Chemical characterization, synthesis and distribution of proteinase inhibitor in newborn rat epidermis

214

Biochimica et Biophysica Acta, 632 (1980) 214--226 © Elsevier/North-Holland Biomedical Press

BBA 29375 CHEMICAL CHARACTERIZATION, SYNTHESIS AND DISTRIBUTION OF PROTEINASE INHIBITOR IN NEWBORN RAT EPIDERMIS

T O S H I H I K O H I B I N O , K I M I E F U K U Y A M A * a n d W I L L I A M L. E P S T E I N

Department of Dermatology, University of California School of Medicine, San Francisco, CA 94143 (U.S.A.) (Received January 31st, 1980)

Key words: Thiol; Proteinase inhibitor synthesis; Enzyme localization; (Rat epidermis)

Summary A protein solubilized in Tris-HC1/saline buffer from keratinized cells of newborn rat epidermis exhibited inhibitor activity to papain and ficin, but not to trypsin, cathepsin D and pepsin. This protein was purified from keratinized cells as well as nonkeratinized and germinative cells by means of IgG affinity chromatography. The inhibitors extracted from all cell layers were immunologically identical and had a molecular weight of approximately 12 500 -+ 500. Since amino acid analysis showed that the inhibitor contains about 35 residues of glycine per tool, [3H]glycine was used to investigate synthesis of the protein. The inhibitor from nonkeratinized and germinative cells was radioactively labeled by 2 h after injection and appeared in keratinized cells by 48 h after injection. Indirect immunofluores'cence microscopy demonstrated in situ distribution of the protein in the entire epidermis, and the protein localized by the plasma membrane in granular cells and diffusely in keratinized cells was shown to be insoluble in Tris-HC1 saline buffer. The results indicate that a thiolproteinase inhibitor is synthesized in epidermal cells during keratinization and is retained as part of the cytoplasmic structure.

Introduction Keratinized cells of rat epidermis, the outermost layer of the skin, contain a large number of proteins produced during differentiation. Previously, Shimizu et al. [ 1] extracted these proteins from newborn rats and fractionated them by means of isoelectrie precipitation and gel filtration. While 75% of these extract* To whom correspondence should be addressed.

215 able proteins were fibrous and precipitated at pH 6.3, 4% were acid-soluble and contained several proteins. When rabbits were injected with a component of the acid~oluble fraction separated with Sephadex G-200 column chromatography, mono-specific antibody directed to one protein was produced. Using this antibody with affinity chromatography we purkfied the small molecular weight protein from a Tris-HC1 saline buffer-soluble fraction of keratinized cells. In order to further characterize this protein we undertook a variety of preliminary experiments including its inhibitor activity to enzymes, since the existence of a thiol-proteinase inhibitor in rat skin had been reported [2,3]. The purified protein demonstrated inhibitory activity to papain and ficin. In this paper we report: 1, isolation and chemical characterization of the epidermal proteinase inhibitor; 2, demonstration of biosynthetic sites of epidermal proteinase inhibitor by detection of [3H]glycine incorporatidn into inhibitor purified with IgG affinity chromatography; and 3, immunohistochemical identification of epidermal proteinase inhibitor in epidermal cells. Materials

2-day.old rats (Sprague-Dawley strain, originally obtained from Charles River Laboratories) randomly bred in this laboratory were used. The following chemicals were obtained from the companies indicated in parentheses: [2-3H]glycine (23 Ci/mmol) (Amersham Corp.); CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals); phenylmethylsulfonyl fluoride, a-N-benzoyl-DL-arginine-p-nitroanilide (Bz-Arg-NA), trypsin (type IV), papain (type III), ficin, cathepsin D, pepsin, chymotrypsinogen, myoglobin and pancreatic trypsin inhibitor ( Sigma Chemical Co.); dithiothreitol, cytochrome c (Calbiochem); sodium dodecyl sulfate (SDS) (Pierce); a-chymotrypsin (Worthington Biochemical); PPO (2,5-diphenyloxazole) (Eastman Kodak Co.); trichloroacetic acid (Mallinckrodt); Triton X-100 (Packard); Freund's incomplete adjuvant and killed Mycobacterium tuberculosis (Difco Laboratories); fluorescein isothiocyanate-conjugated goat antirabbit IgG (Hyland Co.). All other chemicals were of reagent grade, purchased from various chemical sources. Methods

Injection of radioisotopes The animals were killed at 2, 6, and 48 h after intradermal injection of [3H]glycine (50 ~Ci/0.1 ml saline).

