THE OF BIOLOGICAL CHEMISTRY Vol. 255. No. 12. of June … · · 2001-09-05were decalcified with...

THE J O U R N A L OF BIOLOGICAL CHEMISTRY Vol. 255. No. 12. I s u e of June 25, pp. 5931-5942. I980 Prmted in 11.S.A.

Noncollagenous Proteins of Dentin A RE-EXAMINATION OF PROTEINS FROM RAT INCISOR DENTIN UTILIZING TECHNIQUES TO AVOID ARTIFACTS*

(Received for publication, August 20, 1979, and in revised form, December 20, 1979)

Anders Linde,+ Meera Bhown, and William T. Butler8 From the Institute of Dental Research and the Department of Biochemistry, University of Alabama in Birmingham, University Station, Birmingham, Alabama 35294

Noncollagenous proteins (NCPs) were obtained from rat dentin using several precautionary measures to prevent artifactual degradation and losses of the proteins.

Prior to demineralization, rat incisor dentin was extracted with 4 M guanidine hydrochloride (GdmCl) containing enzyme inhibitors. The only major component extracted with GdmCl was a proteoglycan fraction. Most of the NCPs were extracted when the incisors were decalcified with an EDTA solution containing protease inhibitors. The EDTA extract contained four types of macromolecules: acidic glycoproteins, y-carboxyglutamic acid (G1a)-containing proteins, phosphoproteins, and proteoglycans. With two exceptions, the apparent molecular weights of these NCPs were greater than 50,000. Our observations contrast sharply with the results obtained by others for human dentin NCPs and suggest that artifactual degradation and losses of some NCPs occurred in these previous studies.

The organic phosphate-containing fraction was biphasic when the EDTA extract was chromatographed on DEAE-cellulose. Rechromatography of this fraction by two different procedures separated the material into a complex glycoprotein-containing fraction and, a single rat incisor phosphoprotein peak (RIP). Thus, the earlier interpretation by others that the biphasic nature of the RIP-containing fraction represents two widely differing phosphoprotein species was premature.

Highly purified RIP, prepared by passage through a sulfonated polystyrene column, contained no cysteine, valine, methionine, leucine, phenylalanine, or arginine, and the level of phosphoseryl residues was higher than for any previous report. When this preparation of phosphoprotein was dephosphorylated (dP-RIP) and rechromatographed on DEAE-cellulose, a partial separation into two fractions was observed. Automated Edman degradation of fraction dP-RIP suggested the presence of two NH2-terminal sequences: Asp-Asp- Asp-Asn and Asp-Asp-Pro-Asn. However, this material displayed a single protein band when applied to 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. The apparent molecular weight of dP-RIP, compared to

Grants DE-02670 and DE-05092. The costs of publication of this * This research was supported by National Institutes of Health

article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of an International Research Fellowship from the Na- tional Institutes of Health. Present address, Laboratory of Oral Bi- ology, University of Goteborg, Goteborg, Sweden.

0 To whom reprint requests should be addressed.

standard globular proteins, was about 72,000. The data suggest that rat dentin contains at least two major molecular species of RIP that are closely related in size and structure. In addition, preliminary evidence suggested that other minor forms of related phosphoproteins may exist.

Many researchers hypothesize that the organic components of calcified tissues are essential for the formation of the calcium phosphate mineral in these tissues. The organic matrix of bone and dentin is approximately 85 to 90% type I collagen, the level of variation depending upon which species and anatomical location is analyzed. The earlier emphasis was upon the role played by collagen fibers in initiating and controlling crystal growth of the inorganic phase during the formation of mineralized tissues (l), but more recently the interest has shifted to the roles played by NCPs’ in these processes.

One class of NCPs present in dentin is a phosphoprotein in which phosphoserine and aspartic acid account for about 80% of the amino acid residues (2-4). In rat incisor dentin, the phosphoprotein fraction constitutes about half of the NCPs. Phosphate-containing proteins are also present in bone and enamel (5-7). Another protein with unusual properties is the y-carboxyglutamic acid (Gla)-containing protein found in bone (8-10). Like prothrombin, this protein is dependent upon an adequate vitamin K supply for carboxylation of glutamate residues to form Gla (11,12). The Gla-containing bone protein, referred to by some investigators as osteocalcin, has a low molecular weight (approximately s o 0 0 ) and comprises about 10% of the NCPs in bovine and chicken bone. Proteoglycans are also present in bone and dentin (13,14) but have not been characterized rigorously.

A fourth class of NCPs in bone and dentin are the acidic glycoproteins (13). The sialoprotein of bone (15) has received considerable attention. Leaver and co-workers have published a number of papers dealing with the NCPs of human dentin (13, 16-18) and the picture that emerges is complicated. An early publication described 20 distinct NCP components (16), while, in later studies (17, la), 3 “anionic glycoproteins” and 12 “less acidic” proteins were found. Most of these 15 components had molecular weights in the 12,000 to 15,000 range; only three of them had molecular weights greater than 15,000, with the largest being 26,000. The similarity of amino acid and carbohydrate compositions for four “less acidic glycoproteins”

’ The abbreviations used are: NCPs, noncollagenous proteins; GIs, y-carboxyglutamic acid; RIP, rat incisor phosphoprotein; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; dP-RIP, dephosphorylated phosphoprotein; Pth, phenylthiohydantoin.

5931

5932 Noncollagenous Proteins of Rat Dentin

was thought to be due to the pooling of samples from many different individuals (18). When the large number of similar NCPs from human dentin was chromatographed on DEAE- cellulose, almost no separation was achieved. In contrast to other workers (19), Leaver and his colleagues were unable to find the highly phosphorylated phosphoprotein in human dentin.

Some of the confusing results cited above, as well as other inconsistencies with regard to NCPs obtained by different workers (20), could be explained by artifactual proteolysis during extraction and chromatography or because of the use of other questionable techniques. Proteases are present in the tissues and could be included in the extracts. For example, cathepsin D is known to be present in odontoblasts and predentin (21, 22). The extended periods necessary for complete demineralization of tissues would allow ample time for proteolysis. It has been argued that the high levels of EDTA used for demineralization would inhibit proteolysis. Further column fractionation procedures have been performed at room temperature in the absence of protease inhibitors, and some degradation could occur at this step. Intense mechanical dis- integration of bone and dentin to powders, performed by some investigators prior to any extraction or decalcification techniques (14-18), could result in mechanical breakdown of some of the organic components. And finally, we suspect that low molecular weight proteins have been lost because of the use of dialysis membranes without specific molecular weight cutoff limits.

These objections have led us to a comprehensive reassess- ment of the nature of the NCPs of rat dentin with precautionary measures to overcome them. Similar precautionary extraction procedures have been used by Termine et al. (23) in studies on the proteins of fetal bovine enamel and dentin. The results reported here show that rat dentin contains phosphoproteins, acidic glycoproteins, proteoglycans, and low molecular weight, Gla-containing proteins. Only a small number of major acidic glycoproteins, with apparent molecular weights greater than 50,000, was obtained, in contrast to the observations cited above. We also report further studies on the purification and molecular structure of the rat incisor phosphoprotein (RIP).

EXPERIMENTAL PROCEDURES

Materials-Frozen rat heads were purchased from Pel-Freez Inc., Rodgers, AR. Pepstatin, alkaline phosphatase preparations (P-0762 and P-4502), and soybean trypsin inhibitor were obtained from Sigma Chemical Co., St. Louis, MO. Spectrapor 3 dialysis tubing with a nominal molecular weight cutoff limit of 3500 was from Spectrum Medical Industries Inc., Los Angeles, CA. Protein standards for cali- bration of polyacrylamide gels, QAE-Sephadex A-25, and Sephadex G-25, G-75, and (3-200 resins were from Pharmacia Fine Chemicals, Piscataway, NJ. DEAE-cellulose (DE52) was purchased from What- man Ltd., Springfield Mill, Maidstone, Kent, United Kingdom, or from Sigma. AG MP-50 cationic exchange resin was bought from Bio- Rad Laboratories, Richmond, CA. Supplies for the Technicon Autoanalyzer were purchased from Technicon Instrument Co., Tar- rytown, NY.

