Eur. J. Biochem. 92, 183-188 (1978)

Type-I Trimer and Type-I Collagen in Neutral-Salt-Soluble Lathyritic-Rat Dentine Marissa WOIILLEBE and David J. CARMICHAEL Department of Oral BIology, Faculty of Dentistry, University of Alberta, Edmonton (Received June 5 , 1978)

Triple-helical collagen molecules have been obtained from EDTA-demineralized lathyritic rat incisors by neutral buffer extraction. Component a chains, isolated by sequential ion-exchange and gel-filtration chromatography, were shown to be cclI and a2 chains by cyanogen bromide peptide analysis. The a1I:aZ chain ratio was approximately 3 : 1, which is greater than expected for type I collagen. The excess of cclI chains over that required for type I collagen was due to the presence of type I trimer molecules. Fractional salt precipitation separated type I collagen from type I trimer. It is not known at present if type I trimer synthesis also occurs in normal rat tissues.

Collagen is a triple-helical molecule made up of three component a chains. At least four distinct collagen types have been found in vertebrate connective tissues [l]. These collagen types, which have some degree of tissue specificity, differ from each other in their CI chain composition. The most thoroughly studied collagen type, having a chain composition [al(I)]2az, is designated type I. Types 11, I11 and IV are trimers of al(II), al(II1) and al(IV) respectively. Biochemical characterization of dentine collagen has been hindered because of its extreme resistance to solubilization [2-61, a property attributed to a high degree of intermolecular cross-linking and/or an extensive fibre weave. As a consequence, initial approaches to the macromolecular characterization of dentine were essentially degradative in nature and based on a careful analysis of the peptides released by cyanogen bromide digestion. Volpin and Veis [7] suggested that, in addition to type I collagen, type I11 collagen was also present in dentine. In later work, Scott and Veis [8] were unable to demonstrate the presence of type I11 peptides in cyanogen bromide digests of insoluble bovine dentine, using an analytical gel electrophoretic method, and concluded that little or no type 111 collagen was present. Rat dentine collagen was also shown to be essentially type I by Butler and DeSteno [4]. Another approach to the biochemical characterization of dentine is to examine the soluble collagen from lathyritic animals. Experimental lathyrism [9] results in the enhancement of tissue collagen solubility due to the inhibition of collagen cross-linking by the lathyrogen, 8-aminopropionitrile. Although young

rats fed with p-aminopropionitrile are unable to survive on the lathyrogenic diet [4], older animals can [5]. An earlier report [5] from this laboratory showed that a soluble collagen fraction can be obtained from the dentine of lathyritic rats by guanidinium chloride extraction. In the present work we report the isolation and characterization of an undenatured soluble collagen fraction obtained by extraction of EDTA-demineralized lathyritic rat incisors with a Tris/NaCl buffer at neutral pH. An abstract of preliminary results has been reported elsewhere [lo].

METHODS Lathyrism Male Wistar rats were rendered lathyritic by maintaining them for 70 days on a diet of Purina rat chow supplemented with 0.4 % 8-aminopropionitrile fumarate by weight. Dentine Matrix Collagen Preparation The upper and lower incisors were removed from fresh mandibles of lathyritic and normal rats and immediately soaked in 0.15 M NaCl. Following reinoval of adhering soft tissue, bone and pulp, the incisors were washed with fresh 0.025 M EDTA, 0.15 M NaCl, pH 7.4 for three 24-h periods. Demineralization was carried out by shaking with 0.5 M EDTA, pH 7.5, for three weeks, changing the solvent daily. The demineralized incisors were washed free of EDTA and

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Collagen Types in Lathyritic-Rat Dentine

homogenized by shredding in water, using a Virtis 45 homogenizer at full speed for 30 min, and finally lyophilized. All operations during dentine matrix collagen preparation and subsequent extractions were carried out at 4 "C.

was diluted 10-fold with water and lyophilized. The CNBr peptide mixture was dissolved in 0.15 M acetic acid, filtered and lyophilized.