Separation of epidermal cells Skin was removed and the epidermis separated from dermis by incubating in 0.24 M NI-LC1 (pH 9.0) for 10 min at 4~C. The epidermal sheets were washed and immersed in 0.02 M Tris.HC1 buffer (pH 8.0) containing 0.14 M NaC1, 0.0033 M CaCI2 and 10 pg/ml of phenylmethylsulfonyl fluoride for 15 min at 4°C. They were agitated gently and detached basal cells (germinative cells) and spinous cells were removed, frozen, and kept at --80~C. The remaining epidermal sheets were kept in 0.02 M Tris-HC1 buffer (pH 8.0), containing 0.25 M sucrose, 0.0033 M CaC12 and 10 ~g/ml phenylmethyl-

216 sulfonyl fluoride, for 1 h at 4°C. Each epidermal sheet was then placed on a glass plate and the remaining spinous cells and granular cells (nonkeratinized cells) were scraped off gently with a spatula. The epidermal sheets were scraped slightly harder to obtain a mixture of granular and cornified cells, which was then discarded. Keratinized cells remaining on the tape were collected by firm scraping. They were frozen and kept at --80°C. Purity of each cell fraction was monitored by preparing paraffin blocks and hematoxylin- and eosin-stained sections.

Isolation of proteins Germinative cells. Samples from approximately 100 rats were defrosted and the cell debris removed by centrifugation (International Refrigerated Centrifuge, Model 20) at 40 000 X g for 1 h. The supernatant was dialyzed at 4°C for 3 days against 10 #g/ml phenylmethylsulfonyl fluoride added to double-distilled water. Resulting precipitate was removed by centrifugation at 40 000 X g for 20 rain at 4°C, and the supernatant was lyophilized. At room temperature the lyophilized sample was extracted with 0.12 M HC104 (1 ml/5 rag) for 1 h and the supernatant, separated by centrifugation at 30 000 X g for 20 rain, was chilled. With 1 M Na2CO3, pH was titrated to 4.5 and the resulting precipitate (pH 4.5 precipitate), after being kept overnight at 0°C, was removed by centrifugation at 30 000 X g for 20 rain at 4°C. The supernatant (pH 4.5 supernatant) was dialyzed against 0.14 M NaC1 in 0.02 M phosphate buffer, pH 7.2. Nonkeratinized and keratinized cells. Approximately 5 g (wet weight) of each fraction was separately washed twice with phenylmethylsulfonylfluoride (10 /~g/ml) added to 0.01 M Tris-HC1 (pH 8.0). They were homogenized in 0.1 M Tris-HC1 (pH 8.0) containing 0.14 M NaC1 and 10 /~g/ml phenylmethylsulfonyl fluoride with a hand homogenizer and proteins extracted by stirring for 1 h at 4°C. After centrifugation at 30 000 X g, the supernatant (Tris-buffered saline 4°C fraction) was separated and the residue was serially extracted in the same Tris.HC1 buffer at 37°C for 30 min, 4 M urea in 0.05 M phosphate buffer (pH 7.0) at 37°C for 4 h, and 8 M urea in 0.02 M Tris-HC1 (pH 9.0) conraining 0.1 M 2-mercaptoethanol at 37°C for 16 h, as described by Murozuka et al. [4]. All supernatants were dialyzed against double-distilled water and lyophilized and proteins were extracted with 0.12 M HCIO4 titrated to pH 4.5 and both the pH 4.5 supernatant and precipitate for each extract were obtained by the same technique as used for the germinative cells. Sephadex G-50 column chromatography A column of 1.5 × 80 cm pre-equilibrated with 0.14 M NaCl in 0.02 M phosphate buffer, pH 7.2, was used. Blue Dextran 2000, myoglobin and cytochrome c were also eluted as molecular weight markers. Purification of epidermal proteinase inhibitor by affinity chromatography Monospecific antiserum. In preliminary studies the antigen (F IIc fraction) was prepared from keratinized cells using the technqiue reported by Shimizu et al. [1]. Subsequently, proteinase inhibitor Of the karatinized cells was purified from the proteins of pH 4.5 supernatant of the Tris-buffered saline 4°C fraction. The second peak eluted out from Sephadex G-50 column chromatography

