated for extraction and purification. Protein extraction. The stratum corneum from approx. 200 animals was homogenized in 300ml 4 M urea, 0.05 M sodium phosphate (pH 7.1) and 10/~g/ml phenylmethylsulfonylfluoride with a Polytron (Brinkman Instrum.), then stirred overnight at room temperature. The mixture was centrifuged at 27 000 x g for 15 min and the pellet rehomogenized and reextracted for 6 h. The supernatants were pooled and dialyzed vs. distilled H20 with several changes, then concentrated vs. polyethylene glycol. The basic protein and fibrous proteins sometimes precipitated during dialysis although precipitation was generally not complete within 48 h. When this occurred the precipitated material was removed and the supernatant concentrated. The fractions were recombined for chromatography. Column chromatography. The concentrated extract was dialyzed vs. 4 M urea with 0.05 M sodium phosphate (pH 7.1). Any undissolved material (predominently fibrous protein) was removed by centrifugation and the supernatant applied to a 4.8 × 22.5 cm DE-52 column previously equilibrated with the same urea-containing buffer. The column was eluted stepwise at 100 ml/h with 650 ml of the starting buffer, 900 ml 4 M urea with 0.2 M sodium phosphate (pH 7.1), and then 600 ml of 8 M urea with 0.045 M sodium phosphate (pH 7.1) and 0.1 M 2-mercaptoethanol. 10-ml fractions were collected. Absorbance at 280 and 235 nm was monitored. Selected column fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The initial peak (DE-52-I)contained most of the basic protein. This peak was concentrated and dialyzed vs. 0.05 M sodium acetate (pH 5.5) containing 1 M urea and applied to a 1.5 x 27 cm CM-52 column pre-equilibrated with the same buffer. The column was washed with the same buffer containing 0.2 M NaC1, then the basic protein was eluted with a linear gradient 0.18-0.5 M NaC1 in the same buffer. The resulting peak was concentrated by ultrafiltration (Amicon) and stored frozen in small portions. lsoelectricfocusing. Approx. 5 mg protein from the CM-52 peak was dialyzed vs. 10-' M CuC12 and applied to a 110 ml LKB isoelectric focusing column in a sucrose gradient containing 1 ~ ampholite (pH 9-11) as described by the LKB manual.
195 Voltage was 300 V for the first hour then 400 V for the duration of the run. Electrofocusing was continued for 48 h or until the amperage was stable. Fractions of 2.35 ml were collected and absorbance monitored at 280 nm. pH and absorbance at 235 nm were subsequently measured. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracts and column fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to identify the basic protein. Electrophoresis was conducted in 7 ~ gels as previously described [9, 10]. Generally 100/~l of specified column fractions was applied after incubation at 100 °C for 1 min in a final concentration of 4 M urea, 1 ~ sodium dodecyl sulfate, 1 ~ 2-mercaptoethanol. Immunoelectrophoresis. Rocket immunoelectrophoresis [11 ] was performed in 1 ~ agarose containing 0.5 ~ Triton X-100, 0.02 M Veronal (pH 8.4), and antibody to the light chain of newborn rat fibrous protein. Electrophoresis was carried out at 6 V/cm for 4 h with 0.04 M Veronal buffer. Biochemical analyses. Protein was determined by the method described by Lowry et al. [12], or Bramhall et al. [13] for samples containing mercaptoetb,anol and 8 M urea. Bovine serum albumin was used as standard. Amino acid analysis was determined on duplicate samples of the CM-52 peak. Samples were dialyzed, lyophilized and hydrolyzed in 6 M HC1 at 110 °C for 24 h. Amino acid composition was determined on a Beckman 120C amino acid analyzer. The carbohydrate content was determined by the orcinal method for ribose [14], anthrone for neutral hexose [15], and modified Elson-Morgan for hexosamine [16]. Materials. Protein standards for molecular weight determinations were as follows: Bovine serum albumin (66 000, Sigma), ovalbumin (46 000, Sigma), chymotrypsinogen (25 700, Sigma), cytochrome c (12 500, Schwarz/Mann), bovine ~,globulin (53 000 and 24 000, Armour Pharmaceutical). Dialysis membrane, molecular weight cut off 3500, was obtained from Spectrum Medical Industries, Inc. Urea was made up as an 8.8 M stock solution, deionized with a mixed bed ion-exchange resin, and stored in the cold. All other chemicals were reagent grade. RESULTS
Stratum corneum isolation and extraction Incubation of epidermis with buffered 1 ~ sodium dodecyl sulfate resulted in separation and dissolution of the lower cell layers leaving the stratum corneum intact with only a few ceils of the granular layer still adhering, as shown in Fig. 1. This brief sodium dodecyl sulfate treatment in the absence of homogenization should minimize extraction of stratum corneum contents. Then the stratum corneum was homogenized, extracted in trial experiments with 4 M urea for 18 h, then with 8 M urea with reducing agents for 18 h as previously described for fibrous proteins [4]. The extracts and residue were dialyzed, assayed for total protein, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2). The 1 ~o sodium dodecyl sulfate extract of the lower cells contains many polypeptides (Fig. 2, gel A), but the stratum corneum contains primarily the two fibrous proteins and a third band, the stratum corneum basic protein [4, 7]. The basic protein is preferentially extracted
196
Fig. 1. Skin and isolated stratum corneum. 5-ttm paraffin embedded sections stained with hemotoxylin and eosin, ×200. (A) Newborn rat skin: the stratum corneuna is eosinophilic in contrast to the keratohyalin granules which are stained darkly with hematoxylin. (B) Stratum corneum after incubation of epidermis for 45 rain in 0.05 M potassium phosphate (pH 7.1) containing 1 ~ sodium dodecyl sulfate. The entire section is eosinophilic. by 4 M urea (Fig. 2, gel B) and the fibrous proteins by 8 M urea (Fig. 2, gel C), although the same proteins remain in the residue (Fig. 2, gel D), suggesting incomplete extraction. Some o f the proteins in the 1 ~ sodium dodecyl sulfate extract correspond on the gels to proteins extracted from the stratum corneum. These proteins may be derived from the lower cell layers or the stratum corneurn. Partial extraction o f the stratum c o r n e u m by 1 ~ sodium dodecyl sulfate cannot be ruled out. The 4 M urea extract o f this trial experiment and large scale extractions contained an average o f one-third o f the total recovered epidermal proteins or one half of the stratum corneum protein, o f which roughly 25 ~ migrated as the third band on polyacrylamide gels.