Extraction Solutions-Tissues were extracted with the following guanidine HC1 solution (GdmCI): 4 M guanidine HCI containing 50 mM sodium acetate, 10 mM Na,EDTA, 100 mM 6-aminohexanoic acid, 5 mM benzamidine/HCl, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ liter of soybean trypsin inhibitor, and 5 mg/liter of pepstatin. The phenylmethylsulfonyl fluoride was dissolved in propranol (1 mg/ml) and slowly added to the mixture. Pepstatin was dissolved (0.5 mg/ml) in denatured ethanol (Fischer A-407), and 10 ml of this were added to each liter of the mixture. Finally, the pH was adjusted to 6.5.

The EDTA demineralization solution contained 20 mM Tris, 0.25 M NaEDTA, 100 mM 6-aminohexanoic acid, 5 mM benzamidine/HCl, 1 mM sodium iodoacetate, 1 m phenylmethylsulfonyl fluoride, 1 mg/ liter of soybean trypsin inhibitor, and 5 mg/liter of pepstatin. Phen- ylmethylsulfonyl fluoride and pepstatin were added to this mixture in

the same way as to the GdmCl solution. The pH was adjusted to 7.4, with 6 N HCI.

Dissection and Preparation of Denfin-In each preparation described below teeth from 100 frozen rat heads were used. The heads were thawed at 2°C overnight and the mandibular and maxillary incisors were removed and scraped on the outside to remove any adhering soft tissue. The dental pulps in the teeth were removed with an endodontic file, and cold GdmCl extraction solution was used in a syringe to flush the inside of the pulp chamber to remove tissue remnants. In most preparations, the soft pre-enamel, located proxi- mally on the convex side of the incisors (Fig. l) , was scraped off as completely as possible and discarded. The tooth was then cut transversally at the demarcation line between the fully mineralized enamel and the pre-enamel. In this way two portions of mineralized tissue were obtained, a proximal segment denoted Dentin and the larger, distal Enamel + Dentin portion.

In some experiments, the adhering enamel organ was carefully wiped away with a soft paper towel and the soft pre-enamel was left in place. The tooth was then cut transversally at the reddish demarcation line between the hard enamel and the soft pre-enamel. The two portions of tissue were denoted Basal (which included dentin and pre-enamel) and the distal Enamel + Dentin portion. All the tissues obtained were washed briefly with the 4 M GdmCl solution, and the Enamel + Dentin tissue was cut into smaller pieces by means of a bone cutter. The tissues were kept in ice-cold solutions during the whole dissection procedure.

First GdmCl Extraction-Prior to demineralization, extraction of the tissues was performed on a magnetic stirrer at 2°C with daily changes of 4 M GdmC1. Tissue from Enamel + Dentin, Dentin, and Basal areas were extracted with six 80-ml changes, three 40-ml changes, and four 40-ml changes of GdmCl, respectively. The succes- sive extracts of a tissue were generally combined. In one preparation, however, the six extracts from Enamel + Dentin and the three from Dentin were processed separately. The GdmCl extracts, including a small amount of insoluble material, were placed in Spectrapor 3 dialysis tubing and dialyzed against seven changes of distilled water for 7 days at 2°C. After dialysis, the precipitates were removed by centrifugation at 2000 X g for 10 min and discarded and the super- natants were lyophilized.

EDTA Demineralization a n d Extraction-After GdmCl extraction, the tissues were washed briefly with water, transferred to Spec- trapor 3 dialysis bags, and dialyzed against the EDTA demineralization solution a t 2°C. The content of calcium in the dialysate was monitored by atomic absorption spectrophotometry. The EDTA solution was changed every other day until no calcium could be detected; in general, six changes of 800 ml were necessary for complete demineralization of the Dentin and the Basal materials while 12 changes were utilized for the Enamel + Dentin portion. The material was then dialyzed for 2 more days against an additional 800-1111 portion of the EDTA solution, containing 1 M NaC1. After the dialysis bags were opened, the demineralized tissues were washed twice with a small amount of 1 M NaC1. The washes were combined with the EDTA extract from the dialysis bags. To remove EDTA and enzyme inhibitors, 25-1111 portions of the extract were desalted on a Sephadex G-25 column (5 X 75 cm), eluted with water, and lyophilized.

Ion Exchange Chromatography-The GdmCl extracts and the EDTA extracts were chromatographed on DEAE-cellulose columns (2.6 x 11 to 16 cm), using 20 mM Tris-HCI buffer, pH 8.0 at 2°C (24). Generally the columns were eluted with a linear gradient of 0 to 0.7 M NaCl over a total volume of 3000 ml. At least one chromatograph from each type of extract was run with the gradient extending to 1 M NaCl in order to insure complete removal of the protein components

x I

FIG. 1. Schematic drawing showing a longitudinal section of maxillary rat incisor. Incisors were usually divided according to the dashed line to provide the two tissue pools, Enamel + Dentin and Dentin. In other studies the pre-enamel was left in place to obtain a so-called Basal tissue, containing dentin and pre-enamel.

Noncollugenous Proteins of Rut Dentin 5933

from the column. The six GdmCl extracts from Enamel + Dentin portions and the three from Dentin portions that were treated separately were all run on DEAE-cellulose columns (1.6 X 11 cm) eluted with a linear gradient of 0 to 0.5 M NaCl over a total volume of 1000 ml.

Rechromatography of RIP was performed a t 2°C on DEAE-cellulose columns (2.6 X 15 cm or 1.6 X 15 cm) using the buffer designated above. Elution conditions were with a linear gradient from 0 to 0.7 M NaCl over a total of 2000, 3000, or 4000 ml or from 0.2 to 0.5 M NaCl over 2000 ml.

Two Gla-containing fractions were separated by chromatography on a column (1.5 X 10 cm) of QAE-Sephadex A-25 equilibrated in 20 mM Tris-HCI buffer, pH 8.0, at 2°C. Samples of 10 to 15 mg were applied and the column was eluted at 30 ml/h with a linear gradient of 0.1 M to 0.4 M NaCl over a total volume of 2000 ml.

All columns were continuously monitored at 224 nm. In some runs the organic phosphate and uronic acid contents of the fractions were determined as described below.

AG MP-50 Chromatography-The procedure for AG MP-50 column chromatography was a modification of the procedure of Munks- gaard et al. (25). Prior to use, the resin was rinsed in boiling ethanol for 15 min; it was then washed successively with 0.5 M NaOH, water until the pH of the washes was neutral, with 0.5 M HC1, and finally with water. The AG MP-50 column (1.6 X 140 cm) was equilibrated by washing with 3 bed volumes of dilute HCI, pH 1.75. The sample,

of 5 ml/h and 90 min/fraction. When the RIP peak appeared, the dissolved in 7.5 ml, was eluted with the same HCI solution a t a rate

eluting solution was changed to 0.2 M NaOH. The material in the first peak was either dialyzed and lyophilized or lyophilized without desalting. Because of the possible presence of small proteins and peptides, the second peak was lyophilized directly after noting that the pH was neutral or below.

Dephosphorylation of RIP-Highly purified RIP (ie. 32 mg of Fraction Da from AG MP-50) was dissolved in 40 ml of 0.1 M Tris- HCI buffer, pH 8.0, and placed in a shaking water bath at 37°C with 50 units of bovine intestinal alkaline phosphatase insolubilized by attachment to agarose beads (Sigma P-0762). After 24 h, the enzyme was removed by centrifugation for 10 min a t 1700 X g and the solution dialyzed and lyophilized. The remaining organic phosphate content was assayed in each preparation as outlined below.