TrislNaCI Extract ion

Collagen chains were electrophoresed on 5 % gels [14] and cyanogen bromide peptides were electrophoresed on 7.5% gels [8]. Gels were stained with Coomassie blue.

The dentine matrix collagens (lathyritic and normal) were extracted with fresh portions of 1.O M NaCl, 0.05 M Tris, pH 7.4 (150 ml buffer/g of matrix collagen) for three 24-h periods. The neutral salt extracts, as well as the residues, were dialyzed versus water and lyophilized. Isolation of Collagen Chains

Collagenous proteins in the neutral salt extract were separated from acidic macromolecules by passage through aDEAE-cellulose column ( 2 . 6 12cm) ~ equilibrated with 0.2 M NaCl, 0.05 M Tris, pH 7.4 [ l l ] at 6 "C. The unretarded material was desalted by dialysis, lyophilized and redissolved in 0.02 M NaCI, 0.03 M Tris, 2 M urea, pH 7.4, and applied to a DEAE-cellulose column (2.6 x 12 cm) equlibrated at 6 "C with the same buffer used for dissolving the sample, and then eluted with a superimposed linear salt gradient from 0.02 to 0.2 M NaCl over a total volume of 800 ml. Under these conditions, procollagen chains are separated from collagen chains [12]. Pooled fractions were desalted by dialysis and lyophilized. The unretarded material from the second DEAEcellulose chromatography was chromatographed on CM-cellulose (1.6 x 12 cm) equilibrated with 0.03 M sodium acetate, 1 M urea, pH 4.8 and eluted with a superimposed linear salt gradient from 0 to 0.12 M NaCl over a total volume of 400 ml at 45 "C. Pooled fractions were desalted by dialysis and lyophilized. Individual peaks obtained by CM-cellulose chromatography were chromatographed on BioGel A 1.5 m, 200- 400 mesh (2.6 x 106 cm) equilibrated and eluted with 1 M CaClZ, 0.05 M Tris, pH 7.4 [13]. Pooled fractions were desalted by dialysis and lyophilized. All chromatographic separations were monitored by absorbance at 230 nm. The identity of the various peaks were established by sodium dodecylsulfate/ polyacrylamide gel electrophoresis. Cyanogen Bromide Cleavage

Collagen ci chain (2 mg) was digested with 1 ml of a freshly prepared solution of cyanogen bromide in deaerated 70 % formic acid (13 mg/ml) in a NZflushed stoppered test tube for 4 h at room temperature, with continuous stirring. The reaction mixture

Sodium Dodecylsulfate/Polyacrylamide Gel Electrophoresis

Fractional Salt Precipitation

Neutral-salt-soluble dentine was dissolved in 0.16 M sodium phosphate buffer, pH 7.2. The sodium chloride concentration was gradually increased by the addition of 4.4 M NaCl and stirring for 24 h. A precipitate formed at 2.4 M NaCl and was collected by centrifugation at 25 300 x g for 30 min. The 2.4 M NaCl supernatant was dialyzed vs 0.01 M NaHzP04, yielding a precipitate which was collected by centrifugation at 25 300 x g for 30 min. Both 2.4 M NaCl and 0.01 M NazHP04 precipitates were redissolved in 0.16 M sodium phosphate buffer, pH 7.2, and reprecipitated. The precipitates were suspended in and dialyzed against distilled water and lyophilized. All operations were carried out at 4 "C. Limited Pepsin Digestion

Pepsin digestion was carried out to distinguish between randomly coiled chains and native triplehelical molecules [ 151. Collagen samples were digested with pepsin in 0.5 M acetic acid (1 mg/ml) at a collagen :pepsin ratio of 50 : 1 for 18 h at 4 "C. The reaction was stopped by adjusting the pH to 8 with 0.5 M NaOH. Following lyophilization, the pepsin digests were dissolved in 0.01 M phosphate, 0.2% sodium dodecylsulfate, 2 M urea, pH 7.2 and electrophoresed on 5 % gels.