217

Fig. 1. Comparative i m m u n o e l e c t r o p h o r e s i s of purified inhibitor (well 1) and crude epidermal protein extracted in 0.1 M Trls-HC1/0.15 M NaCI, pH 8.0 (well 2). The t h r o u g h contained IgG from a rabbit i m m u n i z e d with purified inhibitor. The plate was stained with Coomassie brilliant blue.

was applied on IgG affinity column as described below, and inhibitor absorbed on the column was used as an antigen. We raised antibody in three rabbits against Shimizu's F IIc fraction and in two rabbits with the purified inhibitor, using Freund's incomplete adjuvant. IgG purified from antisera of all animals [5] used in this experiment crossreacted and was only reacted with proteinase inhibitor, as shown in Fig. 1. Preparation of IgG affinity column. Purified IgG, 300 mg, was coupled with CNBr-activated Sepharose 4B according to the manufacturer's instructions. Approximately 10 mg IgG were found to be coupled with each ml of the Sepharose beads. The IgG-coupled Sepharose 4B was packed in a column (in a typical experiment, a 1.5 × 15 cn column was packed with 25 ml of the IgGcoupled Sepharose). Purification of epidermal proteinase inhibitor, pH 4.5 supernatants prepared from germinative, nonkeratinized, and keratinized cells and dialyzed against 0.14 M NaC1 in 0.02 M phosphate buffer, pH 7.2, were applied separately to the IgG affinity column and eluted with 0.5 M NaC1 in 0.02 M phosphate buffer (pH 7.2). Concentrations of antigen which produced a precipitin line in the middle of antigen-antibody wells with 1 mg/ml rabbit anti-inhibitor IgG were determined and about 10 times less concentration was used for each application (in a typical experiment, about 10 mg protein was used). Nonadsorbed proteins were monitored by measuring absorbance at 230 nm and the column was washed with the same buffer for an additional 30 min after the protein elution was completed. Proteins adsorbed in the column were disassociated with 0.1 M glycine-HC1 buffer (pH 2.3). The absorbance of the eluates was read at 230 nm and neutralized with 1 M glycine-NaOH buffer (pH 11). All procedures were done at 4°C.

Gel electrophoresis Disc gel electrophoresis was carried out by the procedure of Ornstein [6] and Davis [7], using 7% gels. SDS polyacrylamide gel electrophoresis was performed by the method of Weber and Osborn [8], using 7.5 and 15% gels. Chymotrypsinogen, myoglobin, a-chymotrypsin, cytochrome c, and pancreatic trypsin inhibitor were used as standards for calculating molecular weight.

Inhibitor assay Papain, pepsin and ficin were each dissolved in water. Trypsin was dissolved