In a second method, 56 mg of RIP was dissolved in 70 ml of 0.1 M Tris-HCI buffer, pH 8.0, and 200 p1 of a suspension of calf intestinal alkaline phosphatase (Sigma P-4502) was added. After incubation as above, the solution was dialyzed and lyophilized. No difference in dephosphorylation efficiency could be detected between the two methods. In the final step of the second procedure, the dephosphorylated material was chromatographed on a DEAE-cellulose column to remove the phosphatase.

Electrophoresis-Polyacrylamide gel electrophoresis (PAGE) was run in 15% or 7.5% gels according to the method of Maurer (26). A Tris/glycine electrode buffer, pH 8.3, was used, and the gels were stained with either Coomassie brilliant blue or Alcian blue.

(SDS-PAGE) was run in 7.5% gels essentially as described by Furth- Polyacrylamide gel electrophoresis using sodium dodecyl sulfate

mayr and Timpl (27). The buffer was 0.1 M phosphate buffer, pH 7.2, containing 0.1% SDS. The gels were fixed in 50% trichloroacetic acid and stained with Coomassie brilliant blue. After destaining, the gels were scanned with a Gilford 240 spectrophotometer at 550 nm.

Amino Acid Analysis-Amino acid analyses were performed using a Beckman 121 M analyzer as described (28). Normally the samples were hydrolyzed in sealed tubes containing 6 M constant-boiling HCI for 24 h a t 108°C in an oil bath. Phosphoprotein-containing samples were treated differently: to insure complete hydrolysis of phosphoserine, hydrolysis was performed in open, acid-cleaned test tubes in an atmosphere saturated with 6 M HCI in an evacuated desiccator. The desiccator was placed in a constant temperature oven at 108°C for 24 h. Due to the unusual composition of the phosphoproteins (with high levels of serine and aspartic acid and low amounts of other amino acids), the calculations had to be based on at least two different dilutions of the same sample. Since glutamic acid and glycine were present in intermediate amounts, these amino acids served as internal references. To account for the different destruction rates of serine and phosphoserine (Pse) during hydrolysis, the expression of Rich- ardson et al. (29) Ser,,h, = 0.764.Pse + 0.938.Ser, was used.

Gla analyses of the appropriate fractions were kindly performed by Dr. Peter V. Hauschka, Harvard Medical School. Samples were hydrolyzed in 2 M KOH, neutralized, and analyzed with a Beckman 121 M amino acid analyzer (30).

Phosphate Analysis-Samples were hydrolyzed in 0.2 M NaOH a t 37°C in a water bath for 24 h to detect organic phosphate or in constant-boiling 6 M HCI as described for amino acid analysis, to determine total phosphate.

To measure phosphate, an automated technique, based on the manual method of Hurst (31) was developed for the Technicon Autoanalyzer. The solutions used were: Solution 1, molybdate re- agent: 8.6 mM ammonium molybdate and 2 ml/liter of Wetting Agent A in 0.63 M H2SOI; Solution 2, reducing solution: 15.4 mM hydrazine sulfate and 0.9 mM SnC12 and 0.51 M H2SO4. Although these reagents were prepared fresh, it is possible to preserve the molybdate solution for several weeks at 2°C. The general outline of the method is shown in Fig. 2.

Sequence Analysis-For determination of the NHr-terminal sequence of the dephosphorylated phosphoprotein (dP-RIP), 7 mg of the protein was placed in the spinning cup of the Beckman 890C Sequencer and reacted with 2-amino-1,5-naphthalenedisulfonic acid in the presence of N-ethyl-N’-(dimethylaminopropy1)carbodiimide as described (32). Automated Edman degradation was then performed using 0.5 M Quadrol and Beckman Program No. 030176 (see Ref. 33). Pth-derivatives of amino acids were initially analyzed by thin layer chromatography using Solvents XM and modified E of Inagami and Murakami (34). They were then identified and quantitated by high performance liquid chromatography on a Waters mnJ” “- ~ .,quid chromatograph by the method of Bhoul- . -0,.

Carbohydrate Analyses-U, . was determined on the Technicon Autoanalyzer by a carbazole method as described by Ford and Baker (36) using o-glucuronic acid as a standard. Hexose was determined by the sulfuric acid-orcinol method of Winzler (37) using galactose as a standard. Glucosamine and galactosamine were quan- titatively determined by the method of Ford and Baker (36). In addition, glucosamine was qualitatively detected in protein hydrolysates during amino acid analyses (see above).

RESULTS

Extraction of Undemineralized Tissues with GdmC1--In order to determine whether proteins were extractable prior to removal of mineral from the calcified tooth structures, the tissues were fist extracted with several changes of the GdmCl solutions. We reasoned that this procedure would also remove any remaining cellular elements as well as proteins from any soft pre-enamel as shown by Termine et al. (23). The incisors were divided into anatomical sections (see Fig. 1 and “Exper- imental Procedures”) to gain insight into the tissue origins of the extracted proteins; in this way any protein components originating from enamel or pre-enamel could be recognized.

1 A 2 50 Woter c/

m

0 - ag 0:

0

Colorimeter Recorder Flowcell I 5rnm Fllter 6 6 0 n m

Sampler 40 sornples /h

Phosphate. mM

FIG. 2. Automated assay for the measurement of phosphate. Phosphate was assayed by a modification of the molybdate method of Hurst (31) adapted to the Technicon Autoanalyzer. The compositions of the molybdate solution and the reducing solution are given under “Experimental Procedures.” The Technicon tubings designated in the figure were (I) 0.081 standard, (2) 0.020 standard, (3) 0.065 acidflex, (4 ) 0.045 standard, (5) 0.056 acidflex, (6) 0.081 standard. The nominal flow rates (milliliters per min) for each of these are alongside the individual numbers. The resulting color was measured a t 660 nm in a 1.6-mm Technicon N tubular flowcell. When run a t a rate of 40 samples/h, only 160 p1 of sample was used for each analysis. As seen in the right-hand side of the figure, the color yield was linear up to at least 0.7 mM phosphate, but due to the high absorbance, the practical working range of the method is 0.01 to 0.5 mM phosphate.


The combined GdmCl extracts yielded several UV-absorbing fractions when chromatographed on DEAE-cellulose (Fig. 3) and a proteoglycan peak (VII) that eluted at about 0.5 M NaCI. The small amount of phosphate occurring just prior to 0.1 M NaCl in the gradient was inorganic phosphate, since it was detectable without alkaline hydrolysis. No organic phosphate-containing protein peaks were observed.

Some differences were apparent when the six individual extracts of Enamel + Dentin and the three from Dentin were processed separately and then chromatographed on DEAE- cellulose. In the first Enamel + Dentin extract, two asymmetric peaks (V and VI, eluting between 0.12 and 0.2 M NaCl) were observed, but they were absent as early as the thud GdmCl extraction. Since this material was essentially absent from all Dentin extracts, it probably originated from enamel.

In several experiments, the soft pre-enamel was left intact on the proximal dentin and referred to as the Basal portion. When the pooled Basal GdmCl extracts were chromatographed on DEAE-cellulose, a more complicated pattern was obtained. Although the same peaks were observed as in Fig. 3, an additional large asymmetric peak appeared between Peaks I1 and 111. Another major difference was that two major UV-absorbing peaks were present in the elution positions of V and VI; these fractions had an absorbance similar to that of Peak IV. The protein components present in Fractions IV, V, and VI revealed amino acid compositions typical of enamel proteins with high amounts of proline (23). Since these constituents probably originated in pre-enamel, the contamina- tion of Dentin, preparations (as in Fig. 3) with material from pre-enamel appears to be negligible.

Fractions I through IV of Fig. 3 contained negligible protein. Extensive evaluation of the material of Fraction 111 revealed that it contained no organic phosphate, hexoses, or uronic acid, and it had no appreciable 260 nm absorbance. It thus appears that the only major protein-containing fraction attrib- utable to dentin and extractable with GdmCl prior to demineralization is proteoglycan (Peak VII, Fig. 3).