RESULTS Tris/NaCl extraction solubilized 6.2 % of the decalcified lathyritic rat incisor matrix as non-dialyzable collagenous material. Dodecylsulfate/polyacrylamide gel electrophoresis of this material revealed that the majority of the collagen was present in the form of ci components and that limited pepsin digestion of this fraction did not change the electrophoretic pattern to any significant degree (Fig. 1). The neutral-saltsoluble dentine collagen was, therefore, extracted in an undenatured form. It should be noted that subsequent extraction of the Tris/NaCl residue with the denaturant guanidinium chloride yielded an addition-

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M. Wohllebe and D. 3 . Carmichael

Fig. 1. Dodecyl.~ulfate/pol~acrylamide gel electrophoretograms of'acid-soluble rat skin collugen ( a ) before ( b ) after treatment with pepsin; neutral-salt-soluble lathyritic rat dentine ( c ) before and ( d ) after treatment with pepsin. Samples dissolved in 2 M urea, 0.2 "/, sodium dodecylsulfate, 0.01 M phosphate buffer, pH 7.2 at a concentration of 2 mg/ml were denatured at 50 'C for 30 min prior to application of 10-p1 aliquots to 5 % gels. Electrophoresis was carried out at a constant current of 6 mA/tube for 6 h using 0.1 sodium dodecylsulfate, 0.10 M phosphate buffer, pH 7.2 as the running buffer. Migration was towards the anode (bottom). Gels were stained with Coomassie blue

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a1 soluble dentine fraction, the major component of which was a chains, which was easily degraded by pepsin (Wohllebe and Carmichael, unpublished results). The presence of two minor bands, P-1 and P-2, exhibiting slightly lower mobilities than the a1 chain and another diffuse band, P-3, exhibiting slightly higher mobility than the a2 chain was also evident from the dodecylsulfate/polyacrylamide gel electrophoretogram of neutral-salt-soluble dentine. Following pepsin digestion, bands P-1 and P-2 appeared to have been converted into smaller fragments but still with slightly lower mobilities than the a1 chain; band P-3 remained unchanged. Identification of these minor bands is under investigation. The neutral-salt-soluble extract from lathyritic dentine was passed through DEAE-cellulose to remove acidic noncollagenous macromolecules [ 111. Since dodecylsulfate/polyacrylamidegel electrophoresis had suggested the presence of partially degraded pro-a chains, the collagenous material not retained by DEAE-cellulose was subjected to a second DEAEcellulose fractonation (Fig. 2) under conditions shown previously [12] to separate collagen a chains from

p 10

20

30

40

50

60

70

Fraction number Fig. 2. DEAE-callulose chromatogram of collagenous fraction oj neutral-salt-soluble dentine. The column (2.6 x 12 cm) was eluted at 6 "C initially with 0.02 M NaC1, 0.03 M Tris, 2 M urea, pH 7.2, and then with the same buffer superimposed with a linear gradient from 0.02 to 0.20 M NaCl over a total volume of 800 ml. 10-ml fractions were collected

186

Collagen Types in Lathyritic-Rat Dentine

procollagen. The unretarded peak from the second DEAE-cellulose fractionation was separated into its component a chains by CM-cellulose chromatography under denaturing conditions. As seen in Fig. 3 , the C I :~C I ~ratio of neutral-salt-soluble dentine is greater than that exhibited by acid-soluble rat skin collagen. The peaks labelled 2 and 4 were further separately

