Calcif. Tissue Int. 29, 251-256 (1979)
Calcified Tissue International '~. 1979 by Springer-Verlag
Cathepsin D: Ultra-Immunohistochemical Localization in Dentinogenesis H. Nygren, B. Persliden, H-A. Hansson, and A. Linde Laboratory of Oral Biology, Department of Histology, University of GOteborg, Fack, S-400 33, Grteborg, Sweden
Summary. Cathepsin D was purified from rat liver using a new affinity chromatographic method, based on the coupling to the specific inhibitor pepstatin. This preparation was used for the production of specific antibodies from rabbit. The purified IgG fraction was conjugated to horseradish peroxidase in a two-step coupling procedure and used for electron microscopic immunohistochemistry of the odonloblast-predentine region of the rat incisor. Precipitates, indicating the presence of cathepsin D, were seen in the odontoblast, odontoblast process, and in the extracellular unmineralized matrix, the predentine. The observations are discussed in relation to proteoglycan degradation at the mineralization front simultaneous with crystal tbrmation, and in relation to the function oflysosomal enzymes in the turnover of connective tissue. Key words: Cathepsin -- Calcification -- Dentinogenesis -- Proteoglycans.
Introduction The degradation of proteoglycans (PGs) in different connective tissues has been suggested to be due to enzymatic activity. The acid endopeptidase cathepsin D (EC 3.4.23.5) has frequently been discussed since this enzyme seems to be of crucial importance for PG degradation in, e.g., cartilage of different species [ I-3]. Not many studies have been published on acid hydrolases in hard tissues, and their distribution has most often been considered in connection with resorptive processes. Some reports, however, have demonstrated the presence of and/or discussed the possible function of acid hydrolases in bone and dentine formation [4-13]. Send offprint requests to A. Linde at the above address.
Disappearance of a considerable portion of the PGs present in the unmineralized matrix simultaneous with mineral formation has been shown for several mesenchymal calcified tissues using different techniques [8, 11, 14-19]. The functional significance of this PG disappearance in the calcification process is not known. The only statement that can be made at the present is that due to the physicochemical properties of PGs [20], this change in the composition of the organic matrix should be important. In order to study the possible enzymatic degradation of PGs in the dentinogenically active rat incisor, cathepsin D was studied by immunohistochemical technique, using fluorescein isothiocyanate (FITC) labeled rabbit anti-rat cathepsin D IgG on the light microscopic level [ 13]. Cathepsin D was found to be located in the odontoblastema and over the predentine. Due to the limited resolution it was not possible to further characterize the distribution of cathepsin D. The present study, using electron microscopic immunohistochemistry, was undertaken to elucidate the subcellular distribution of cathepsin D in the odontoblast-predentine area of dentinogenically active rat incisors. Presumably this might also give some hint as to the localization for PG degradation in the tissue, whether it is an extracellular or intracellular event.
Materials and Methods
Cathepsin D Purification Cathepsin D was purified from rat liver by aff• chromatography on pepstatin-coupled AH-Sepharose 4B as described by Linde and Persliden [22]. A crude enzyme preparation was obtained from rat liver homogenate [ 13] and applied to the affinity chromatographic column, previously equilibrated with 0.1 M N.acitrate-HCl buffer, pH 3.0, containing 0.5 M NaC!. The column
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Ten milligrams of HRPO (Sigma Chemical Co., St. Louis, Missouri, USA) was reacted with 0.5 ml of 0.2% charcoal-filtered glutaraldehyde (Polaron Equipment Ltd., Liverpool, England) in 0.1 M borate buffer, pH 9.5, for 2 h at room temperature. The reacted HRPO was separated from free glutaraldehyde on a Sephadex G-25 column (0.7 • 12 cm) equilibrated with 0.15 M NaC1. The HRPO-glutaraldehyde complex was in a second step reacted with 5 mg IgG at pH 9.5 for 25 h in a total incubation volume of 2 ml in the cold. The reaction was terminated by the addition of 0. I ml 0.2 M lysine. The IgG-HRPO conjugate was separated from free IgG and HRPO on a Sephacryl S-200 Superfine column (1.6 • 100 cm) eluted with PBS buffer, pH 7.2, at a flow rate of 4 ml/h [25]. The IgG-HRPO conjugate was stored frozen in portions of 0.5 ml at -22~ prior to the immunohistochemical experiments.