Column chromatography The dialyzed and concentrated 4 M urea extract from approx. 200 newborn
197
Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of newborn rat epidermis. Minced epidermis was stirred with 0.05 M sodium phosphate (NaPO4) (pH 7.1) containing 1 ~ sodium dodecyl sulfate for 45 min, then centrifuged at 25 000 x g. The residue (stratum corneum) was homogenized and extracted with 4 M urea in 0.05 M sodium phosphate for 18 h, then centrifuged at 25 000 x g. The pellet was re-extracted with 8 M urea plus 0.1 M 2-mercaptoethanol plus 1 mM dithiothreitol in the same buffer for 18 h, then centrifuged at 25 000 x g. The final insoluble residue was resuspended in the same buffer containing 8 M urea. Extracts were dialyzed and run on sodium dodecy! sulfate-polyacrylamide gels. A, sodium dodecyl sulfate extract; B, 4 M urea extract; C, 8 M urea extract; D, residue after 8 M urea extraction; E, molecular weight standards; I, bovine serum albumin; 1I, ovalbumin; Ill, chymotrypsinogen; IV, cytochrome c.
r a t skins was a p p l i e d to a DE-52 c o l u m n previously equilibrated in 0.05 M s o d i u m p h o s p h a t e c o n t a i n i n g 4 M urea. The c o l u m n was eluted stepwise by increasing the c o n c e n t r a t i o n o f buffer a n d urea. The elution profile, m o n i t o r e d at 280 a n d 235 nm, is seen in Fig. 3a. S o d i u m d o d e c y l s u i f a t e - p o l y a c r y l a m i d e gels o f the c o l u m n p e a k s are seen in Fig. 3b. The basic p r o t e i n was recovered in p e a k I ( u n a d s o r b e d material) a l t h o u g h some p r o t e i n with similar m o b i l i t y on the gels was identified in p e a k II. F i b r o u s p r o t e i n s were eluted in peaks II and III. Peak I f r o m the DE-52 c o l u m n was c o n c e n t r a t e d a n d dialyzed vs. 0.05 M s o d i u m acetate ( p H 5.5) c o n t a i n i n g 1 M urea. H a l f o f this m a t e r i a l was a p p l i e d to a C M - 5 2 c o l u m n a n d the basic p r o t e i n eluted with a linear g r a d i e n t o f 0.18-0.5 M N a C l in the same buffer. The elution profile is shown in Fig. 4a. Electrophoresis o f the c o m b i n e d p e a k fractions on s o d i u m dodecyl s u l f a t e - p o l y a c r y l a m i d e gel resulted in one m a j o r b a n d with several very m i n o r b a n d s o f greater mobility. T h e m o l e c u l a r weight o f the m a j o r b a n d is a p p r o x . 50 000 when c o m p a r e d with p r o t e i n s t a n d a r d s on the gels. The same result is o b t a i n e d in the presence o r absence o f 2 - m e r c a p t o e t h a n o l .
198 1.5-
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lsoelectric focusing C o l u m n isoelectric focusing was conducted in a n effort to purify the protein more completely as well as to measure the isoelectric point. The C M - 5 2 purified protein was dialyzed vs. 10 -5 M CuC12, a n d applied in the sucrose gradient c o n t a i n i n g ampholite, p H 9-11. Protein peaks were eluted at p H 10.5, 10.0, 9.60, 9.38 (Fig. 5a). Protein yield was 5, 16, 32 a n d 18 % in peaks I - I V , respectively. The m a j o r fraction was that with pI 9.60. Duplicate runs showed similar elution patterns a n d pI values.
199
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Fig. 4. (a) Ion-exchange chromatography on CM-52 cellulose. Peak I from the DE-52 column was concentrated, dialyzed vs. starting buffer, and applied to a 1.5 x 27 cm column pre-equilibrated with 0.05 M sodium acetate (pH 5.5) containing 1 M urea. The column was washed with starting buffer containing 0.2 M NaCI, then the protein eluted with a 0.18-0.5 M NaCI gradient in the same buffer. 6-ml fractions were collected. Fractions 53-71 were combined for the peak. (b) Sodium dodecyl sulfate-po!yacrylamide gel electrophoresis of the peak from a. The polypeptides of less than 50 000 daltons were not separated by this procedure and a very low molecular weight species migrating faster than cytochrome c appeared. The peak fractions were pooled, dialyzed and concentrated vs. polyethylene glycol, then re-analyzed on sodium dodecyl sulfate gels (Fig. 5b). The predominant polypeptide recovered was smaller than cytochrome c. The original dialyzed sample applied to the column did not contain this low molecular weight fragment. Thus, the exposure to high pH, low ionic strength, or the time required at 5-10 °C for the focusing and subsequent procedures led to breakdown of the protein.
Amino acid composition Amino acid analysis of the CM-52 purified protein revealed a high content of glycine, glutamic acid, serine, arginine and a relatively high histidine content. These five amino acids comprised 74 % of the total residues. The protein has a low content of amino acids with non-polar side chains. It contains no cysteine or lysine. The amino acid composition is shown in Table I. The composition of the fibrous proteins of the stratum corneum [4] is also shown for comparison.
Carbohydrate analysis Carbohydrate analysis on 1-mg samples revealed no ribose, less than 0.1% hexosamine and less than 0.1% neutral hexose.
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Fig. 5. (a) Column isoelectric focusing of the CM-52 peak. 5 mg protein was applied in a sucrose gradient containing 1% ampholites (pH 9-11). Voltage was 300 V for 1 h, then 400 V for 47 h. 2.35ml fractions were collected and absorbance at 280 nm was monitored. Absorbance at 235 nm was measured in samples diluted 10-fold. Fractions were combined for peaks I-IV, as indicated. The peaks were dialyzed vs. 0.05 M sodium phosphate (pH 7.1) to remove ampholites then concentrated. (b) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pooled, concentrated peaks, from a. Arrow indicates position of 50 000 dalton species.