Dentin Proteins Solubilized during EDTA Demineraliza- tion-In order to study the proteins within the mineralized matrix, the tissues were demineralized completely with EDTA solutions containing protease inhibitors. Since some proteins might arise from fully mineralized enamel, the extracts taken from different anatomical locations of the rat incisor (Fig. 1) were processed separately. Identical results were obtained when Enamel + Dentin or Dentin extracts were analyzed by DEAE-cellulose chromatography or by PAGE, indicating that the putative enamel proteins were either absent or present at

0 500

E L U T I O N VOLUME ( m 0

FIG. 3. DEAE-cellulose chromatography of Dentin GdmCl extract. The GdmCl extract (82 mg) was applied to a DEAE-cellulose column (2.6 X 13 cm) equilibrated with 20 mM Tris-HCI buffer, pH 8.0 at 2°C. The column was eluted with a linear NaCl gradient from 0 to 0.7 M over a total volume of 2000 ml in the above buffer at a rate of 40 ml/h. Phosphate and uronic acid determinations were performed for all fractions, but only positive findings are denoted. This chromatogram is representative of GdmCl extracts of Dentin and of Enamel + Dentin. The elution positions of Fractions V and VI, seen in early extracts of Dentin + Enamel and Basal tissues, are denoted.

FIG. 4. Polyacrylamide gel electrophoresis (PAGE) of DEAE-cellulose fractions from EDTA extracts of dentin. PAGE was run at 1 mA/tube with a Tris/glycine electrode buffer, pH 8.3 (25°C). and stained with Coomassie brilliant blue or Alcian blue. Gels a to e are 15% gels; Gel f is 7.5%. The gels are from left to right: (a ) total EDTA extract of Enamel + Dentin stained with Coomassie brilliant blue; ( b ) total EDTA extract of Enamel + Dentin stained with Alcian blue; (c ) Fraction B (Fig. 6) stained with Coomassie brilliant blue; (d) Fraction C stained with Coomassie brilliant blue; ( e ) Fraction D stained with Alcian blue; ( f ) Fraction E stained with Coomassie brilliant blue.

undetectable levels. Furthermore, no differences were observed between DEAE-cellulose chromatograms of extracts from Basal tissues and those from Dentin. Presumably the proteins in the soft pre-enamel were removed totally by initial GdmCl extraction. In view of these considerations, we con- clude that all the EDTA-extractable protein components were derived from mineralized dentin.

When run on 15% PAGE, the EDTA extracts revealed several components stainable with Coomassie brilliant blue (Fig. 4a). One of these proteins moved with the tracking dye. When stained with Alcian blue, one intensely staining band was seen in the central region of the gel and one faint band at the front (Fig. 4b) .

Several Coomassie brilliant blue-staining components were observed with the crude EDTA extracts of Enamel + Dentin or Dentin run on 7.5% SDS-PAGE (Fig. 5). Compared to standard globular proteins, all visible protein components except one (apparent molecular weight 22,000) displayed apparent molecular weights above 50,000. The low molecular weight Gla-containing proteins (see below) migrated with the solvent front in these gels.

Five major UV-absorbing fractions (A through E ) were obtained when the EDTA extracts were chromatographed on DEAE-cellulose (Fig. 6). Even when the gradient was extended to 1 M NaCl, nothing was eluted later than Fraction E. These five fractions were separated as in Fig. 6 or better in all chromatograms. The complexity of Fractions B, C, and D indicated that each contained more than one component. Only Fraction E contained uronic acid and Fraction D exhibited a large content of organic phosphate.

Fraction A (Fig. 6) contained little protein after dialysis and gave no Coomassie brilliant blue detectable bands on 15% PAGE. In some runs it yielded small amounts of amino acids after acid hydrolysis. The low levels of material consistently found in Fraction A suggest that it does not represent a major macromolecular component; therefore it was not further studied.

Electrophoresis of Fraction B material revealed a major component with a slightly higher mobility than a second,

Noncollagenous Proteins of Rat Dentin 5935

minor component (Fig. 4c). The amino acid composition of this fraction was characterized by relatively high levels of aspartic acid, glutamic acid, and glucosamine (Table I). There was some variation of this composition between different preparations; therefore, the data of Table I are given as mean values f S.D. (n = 4). These data show that Fraction B primarily contains two components which are acidic glycoproteins.

The size of Fraction B in Fig. 6 should not be viewed as a reflection of the relative proportions of the two glycoproteins, because the predominant UV-absorbing moiety of this fraction was EDTA. For unknown reasons, it is difficult to remove all of the EDTA from the mixture of proteins in the extract by dialysis or by desalting with gel columns. In separate experi-

0 20 40 60 Migration Distance (mm)

FIG. 5. SDS-PAGE of EDTA extract from Enamel + Dentin. Crude EDTA extract (unreduced) was applied to 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gels. Electrophoresis was run at 6 mA/ gel in 0.1 M phosphate buffer, pH 7.2, containing 0.1% SDS as the electrode buffer. The gels were scanned in a Gilford 240 spectrophotometer at 550 nm. The upper graphs shows a scan of the Pharmacia “low molecular weight” standards (from left to right): phosphorylase b (94,000), bovine serum albumin (67,000). ovalbumin (43,000), car- bonic anhydrase (30,000). soybean trypsin inhibitor (20,000), and a- lactalbumin (14,400). The scan of the EDTA extract, as seen in the lower graph, shows that all components except one displayed molecular weights above 50,000. One component migrated with an apparent molecular weight of 22,000. Since we have observed that the y- carboxyglutamic acid (Gla)-containing proteins from rat incisor dentin show an apparent molecular weight of 10,OOO to 12,000 on 158 SDS- PAGE, this protein occurred in the small band running with the front.

ments, utilizing the usual DEAE-cellulose chromatographic conditions, EDTA eluted in the position of Fraction B.

The asymmetry of Fraction C suggested that it contained several components, a fact borne out by PAGE (Fig. 4d). The amino acid composition of this fraction was similar to that of Fraction B, with high levels of aspartic acid, glutamic acid, and glucosamine. One notable difference was the presence of hydroxyproline, accounting for about 1% of the amino acids (Table I). Using gel electrophoresis, two major bands and several minor ones were observed (Fig. 4d). One of the prominent bands moved near the tracking dye and the other migrated only about one-third of the distance of the gel and in close proximity to the two bands from Fraction B.

The major band which migrated with the tracking dye has been identified as a Gla-containing protein or proteins, similar to those found in chicken and bovine bone (8, 10). It was separated from the higher molecular weight components by submitting either Fraction C or the original EDTA extract to gel chromatography on Sephadex G-50 (results not shown). The Gla-containing protein(s) were well included in the gel, while other constituents were eluted in the void volume. Compared to the positions of globular standards chromatographed on the same column in separate experiments, the Gla-containing protein(s) eluted at molecular weights of 10,000; 15% SDS-PAGE gave an apparent size of 10,000 to 12,000.

The low molecular weight, Gla-containing material from Sephadex G-50 was next submitted to ion exchange chromatography. When chromatographed on QAE-Sephadex A-25 this fraction separated into two symmetric UV-absorbing peaks of similar size (data not shown). There was no apparent difference in the amino acid compositions of the two fractions. Each composition was similar to that of the bovine Gla- containing protein from bone (i.e. the composition deduced from the amino acid sequence (38)) and included about 3 residues of Gla/51 residues. The NH2 terminus of the later eluting fraction from QAE-Sephadex chromatography, determined by automated Edman degradation, was Tyr-Leu-Asn- X-Gly-Leu-Gly-Ala-Hyp-Ala-Pro-Tyr-Pro-Asp-Pro-Leu. This sequence is almost identical to that reported by Price et al. (38) for the bovine bone protein,

The important distinction about Fraction D was its high content of organic phosphate (Fig. 6). This observation along with its unusual amino acid composition (Table I) indicated the presence of the highly phosphorylated rat incisor phosphoprotein (RIP) in Fraction D (2-4). On 15% PAGE, Fraction D yielded a weakly staining band migrating with the front, and a prominent band that migrated almost half of the gel length (Fig. 4e). These bands corresponded to the Alcian blue- staining bands in the crude EDTA extract (Fig. 46).