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Fig. 1. Elution profile from separation of the different reaction products after the coupling procedure with HRPO. The Sephacryl S-200 Superfine column (I.6 • 100 cm) was eluted with PBS buffer, pH 7.2, at flow rate of 4 ml/h and with fraction volumes of 3 ml. Protein was estimated as absorbance at 280 nm (--), and HRPO was measured by absorbance at 403 nm (..... ). Fractions indicated ( ) were pooled and referred to as IgG-HRPO conjugate, a, IgG-HRPO conjugate: b, unlabeled IgG: c, unbound HRPO
was washed with the equilibration buffer and the enzyme was eluted in one single fraction with 0.1 M NaHCO.~, pH 7.0, containing 0.5 M NaCI. The purity of the cathepsin D preparation was tested by electrophoresis on 7% polyacrylamide gels. DEAE- and CM-cellulose ion-exchange chromatography, and molecular sieve chromatography on Sephacryl S-200 Superfine [13].
Preparation qf Antibodies One milligram of the purified enzyme, dissolved in 1 ml phosphate-buffered saline (PBS buffer), pH 7.2, was injected subcutaneously together with 1 ml of Freund's complete adjuvant once weekly for 4 weeks into albino rabbits. Six weeks after the last injection, the same amount of enzyme alone was injected intravenously. Ten days after this injection, blood was withdrawn from the marginal ear vein. The serum was fractionated on a QAE-Sephadex A-50 ion-exchange column (equilibrated with 0.1 M Tris-HCl buffer, pH 6.5) by a NaCI gradient from 0.0-0.1 M NaCI in the equilibration buffer. The IgG fraction obtained was transferred to PBS buffer, pH 7.2, by buffer exchange on a Sephadex G-25 column and tested immunoelectrophoretically [21] against the antigen [13, 22].
HRPO Conjugation The coupling reaction for horseradish peroxidase (HRPO) to lgG was modified after Avrameas and Ternynck [23]. Several changes were, however, made to optimize the reaction [24], and therefore the method will be given in detail.
lmmunohistochemistry For the immunohistochemical study. Sprague-Dawley rats (body weight 200 g) were perfused through the aorta with 200 ml of 4% lbrmaldehyde in 0.15 M cacodylate buffer, pH 7.2. containing 0.1% picric acid, for 30 rain. The apical two-thirds of the maxillary incisors were dissected out according to Linde [26] and fractured. The dentine fragments thus obtained (with adhering odontoblasts) were then immersed in the same fixative for an additional 18 h and washed in the cacodylate buffer. The specimens were incubated for 12 h with the rabbit antirat cathepsin D IgG-HRPO conjugate, obtained 'after molecular sieve chromatography in PBS buffer, pH 7.2, containing 1% albumin. After incubation with antibodies, the cells and the fractured dentine pieces were rinsed in the cacodylate buffer for 12 h and in 0.15 M cacodylate buffer, pH 5.0, for 30 min. After rinsing, incubation of the specimens was performed in 0.5 mg/ml of 3,3-diaminobenzidine (British Drug House, Poole, England) in 0.15 M cacodylte buffer, pH 5.0, containing 0.01% H202 for 30 min. The specimens were then rinsed in 0.15 M cacodylate buffer, pH 7.2. postfixed in 2% OsO4 in the cacodylate buffer for 2 h, dehydrated in ethanol, and embedded in Epon. Thick section ( 1 p.m/were taken for orientation. Thin sections (500 A) were cut on an LKB Ultratome III ultramicrotome and examined in a JEOL 100 C electron microscope. As controls, specimens were incubated with 3.3-diaminobenzidine and H,_O2 without any prior incubation with the conjugated antibodies towards cathepsin D. Also, specimens were incubated with rabbit anti-goat IgG-HRPO at the same protein concentrations as with anti-cathepsin D IgG-HRPO.
Results
T h e c a t h e p s i n D p r e p a r e d by the p r e s e n t affinity c h r o m a t o g r a p h y t e c h n i q u e w a s s h o w n to b e p u r e b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s , i o n - e x c h a n g e chromatography on DEAE- and CM-cellulose and m o l e c u l a r s i e v e c h r o m a t o g r a p h y o n S e p h a c r y l S200 S u p e r f i n e . F u r t h e r m o r e , w h e n t e s t e d b y i m m u n o e l e c t r o p h o r e s i s , the c a t h e p s i n D w a s s h o w n to be immunologically homogeneous showing just one p r e c i p i t a t i o n l i n e [22].