201 TABLE I AMINO ACID COMPOSITION OF STRATUM CORNEUM PROTEINS" Basic protein
Cystine (half) Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
0 0 7.9 13.9 3.6 5.7 17.3 20.5 2.8 14.4 11.8 0.6 0 1.4 0 trace 0
Fibrous protein** Heavy chain
Light chain
0.8 7.1 0.8 7.4 8.9 2.8 9.5 14.3 2.0 11.5 4.0 3.2 4.9 3.6 5.9 2.2 4.8 1.5
0.8 4.6 0.7 5.8 8.2 3.2 11.9 17.9 0.6 15.1 2.8 1.7 2.8 2.8 7.7 5.5 3.6 2.3
Residues/100 residues. "* Ref. 4.
Immunoelectrophoresis The basic protein preparation was tested for cross-reactivity or contamination with the fibrous proteins by rocket immunoelectrophoresis using antibody to the newborn rat fibrous protein (light chain) [4]. The basic protein migrates toward the cathode at the p H used (pH 8.4) whereas the fibrous proteins migrate toward the anode. 25 b~g of the CM-52 purified protein showed no reactivity with the antibody when electrophoresed toward the cathode (to test for cross-reactivity) or toward the anode (to test for contamination) under conditions which showed detectable reaction with 0.25/~g of the light chain of the fibrous protein. DISCUSSION A protein has been isolated and partially purified from the stratum corneum of newborn rats which differs in several respects from the fibrous proteins isolated from the same cell layer. The most distinctive characteristic of this protein is its basic nature; accordingly I suggest that it be called stratum corneum basic protein until its function is identified and it can be given a more suitable descriptive name. The purification of the protein is based on its basic nature. However, neither the original quantity nor the purification factor can be determined because of the lack of a specific assay for the protein. The relative amount and the relative purity were assessed by migration on sodium dodecyl sulfate-polyacrylamide gels. The final preparation used for characterization was free of fibrous protein as judged by (1) rocket immunoelectrophoresis using antibody to fibrous protein and (2) sodium
202 dodecyl sulfate-polyacrylamide gel electrophoresis of 50 #g of the CM-52 peak. It contained some polypeptides of lower molecular weight than the 50 000 daltons band. These polypeptides could not be removed by isoelectric focusing and their prominence increased with time of storage; thus they probably represent breakdown products. Attempts to further purify the protein by isoelectric focusing were unsuccessful due to its instability under the conditions required. Four isoelectric protein peaks were found after column isoelectric focusing. The isoelectric point of the most prominent peak was 9.60. The basic protein was previously identified as a component in extracts of stratum corneum obtained by the EDTA trypsinization procedure of Stern and coworkers [4, 7, 17]. During the purification of fibrous protein by Huang et al. [4], this basic protein was shown to have a low content of sulfhydryl groups which could be derivatized by iodoacetic acid and was considered to be a non-fibrous protein on that basis. The present study has shown that the protein can be extracted with lower concentration of urea in the absence of reducing agents, that it does not bind to DE-52 in contrast to the fibrous proteins, and that its migration on sodium dodecyl sulfatepolyacrylamide gels is the same in the presence or absence of reducing agent. The amino acid compositions of these three major proteins of the stratum corneum have some similarities. All are rich in glycine, serine, and glutamic acid. However, a large proportion of the glutamic acid must be present as the amide in the basic protein. The basic protein has a much higher proportion of histidine, arginine, and alanine than the fibrous proteins; and it has a lower proportion of cysteine, lysine, aspartic acid, and non-polar amino acids than the fibrous proteins. The amino acid composition of the stratum corneum basic protein is very similar to the histidine-containing protein isolated by Bernstein and coworkers from the granular layer of newborn rat epidermis [18, 19] and from keratohyalin [8]. In addition, the molecular weight of the basic protein is similar to one component of potassium phosphate-extracted keratohyalin [7, 8]. Similar histidine-containing proteins have also been isolated from other keratohyalin preparations by Tezuka and Freedberg [20] and Ugel and Idler [21]. Further studies are in progress to determine if the stratum corneum basic protein is derived from keratohyalin. Early in this work it was determined that the epidermal cell separation technique utilizing buffered EDTA then trypsinization resulted in stratum corneum preparations with variable yields of the basic protein. This variability is now thought to be due to sensitivity of this protein to EDTA and possibly to trypsin. The purified basic protein can be dialyzed vs. 1 mM EDTA or 1 mM solutions of CuC12, MnC12, MgC12, ZnCI2, and CaCI2. Stability vs. EDTA and some ions has also been tested in the presence of 4 M urea or in buffers at pH 7 or pH 5 with no change in its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels. Thus, stabilization of the basic protein by metal ions has not been demonstrated directly. However, we have not ruled out stabilization by very tightly bound divalent cations. These results suggest that the sensitivity of the basic protein to EDTA in situ may be due to a metalinhibited protease. This in situ sensitivity may explain why this protein was not identified in previous studies in which EDTA-containing buffers were used [22]. Another explanation for the apparent absence of the basic protein is its low absorbance at 280 nm, the wavelength generally used to follow chromatographic profiles. A protein of similar molecular weight was identified by Matoltsy [23] in sodium