218 in 1 mM HCI and cathepsin D was dissolved in 0.1 M citrate phosphate buffer (pH 3.0). Each enzyme was used within 2 days. Proteinase inhibitor was added to enzymes and buffers at 22°C. 10 rain later, assays were initiated by adding substrates and incubated at 37°C for an additional 10 min. Inhibitor activities to papain, ficin and trypsin were measured using Bz-ArgNA hydrolysis [9]. For papain or ficin, inhibitor (0.1 ml) serially diluted was mixed with 0.1 ml of the enzyme (0.1 mg/ml) and 0.1 ml 0.2 M Tris-HC1 (pH 7.5) containing 0.004 M dithiothreitol and 0.008 M EDTA. For trypsin, it was mixed with 0.1 ml of 0.2 M Tris-HC1 (pH 9.0) containing 0.01 M CaC12 and 0.1 ml trypsin (0.05 mg/ml). Bz-Arg-NA was dissolved in dimethyl sulfoxide and diluted with an appropriate buffer to make concentrations of 0.02 M for papain and ficin, and 0.005 M for trypsin. The reactions were stopped by adding 0.4 ml 30% acetic acid and absorbance of the reaction products was read at 405 nm. In separate experiments, inhibitor was dissolved in glycineHC1 buffer (pH 2.0), citrate-phosphate buffer (pH 7.0 and 8.0) and glycineNaOH buffer (pH 9.0, 10.0 and 11.0) in order to test its stability at the various pHs. Each buffer concentration was 0.05 M. Incubation mixtures consisted of 0.2 ml Tris-HCl (pH 7.5) containing 0.004 M dithiothreitol and 0.008 M EDTA, 0.05 ml of a papain solution (0.2 mg/ml) and 0.05 ml proteinase inhibitor (0.08 mg/ml) in the various buffers. Assays were carried out as described above. Enzyme activities were calculated from extinction coefficient for the absorption of p-nitroaniline at 405 nm (E 10 500). 1 unit of enzyme was expressed as the amount that would hydrolyze 1 mM of Bz-Arg-NA per min. 1 inhibitor unit was designated as the amount of protein that inhibits 2 units of enzyme activity by 50%. Inhibition of the papain proteolytic activity by epidermal proteinase inhibitor was also measured by means of casein as a substrate with a modification of the procedure described by Arnon and Shapira [10]. The incubation mixture consisted of 0.5 ml of a Tris-HC1 buffer, 0.25 ml of a papain solution (0.03 mg/ ml) and 0.25 ml of proteinase inhibitor solution. The reaction was started by adding 1 ml 0.5% casein in water (w/v) and stopped by adding 3 ml 5% trichloroacetic acid (w/v). The precipitates formed were removed by centrifugation and assay products in the supernatants were read at 280 nm. The readings were corrected for a blank solution which contained inhibitor with no papain or with papain but without incubation at 37°C. Inhibitor activity to cathepsin D was assayed by the method based on Anson [11]. 0.2 ml cathepsin D (0.03 mg/ml) in 0.1 M citrate phosphate buffer (pH 3.0) was mixed with various concentrations of inhibitor. 0.4 ml 2.5% bovine hemoglobin in water (w/v) was used as a substrate and reaction was stopped by adding 0.9 ml 5% trichloroacetic acid. The reaction product was measured as described below. A modification of the procedure described by Rajagopalan et al. [12] was used to assay epidermal proteinase inhibitor against pepsin activity. Incubation mixture contained 0.1 ml pepsin in water (0.05 mg/ml) and 0.1 ml of an inhibitor solution. 1 ml of acidified hemoglobin (pH 1.7) was used as a substrate and the reaction was stopped by adding 5 ml 4% trichloroacetic acid. Reaction products were measured as described in the papain inhibition assay using casein as the substrate.

219 Enzyme activity was expressed as a unit that causes adsorbance at 280 nm to increase by 1.0 in 10 rain in excess of blank reading.

Kinetics Kinetic assays were carried out under the following conditions. The reaction mixture and procedures were identical to those described in inhibition of papain activity with Bz-Arg-NA as a substrate. Different concentrations of inhibitor (10, 20 and 40 /~g/ml) and Bz-Arg-NA (2.5, 5, 10 and 20 raM)were used. Other chemical procedures (a) Radioactivity. This was measured by a Beckman liquid scintillation counter (Model LS-150) after the addition of 10 ml toluene scintillation fluid (15 g PPO/2 1 toluene/1 1 Triton X-100) into 0.1 ml of each sample. (b) Protein concentrations were determined by the method of Lowry et al. [13] using bovine serum albumin as a standard for all fractions except that of epidermal proteinase inhibitor dissociated from IgG affinity chromatography. The inhibitor concentration was calculated using the following formula: Inhibitor concn.=

absorbance at 230 nm 2.56 (absorbance of I mg/ml inhibitor)

(c) For amino acid analysis, purified EPI obtained from keratinized cells was hydrolyzed in constant boiling distilled 6 N HC1 for 24 h under vacuum at 110°C and analyzed with a Beckman 119 amino acid analyzer.