H. Nygren et al.: Cathepsin D in Dentinogenesis
253
Fig. 2. Central part of odontoblast incubated with anti-cathepsin D IgG-HRPO. HRPO activity, indicating the presence of cathepsin D. is seen in a lysosome-like vesicle (arrow). • 12,000. Fig. 3. Proximal part of odontoblast process, i.e., within the inner third of the predentine. Incubation with anti-cathepsin D IgG-HRPO. Precipitates are located within elongated vesicles (arrows). x 12,000. Fig. 4. Distal portion of odontoblast process, i.e., in the outer third of the predentine, with surrounding predentine. Intense precipitates (arrows), indicating the presence o f c a t h e p s i n D. can be seen close to the cell membrane. • 15,000. Fig. 5. Odontoblast process, incubated with peroxidase-coupled anti-cathepsin D IgG. Positive reactions are seen in granules within the process, x 12,000
254
Fig. 6. Portion of newly formed predentine from fractured rat incisor tooth. Precipitates (arrows) are present in the extracellular matrix of the predentine. • 30,000
By separating the I g G - H R P O reaction products on Sephacryl S-200, it was possible to completely remove contaminating IgG and HRPO from the I g G - H R P O conjugate (Fig. 1). Generally, the yield of conjugated rabbit anti-rat cathepsin D with HRPO was in the range of 40% as judged by the two IgG fractions obtained by molecular sieve chroma-
H. Nygren et al.: Cathepsin D in Dentinogenesis tography, i.e., fraction a (IgG-HRPO) expressed in percent of total IgG (fraction a + b). The protein concentration in the conjugate was found to be approximately 80/zg IgG/ml and 20/xg HRPO/ml. The values were obtained by optical density measurements at 280 nm and 403 nm for IgG and H R P O , respectively, using the extinction coefficients 14.9 for rabbit IgG and 14.5 for HRPO. Precipitates, indicating the presence of cathepsin D, were seen in lysosome-like vesicles in the odontoblast Golgi region (Fig. 2). In the proximal part of the odontoblast processes, i.e., within the inner third of the predentine, electron-dense precipitates were seen in elongated vesicles (Fig. 3). In the more distal part of the odontoblast process, approximately in the outer third of the predentine, dense precipitates were seen inside the processes and in close relation to the cell membrane. In this area it was not possible to detect any vesicle-like structures in connection with the electron-dense particles (Figs. 4 and 5). For technical reasons (since the sections were not demineralized), it was not possible to study cathepsin D localization in the cell process further out in the dentine. Extracellular localization of cathepsin D in the predentine matrix was indicated by electron-dense precipitates in the areas between odontoblast processes (Fig. 6), predominantly in the proximal third of the predentine. Control incubations with 3,3-diaminobenzidine and H202, but without I g G - H R P O conjugate added, showed a scattered distribution of small precipitates randomly localized or related to mitochondria (Fig. 7). Incubation of odontoblasts with control serum (anti-goat-IgG-HRPO) showed no precipitates not seen in the control specimens with 3,3-diaminobenzidine and H~O_~only.
Fig. 7. Control section of odontoblast incubated with 3.3diaminobenzidine without the addition of anti-cathepsin D IgG-HRPO. Precipitates can be seen in mitochondria (arrow). Scattered distribution of small precipitates (arrows) can be seen in the cytoplasm. • 18.000
H. Nygren et al.: Cathepsin D in Dentinogenesis
Discussion
Molecular sieve c h r o m a t o g r a p h y for the separation of the I g G - H R P O conjugate from the other constituents in the reaction mixture was described by B o o r s h m a and Kalsbeck [25]. In this way excess free IgG and H R P O are separated from the rabbit anti-rat cathepsin D IgG H R P O preparation. This is of vast importance for avoiding nonspecific staining and thus for the interpretation of precipitates occurring in the specimen. Incubating with only 3,3-diaminobenzidine and H202 was undertaken in order to identify any intracellular peroxidase activity. This could be seen in mitochondria of the odontoblasts. Nonspecific activity, i.e., nonspecific adsorption of I g G - H R P O conjugates to tissue c o m p o n e n t s , was controlled by incubation of the specimens with rabbit anti-goat I g G - H R P O . This type of control revealed no nonspecific electron-dense precipitates in addition to those seen in the other control incubations with 3,3diaminobenzidine and H.,O., only. Thus precipitates occurring in regions where 3,3-diaminobenzidine control incubations revealed no precipitates were taken as an indication for the presence of cathepsin D in the tissue. The use of fixative in immunohistochemistry is a double-edged procedure. In the present study 4% formaldehyde was used as perfusion and immersion fixative. Although this technique destroyed a large portion of the