For some reason, this phosphoprotein-containing peak FIG. 6. DEAE-cellulose chroma-

” ’ tography of the Enamel + Dentin extract. The crude EDTA extract (98 mg) was applied to a DEAE-cellulose column (2.6 X 15 cm) equilibrated with 20 mM Tris-HC1 buffer, pH 8.0. The col- - umn was eluted at 2°C at a rate of 50 - ml/h with a linear gradient of 0 to 0.7 M - Z -O NaCl over a total volume of 3000 ml.

. - 6 Only the positive findings for organic o a phosphate are denoted in the figure. It

should be noted that the material ap-

usual amount; this procedure was inten- tional so that Peak E could be readily

smaller sample sized yielded better separation of Fractions B, C , and D.

’

” 1 .03

- - v c

-01

-0 visualized. The routine application of LO

n plied to the column was about twice the


emerged from DEAE-cellulose at a lower NaCl concentration than noted in our previous studies (3, 4). Whether this was a difference brought about by using protease inhibitors in the preparations is unknown. The results could not be attributed to differences in the capacities of the DEAE-cellulose ion exchangers, since the elution positions for Peak D did not vary when different preparations and even different commer- cial sources of the resin were used.

Fraction E constituted only a minor amount of the total

TABLE I Composition of fractions from DEAE-cellulose chromatography of

EDTA extracts of Enamel + Dentin Values are residues per thousand and are mean values f S.D. for

four analyses, except for Fraction E. Fraction"

Amino Acid B C D E

Hydroxyproline l o + 1 Aspartic acid 120f 9 161 f 12 363 f 14 152 Threonine 5 8 f 2 4 7 f 2 1 7 f 2 55 Serine 71 f 8 7 3 f 12 460k 10 108 Glutamic acid 132f 13 149% 14 5 6 f 6 137 Proline 8 5 f 6 7 9 f 5 9 f 1 56 Glycine 8 2 f 9 101 f 7 3 2 f 3 99 Alanine 9 5 f 1 2 7 0 f 4 1 3 f 2 64 Cysteine 2 4 f 3 2 3 % 4 l f l I1 Valine 6 3 f 3 3 8 f 5 5 f 1 41 Methionine 5 f 2 5 f l 1 f 1 11 Isoleucine 3 0 f 4 2 9 f 2 4 f l 31 Leucine 7 4 f 9 6 7 f 2 6 f 1 95 Tyrosine 1 8 % 2 3 2 f 3 4 f l 35 Phenylalanine 4 2 f 5 1 9 f 2 3 f 1 25 Lysine 3 5 f 3 30f3 1 1 f 3 36 Histidine 2 3 f 3 3 4 f 3 8 f 2 19 Arginine 44 f 10 3 5 f 1 8 f 2 27

Glucosamine Presenth Presentb Presenth 21' Galactosamine nd" nd" nd" 203'

I' See the text and Fig. 6 for explanation of these symbols. Detected on the amino acid analyzer after hydrolysis in 6 N HC1

at 108°C for 24 h. ' Determined by the method for Ford and Baker (36). " Not determined.

EDTA extract as judged by the 224 nm absorbance (Fig. 6 ) . The material in this peak was excluded from 7.5% PAGE (Fig. 4f ), and contained large amounts of uronic acid. In the amino acid analysis (Table I) both glucosamine and galactosamine were seen. Thus, Fraction E contained proteoglycan, mainly consisting of galactosaminoglycans. The ratio between galactosamine and glucosamine, as observed with the amino acid analyzer, was about 101. However, one must remember that a substantial and differential degradation of aminosugars takes place during standard hydrolysis conditions for proteins. Nevertheless, compared to cartilage proteoglycans, the ratio of hexosamines to serine appeared to be relatively low.

Rechromatography of Phosphoprotein-containing Peak- Fraction D from Fig. 6 was biphasic, an observation consistent with the results of Dimuzio and Veis (39). Although this partial separation into two fractions, with the later eluting portion present in lesser quantities, was a consistent finding, the relative proportions of the two varied considerably. It should be noted that the organic phosphate content did not follow the contours of the 224 nm absorbance curve.

In order to gain insight into the possible presence of two different phosphoproteins as postulated by Dimuzio and Veis (39), we divided the contents of Fraction D from two preparations into two separate parts, a and p, representing the early and later eluting portions, respectively. The compositions of a and p fractions (Table 11) were quite similar. The principal differences were that a consistently gave slightly elevated total serine and lowered glutamic acid values, relative to the p peak. Consistent with the impression obtained from the phosphate analysis of the fractions in Fig. 6, the a fraction displayed a higher degree of serine phosphorylation (88%) than p (64%).

In an attempt to resolve the two putative phosphoproteins present in the total RIP fraction of Fig. 6, Fraction D was rechromatographed on DEAE-cellulose using a slightly lower rate of elution and a shallower gradient (Fig. 7). Instead of a better resolution into two distinct phosphate-containing fractions, the procedure separated the material into a complex fraction ( y ) devoid of organic phosphate and a single phosphoprotein fraction that eluted at almost 0.3 M NaCl. The

TABLE I1 Amino acid analysis of different RIP fractions

The values are given in residues per thousand. They are mean values of two determinations except for the y fraction, which is the mean value f S.D. for four determinations.

Amino acid Fraction"

(1 B Y ~

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

363 16

486 41 7

29 12 1 5 1 4 5 3 3

12 8 5

353 17

459 66 10 32 12 tr"

4 1 4 4 4 3

12 9

11

149 f 20 65 f 5

115 f 24 128 f 11 53 f 7

112 f 23 78 & 19 8 * 5

55 f 13 1 f 2

34 f 6 54 f 13 23 f 7 30 f 12 51 f 13 17 f 2 31 f 10

Glucosamine' Present Present Present

6 c Da Db D C

373 373 395 30 1 325 9 11 9 21 25

545 514 531 416 376 23 36 21 101 103 tr" tr" tr" 15 18 19 23 21 46 50 6 I1 6 22 25

trh 2 1 5 9 1 1 3 1 2 2 4 5 1 2 10 10 2 3 1 8 8 1 1 8 5

10 11 9 15 16 6 8 7 9 10 2 3 16 16

Present Present

'' See the text and Figs. 7. 8, and 11 for explanation of these designations. - Trace. ' Detected on the amino acid analyzer.


phosphate content closely followed the absorbance curve and the ratio of phosphate to was greater than for the biphasic RIP fraction of Fig. 6. Utilization of several different gradient elution schemes, including that for the original chromatogram (Fig. 6), gave essentially the same results and no resolution of the phosphoprotein peak into two distinct components.

The amino acid compositions of the y fraction (Table 11) which showed some variation for different preparations, re- sembled that for the acidic glycoproteins in Fractions R and C of Fig. 6. Relatively high levels of aspartic acid, serine, glutamic acid, and glycine were present and glucosamine was a conspicuous component. One of the most notable features of the composition was the presence of relatively large amounts of the hydrophobic and basic amino acids.

The RIP fraction of Fig. 7 was also divided into two portions, 6 and E prior to analysis in order to determine whether any partial separation into separate molecular species had occurred. The compositions of the 6 and E fractions (Table 11) proved to be almost identical, but small differences in the serine (plus phosphoserine). glutamic acid, and alanine values were consistent findings. In addition, the organic phosphate content of the E fraction was slightly lower than in 6.

Compared to the crude RIP (Le. Fraction D, Table I), the S and E fractions were highly enriched in phosphoserine, glutamic acid had decreased (note that the ratio of glutamic acid to glycine was closer to unity), and the levels of most hydrophobic amino acids were diminished sharply. Unlike the crude RIP, no glucosamine could be detected with the amino acid analyzer in the S and E fractions. These differences thus reflect the removal of the glycoprotein material recovered in the y fraction. Furthermore, the amino acid compositions showed a trend toward the simpler composition obtained by Munksgaard et al. (25); the data clearly show that noncova- lently bound glycoproteins were associated with the phosphoprotein peak of Fig. 6. Since the removal of the glucosamine- containing y fraction from the phosphoprotein was attained at pH 8 and 2”C, it is unlikely that the two were bound by a covalent linkage.