Indirect immunofluorescence microscopy Skin of newborn rat was frozen in liquid nitrogen, cut at 4/~m, and placed on slides. Some slides were fixed in 2% paraformaldehyde buffered with 0.02 M phosphate (pH 7.2). Others were sequentially treated for I h with 0.14 M NaC1 in 0.1 M Tris-HC1 buffer, pH 8.0, at 4°C, the same buffer at 37°C, 1 M potassium phosphate (pH 7.0) and 4 M urea in 0.05 M phosphate buffer (pH 7.0). At each step at least six slides were fixed. These were washed in three changes of 0.14 M NaC1 in 0.02 M phosphate buffer, pH 7.2, air-dried and reacted with several dilutions of rabbit anti-inhibitor IgG at 22°C for 30 rain. The excess IgG was removed and the sections were washed in three changes of the buffer and stained with fluorescein isothiocyanate conjugated-labeled goat anti-rabbit IgG at 22°C for 30 rain. The sections were examined with a Zeiss fluorescence microscope. Results

Purification of proteinase inhibitor from keratinized cells. Table I demonstrates the recovery of inhibitor activity in extracts of keratinized cells during the purification procedure. About 91% of the inhibitor activity was found in the Tris-buffered saline 4°C fraction. An increase of approximately 3-fold in specific activity was seen in the pH 4.5 supernatant. Sephadex G-50 column chromatography of the pH 4.5 supernatant showed three peaks. A further increase in specific activity was found in the second peak. After chromatogra-

220

TABLE I SUMMARY OF EPIDERMAL PROTEINASE INHIBITOR NONKERATINIZED AND GERMINATIVE CELLS Purification steps

PURIFICATION

FROM

Specific activity ( X 1 0 -3 )

KERATINIZED,

Protein (mg)

Inhibitor unit (IU)

Total activity (rag f l U )

Yield (%)

Total extractable protein K e r a t i n i z e d cells N o n k e r a t i n i z e d cells G e r m i n a t i v e cells

605 509 224

176.4 442.6 118.5

3.43 1.15 1.89

5.67 2.26 8.44

T r i s o b u f f e r e d s a l i n e 4°C f r a c t i o n K e r a t i n i z e d cells N o n k e r a t i n i z e d cells

296 135

94.8 195.6

3.12 0.69

10.50 5.11

90.9 58.3

1.9 2.3

100 100 100

Purification factor

1 1 1

pH 4.5 supernatant K e r a t i n i z e d cells N o n k e r a t i n i z e d cells G e r m i n a t i v e cells

59.4 8.7 7.7

35.6 59.3 26.7

1.67 0.15 0.29

28.10 17.10 37.50

48.7 13 15.2

4.9 7.6 4.4

S e p h a d e x G ° 5 0 , p e a k II K e r a t i n i z e d cells

15.5

17.8

0.871

56.2

25.4

9.9

4,8 0.7 1.6

5.9 5.9 5.7

0.814 0.12 0.28

169.5 171.4 175.4

23.7 10.4 14.8

29.8 75.8 20.8

Aff'mity column-adsorbed K e r a t i n t z e d cells N o n k e r a t i n i z e d cells G e r m i n a t i v e cells

phy of the second peak on an IgG affinity column, a further 3-fold increase in inhibitor activity was obtained. The overall purity was increased by 30-fold. The IgG affinity column-purified protein (epidermal proteinase inhibitor) showed a single protein band on both disc gel electrophoresis and SDS gel electrophoresis. The molecular weight was estimated to be about 13 000. Inhibitor assays. Papain activity was 13.5 mU on Bz-Arg-NA and 0.5 U on casein. Ficin and trypsin activities were 8.2 mU and 35.2 mU respectively. Cathepsin activity was 0.3 U, while pepsin activity was 0.5 U on hemoglobin. Epidermal proteinase inhibitor demonstrated a linear inhibition of both papain and ficin, up to about 90% inhibition (Fig. 2). Units were calculated to be 5.9

a

'~

80 .~

80-

60.

60.

40

40.

20,

2o.

0

.

.

20

40

.

. 60

. 80

. 100

,

,

0

10 20 I n h i b i t o r (~g/ml)

,

30

~,~ ,;; 40

100

,

120

Fig. 2. E f f e c t o f i n h i b i t o r o n five p r o t e i n a s e a c t i v i t i e s a t d i f f e r e n t c o n c e n t r a t i o n s u s i n g (a) B z - A x g - N A o r (b) c a s e i n as s u b s t r a t e . A c t i v i t i e s o f p a p a i n (o o ) , (¢ -') a n d f l c i n (E -') w e r e i n h i b i t e d , b u t t h o s e o f t r y p s i n (A A), c a t h e p s i n D (o o) a n d p e p s i n (4 4) were not.