antigen amount in the tissue specimens, it made it possible to locate the immunoprecipitate to known morphological structures. It should, however, be e m p h a s i z e d that the method can be regarded as only qualitative due to the fixation technique. Electron-dense precipitates, indicating the presence of cathepsin D, were seen in the Golgi region of the odontoblasts as well as in elongated vesicles in the odontoblast processes. Also, the e n z y m e was found to be localized in close relation to the cell m e m b r a n e of the odontoblast process. These resuits m a y be taken to indicate a transport of cathepsin D f r o m Golgi region of the odontoblast throughout the process in vesicle-like structures, probably derived from the Golgi apparatus. The extracellular distribution of cathepsin D in the predentine, preferentially in the proximal third of the predentine matrix, is of interest since this would implicate an extracellular cathepsin D function. This finding is in agreement with those of Poole et al. [27]. These authors studied cathepsin D localization in an in vitro chick bone organ culture. An extracellular distribution of cathepsin D, probably due to cell secretion, was found. The localization of cathepsin D has also been studied in
255
growing rat incisor by F I T C technique [13]. An intense fluorescence o v e r the o d o n t o b l a s t e m a and over the predentine was found, but no conclusions could be drawn whether this FITC-staining was due to intra- or intra- and extracellular distribution of the enzyme. An extracellular secretion of lysosomal e n z y m e s has been discussed in relation to the degradation of extracellular matrix m a c r o m o l e c u l e s [28, 29]. According to Dingle [29], the matrix c o m p o n e n t s would be initially degraded in the extracellular space followed by endocytosis and complete digestion in the lysosomal system of the cells. The present findings support the theory of an initial extracellular function of cathepsin D. It may be speculated that this is a m e c h a n i s m c o m m o n to different connective tissues. The localization of high activity of cathepsin D in the dentinogenic region [12] is strong evidence for an important function in the degradation of PGs taking place simultaneously with crystal formation at the mineralization front. The results of Poole et al. [27] indicate that this process occurs also in bone formation. Further degradation of PG constituents, be it extra- or intracellular, m a y be brought about by the combined action of other acid hydrolases, earlier shown to be present in dentinogenically active odontoblasts [10]. The functional importance of the removal of PGs is not known at present. It may be as simple as to provide space for mineral crystals. It may also be in order to r e m o v e m a c r o m o l e c u l e s with a strong polyanionic character after having fulfilled some role as inhibitors of mineral formation or in the extracellular aggregation of collagen.
Acknowledgments. We are indebted to Dr. Neil Barclay for fruitful discussion. We gratefully acknowledge the excellent technical assistance of Ms. I. Haag, Ms. A. H6glund, and Ms. U. Svedin. The investigation was supported by The Swedish Medical Research Council, The Faculty of Odontology, University of G6teborg, and The Magnus Bergvall Foundation.
References 1. Dingle. J.T., Barrett, A.J.. Weston, P.D.: Characteristics of immunoinhibition and the confirmation of a role in cartilage breakdown, Biochem. J. 123:1-13, 1971 2. Dingle, J.T., Barrett, A.J.. Poole, A.R.. Stovin, P.: Inhibition by pepstatin of human cartilage degradation, Biochem. J. 127:443-444, 1972 3. Woessner, J.F.: Purification of cathepsin D from cartilage and uterus and its action on the protein-polysaccharide complex of cartilage, J. Biol. Chem. 248:1634-1642, 1972 4. Vaes, G., Jacques, P.: Studies on bone enzymes: the assay
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5.
6. 7.
8.
9.
10.
11. 12.
H. Nygren et al.: Cathepsin D in Dentinogenesis
of acid hydrolases and other enzymes in bone tissue, Biochem. J. 97:380-388, 1965 Vaes. G., Jacques, P.: Studies on bone enzymes: distribution of acid hydrolases, alkaline phenylphosphatase, cytochrome oxidase and catalase in subcellular fraction of bone tissue homogenates, Biochem. J. 97:389-392, 1965 Vaes. G.: Hyaluronidase activity in lysosomes of bone tissue. Biochem. J. 103:802-804, 1967 Suga, S.: Histochemical observation of proteolytic enzyme activity in the developing dental hard tissue of rat, Arch. Oral Biol. 15:555-558. 1970 Baylink. D., Wergedal. J., Thompson, E.: Loss of proteinpolysaccharides at sites where bone mineralization is initiated, J. Histochem. Cytochem. 20:279-292, 1972 Engstr6m. C., Linde, A., Persliden. B.: A fluorimetric study of fl-glucuronidase and/3-galactosaminidase from the pulp of the rat incisor, Arch. Oral Biol. 17:1421-1430, 1972 Engstr6m, C.. Linde, A., Persliden. B.: Acid hydrolases in the odontoblast-predentin region of dentinogenically active teeth, Scand. J. Dent. Res. 84:76-81, 1976 Hjerpe, A., Engfeldt, B.: Proteoglycans of dentine and predentine Calcif. Tissue Res. 22:173-182, 1976 Linde. A., Persliden, B.: Cathepsin D activity in isolated odontoblasts, Calcif. Tissue Res. 23:33-38, 1977