In order to reproduce and extend the observations of Munksgaard et al. (25), crude RIP material from Fig. 6 was passed through a sulfonated polystyrene (AG MP-50) column at 2°C (Fig. 8). After the initial phosphoprotein fraction designated Da, eluted in the initial dilute HCl solution, the column was washed with 0.2 M NaOH to remove a second peak, called Db. Since the solution containing Db was either slightly acidic or neutral in the chromatograms, no alkaline elimination of organic phosphate could have occurred. Peak

R I 1 4

6 ELUTION VOLUME Iml) 360 600 900

FIG. 7. Rechromatography of RIP on DEAE-cellulose. Thirty-five milligrams of crude HIP, i.e. Fraction D in Fig. 6, were applied to a DEAE-cellulose column (2.6 X 16 cm). The column was equilibrated with a 20 mM Tris-HCI buffer, pH 8.0 at 2°C. The material was eluted with a linear gradient of 0.2 to 0.5 M NaCl over a total of 2000 ml in the same buffer at a rate of 40 ml/h. Organic phosphate was measured in each fraction but only the positive results are denoted. The major peak was arbitrarily divided into two parts. f i and E .

Fraction Number FIG. 8. Rechromatography of RIP on AG MP-50. Material (29

mg) from the crude HIP fraction (Fraction 11, Fig. 6) was dissolved in 7.5 ml of dilute HCI, pH 1.75, and chromatographed as described under “Experimental Procedures.”

e

FIG. 9. Fifteen per cent PAGE of different phosphoprotein preparations. See the legend to Fig. 4 for details. The gels were stained with either Alcian blue (Gels a to d ) or Coomassie brilliant blue (Gel e ) . The gels are from left to right: (a) the total EDTA extract; ( b ) the crude HIP; i.e. Fraction D, Fig. 6; ( c ) Fraction Da; ( d ) Fraction Db; ( e ) Fraction Db stained with Coomassie brilliant blue.

Da contained 92% of the organic phosphate and Db only 8%. The amino acid composition of Fraction Da (Table 11) was

similar to those of Components 6 and E , but contained no valine, methionine, leucine, phenylalanine, or arginine, and the glutamic acid and glycine values were the same. The results were almost identical with the compositions reported by Munksgaard et al. (25); however we have noted that low amounts of isoleucine and tyrosine are consistently present in the preparation. In addition, the degree of phosphorylation in Fraction Da was higher than reported previously; 85 to 9O”r (mean value 87%) of the seryl residues were phosphorylated.

Fraction Db contained organic phosphate and was rich in serine, aspartic acid, and glutamic acid (Table 11) but, in contrast to Fraction Da, it also contained glucosamine and significant amounts of all the hydrophobic amino acids, as well as arginine and traces of cysteine.

When the total EDTA extract of rat incisor dentin was run on PAGE and stained with Alcian blue, one intense band was observed migrating about one-third of the gel length (Fig. 9a). Immediately below this was a weakly staining band not well separated from the major one, and at the buffer front was a


third, faint band. The crude RIP fraction from DEAE-cellulose in Fig. 6 gave essentially the same results (Fig. 9b), although the band at the front seemed to be comparatively increased in intensity.

Electrophoresis of the purified RIP fraction from the AG MP-50 column (Fraction Da, Fig. 8) revealed one band at the front and one intensely staining band migrating approximately one-third of the gel length with a weak band below it (Fig. 9c). When the gels of fraction Da were stained with Coomassie briUiant blue, no bands were visible.

Alcian blue-staining of Fraction Db revealed that the major acidic components were at the buffer front (Fig. 9d); there was also a prominent band near the first third of the gel that displayed a slightly higher mobility than the major band in either the total EDTA or the crude RIP and Da fractions. Its

0 3 - 0 2 =

01 ; 3 - - 0

0 0 500

Elutlon Volume (mll FIG. 10. Rechromatography of Fraction Da on DEAE-cellu-

lose. Fraction Da (Fig. 8,11 mg) was rechromatographed on a DEAE- cellulose column (1.6 X 15 cm) equilibrated with a 20 mM Tris-HCI buffer, pH 8.0 at 2°C. The column was eluted at a rate of 40 ml/h with a linear gradient of 0 to 0.7 M NaCl over a total volume of 2000 ml in the above buffer.

T I 1 Dc t o 3 I c

g 10-

-01 : 2 -01 z”

“051 d N N I - 0 2 =

- E

$ 0 5 -

e 5:

E - 0 3 v -

0

2 0 - ; n - 0 - 0

a 1

0 500 1000 Elution Volume (ml)

FIG. 11. Rechromatography of Fraction Db on DEAE-cellulose. Fraction Db (Fig. 8, 29 mg) was chromatographed on DEAE- cellulose as in Fig. 10. Since Fraction Db was lyophilized without prior desalting, it still contained salt and was thus dissolved in 10 ml of buffer before application to the column. Organic phosphate was found in the major peak (Dc), but no phosphate was detected else- where in the chromatogram.

2 0

E d c

N N 10

0

mobility more nearly corresponded to that of the weaker staining band in these preparations. Several weakly staining bands were also visible. One of these components corresponded in mobility to the major band in the RIP preparations (see above). If polyacrylamide gels from Fraction Db were stained with Coomassie brilliant blue (Fig, 9e), several bands in the lower part of the gel were observed. The band migrating to the front was also stained with Coomassie brilliant blue, while the major band in the upper part of the gel was not stained.

For further characterization, the materials from Peaks Da and Db were each rechromatographed on DEAE-cellulose. As shown in Fig. 10, the phosphoprotein from Fraction Da eluted as a single component a t the expected NaCl concentration. The AG MP-50 chromatographic step had thus eliminated the tendency for this material to partially subdivide into two peaks (as shown in Fig. 6), concomitant with the removal of protein material into Fraction Db.

When Fraction Db was rechromatographed, this material was separated into several components (Fig. 11) with a major peak (Dc) eluting at a slightly lower NaCl concentration than that for RIP. Fraction Dc was associated with an organic phosphate peak, but the relative amount of phosphate was lower than usually observed for purified RIP (see Fig. 7). The amino acid composition of Fraction Dc was similar to that of Db (Table 11). Only 13% of the seryl residues of Dc were phosphorylated.

It is possible that the phosphate-containing component of Fraction Dc represents a minor phosphoprotein component and corresponds to the major Alcian blue-staining protein band observed when Fraction Db was subjected to PAGE (Fig. 9d) . This component cochromatographs with the major RIP on DEAE-cellulose (Figs. 6 and 7) but is retained by the AG MP-50 resin, effecting its separation from the phosphoprotein of Da (Fig. 8).

Dephosphorylation of RIP-Purified RIP (i.e. Fraction Da, Fig. 8), was incubated with two different alkaline phosphatase preparations and with each, 99 to 100% dephosphorylation was achieved. When the incubation mixture was chromatographed on DEAE-cellulose (Fig. 12), it separated into two minor fractions (X and p) and a major biphasic one (dP-RIP). The amino acid analysis of Fraction X (Table 111) was unlike that of RIP and showed that this material was probably derived from the alkaline phosphatase preparation. On the other hand, Fraction p had a composition expected for a dephosphorylated dentin phosphoprotein; in contrast to Da and dP-RIP, it contained significant amounts of cysteine, valine, leucine, phenylalanine, and arginine, and the serine content was relatively low. Similar to Fraction Dc (see above), p may represent a minor, distinct dentin phosphoprotein.