1

3

1

Fraction number

Fig. 3. CM-cellulose chromalogrum of unreturded collagenou,s peak and acid-soluble rat skin collagen (-----I. The from Fig.2 i-) column ( 1 . 6 ~12 cm) was eluted at 45'C with 0.03 M sodium acetate, 1 M urea, pH 4.8, superimposed with a linear gradient from 0 to 0.12 M NaCl over a total volume of 400 ml. 11-ml fractions were collected

chromatographed on BioGel A 1.5 m, yielding electrophoretically homogeneous CI chains. To further characterize the isolated dentine a chains, they were cleaved with cyanogen bromide. The cyanogen bromide peptide patterns on dodecylsulfate/polyacrylamide gels were then compared with those of authentic rat skin a11 and a2 chain cyanogen bromide peptides. It was evident that the skin and dentine CI chains exhibited the same cyanogen bromide peptides, thus establishing the identity of the dentine a chains as a11 and a2 (Fig. 4). Since only a11 and a2 chains could be identified in the neutral-salt-soluble dentine, it was appropriate to identify the cause of the variation from the a1 :a2 ratio of 2: 1 normally expected in a type I collagen. Following removal of the acidic noncollagenous macromolecules, fractional precipitation [ 161 of neutral-salt-soluble dentine was carried out. Some 60 %, of the material precipitated at 2.4 M NaCI, and another 10 % was precipitated when the 2.4 M NaCl supernatant was dialyzed versus 0.01 M Na2HP04. The dodecylsulfate/polyacrylamide gel electrophoretograms of the material obtained during fractional precipitation are shown in Fig. 5. The 2.4 M NaCl precipitate exhibited a subunit composition very similar to that of acid-soluble rat skin collagen. The 0.01 M Na2HP04 precipitate was considerably enriched in a11 chains as well as bands P-1 and P-2.

Fig. 4. Dodecylsu1~ate~~)olyucrylamide gt.1 electrophoreiojirams of cyanojien bromide peptidesfrom ( a ) rat skin r l l , ( h ) rat dentine 1 1 I , i c ) rat skin ci2 and ( d ) rat dentine z2. Samples dissolved in 2 M urea, 0.2'%, sodium dodecylsulfate, 0.01 M phosphate, pH 7.2 were denatured at 50 'C for 30 min prior to application of 10.~1aliquots to 7.5 gels. Electrophoresis was carried out at a constint current of 1 mA,tube for 20 min, 3 mA/tube for 40 min and 6 mA/tube for 3 h, using 0.1 7: sodium dodecylsulfate, 0.05 M phosphate buffer, pH 7.2 as the running buffer. Migration was towards the anode (bottom). Gels were stained with Cootnassie blue

M. Wohllebe and D. J. Carmichael

gel electrophoretograms of neutral-.sult-soluble Iathyritic rat dentine ( a ) before and ( b ) after pepsin ; Fig. 5. Dodecylsulfate~polyacr~~lamide 2.4 M NaCI precipitate (from neutral-salt-soluble lathyritic' rat dentine) (c) before and ( d ) afier pepsin; 0.01 M Nu~HPOA.precipitate (from neutral-salt-.~olublelarhyritic rat dentine) ( e ) before and if) after pep.vn. Electrophoresis conditions are as described for Fig. 1

Band P-3 was present in both 2.4 M NaCl and 0.01 M Na2HP04 precipitates. The a11chains in the 0.01 M Na2HP04 precipitate were most likely present as [ C ( ~ ( T ) ] ~ molecules rather than as randomly coiled chains since the material from which it was obtained had been shown to be undenatured. Furthermore, the alI chains remained unchanged following pepsin digestion of the 0.01 M Na2HP04 precipitate. Some [al(I)]2az molecules were probably still present, accounting for the presence of a2 chains in the 0.01 M NazHP04 precipitate. Other workers [17,18] have previously observed that it is difficult to achieve complete separation of type 1 collagen from type I trimer.