13. Linde, A., Persliden, B., R6nnbiick, L.: Cathepsin D: Purification from rat liver and immunohistochemical demonstration in rat incisor. Acta Odont. Scand. 36:117-126. 1978 14. Campo, R.D., Dziewiatkowski, D.D.: Turnover of the organic matrix of cartilage and bone as visualized by autoradiography, J. Cell. Biol. 18:19-29, 1963 15. Fiore-Donno, G.. Baume, L.-J.: Etude histochemique de la dentinogen~se humaine, Helv. Odont. Acta 10:141-185. 1966 16. Hirschman, A., Dziewiatkowski, D.D.: Protein-polysaccharide loss during endochondral ossification: Immunochemical evidence, Science 154:393-395, 1966 17. H6hling. H.J., Nicholson, W.A.P., Schreiber. J., Zessack, U.: The distribution of some elements in predentine and dentine of rat incisor, Naturwissenschaften 59:423, 1972
18. Pugliarello. M.C., Vittur, F.. de Bernard, B., Bonucci. E., Ascenzi, A.: Chemical modifications in osteones during calcification, Calcif. Tissue Res. 5:108-114, 1970 19. Lennox, D.W.. Provenza, D.V.: Mucopolysaccharides in odontogenesis. Histochemical and autoradiographic study, Histochemie 23:328-341, 1970 20. Laurent, T.C.: The ultrastructure and physical-chemical properties of interstitial connective tissue. Pflfigers Arch. 336:S 2 I-S 33, 1972 21. Campbell, D.H., Garvey, J.S., Cremer, N.E., Sussdorf, D.H.: lmmunoelectrophoresis. In: Methods in Immunology, pp. 149-155. W.A. Benjamin, New York, 1963 22. Linde, A., Persliden. B.: Purification of cathepsin D by AHSepharose affinity chromatography, Prep. Biochem. 8:231240, 1978 23. Avrameas, S., Ternunck, T.: Peroxidase labeled antibody and Fab conjugates with enhanced intracellular penetration, Immunochemistry 8:1175-1179, 197 I 24. Molin, S.-O.. Nygren, H., Dolonius, L.: A new method for the study of glutaraldehyde-induced crosslinking properties in proteins with special reference to the reaction with aminogroups. J. Histochem. Cytochem. 26:412-414. 1978 25. Boorshma, D.M., Kalsbeck, G.L.: A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method, J. Histochem. Cytochem. 23:200207, 1975 26. Linde. A.: A method for the biochemical study of enzymes in the rat odontoblasts during dentinogenesis, Arch. Oral Biol. 15:1209-1212, 1972 27. Poole, A.R., Hembry, R.M., Dingle, J.T.: Extracellular localization of cathepsin D in ossifying cartilage, Calcif. Tissue Res. 12:313-321, 1973 28. Dingle, J.T.. Fell, H.B., Glauert, A.M.: Endocytosis of sugars in embryonic skeletal tissues in organ culture, J. Cell. Sci. 4:139-154, 1969 29. Dingle, J.T.: Secretion of enzymes into the pericellular environment. Philos. Trans. R. Soc. Lond. 271:315-324, 1975
Received September 5, 1978 / Revised July 9, 1979 / Accepted July 11, 1979
Calcified Tissue International '~. 1979 by Springer-Verlag
Cathepsin D: Ultra-Immunohistochemical Localization in Dentinogenesis H. Nygren, B. Persliden, H-A. Hansson, and A. Linde Laboratory of Oral Biology, Department of Histology, University of GOteborg, Fack, S-400 33, Grteborg, Sweden
Summary. Cathepsin D was purified from rat liver using a new affinity chromatographic method, based on the coupling to the specific inhibitor pepstatin. This preparation was used for the production of specific antibodies from rabbit. The purified IgG fraction was conjugated to horseradish peroxidase in a two-step coupling procedure and used for electron microscopic immunohistochemistry of the odonloblast-predentine region of the rat incisor. Precipitates, indicating the presence of cathepsin D, were seen in the odontoblast, odontoblast process, and in the extracellular unmineralized matrix, the predentine. The observations are discussed in relation to proteoglycan degradation at the mineralization front simultaneous with crystal tbrmation, and in relation to the function oflysosomal enzymes in the turnover of connective tissue. Key words: Cathepsin -- Calcification -- Dentinogenesis -- Proteoglycans.
Introduction The degradation of proteoglycans (PGs) in different connective tissues has been suggested to be due to enzymatic activity. The acid endopeptidase cathepsin D (EC 3.4.23.5) has frequently been discussed since this enzyme seems to be of crucial importance for PG degradation in, e.g., cartilage of different species [ I-3]. Not many studies have been published on acid hydrolases in hard tissues, and their distribution has most often been considered in connection with resorptive processes. Some reports, however, have demonstrated the presence of and/or discussed the possible function of acid hydrolases in bone and dentine formation [4-13]. Send offprint requests to A. Linde at the above address.