-0 3

- FIG. 12. DEAE-cellulose chroma- : tography of dephosphorylated RIP. : Purified KIP, Fraction Da (Fig. 8), was

-02 - dephosphorylated with intestinal alka-

- perimental Procedures.” After dialysis and lyophilization, 42 mg of this material

-01 Z were applied to a DEAE-cellulose column (2.6 X 14 cm) equilibrated and eluted by the procedure described in Fig. 10.

v 3 line phosphatase as described under “Ex-

-0

Elution Volume (ml)


The major fraction, dP-RIP, eluted at approximately the Same place in the gradient as crude RIP and Fraction Da (Figs. 6 and 10). The partial fractionation of Dp-RIP into two peaks suggested that at least two components were present. When subjected to 7.5% SDS-PAGE, dP-RIP gave a single, sharp band with no indications that two components were present (data not shown). The amino acid composition of dP- RIP (Table 111) was almost identical with that of Fraction Da.

When dP-RIP was run on 7.5% SDS-PAGE, along with a set of standard globular proteins, an apparent molecular weight of 72,000 (71,000 to 73,000 for two separate experiments) was determined. It was observed that the dP-RIP band disappeared during the destaining procedure; in order to preserve this protein band, the gels were incompletely de- stained prior to scanning them. To calculate the molecular weight of RIP from this value, the contribution of phosphate groups must be added; this calculation showed the size of RIP to be approximately 100,000. However one should realize that the binding of SDS to a substance such as dP-RIP, which contains large quantities of negatively charged side chains, may be far from ideal. Thus its mobility might be relatively low and the molecular weight estimates would be high. An example of such an anomalous behavior is that observed for collagen peptides, which exhibit lower mobilities on SDS- PAGE than globular proteins of comparable molecular weights (27).

Sequence data for the first six cycles of Edman degradation of fraction dP-RIP are given in Table IV. The preparation yielded only Pth-derivatives of aspartic acid at each of the first two cycles. The recovery of this derivative was that expected for 7 mg of a polypeptide of 55,000 to 68,000 daltons (assuming that the absolute yield of the Pth-derivative of aspartic acid was 40 to 50%). These data show that the material of dP-RIP had a single NHa-terminal Asp-Asp sequence. On the other hand, derivatives of both aspartic acid and proline were observed at Cycle 3 and, at Cycle 4, Pth- derivatives of aspartic acid and asparagine (but no Pth-derivative of proline) were noted. After Cycle 4, increasing levels of Pth-derivatives of aspartic acid and of serine were found, as itlustrated in Table IV for Cycles 5 and 6. It is likely that, after several treatments with the reagents used for Edman degradation, some breakdown of the highly acidic polypeptide

TABLE I11 Amino acid analyses of fractions obtained from DEAE-cellulose

chromatography of enzymatically dephosphorylated RIP Values are given in residues per thousand and are the mean of two

determinations. Fraction"

Amino acid h !J dP-RIP

Aspartic acid 132 292 402 Threonine 75 28 9 Serine 58 347 516 Glutamic acid 108 85 22 Proline 66 22 2 Glycine 96 55 21 Alanine 119 33 8 Cysteine 7 8 Valine 81 19 Methionine 12 Isoleucine 30 12 2 Leucine 72 29 Tyrosine 33 8 2 Phenylalanine 31 13 Lysine 38 33 9 Histidine 17 a 7 Arginine 24 7 Glucosamine Present

I' See Fig. 12 and the text for explanations of these designations.

TABLE IV Summary of identification and recovery of Pth-derivatives of amino acids from automated Edman degradation of dP-RIP

The positive identification of a Pth-derivative of an amino acid is denoted by the name of the parent amino acid under the identication method. The level of background Pth-derivatives of amino acids was less than 3 nmol.

Identification method

Cycle Thin layer" High Per- Conclusion chromatog- l ~ u ~ ~ ~ - Recovery"

raphy matography

4 Asn

nmof

ASP 39.3

ASP 39.6

ASP 42.8 Pro 31.8

Asn 20.3 ASP 30.9

ASP 53.4 Asn 13.6 Ser 18.9

ASP 61.8 Ser 16.0

" Separation was in Solvent XM of Inagami and Murakami (34). Identities of the Pth-derivatives of amino acids were aided by spraying the thin layer chromatograms with ninhydrin. Confirmation of Pth- derivative of aspartic acid was in the modified E solvent (34).

Recoveries were calculated from the peak heights of the Pth- derivatives of amino acids on the high pressure liquid chromatography runs.

chains occurred, exposing new NH, termini; therefore, the information obtained after Cycle 4 were impossible to inter- pret. A reasonable conclusion for the above data is that the components of Fraction dP-RIP have at least two NH2-ter- mind sequences: Asp-Asp-Asp-Asn- and Asp-Asp-Pro-Asn.

DISCUSSION

The long range objective of our research is to understand the molecular mechanisms involved in the formation of mineralized tissues. The choice of dentin as a model for studying this process is based upon the facts that, in dentinogenesis, only one cell type, the odontoblast, is involved, and that there appear to be no biodegradative or remodeling processes taking place. Thus, although some of the mechanisms preceding dentin formation may be similar to those in bone and cementum, the system is relatively simpler. The specific model chosen is the rat incisor which, unlike most teeth, is contin- ually erupting and forming dentin.

Osteogenesis and dentinogenesis probably involve the formation and translocation of phosphoproteins and glycoproteins (40-42). The evidence to support this possibility is mainly from autoradiographic studies on bone and dentin. For example, Weinstock and Leblond (40) demonstrated that [33P]phosphate-labeled material (presumably the phosphoprotein) reached the predentin-dentin junction within 90 min after in vivo injection of the radiolabel. The short time suggested that the phosphoprotein was deposited selectively at the mineralization site and was involved in the transformation of predentin to dentin. Despite the exciting implications of these experiments, delineation of the biochemical events involved in the formation of bone, dentin, and cementum is impossible, because the exact nature of all the macromolecules within these calcified structures is unknown. The experiments


described in this communication represent some of our efforts to completely characterize the NCPs of dentin.

In our endeavors to study the proteins of mineralized tissues, we have striven to eliminate techniques which might produce artifacts or result in the loss of protein constituents. All extraction solutions contained a series of inhibitors to counteract the action of proteases. Another precaution that we have utilized is to carefully break calcified structures into small pieces rather than subject them to mechanical grinding or powdering prior to decalcification. Still another precaution, the use of dialysis tubing with molecular weight cutoff limits of 3500 eliminated the loss of low molecular weight components.

Initial GdmCl extractions were used to determine whether proteins could be removed prior to demineralization from such tissues as predentin, enamel, or pre-enamel (23) or from odontoblastic processes or other soft connective tissues still adhering to the teeth. The only major proteinaceous material extractable from dentin with GdmCl prior to demineralization was proteoglycan. I t should be noted that a second portion of proteoglycan was present in the EDTA solutions after decalcification. Further studies on the nature and origin of these two proteoglycan fractions will be particularly interesting in view of earlier observations suggesting that the quality and quantity of glycosaminoglycans in predentin are different from that in dentin (43).

During demineralization of rat incisor tissues with EDTA solutions, several macromolecular constituents derived from dentin were solubilized. The proteinaceous material recovered in this extract falls into four categories: acidic glycoproteins, Gla-containing proteins, phosphoproteins, and proteoglycans. One striking observation was that only a few major protein components could be obtained from rat dentin and that most of these had apparent molecular weights above 50,000. The two exceptions were a small amount of material of 22,000 molecular weight (Fig. 5), and the Gla-containing proteins. These results contrast sharply with those of Leaver and his associates (13, 16-18) who found a large number of glycoproteins with lower molecular weights. I t is possible that some proteolysis had taken place in their preparations, since protease inhibitors were not used. A strict comparison cannot be made between our observations and the earlier studies of the Leaver group because while we have studied rat incisor proteins, they used human dentin for most of their investigations. Nevertheless, in a more recent investigation Smith and Leaver (44) have extracted the NCPs from rabbit incisors with techniques similar to those used with human dentin and again a complex mixture of glycoproteins was obtained.