DISCUSSION The present results demonstrate that by a suitable choice of extraction procedure, native triple-helical collagen molecules can be obtained from lathyritic rat incisors, thus allowing the isolation and characterization of the CI chain components as well as an analysis of collagen types of dentine matrix. Although only C I ~ ( Iand ) a2 chains were found in the neutral-saltsoluble dentine collagen they were present in a chain ratio of approximately 3: 1, which was greater than the expected ratio if only type 1 collagen were present. The excess of al(I) chains are present as [a1(1)]3 molecules which are separable from type I collagen by fractional precipitation. The conclusion that type I trimer accounts for the excess of al(I) chains over that required for type I

collagen is supported by the following arguments. Non-denaturing extraction conditions were used and the collagenous material in the neutral-salt-soluble extract of lathyritic dentine matrix was resistant to limited pepsin digestion. At no time during the isolation and purification procedures was the collagenous material exposed to conditions which would favor renaturation in vitro [19] o f any randomly coiled chains present. Only native molecules are precipitated from salt solutions by dialysis versus 0.01 M Na2HP04

POI.

Type I trimer was originally isolated from chick chondrocytes grown in culture in the presence of 5-bromodeoxyuridine [21]. Later work demonstrated its synthesis in vitvu by several types of cultured cells [17,18,22-261. The procollagen synthesized by a teratocarcinoma-derived cell line was closely related to type I trimer procollagen [27]. Type I trimer synthesis in vivo by a polyoma-virus-induced mouse tumor [28] has been reported. Of relevance to the present study is the recent report [29] showing that the collagen synthesized by mouse tooth germs in organ culture in ascorbate deficiency, as well as in the presence of ascorbate, exhibits a n a1 :a2 chain ratio greater than 2. Although no attempt to characterize the a1 chains was made, they were most likely cxll chains, in the light of very recent findings [30] that rat incisor odontoblasts in organ culture synthesize both type I trimer and type I collagen. All the reported instances of type 1 trimer synthesis are by cells in culture and cells with presumably

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M. Wohllebe and D . J . Carmichael: Collagen Types in Lathyritic-Rat Dentine

altered metabolic states. This poses a question as to the significance of type I trimer synthesis. Is type I trimer formed only as a result of breakdown of normal regulatory controls of collagen biosynthesis in response to trauma? Embryonic chick calvaria in organ culture synthesize only type I trimer procollagen in the presence of a&-dipyridyl, but if these calvaria are transferred to a,a’-dipyridyl-deficient medium during a pulsechase experiment, the procollagen extracted from the tissue at the end of the chase period is only type I procollagen [26]. It was suggested that a,a’-dipyridyl interfered with the assembly of pro-a11 and pro-a2 into type I procollagen by a chain-selecting mechanism such that only pro-a11 chains were able to come together and form type I trimer procollagen, and that removal of up’-dipyridyl from the medium during the chase period allowed this chain-selecting mechanism to function normally again, thus producing type I procollagen. It is quite plausible that in this study, type I trimer synthesis in lathyritic rat incisors is a consequence of long-term exposure of odontoblasts to P-aminopropionitrile. In addition to its usual inhibitory effect on lysyl oxidase, P-aminopropionitrile may also have exerted some influence on gene expression by odontoblasts. A recent report [31] also showed type I trimer synthesis by lathyritic chick embryos in vivo. To this time, no experimental data has been obtained which would indicate type I trimer synthesis by normal tissues in vivo. By using specific antibodies against [a1(1)]3 [32], we are proceeding with an immunohistological detection and localization of [a1(I)]3 in normal rat incisor predentine and dentine, as well as in other normal tissues. This study was supported by the Medical Research Council of Canada.

REFERENCES 1. Miller, E. J. (1976) Mu/. CeN. Biochem. 13, 165-191. 2. Veis, A. & Schleuter, R. J. (1964) Biochemistry, 3,1650-1657.