Disappearance of a considerable portion of the PGs present in the unmineralized matrix simultaneous with mineral formation has been shown for several mesenchymal calcified tissues using different techniques [8, 11, 14-19]. The functional significance of this PG disappearance in the calcification process is not known. The only statement that can be made at the present is that due to the physicochemical properties of PGs [20], this change in the composition of the organic matrix should be important. In order to study the possible enzymatic degradation of PGs in the dentinogenically active rat incisor, cathepsin D was studied by immunohistochemical technique, using fluorescein isothiocyanate (FITC) labeled rabbit anti-rat cathepsin D IgG on the light microscopic level [ 13]. Cathepsin D was found to be located in the odontoblastema and over the predentine. Due to the limited resolution it was not possible to further characterize the distribution of cathepsin D. The present study, using electron microscopic immunohistochemistry, was undertaken to elucidate the subcellular distribution of cathepsin D in the odontoblast-predentine area of dentinogenically active rat incisors. Presumably this might also give some hint as to the localization for PG degradation in the tissue, whether it is an extracellular or intracellular event.
Materials and Methods
Cathepsin D Purification Cathepsin D was purified from rat liver by aff• chromatography on pepstatin-coupled AH-Sepharose 4B as described by Linde and Persliden [22]. A crude enzyme preparation was obtained from rat liver homogenate [ 13] and applied to the affinity chromatographic column, previously equilibrated with 0.1 M N.acitrate-HCl buffer, pH 3.0, containing 0.5 M NaC!. The column
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Ten milligrams of HRPO (Sigma Chemical Co., St. Louis, Missouri, USA) was reacted with 0.5 ml of 0.2% charcoal-filtered glutaraldehyde (Polaron Equipment Ltd., Liverpool, England) in 0.1 M borate buffer, pH 9.5, for 2 h at room temperature. The reacted HRPO was separated from free glutaraldehyde on a Sephadex G-25 column (0.7 • 12 cm) equilibrated with 0.15 M NaC1. The HRPO-glutaraldehyde complex was in a second step reacted with 5 mg IgG at pH 9.5 for 25 h in a total incubation volume of 2 ml in the cold. The reaction was terminated by the addition of 0. I ml 0.2 M lysine. The IgG-HRPO conjugate was separated from free IgG and HRPO on a Sephacryl S-200 Superfine column (1.6 • 100 cm) eluted with PBS buffer, pH 7.2, at a flow rate of 4 ml/h [25]. The IgG-HRPO conjugate was stored frozen in portions of 0.5 ml at -22~ prior to the immunohistochemical experiments.
~5o volume(m[)
Fig. 1. Elution profile from separation of the different reaction products after the coupling procedure with HRPO. The Sephacryl S-200 Superfine column (I.6 • 100 cm) was eluted with PBS buffer, pH 7.2, at flow rate of 4 ml/h and with fraction volumes of 3 ml. Protein was estimated as absorbance at 280 nm (--), and HRPO was measured by absorbance at 403 nm (..... ). Fractions indicated ( ) were pooled and referred to as IgG-HRPO conjugate, a, IgG-HRPO conjugate: b, unlabeled IgG: c, unbound HRPO
was washed with the equilibration buffer and the enzyme was eluted in one single fraction with 0.1 M NaHCO.~, pH 7.0, containing 0.5 M NaCI. The purity of the cathepsin D preparation was tested by electrophoresis on 7% polyacrylamide gels. DEAE- and CM-cellulose ion-exchange chromatography, and molecular sieve chromatography on Sephacryl S-200 Superfine [13].
Preparation qf Antibodies One milligram of the purified enzyme, dissolved in 1 ml phosphate-buffered saline (PBS buffer), pH 7.2, was injected subcutaneously together with 1 ml of Freund's complete adjuvant once weekly for 4 weeks into albino rabbits. Six weeks after the last injection, the same amount of enzyme alone was injected intravenously. Ten days after this injection, blood was withdrawn from the marginal ear vein. The serum was fractionated on a QAE-Sephadex A-50 ion-exchange column (equilibrated with 0.1 M Tris-HCl buffer, pH 6.5) by a NaCI gradient from 0.0-0.1 M NaCI in the equilibration buffer. The IgG fraction obtained was transferred to PBS buffer, pH 7.2, by buffer exchange on a Sephadex G-25 column and tested immunoelectrophoretically [21] against the antigen [13, 22].
HRPO Conjugation The coupling reaction for horseradish peroxidase (HRPO) to lgG was modified after Avrameas and Ternynck [23]. Several changes were, however, made to optimize the reaction [24], and therefore the method will be given in detail.