221 for papain and 8.8 for ficin, indicating that epidermal proteinase inhibitor inhibited papain more than ficin. No inhibition of trypsin, pepsin or cathepsin D was observed. When t w o substrates were compared for inhibitor activity on papain, there was no significant difference in the a m o u n t of inhibitor used: 50% inhibition of 1 mg papain was seen with 0.4 mg inhibitor for Bz-Arg-NA and 0.36 mg for casein. The total epidermal proteinase inhibitor activity was retained b e t w e e n pH 2 and 11 during the incubation at 37°C for 20 min. Inhibitor was also tested for stability at various temperatures. At pH 7.0 it was stable between 37 and 80°C. After 10 min incubation at 100°C, 46% activity was lost. Kinetic assay. The double reciprocal plots of Lineweaver and Burk [14] were used for the analysis of kinetics. Proteinase inhibitor showed the noncompetitive inhibition mechanism and reduced the apparent value of velocity b u t had no effect on Km. An inhibitor constant (Ki) was also determined by the m e t h o d of Dixon [15]. The Ki value for epidermal proteinase inhibitor with Bz-Arg-NA as the variable substrate was 1.6 • 10 -9 M and the intersection was on the abscissa. Amino acid composition, The inhibitor has a very high glycine residue content (35/mol). Glycine, glutamine, aspartate and lysine residues constitute 75 of the 125 residues.

Purification of inhibitor from nonkeratinized and germinative cells by IgG affinity chromatography. Protein concentrations and inhibitor activity measured in each purification procedure of epidermal proteinase inhibitor from nonkeratinized cells and germinative cells are compared in Table I. Among the total extracts, the highest specific activity was found in the germinative cells. The percent of proteins recovered in pH 4.5 supernatants of nonkeratinized cells or germinative cells was much smaller than that of keratinized cells, and increases of the specific activity were achieved. After purification by IgG affinity column chromatography, the specific activities found in all cell samples were almost identical. Fig. 3 shows gel patterns of pH 4.5 supernatants obtained from germinative, nonkeratinized and keratinized cells which were applied on IgG affinity columns along with those of protein absorbed and eluted o u t by glycine buffer. The antigenic protein (epidermal proteinase inhibitor) purified from different cells showed essentially an identical migration pattern. Agar double diffusion technique with rabbit anti-epidermal proteinase inhibitor IgG was used to test their immunological relationship. Purified epidermal proteinase inhibitors from keratinized, nonkeratinized and germinative cells showed a line of identity (Fig. 4). Serum from n e w b o r n rat did n o t show the reactivity. Incorporation of [3H]glycine into inhibitor. Both water-soluble fractions of germinative cells and Tris-buffered saline 4°C fractions of nonkeratinized cells obtained 2 and 6 h after injection of [3H]glycine were radioactively labeled. Radioactivity was detected in all protein fractions during inhibitor purification; radioactivity in inhibitors purified b y IgG column chromatography was expressed as percent of that in Tris-buffered saline 4°C fraction (or water-soluble) (Table II). Specific radioac.tivity in proteins eluted o u t at pH 7.2 and in inhibitor were compared. The ratio was 1 : 0.24 in nonkeratinized cells, whereas it was 1 : 1.9

222

~a

b

C

d

e

f

Fig. 3. SDS p o l y a e r y l a m i d e (7.5%) gel electrophoreais of proteins in pH 4.5 s upe rna t a nt s of germinative (a), nonkeratLnized (c) and k e r a t i n i z e d (e) cells and purified e pi de rma l proteinase i n h i b i t o r from those fractions (b, d, and f), respectively. Fig. 4. A double diffusion plate which d e m o n s t r a t e s an i m m u n o l o g i c a l relationship of proteinase inhibitots purified fro m pH 4.5 s u p e r n a t a n t s of germinative (well 1), n o n k e r a t i n i z e d (well 2), and ke ra t i ni z e d (well 3) ceils. R a b b i t anti-inhibitor IgG is in the center well.