3. Schleuter, R. J. & Veis, A. (1964) Biochemistry, 3, 1657-1665. 4. Butler, W. T. & DeSteno, C. V. (1971) Ahstr. 49th Gen. Session, lnt. Ass. Dent. Res., p. 87. 5. Carmichael, D. J., Dodd, C. M. & Nawrot, C. F. (1974) Culcif. Tissue Res. 14, 177- 194. 6. Carmichael, D. J., Dodd, C. M. & Veis, A. (1977) Biochim. Biophys. Actu, 491, 177 - 192. 7. Volpin, D. & Veis, A. (1973) Biochemistry, 12, 1452-1464. 8. Scott, P. G. &Veis, A. (1976) Connect. Tissue Res. 4 , 117- 129. 9. Tanzer, M. L. (1965) Int. Rev. Connect Tissue Res. 3, 91 -112. 10. Wohllebe, M. & Carmichael, D. J. (1978) Absfr. 515th Gen. Session, Int. Ass. Dent. Res., p. 279. 11. Miller, E. J. (1971) Biochemistry, 10, 1652- 1658. 12. Byers, P. H., McKenney, K. H., Lichtenstein, J. R. & Martin, G. R. (1974) Biochemistry, 13, 5243-5248. 3 3. Piez, K. A. (1968) Anal. Biochem. 26, 305-312. 14. Furthmayr, H. &Timpl, R. (1971) Anal. Biochem. 41,510-516. 15. Layman, D. L., McGoodwin, E. B. & Martin, G. R. (1971) Proc. Nut1 Acad. Sci. U.S.A. 68, 454-458. 16. Trelstad, R . L., Catanese, V. M. & Rubin, D. F. (1976) Anal. Biochem. 71, 114-118. 17. Benya, P. D., Padilla, S. R. & Nimni, M. E. (3977) Biochemistry, 16, 865 - 872. 18. Narayanan, A. S. & Page, R. C. (1976) J . Biol. Chem. 251, 5464 5471, 19. Tkocz, C. & Kuhn, K. (1969) Eur. J . Biochem. 7,454-462. 20. Trelstad, R. L., Kang, A. H., Toole, B. P. & Gross, J. (1972) J . Biol. Chem. 247, 6469 - 6473. 21 Mayne, R., Vail, M. S. & Miller, E. J. (1975) Proc. Nutl Acad. Sci. U.S.A. 72,4511-4515. 22. Mayne, R., Vail, M. S., Mayne, P. M. & Miller, E. J. (1976) Proc. Nutl Acud. Sci. U.S.A. 73, 1674-1678. 23 Mayne, R., Vail, M. S. & Miller, E. J. (3976) Dev. Biol. 54, 230 - 240. 24 Daniel, J. C. (1976) Cell Differ. 5, 247-253. 25 Mayne, R., Vail, M. S. & Miller, E. J. (1978) Biocliemistry, 17, 446 - 452. 26 Muller, P. K., Meigel, W. N., Pontz, B. F. & Raisch, K. (1974) Hoppe-Seyler’s 2. Physiol. Chem. 355, 985- 996. 27 Little, C. D., Church, R. L., Miller, R. A . & Ruddle, F. H. (1977) Cell, 10,287-295. 28 Moro, L. & Smith, B. D. (1977) Arch. Biochem. Biophys. 182, 33-41. 29 Schiltz, J. R., Rosenbloom, J. & Levenson, G. E. (1977) J . Embryol. E.xp. Morphol. 37, 49- 57. 30 Munksgaard, E. C., Rhodes, M., Mayne, R. & Butler, W. T. (1978) Eur. J . Biochem. 82, 609-617. 31 Jimenez, S. A., Bashey, R. I., Benditt, M. & Yankowski, R. (1977) Biochem. Biophys. Res. Commun. 78, 1354- 1361. 32 Hahn, E., Timpl, R. & Miller, E. J. (1974) J . Inzmunol. 113, 421 -423. -

M. Wohllebe and D. J. Carmichael, Department of Oral Biology, Faculty of Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2N8