lmmunohistochemistry For the immunohistochemical study. Sprague-Dawley rats (body weight 200 g) were perfused through the aorta with 200 ml of 4% lbrmaldehyde in 0.15 M cacodylate buffer, pH 7.2. containing 0.1% picric acid, for 30 rain. The apical two-thirds of the maxillary incisors were dissected out according to Linde [26] and fractured. The dentine fragments thus obtained (with adhering odontoblasts) were then immersed in the same fixative for an additional 18 h and washed in the cacodylate buffer. The specimens were incubated for 12 h with the rabbit antirat cathepsin D IgG-HRPO conjugate, obtained 'after molecular sieve chromatography in PBS buffer, pH 7.2, containing 1% albumin. After incubation with antibodies, the cells and the fractured dentine pieces were rinsed in the cacodylate buffer for 12 h and in 0.15 M cacodylate buffer, pH 5.0, for 30 min. After rinsing, incubation of the specimens was performed in 0.5 mg/ml of 3,3-diaminobenzidine (British Drug House, Poole, England) in 0.15 M cacodylte buffer, pH 5.0, containing 0.01% H202 for 30 min. The specimens were then rinsed in 0.15 M cacodylate buffer, pH 7.2. postfixed in 2% OsO4 in the cacodylate buffer for 2 h, dehydrated in ethanol, and embedded in Epon. Thick section ( 1 p.m/were taken for orientation. Thin sections (500 A) were cut on an LKB Ultratome III ultramicrotome and examined in a JEOL 100 C electron microscope. As controls, specimens were incubated with 3.3-diaminobenzidine and H,_O2 without any prior incubation with the conjugated antibodies towards cathepsin D. Also, specimens were incubated with rabbit anti-goat IgG-HRPO at the same protein concentrations as with anti-cathepsin D IgG-HRPO.
Results
T h e c a t h e p s i n D p r e p a r e d by the p r e s e n t affinity c h r o m a t o g r a p h y t e c h n i q u e w a s s h o w n to b e p u r e b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s , i o n - e x c h a n g e chromatography on DEAE- and CM-cellulose and m o l e c u l a r s i e v e c h r o m a t o g r a p h y o n S e p h a c r y l S200 S u p e r f i n e . F u r t h e r m o r e , w h e n t e s t e d b y i m m u n o e l e c t r o p h o r e s i s , the c a t h e p s i n D w a s s h o w n to be immunologically homogeneous showing just one p r e c i p i t a t i o n l i n e [22].
H. Nygren et al.: Cathepsin D in Dentinogenesis
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Fig. 2. Central part of odontoblast incubated with anti-cathepsin D IgG-HRPO. HRPO activity, indicating the presence of cathepsin D. is seen in a lysosome-like vesicle (arrow). • 12,000. Fig. 3. Proximal part of odontoblast process, i.e., within the inner third of the predentine. Incubation with anti-cathepsin D IgG-HRPO. Precipitates are located within elongated vesicles (arrows). x 12,000. Fig. 4. Distal portion of odontoblast process, i.e., in the outer third of the predentine, with surrounding predentine. Intense precipitates (arrows), indicating the presence o f c a t h e p s i n D. can be seen close to the cell membrane. • 15,000. Fig. 5. Odontoblast process, incubated with peroxidase-coupled anti-cathepsin D IgG. Positive reactions are seen in granules within the process, x 12,000
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Fig. 6. Portion of newly formed predentine from fractured rat incisor tooth. Precipitates (arrows) are present in the extracellular matrix of the predentine. • 30,000
By separating the I g G - H R P O reaction products on Sephacryl S-200, it was possible to completely remove contaminating IgG and HRPO from the I g G - H R P O conjugate (Fig. 1). Generally, the yield of conjugated rabbit anti-rat cathepsin D with HRPO was in the range of 40% as judged by the two IgG fractions obtained by molecular sieve chroma-
H. Nygren et al.: Cathepsin D in Dentinogenesis tography, i.e., fraction a (IgG-HRPO) expressed in percent of total IgG (fraction a + b). The protein concentration in the conjugate was found to be approximately 80/zg IgG/ml and 20/xg HRPO/ml. The values were obtained by optical density measurements at 280 nm and 403 nm for IgG and H R P O , respectively, using the extinction coefficients 14.9 for rabbit IgG and 14.5 for HRPO. Precipitates, indicating the presence of cathepsin D, were seen in lysosome-like vesicles in the odontoblast Golgi region (Fig. 2). In the proximal part of the odontoblast processes, i.e., within the inner third of the predentine, electron-dense precipitates were seen in elongated vesicles (Fig. 3). In the more distal part of the odontoblast process, approximately in the outer third of the predentine, dense precipitates were seen inside the processes and in close relation to the cell membrane. In this area it was not possible to detect any vesicle-like structures in connection with the electron-dense particles (Figs. 4 and 5). For technical reasons (since the sections were not demineralized), it was not possible to study cathepsin D localization in the cell process further out in the dentine. Extracellular localization of cathepsin D in the predentine matrix was indicated by electron-dense precipitates in the areas between odontoblast processes (Fig. 6), predominantly in the proximal third of the predentine. Control incubations with 3,3-diaminobenzidine and H202, but without I g G - H R P O conjugate added, showed a scattered distribution of small precipitates randomly localized or related to mitochondria (Fig. 7). Incubation of odontoblasts with control serum (anti-goat-IgG-HRPO) showed no precipitates not seen in the control specimens with 3,3-diaminobenzidine and H~O_~only.