203 dodecyl sulfate-polyacrylamide gels of stratum corneum extracted with buffered sodium dodecyl sulfate. This protein was more easily extracted at acidic pH as would be expected for a basic protein. What are the possible functions of the stratum corneum basic protein? The protein is an extremely polar, highly positive-charged molecule devoid of sulfhydryl groups and carbohydrate. These properties suggest that it is not membrane protein or an extracellular protein. We can also rule out the dense peripheral band on the inner aspect of the cell membrane which has been shown to contain sulfhydryl groups by histochemistry [24], autoradiography [25, 26], and biochemical analysis [27]. Another possible function of the basic protein is that of the matrix substance in the stratum corneum. Morphologic studies suggest that the stratum corneum matrix may be derived from keratohyalin, whereas the fibrous proteins are derived from tonofilaments [1]. The basic protein is similar to a histidine-containing protein of keratohyalin in both molecular weight and amino acid composition, suggesting it could be derived from keratohyalin. Thus, the properties of the stratum corneum basic protein isolated here suggest that it is a good candidate for a matrix substance. Tezuka [28] has recently isolated a presumed matrix protein from human plantar stratum corneum which differs in its amino acid composition from that reported here. Tezuka's protein has an amino acid composition similar to that of fibrous protein. Antibody to his material reacts with keratohyalin granules in situ, an observation consistent with the proposed keratohyalin granule origin of matrix protein. However, antibody to fibrous protein has previously been shown to react with keratohyalin granules presumably due to the presence of tonofilaments in and around these granules [4, 29]. Thus, the role of Tezuka's protein as matrix has not been unequivocally demonstrated. In conclusion, a basic protein has been isolated from newborn rat stratum corneum. The protein is a major component of stratum corneum, but differs from the fibrous proteins; it is similar to a keratohyalin protein. The basic stratum corneum protein has properties which are consistent with its possible function as a stratum corneum interfilamentous matrix protein. ACKNOWLEDGEMENTS This work was supported by U.S. Public Health Service Grant Nos. DE-04660 and DE-02600, Center for Research in Oral Biology. The author would like to express her appreciation to Ms. Sue Ling for her excellent technical assistance and to Dr. Irving B. Stern for discussion and advice on the manuscript. REFERENCES 1 Brody, I. (1959) J. Ultrastruct. Res. 2, 482-511 2 Baden, H. P. and Bonar, L. (1968) J. Invest. Dermatol. 51, 478483 3 Lee, L. D., Fleming, B. C., Waitkus, R. F. and Baden, H. P. (1975) Biochim. Biophys. Acta 412, 82-90 4 Huang, L.-Y., Stern, I. B., Clagett, J. A. and Chi, E. Y. 0975) Biochemistry 14, 3573-3580 5 Steinert, P. M. (1975) Biochem. J. 149, 3948 6 Steinert, P. M. and Idler, W. W. (1975) Biochem. J. 151, 603-614 7 Dale, B. A. and Stern, I. B. 0975) J. Invest. Dermatol. 65, 223-227
204 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Sibrack, L. A., Gray, R. H. and Bernstein, I. A. (1974) J. Invest. Dermatol. 62, 394-405 Dale, B. A. and Stern, 1. B. (1975) J. Invest. Dermatol. 65, 220-222 Dunker, A. K. and Rueckert, R. R. (1969) J. Biol. Chem. 244, 5074-5080 Laurell, C.-B. (1966) Anal. Biochem. 15, 45-52 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 Bramhall, S., Noack, N., Wu, M. and Loewenberg, J. R. (1969) Anal. Biochem. 31, 146-148 Ogur, M. and Rosen, G. (1950) Arch. Biochem. Biophys. 25, 262-276 Scott, Jr., T. A. and Melvin, E. H. (1953) Anal. Chem. 25, 1656-1661 Gatt, R. and Berman, E. R. (1966) Anal. Biochem. 15, 167-171 Stern, 1. B. and Sekeri-Pataryas, K. H. (1972) J. Invest. Dermatol. 59, 251-259 Gumucio, J., Feldkamp, C. and Bernstein, 1. A. (1967) J. Invest. Dermatol. 49, 545-551 Hoober, J. K. and Bernstein, I. A. (1966) Proc. Natl. Acad. Sci. U.S. 56, 594-601 Tezuka, T. and Freedberg, I. M. (1974) J. Invest. Dermatol. 63, 402-406 Ugel, A. R. and Idler, W. (1972) J. Cell Biol. 52, 453-464 Shimizu, T., Fukuyama, K. and Epstein, W. L. (1974) Biochim. Biophys. Acta 359, 389-400 Matoltsy, A. G. (1975) J. Invest. Dermatol. 65, 127-142 Jessen, H. (1973) Histochemie 33, 15-29 Fukuyama, K. and Epstein, W. L. (1969) J. Cell Biol. 40, 830-838 Fukuyama, K. and Epstein, W. L. (1975) J. Ultrastruct. Res. 51, 314 325 Matoltsy, A. G. (1974) J. Invest. Dermatol. 62, 343 (abstract) Tezuka, T. (1975) Acta Dermatovenerol. (Stockholm) 55, 401-412 Dale, B. A., Stern, I. B., Rabin, M. and Huang, L.-Y. (1976) J. Invest. Dermatol. 66, 230-235
PURIFICATION AND CHARACTERIZATION OF A BASIC PROTEIN FROM THE STRATUM CORNEUM OF M A M M A L I A N EPIDERMIS
BEVERLY A. DALE
Department of Periodontics, SM-44, School of Dentistry, University of Washington, Seattle, Wash. 98195 (U.S.A.) (Received August 19th, 1976)
SUMMARY
A basic protein has been isolated and purified from the stratum corneum of newborn rat epidermis. This protein is referred to as stratum corneum basic protein. It was purified by ion-exchange chromatography on DE-52 and CM-52 cellulose. The protein has a molecular weight of 50 000 on sodium dodecyl sulfate-polyacrylamide gels. It is composed of one polypeptide chain and contains no detectable carbohydrate. The protein has an isoelectric point in the range o f p H 9-10, but decomposes during isoelectric focusing giving rise to a polypeptide of less than 10 000 daltons. Amino acid analysis reveals high quantities of glutamic acid, glycine, serine, arginine and relatively high levels of histidine, with these five amino acids composing 74 ~ of the total residues. The amino acid analysis is very similar to histidinecontaining keratohyalin proteins isolated from the granular layer of epidermis by several investigators. The stratum corneum basic protein differs from fibrous proteins isolated from the same cell layer with respect to net charge, amino acid composition, and molecular weight. The protein does not react with antibody to the fibrous protein. The basic protein has properties which are consistent with its possible function as a stratum corneum interfilamentous matrix protein.