in germative cells. Inhibitor purified from keratinized cells of newborn rats 48 h after injection of [3H]glycine also was radioactive, indicating that epidermal proteinase inhibitor synthesized in the precursor cells is retained in epidermal cells during cell differentiation. TABLE II PERCENT R A D I O A C T I V I T Y R E C O V E R Y IN E P I D E R M A L P R O T E I N A S E A F T E R IgG A F F I N I T Y CHROMATOGRAPHY All n u m b e r s represent average values of a duplicate study. Epidermal proteinase i n h i b i t o r from

Hours after [ 3 H] glycine injection

% Radioactivity Tris-buffered saline 4°C (or H 20-soluble) protein (100%)

pH 4.5 s u p e r n a t a n t prot e i n (100%)

9.45±0.05 6.3 ± 1 . 4

Germinative cells

2 6

2.3 2.3

N o n k e r a t i n i z e d cells

2 6

0.2 0.3

0.75±0.25 0.65±0.05

0.25±0.05

1.15±0.25

Keratinized cells

48

±0.1 ±0.4

223

Indirect immunofluorescence microscopy. The reaction product was seen diffusely over the cytoplasm of nonextracted epidermal cells of newborn rat skin. No reaction was detected in the nuclei, keratohyalin granules, hair follicles or the dermis. The staining reaction was not seen when anti-epidermal proteinase inhibitor IgG or fluorescein isothiocyanate-conjugated goat antirabbit-labeled IgG was replaced by saline. Blocking of the reaction also was possible by reacting the tissue sections with goat anti-rabbit IgG prior to the application of fluorescein isothiocyanate-conjugated goat antirabbit-labeled IgG. After incubation of the tissue sections in 0.1 M Tris-HC1 buffer containing 0.14 M NaC1, pH 8.0, at 4°C, the staining reaction was reduced, particularly from the lower strata of epidermal cells. Incubation in the same buffer at 37°C further removed antigen from the sections, but significant reactivity was retained in the plasma membrane of granular cells and in keratinized cells. Decrease in reactivity continued to be seen in the tissue sections treated with 1 M phosphate buffer, and 4 M urea treatment completely removed reactive proteins. Detection of proteinase inhibitor in different protein extracts by agar diffusion and SDS gel electrophoresis. Fig. 5 summarizes immunological reactivities of rabbit anti-inhibitor IgG with proteins in Tris-buffered saline 4~C fractions and the extractants obtained by the stepwise technique described by Murozuka et al. [4]. The Tris-buffered saline fractions of both keratinized and nonkeratinized cells reacted strongly with the IgG. Proteins of keratinized cells extractable in 1 M phosphate buffer and 4 M urea also showed immunological reactivity to anti-inhibitor IgG indicating that not all the inhibitor was soluble. No reactivity was detected when 1 M phosphate buffer- and 4 M urea-soluble proteins of nonkeratinized cells in crude extracts were placed in agar diffusion wells with anti-inhibitor IgG. However, when pH 4.5 supernatants were tested at a concentration of about 1.0--1.2 mg/ml or when larger antigen wells were

Fig. 5. I m m u n o l o g i c a l reactivity o f pH 4.5 s u p e r n a t a n t s w i t h rabbit anti-inhibitor (center well) by Ouchter lony d o u b l e diffusion technique. In t h e o u t s i d e wells, pH 4.5 s u p e r n a t a n t s prepared f r o m Tris-buffered saline 4°C (1)0 Tris-buffared saline 37°C (2), 1 M p o t a s s i u m p h o s p h a t e (3), a nd 4 M urea in 0.05 M p h o s phate buffer (4) wexe placed, a° Keratinized cells; b° n o n k e r a t i n i z e d cells.

224

a

b

c

d

e £

g

h

Fig. 6. S D S p o l y a c r y l a m i d e ( 7 . 5 % ) gel electrophoresis of proteins in p H 4 . 5 supernatant prepared with each'extractant o b t a i n e d f r o m k e r a t i n i z e d (a---d) a n d n o n k e r a t i n i z e d ( e - - h ) ceils, a a n d e in T B S 4 ° C ; b a n d f i n T r i s - b u f f e r e d s a l i n e 3 7 ° C ; e a n d g in 1 M p o t a s s i u m p h o s p h a t e ; d a n d h in 4 M u r e a i n 0 . 0 5 M

phosphate b u f f e r . A r r o w i n d i c a t e s e p i d e r m a l p r o t e i n a s e i n h i b i t o r .