Fig. 7. Control section of odontoblast incubated with 3.3diaminobenzidine without the addition of anti-cathepsin D IgG-HRPO. Precipitates can be seen in mitochondria (arrow). Scattered distribution of small precipitates (arrows) can be seen in the cytoplasm. • 18.000
H. Nygren et al.: Cathepsin D in Dentinogenesis
Discussion
Molecular sieve c h r o m a t o g r a p h y for the separation of the I g G - H R P O conjugate from the other constituents in the reaction mixture was described by B o o r s h m a and Kalsbeck [25]. In this way excess free IgG and H R P O are separated from the rabbit anti-rat cathepsin D IgG H R P O preparation. This is of vast importance for avoiding nonspecific staining and thus for the interpretation of precipitates occurring in the specimen. Incubating with only 3,3-diaminobenzidine and H202 was undertaken in order to identify any intracellular peroxidase activity. This could be seen in mitochondria of the odontoblasts. Nonspecific activity, i.e., nonspecific adsorption of I g G - H R P O conjugates to tissue c o m p o n e n t s , was controlled by incubation of the specimens with rabbit anti-goat I g G - H R P O . This type of control revealed no nonspecific electron-dense precipitates in addition to those seen in the other control incubations with 3,3diaminobenzidine and H.,O., only. Thus precipitates occurring in regions where 3,3-diaminobenzidine control incubations revealed no precipitates were taken as an indication for the presence of cathepsin D in the tissue. The use of fixative in immunohistochemistry is a double-edged procedure. In the present study 4% formaldehyde was used as perfusion and immersion fixative. Although this technique destroyed a large portion of the antigen amount in the tissue specimens, it made it possible to locate the immunoprecipitate to known morphological structures. It should, however, be e m p h a s i z e d that the method can be regarded as only qualitative due to the fixation technique. Electron-dense precipitates, indicating the presence of cathepsin D, were seen in the Golgi region of the odontoblasts as well as in elongated vesicles in the odontoblast processes. Also, the e n z y m e was found to be localized in close relation to the cell m e m b r a n e of the odontoblast process. These resuits m a y be taken to indicate a transport of cathepsin D f r o m Golgi region of the odontoblast throughout the process in vesicle-like structures, probably derived from the Golgi apparatus. The extracellular distribution of cathepsin D in the predentine, preferentially in the proximal third of the predentine matrix, is of interest since this would implicate an extracellular cathepsin D function. This finding is in agreement with those of Poole et al. [27]. These authors studied cathepsin D localization in an in vitro chick bone organ culture. An extracellular distribution of cathepsin D, probably due to cell secretion, was found. The localization of cathepsin D has also been studied in
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growing rat incisor by F I T C technique [13]. An intense fluorescence o v e r the o d o n t o b l a s t e m a and over the predentine was found, but no conclusions could be drawn whether this FITC-staining was due to intra- or intra- and extracellular distribution of the enzyme. An extracellular secretion of lysosomal e n z y m e s has been discussed in relation to the degradation of extracellular matrix m a c r o m o l e c u l e s [28, 29]. According to Dingle [29], the matrix c o m p o n e n t s would be initially degraded in the extracellular space followed by endocytosis and complete digestion in the lysosomal system of the cells. The present findings support the theory of an initial extracellular function of cathepsin D. It may be speculated that this is a m e c h a n i s m c o m m o n to different connective tissues. The localization of high activity of cathepsin D in the dentinogenic region [12] is strong evidence for an important function in the degradation of PGs taking place simultaneously with crystal formation at the mineralization front. The results of Poole et al. [27] indicate that this process occurs also in bone formation. Further degradation of PG constituents, be it extra- or intracellular, m a y be brought about by the combined action of other acid hydrolases, earlier shown to be present in dentinogenically active odontoblasts [10]. The functional importance of the removal of PGs is not known at present. It may be as simple as to provide space for mineral crystals. It may also be in order to r e m o v e m a c r o m o l e c u l e s with a strong polyanionic character after having fulfilled some role as inhibitors of mineral formation or in the extracellular aggregation of collagen.
Acknowledgments. We are indebted to Dr. Neil Barclay for fruitful discussion. We gratefully acknowledge the excellent technical assistance of Ms. I. Haag, Ms. A. H6glund, and Ms. U. Svedin. The investigation was supported by The Swedish Medical Research Council, The Faculty of Odontology, University of G6teborg, and The Magnus Bergvall Foundation.
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H. Nygren et al.: Cathepsin D in Dentinogenesis
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Received September 5, 1978 / Revised July 9, 1979 / Accepted July 11, 1979