INTRODUCTION
The stratum corneum, or keratinized layer, of mammalian epidermis is composed of flattened cells devoid of organelles but filled with filamentous material embedded in a matrix [1]. The filamentous proteins, referred to as a-keratin, have been biochemically characterized in newborn rat, human, and bovine snout and hoof epidermis [2-6]. In the newborn rat these proteins are composed of polypeptide subunits of approx. 61 000 and 66 000 daltons as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis [4]. A prominent additional component with approximate molecular weight 50 000 was also identified in the sodium dodecyl sulfate gels of the crude extract of the stratum corneum [4, 7]. The quantity of this component suggested that it serves an important role in the stratum corneum, possibly as an interfilamentous matrix protein. The protein was previously shown to have different
194 solubility properties and sulfhydryl content than the fibrous proteins, but was extracted in approximately equal quantities as the fibrous proteins [4]. This component, referred to as stratum corneum basic protein, is the subject of the present study. MATERIALS AND METHODS
Isolation of stratum corneum. The dorsal skin from 1-2-day-old SpragueDawley, Berkeley strain rats was used in these studies. The subcutaneous fat was removed by gentle wiping, then the skins were placed in cold 0.15 M NaCI. Skins were incubated 10 min in cold 0.24 M NH4C1 (pH 9.5) with stirring 30 s at the beginning and end of the incubation period as described by Sibrack et al. [8], after which the epidermis could be separated from the dermis with tissue forceps. The epidermal tissue from about 50 rats was pooled, finely minced, and stirred 45 min in 75 ml 1 sodium dodecyl sulfate with 0.05 M sodium phosphate (pH 7.1) to remove the lower cell layers. Intact stratum corneum was removed by centrifugation at 12 000 × g for 10 min and stored frozen until enough material was accumulated for extraction and purification. Protein extraction. The stratum corneum from approx. 200 animals was homogenized in 300ml 4 M urea, 0.05 M sodium phosphate (pH 7.1) and 10/~g/ml phenylmethylsulfonylfluoride with a Polytron (Brinkman Instrum.), then stirred overnight at room temperature. The mixture was centrifuged at 27 000 x g for 15 min and the pellet rehomogenized and reextracted for 6 h. The supernatants were pooled and dialyzed vs. distilled H20 with several changes, then concentrated vs. polyethylene glycol. The basic protein and fibrous proteins sometimes precipitated during dialysis although precipitation was generally not complete within 48 h. When this occurred the precipitated material was removed and the supernatant concentrated. The fractions were recombined for chromatography. Column chromatography. The concentrated extract was dialyzed vs. 4 M urea with 0.05 M sodium phosphate (pH 7.1). Any undissolved material (predominently fibrous protein) was removed by centrifugation and the supernatant applied to a 4.8 × 22.5 cm DE-52 column previously equilibrated with the same urea-containing buffer. The column was eluted stepwise at 100 ml/h with 650 ml of the starting buffer, 900 ml 4 M urea with 0.2 M sodium phosphate (pH 7.1), and then 600 ml of 8 M urea with 0.045 M sodium phosphate (pH 7.1) and 0.1 M 2-mercaptoethanol. 10-ml fractions were collected. Absorbance at 280 and 235 nm was monitored. Selected column fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The initial peak (DE-52-I)contained most of the basic protein. This peak was concentrated and dialyzed vs. 0.05 M sodium acetate (pH 5.5) containing 1 M urea and applied to a 1.5 x 27 cm CM-52 column pre-equilibrated with the same buffer. The column was washed with the same buffer containing 0.2 M NaC1, then the basic protein was eluted with a linear gradient 0.18-0.5 M NaC1 in the same buffer. The resulting peak was concentrated by ultrafiltration (Amicon) and stored frozen in small portions. lsoelectricfocusing. Approx. 5 mg protein from the CM-52 peak was dialyzed vs. 10-' M CuC12 and applied to a 110 ml LKB isoelectric focusing column in a sucrose gradient containing 1 ~ ampholite (pH 9-11) as described by the LKB manual.
195 Voltage was 300 V for the first hour then 400 V for the duration of the run. Electrofocusing was continued for 48 h or until the amperage was stable. Fractions of 2.35 ml were collected and absorbance monitored at 280 nm. pH and absorbance at 235 nm were subsequently measured. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracts and column fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to identify the basic protein. Electrophoresis was conducted in 7 ~ gels as previously described [9, 10]. Generally 100/~l of specified column fractions was applied after incubation at 100 °C for 1 min in a final concentration of 4 M urea, 1 ~ sodium dodecyl sulfate, 1 ~ 2-mercaptoethanol. Immunoelectrophoresis. Rocket immunoelectrophoresis [11 ] was performed in 1 ~ agarose containing 0.5 ~ Triton X-100, 0.02 M Veronal (pH 8.4), and antibody to the light chain of newborn rat fibrous protein. Electrophoresis was carried out at 6 V/cm for 4 h with 0.04 M Veronal buffer. Biochemical analyses. Protein was determined by the method described by Lowry et al. [12], or Bramhall et al. [13] for samples containing mercaptoetb,anol and 8 M urea. Bovine serum albumin was used as standard. Amino acid analysis was determined on duplicate samples of the CM-52 peak. Samples were dialyzed, lyophilized and hydrolyzed in 6 M HC1 at 110 °C for 24 h. Amino acid composition was determined on a Beckman 120C amino acid analyzer. The carbohydrate content was determined by the orcinal method for ribose [14], anthrone for neutral hexose [15], and modified Elson-Morgan for hexosamine [16]. Materials. Protein standards for molecular weight determinations were as follows: Bovine serum albumin (66 000, Sigma), ovalbumin (46 000, Sigma), chymotrypsinogen (25 700, Sigma), cytochrome c (12 500, Schwarz/Mann), bovine ~,globulin (53 000 and 24 000, Armour Pharmaceutical). Dialysis membrane, molecular weight cut off 3500, was obtained from Spectrum Medical Industries, Inc. Urea was made up as an 8.8 M stock solution, deionized with a mixed bed ion-exchange resin, and stored in the cold. All other chemicals were reagent grade. RESULTS
Stratum corneum isolation and extraction Incubation of epidermis with buffered 1 ~ sodium dodecyl sulfate resulted in separation and dissolution of the lower cell layers leaving the stratum corneum intact with only a few ceils of the granular layer still adhering, as shown in Fig. 1. This brief sodium dodecyl sulfate treatment in the absence of homogenization should minimize extraction of stratum corneum contents. Then the stratum corneum was homogenized, extracted in trial experiments with 4 M urea for 18 h, then with 8 M urea with reducing agents for 18 h as previously described for fibrous proteins [4]. The extracts and residue were dialyzed, assayed for total protein, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2). The 1 ~o sodium dodecyl sulfate extract of the lower cells contains many polypeptides (Fig. 2, gel A), but the stratum corneum contains primarily the two fibrous proteins and a third band, the stratum corneum basic protein [4, 7]. The basic protein is preferentially extracted
196
Fig. 1. Skin and isolated stratum corneum. 5-ttm paraffin embedded sections stained with hemotoxylin and eosin, ×200. (A) Newborn rat skin: the stratum corneuna is eosinophilic in contrast to the keratohyalin granules which are stained darkly with hematoxylin. (B) Stratum corneum after incubation of epidermis for 45 rain in 0.05 M potassium phosphate (pH 7.1) containing 1 ~ sodium dodecyl sulfate. The entire section is eosinophilic. by 4 M urea (Fig. 2, gel B) and the fibrous proteins by 8 M urea (Fig. 2, gel C), although the same proteins remain in the residue (Fig. 2, gel D), suggesting incomplete extraction. Some o f the proteins in the 1 ~ sodium dodecyl sulfate extract correspond on the gels to proteins extracted from the stratum corneum. These proteins may be derived from the lower cell layers or the stratum corneurn. Partial extraction o f the stratum c o r n e u m by 1 ~ sodium dodecyl sulfate cannot be ruled out. The 4 M urea extract o f this trial experiment and large scale extractions contained an average o f one-third o f the total recovered epidermal proteins or one half of the stratum corneum protein, o f which roughly 25 ~ migrated as the third band on polyacrylamide gels.