used, the protein in 1 M phosphate as well as that in 4 M urea showed reactivity demonstrated by a line of identity. Results of SDS gel electrophoresis coincided with the findings with agar diffusion techniques (Fig. 6). They demonstrate the presence of a distinct epidermal proteinase inhibitor band (molecular weight approximately 12 000) in all samples of keratinized cells and in Tris-buffered saline-soluble fractions of nonkeratinized cells. The band was less obvious in 1 M phosphate and 4 M urea extracts. Discussion

A protein (epidermal proteinase inhibitor) that demonstrates inhibitory activity to papain and ficin, but not to trypsin, cathepsin D and pepsin, was isolated from newborn rat epidermis. It has a molecular weight of approximately 12 500 and is a heat stable noncompetitive inhibitor to the enzymes. The general chemical properties of epidermal proteinase inhibitor are similar to other thiol-proteinase inhibitors isolated from chicken egg white [16], bovine nasal cartilage [17], porcine leukocytes [18], the healing skin sites of Arthustype inflammation [19--21], and normal epidermis of rat [22] and human [23]. However, epidermal proteinase inhibitor differs considerably in its unique localization in the skin. The thiol-proteinase inhibitor found in the Arthus-type

225 reaction was thought to be secreted by mononuclear cells [24] or to come from sera [25]. In addition, an SH-proteinase inhibitor from rat epidermis characterized by J~irvinen et al. [22] seemed to be localized in the superficial layers (granular layer) of the skin while epidermal proteinase inhibitor was found throughout the epidermis. Proteinase inhibitors of both germinative and nonkeratinized cells were radioactive at 2 h after injection of [3H]glycine, indicating that: 1, EPI is formed in the epidermis; and 2, synthesis continues to take place in the cells undergoing differentiation. In germinative cells the specific ac.tivity of labeled inhibitor was higher than in other proteins, but in nonkeratinized cells the proteins other than epidermal proteinase inhibitor showed a higher specific activity. Synthesis of protein(s) related to epidermal cell differentiation was considered to account for the [3H]glycine incorporation, as demonstrated previously by autoradiography [26]. Radioactive epidermal proteinase inhibitor appeared in keratinized cells at 48 h after injection of [3H]glycine. Since germinative cells take more than 96 h to become keratinized cells in newborn rats [27], the radioactive inhibitor was considered to be synthesized in upper spinous or granular cells, confirming that inhibitor synthesis continues to occur in epidermal cells as they differentiate. All biological functions of epidermal proteinase inhibitor in newborn rat epidermis remain unknown. A recent study with rat liver cathepsin L showed that epidermal proteinase inhibitor seemed to be involved in a dermal injury caused by a thiol proteinase [28]. It also may be a tissue source for proteinase inhibitor against microorganism invasion, as proposed for the inhibitor by J~irvinen [22]. Another possibility is that epidermal proteinase inhibitor may play a protective role in the epidermis against external proteinases. The plasma membrane of differentiated epidermal cells is modified so that a thickening of the membrane occurs in the upper granular layer by deposition of cystine-rich protein [29,30]. Localization of Tris-buffered saline-insoluble epidermal proteinase inhibitor by the thickened plasma membrane may contribute further to prevention of enzyme destruction of terminally differentiated epidermal cells. A preliminary study showed that the plasma membrane-bound epidermal proteinase inhibitor still demonstrates inhibitor activity to papain and ficin. In the past, morphological and chemical markers for differentiated epidermal cells have induced keratohyalin granules, small lamellated granules, plasma membrane envelope [31]. They have been detected by means of electron microscopy and use of radioisotopes. The observation made in the present study provides an additional marker, i.e., plasma membrane-bound epidermal proteinase inhibitor protein. Since immunofluorescence microscopy is a simple and sensitive technique, application of this new marker in studies of epidermal cells may become useful. Acknowledgements This investigation was supported by Research Grant AM 12433 from the National Institutes of Health, by a special appropriation from the State of California for the study of psoriasis, and by the Skin Disease Research Foundation, San Francisco. Dr. Fukuyama is the recipient of Biomedical Research Grant RR 05355.

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