Column chromatography The dialyzed and concentrated 4 M urea extract from approx. 200 newborn
197
Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of newborn rat epidermis. Minced epidermis was stirred with 0.05 M sodium phosphate (NaPO4) (pH 7.1) containing 1 ~ sodium dodecyl sulfate for 45 min, then centrifuged at 25 000 x g. The residue (stratum corneum) was homogenized and extracted with 4 M urea in 0.05 M sodium phosphate for 18 h, then centrifuged at 25 000 x g. The pellet was re-extracted with 8 M urea plus 0.1 M 2-mercaptoethanol plus 1 mM dithiothreitol in the same buffer for 18 h, then centrifuged at 25 000 x g. The final insoluble residue was resuspended in the same buffer containing 8 M urea. Extracts were dialyzed and run on sodium dodecy! sulfate-polyacrylamide gels. A, sodium dodecyl sulfate extract; B, 4 M urea extract; C, 8 M urea extract; D, residue after 8 M urea extraction; E, molecular weight standards; I, bovine serum albumin; 1I, ovalbumin; Ill, chymotrypsinogen; IV, cytochrome c.
r a t skins was a p p l i e d to a DE-52 c o l u m n previously equilibrated in 0.05 M s o d i u m p h o s p h a t e c o n t a i n i n g 4 M urea. The c o l u m n was eluted stepwise by increasing the c o n c e n t r a t i o n o f buffer a n d urea. The elution profile, m o n i t o r e d at 280 a n d 235 nm, is seen in Fig. 3a. S o d i u m d o d e c y l s u i f a t e - p o l y a c r y l a m i d e gels o f the c o l u m n p e a k s are seen in Fig. 3b. The basic p r o t e i n was recovered in p e a k I ( u n a d s o r b e d material) a l t h o u g h some p r o t e i n with similar m o b i l i t y on the gels was identified in p e a k II. F i b r o u s p r o t e i n s were eluted in peaks II and III. Peak I f r o m the DE-52 c o l u m n was c o n c e n t r a t e d a n d dialyzed vs. 0.05 M s o d i u m acetate ( p H 5.5) c o n t a i n i n g 1 M urea. H a l f o f this m a t e r i a l was a p p l i e d to a C M - 5 2 c o l u m n a n d the basic p r o t e i n eluted with a linear g r a d i e n t o f 0.18-0.5 M N a C l in the same buffer. The elution profile is shown in Fig. 4a. Electrophoresis o f the c o m b i n e d p e a k fractions on s o d i u m dodecyl s u l f a t e - p o l y a c r y l a m i d e gel resulted in one m a j o r b a n d with several very m i n o r b a n d s o f greater mobility. T h e m o l e c u l a r weight o f the m a j o r b a n d is a p p r o x . 50 000 when c o m p a r e d with p r o t e i n s t a n d a r d s on the gels. The same result is o b t a i n e d in the presence o r absence o f 2 - m e r c a p t o e t h a n o l .
198 1.5-
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Fig. 3. (a) Ion-exchange chromatography on DE-52 cellulose. The 4 M urea extract of stratum corneum was dialyzed vs. starting buffer and applied to a 4.8 × 22.5 cm column pre-equilibrated with 0.05 M sodium phosphate (NaPO4) (pH 7.1) containing 4 M urea. The protein was eluted stepwise with 650 ml starting buffer, 900 ml 0.2 M sodium phosphate (pH 7.1) containing 4 M urea, 600 ml 8 M urea plus 0.1 M 2-mercaptoethanol. 10-ml fractions were collected. Absorbance was monitored at 280 and 235 nm. Peak fractions were combined as indicated. (b) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of peak fractions from a.
lsoelectric focusing C o l u m n isoelectric focusing was conducted in a n effort to purify the protein more completely as well as to measure the isoelectric point. The C M - 5 2 purified protein was dialyzed vs. 10 -5 M CuC12, a n d applied in the sucrose gradient c o n t a i n i n g ampholite, p H 9-11. Protein peaks were eluted at p H 10.5, 10.0, 9.60, 9.38 (Fig. 5a). Protein yield was 5, 16, 32 a n d 18 % in peaks I - I V , respectively. The m a j o r fraction was that with pI 9.60. Duplicate runs showed similar elution patterns a n d pI values.
199
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Fig. 4. (a) Ion-exchange chromatography on CM-52 cellulose. Peak I from the DE-52 column was concentrated, dialyzed vs. starting buffer, and applied to a 1.5 x 27 cm column pre-equilibrated with 0.05 M sodium acetate (pH 5.5) containing 1 M urea. The column was washed with starting buffer containing 0.2 M NaCI, then the protein eluted with a 0.18-0.5 M NaCI gradient in the same buffer. 6-ml fractions were collected. Fractions 53-71 were combined for the peak. (b) Sodium dodecyl sulfate-po!yacrylamide gel electrophoresis of the peak from a. The polypeptides of less than 50 000 daltons were not separated by this procedure and a very low molecular weight species migrating faster than cytochrome c appeared. The peak fractions were pooled, dialyzed and concentrated vs. polyethylene glycol, then re-analyzed on sodium dodecyl sulfate gels (Fig. 5b). The predominant polypeptide recovered was smaller than cytochrome c. The original dialyzed sample applied to the column did not contain this low molecular weight fragment. Thus, the exposure to high pH, low ionic strength, or the time required at 5-10 °C for the focusing and subsequent procedures led to breakdown of the protein.
Amino acid composition Amino acid analysis of the CM-52 purified protein revealed a high content of glycine, glutamic acid, serine, arginine and a relatively high histidine content. These five amino acids comprised 74 % of the total residues. The protein has a low content of amino acids with non-polar side chains. It contains no cysteine or lysine. The amino acid composition is shown in Table I. The composition of the fibrous proteins of the stratum corneum [4] is also shown for comparison.
Carbohydrate analysis Carbohydrate analysis on 1-mg samples revealed no ribose, less than 0.1% hexosamine and less than 0.1% neutral hexose.
200
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Fig. 5. (a) Column isoelectric focusing of the CM-52 peak. 5 mg protein was applied in a sucrose gradient containing 1% ampholites (pH 9-11). Voltage was 300 V for 1 h, then 400 V for 47 h. 2.35ml fractions were collected and absorbance at 280 nm was monitored. Absorbance at 235 nm was measured in samples diluted 10-fold. Fractions were combined for peaks I-IV, as indicated. The peaks were dialyzed vs. 0.05 M sodium phosphate (pH 7.1) to remove ampholites then concentrated. (b) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pooled, concentrated peaks, from a. Arrow indicates position of 50 000 dalton species.
201 TABLE I AMINO ACID COMPOSITION OF STRATUM CORNEUM PROTEINS" Basic protein
Cystine (half) Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
0 0 7.9 13.9 3.6 5.7 17.3 20.5 2.8 14.4 11.8 0.6 0 1.4 0 trace 0
Fibrous protein** Heavy chain
Light chain
0.8 7.1 0.8 7.4 8.9 2.8 9.5 14.3 2.0 11.5 4.0 3.2 4.9 3.6 5.9 2.2 4.8 1.5
0.8 4.6 0.7 5.8 8.2 3.2 11.9 17.9 0.6 15.1 2.8 1.7 2.8 2.8 7.7 5.5 3.6 2.3
Residues/100 residues. "* Ref. 4.
Immunoelectrophoresis The basic protein preparation was tested for cross-reactivity or contamination with the fibrous proteins by rocket immunoelectrophoresis using antibody to the newborn rat fibrous protein (light chain) [4]. The basic protein migrates toward the cathode at the p H used (pH 8.4) whereas the fibrous proteins migrate toward the anode. 25 b~g of the CM-52 purified protein showed no reactivity with the antibody when electrophoresed toward the cathode (to test for cross-reactivity) or toward the anode (to test for contamination) under conditions which showed detectable reaction with 0.25/~g of the light chain of the fibrous protein. DISCUSSION A protein has been isolated and partially purified from the stratum corneum of newborn rats which differs in several respects from the fibrous proteins isolated from the same cell layer. The most distinctive characteristic of this protein is its basic nature; accordingly I suggest that it be called stratum corneum basic protein until its function is identified and it can be given a more suitable descriptive name. The purification of the protein is based on its basic nature. However, neither the original quantity nor the purification factor can be determined because of the lack of a specific assay for the protein. The relative amount and the relative purity were assessed by migration on sodium dodecyl sulfate-polyacrylamide gels. The final preparation used for characterization was free of fibrous protein as judged by (1) rocket immunoelectrophoresis using antibody to fibrous protein and (2) sodium
202 dodecyl sulfate-polyacrylamide gel electrophoresis of 50 #g of the CM-52 peak. It contained some polypeptides of lower molecular weight than the 50 000 daltons band. These polypeptides could not be removed by isoelectric focusing and their prominence increased with time of storage; thus they probably represent breakdown products. Attempts to further purify the protein by isoelectric focusing were unsuccessful due to its instability under the conditions required. Four isoelectric protein peaks were found after column isoelectric focusing. The isoelectric point of the most prominent peak was 9.60. The basic protein was previously identified as a component in extracts of stratum corneum obtained by the EDTA trypsinization procedure of Stern and coworkers [4, 7, 17]. During the purification of fibrous protein by Huang et al. [4], this basic protein was shown to have a low content of sulfhydryl groups which could be derivatized by iodoacetic acid and was considered to be a non-fibrous protein on that basis. The present study has shown that the protein can be extracted with lower concentration of urea in the absence of reducing agents, that it does not bind to DE-52 in contrast to the fibrous proteins, and that its migration on sodium dodecyl sulfatepolyacrylamide gels is the same in the presence or absence of reducing agent. The amino acid compositions of these three major proteins of the stratum corneum have some similarities. All are rich in glycine, serine, and glutamic acid. However, a large proportion of the glutamic acid must be present as the amide in the basic protein. The basic protein has a much higher proportion of histidine, arginine, and alanine than the fibrous proteins; and it has a lower proportion of cysteine, lysine, aspartic acid, and non-polar amino acids than the fibrous proteins. The amino acid composition of the stratum corneum basic protein is very similar to the histidine-containing protein isolated by Bernstein and coworkers from the granular layer of newborn rat epidermis [18, 19] and from keratohyalin [8]. In addition, the molecular weight of the basic protein is similar to one component of potassium phosphate-extracted keratohyalin [7, 8]. Similar histidine-containing proteins have also been isolated from other keratohyalin preparations by Tezuka and Freedberg [20] and Ugel and Idler [21]. Further studies are in progress to determine if the stratum corneum basic protein is derived from keratohyalin. Early in this work it was determined that the epidermal cell separation technique utilizing buffered EDTA then trypsinization resulted in stratum corneum preparations with variable yields of the basic protein. This variability is now thought to be due to sensitivity of this protein to EDTA and possibly to trypsin. The purified basic protein can be dialyzed vs. 1 mM EDTA or 1 mM solutions of CuC12, MnC12, MgC12, ZnCI2, and CaCI2. Stability vs. EDTA and some ions has also been tested in the presence of 4 M urea or in buffers at pH 7 or pH 5 with no change in its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels. Thus, stabilization of the basic protein by metal ions has not been demonstrated directly. However, we have not ruled out stabilization by very tightly bound divalent cations. These results suggest that the sensitivity of the basic protein to EDTA in situ may be due to a metalinhibited protease. This in situ sensitivity may explain why this protein was not identified in previous studies in which EDTA-containing buffers were used [22]. Another explanation for the apparent absence of the basic protein is its low absorbance at 280 nm, the wavelength generally used to follow chromatographic profiles. A protein of similar molecular weight was identified by Matoltsy [23] in sodium
203 dodecyl sulfate-polyacrylamide gels of stratum corneum extracted with buffered sodium dodecyl sulfate. This protein was more easily extracted at acidic pH as would be expected for a basic protein. What are the possible functions of the stratum corneum basic protein? The protein is an extremely polar, highly positive-charged molecule devoid of sulfhydryl groups and carbohydrate. These properties suggest that it is not membrane protein or an extracellular protein. We can also rule out the dense peripheral band on the inner aspect of the cell membrane which has been shown to contain sulfhydryl groups by histochemistry [24], autoradiography [25, 26], and biochemical analysis [27]. Another possible function of the basic protein is that of the matrix substance in the stratum corneum. Morphologic studies suggest that the stratum corneum matrix may be derived from keratohyalin, whereas the fibrous proteins are derived from tonofilaments [1]. The basic protein is similar to a histidine-containing protein of keratohyalin in both molecular weight and amino acid composition, suggesting it could be derived from keratohyalin. Thus, the properties of the stratum corneum basic protein isolated here suggest that it is a good candidate for a matrix substance. Tezuka [28] has recently isolated a presumed matrix protein from human plantar stratum corneum which differs in its amino acid composition from that reported here. Tezuka's protein has an amino acid composition similar to that of fibrous protein. Antibody to his material reacts with keratohyalin granules in situ, an observation consistent with the proposed keratohyalin granule origin of matrix protein. However, antibody to fibrous protein has previously been shown to react with keratohyalin granules presumably due to the presence of tonofilaments in and around these granules [4, 29]. Thus, the role of Tezuka's protein as matrix has not been unequivocally demonstrated. In conclusion, a basic protein has been isolated from newborn rat stratum corneum. The protein is a major component of stratum corneum, but differs from the fibrous proteins; it is similar to a keratohyalin protein. The basic stratum corneum protein has properties which are consistent with its possible function as a stratum corneum interfilamentous matrix protein. ACKNOWLEDGEMENTS This work was supported by U.S. Public Health Service Grant Nos. DE-04660 and DE-02600, Center for Research in Oral Biology. The author would like to express her appreciation to Ms. Sue Ling for her excellent technical assistance and to Dr. Irving B. Stern for discussion and advice on the manuscript. REFERENCES 1 Brody, I. (1959) J. Ultrastruct. Res. 2, 482-511 2 Baden, H. P. and Bonar, L. (1968) J. Invest. Dermatol. 51, 478483 3 Lee, L. D., Fleming, B. C., Waitkus, R. F. and Baden, H. P. (1975) Biochim. Biophys. Acta 412, 82-90 4 Huang, L.-Y., Stern, I. B., Clagett, J. A. and Chi, E. Y. 0975) Biochemistry 14, 3573-3580 5 Steinert, P. M. (1975) Biochem. J. 149, 3948 6 Steinert, P. M. and Idler, W. W. (1975) Biochem. J. 151, 603-614 7 Dale, B. A. and Stern, I. B. 0975) J. Invest. Dermatol. 65, 223-227
204 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Sibrack, L. A., Gray, R. H. and Bernstein, I. A. (1974) J. Invest. Dermatol. 62, 394-405 Dale, B. A. and Stern, 1. B. (1975) J. Invest. Dermatol. 65, 220-222 Dunker, A. K. and Rueckert, R. R. (1969) J. Biol. Chem. 244, 5074-5080 Laurell, C.-B. (1966) Anal. Biochem. 15, 45-52 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 Bramhall, S., Noack, N., Wu, M. and Loewenberg, J. R. (1969) Anal. Biochem. 31, 146-148 Ogur, M. and Rosen, G. (1950) Arch. Biochem. Biophys. 25, 262-276 Scott, Jr., T. A. and Melvin, E. H. (1953) Anal. Chem. 25, 1656-1661 Gatt, R. and Berman, E. R. (1966) Anal. Biochem. 15, 167-171 Stern, 1. B. and Sekeri-Pataryas, K. H. (1972) J. Invest. Dermatol. 59, 251-259 Gumucio, J., Feldkamp, C. and Bernstein, 1. A. (1967) J. Invest. Dermatol. 49, 545-551 Hoober, J. K. and Bernstein, I. A. (1966) Proc. Natl. Acad. Sci. U.S. 56, 594-601 Tezuka, T. and Freedberg, I. M. (1974) J. Invest. Dermatol. 63, 402-406 Ugel, A. R. and Idler, W. (1972) J. Cell Biol. 52, 453-464 Shimizu, T., Fukuyama, K. and Epstein, W. L. (1974) Biochim. Biophys. Acta 359, 389-400 Matoltsy, A. G. (1975) J. Invest. Dermatol. 65, 127-142 Jessen, H. (1973) Histochemie 33, 15-29 Fukuyama, K. and Epstein, W. L. (1969) J. Cell Biol. 40, 830-838 Fukuyama, K. and Epstein, W. L. (1975) J. Ultrastruct. Res. 51, 314 325 Matoltsy, A. G. (1974) J. Invest. Dermatol. 62, 343 (abstract) Tezuka, T. (1975) Acta Dermatovenerol. (Stockholm) 55, 401-412 Dale, B. A., Stern, I. B., Rabin, M. and Huang, L.-Y. (1976) J. Invest. Dermatol. 66, 230-235