Another interesting finding was that rat dentin contained a substantial quantity of Gla-containing proteins similar to those found in chicken and bovine bone (8-10). These low molecular weight dentin components separated into two chromatographic fractions when subjected to ion exchange chromatography but the amino acid compositions of the two were indistinguishable. Thus, there appear to be at least two Gla- containing dentin proteins with unknown differences. A similar observation was made by Hauschka and Gallop (45) for the Gla-containing component(s) from chicken bone. The function of Gla-containing proteins in mineralized tissues is unknown; the affinity for calcium ions appears comparatively low while that for hydroxyapatite is high (46). They may play some role in controlling the rate and size of apatite crystals within calcifying tissues.

None of the major proteins uncovered in our studies seemed to have properties identifying them as the NH?-terminal or COOH-terminal propeptides removed from type I procollagen. The NH2-terminal component excised from 0'111) chains con-

tains hydroxyproline and has a molecular weight of above 15,000 (47). The only hydroxyproline recovered in the soluble dentin components was present in the Gla-containing proteins. The COOH-terminal propeptide from the three procollagen chains has a molecular weight of about 105,000 when the interchain disulfide bonds are intact (48). Reduction and alkylation does not affect the appearance of the soluble dentin protein components on SDS-PAGE in a way that would demonstrate such a component. The absence of even small amounts of these collagen propeptides in a tissue with such levels of collagen suggests that a biodegradative mechanism for their removal exists after their function in collagen formation has been fulfilled.

The precautions we have introduced do not seem to affect the overall amino acid composition of RIP, compared to that previously reported. The composition of Fraction Da was similar to that reported by Munksgaard et al. (25) for phosphoprotein preparations obtained without the same degree of care. On the other hand, the level of phosphorylation of seryl residues in the purified RIP (Fraction Da) was 87%) while that found in the earlier studies (25) was only 81%. It is possible that this difference is due to the inhibition of alkaline phosphatase activity present in dentin tissues (49) by the inclusion of enzyme inhibitors.

Using the amino acid composition of the dephosphorylated phosphoprotein (dP-RIP) and the value of 85% for the phosphorylated serines, one can calculate that 26.8% of the protein is phosphate. This value is the highest yet reported and undoubtedly reflects a combination of our efforts to prevent artifactual degradation and to obtain a protein preparation which is highly purified and relatively free from nonphospho- rylated contaminants.

In the present investigation we have noted that certain changes in the amino acid composition characterize the increasing purity of RIP preparations (see Table 11). Aspartic acid and total serine values are slightly increased, the quantity of glutamic acid approaches that of glycine and a number of minor amino acids, i e . cysteine, valine, methionine, leucine, phenylalanine, and arginine, are eliminated entirely. Further- more, glucosamine is reduced to levels undetectable with the amino acid analyzer. Similar conclusions were drawn earlier by Munksgaard et al. (25).

Several different molecular weights for dentin phosphoprotein have been reported in the literature. The apparent molecular weight obtained in our investigations for the dephosphorylated phosphoprotein, dP-RIP, utilizing SDS-PAGE was about 72,000. If the weight of organic phosphate is added, the molecular weight for RIP would be about 100,000. Ter- mine et al. (23) have used preparative techniques similar to our own and obtained a molecular weight of 96,000 for the phosphoprotein from fetal bovine teet.h, when extracts were analyzed with SDS-PAGE in urea. Except for the M , = 65,000 and 71,000 values reported by Dimuzio and Veis (39) for their two RIP peaks, the other molecular weight estimates have been in the 30,000 to 37,000 range (2, 4, 50, 51).

Dimuzio and Veis (39) analyzed RIP extracted during demineralization with EDTA solutions. They observed that, upon DEAE-cellulose chromatography of the dentin extract, the phosphoprotein was found in a biphasic peak and postulated the existence of two distinct phosphoproteins called a- and P-phosphophoryns. These two fractions were further chromatographed in 3 M GdmCl on Sepharose CL-GB yielding elution profdes with several incompletely resolved components. Although no phosphate analyses were performed on the eluates, the overall compositions of the two fractions were clearly that of a dentin phosphoprotein. However, the a- and P-phosphophoryn fractions cont,ained significant quantities of

Noncollugenous Proteins of Rut Dentin 594 1

cysteine, valine, methionine, leucine, phenylalanine, and arginine and were thus more impure than Fraction Da and the proteins prepared by Munksgaard et al. (25). In contrast to the findings of Jontell et al. (4), the a- and P-phosphophoryn fractions contained high levels of hexose (6.9 and 18.0% by weight, respectively) and relatively low levels of phosphorus. Glucosamine was present in the a component while the /3 peak contained both glucosamine and galactosamine. It seems clear that the material in these two peaks was impure, consisting of phosphoprotein contaminated with glycoproteins and possibly proteoglycans.

We have also observed that the RIP occurs in a biphasic peak with the earlier material relatively enriched in organic phosphate (Fig. 6). Rechromatography, either on DEAE-cellulose (Fig. 7) or an AG MP-50 column (Fig. 8), separated the biphasic peak into a complex, glycoprotein-containing fraction and a symmetric phosphoprotein peak. These observations suggest that the chromatographic heterogeneity on DEAE- cellulose observed by Dimuzio and Veis (39) was due to the presence of contaminating proteinaceous material and not to different chromatographic properties of two distinct molecular species of RIP. We therefore suggest that the conclusions of Dimuzio and Veis (39) at this level of investigation were premature.

On the other hand, the following lines of evidence from the present investigation do suggest the existence of more than one dentin phosphoprotein. First, the compositions of two zones (6 and E) of the phosphoprotein fraction from rechromatography on DEAE-cellulose (Fig. 7) differ slightly in total serine and glutamic acid; this trend was also seen for the a and ,B fraction of Fig. 6. Second, the partial separation of the dephosphorylated phosphoprotein preparation into two fractions (Fig. 10) cannot be explained (as above) by the presence of contaminating substances but probably reflects the existence of more than one molecular species. The amount of phosphate remaining after dephosphorylation was less than 1% of the original, and therefore the fractionation in Fig. 10 was not due to phosphoseryl residues. Indeed, the sequence data for dP-RIP (Table IV) indicate that two closely related proteins are present. And finally, the occurrence of aspartic acid and serine-rich proteins in Fractions Dc (Fig. 11) and p (Fig. 12) are probably indicative of minor amounts of phosphoproteins differing in amino acid composition from the major species. Until more specific chemical data are available, it is impossible to determine whether the differences cited above are related to different gene products, to post-transla- tional modifications or to both.

The accurate and complete analysis of proteins with the composition of RIP requires several special techniques. Be- cause phosphoserine, serine, and aspartic acid account for about 90% of the amino acids, two or more dilutions of the hydrolysates must be run on the amino acid analyzer. In our studies, we used glutamic acid and glycine values as internal references; this approach is increasingly important after higher levels of purity are attained, when aspartic acid and phosphoserine amounts are high and those of some other amino acids are quite low. In order to show that a particular amino acid is absent and to compute those amino acids present in small amounts, analyses must be performed with large amounts of the RIP hydrolysates such that the aspartic acid and serine peaks are vastly off-scale. With this type ofanalysis, we have shown that the AG MP-50-purified protein (Da) contains low, but definite amounts of proline, isoleucine, and tyrosine (Table 11); these amino acids were previously reported to be absent from similar preparations of RIP (25).

Another important aspect of phosphoprotein analysis stems from the fact that phosphoseryl and seryl residues are lost at

different rates, during acid hydrolysis. Based on several types of experiments, Richardson et al. (29) have formulated the expression:

Ser,,h, = 0.763 Pse + 0.938 Ser

to account for these different destruction rates, when the phosphoprotein is hydrolyzed for 24 h in 6 N HC1 at 108°C. The phosphoserine in a complete hydrolysate can be determined from the inorganic phosphate content since only mono- phosphate esters of serine are present (2,29). We have found that an automated molybdate assay (Fig. 2) is convenient, highly reproducible, and sensitive enough for this type of analysis. Hydrolysis in an evacuated desiccator was necessary to achieve complete liberation of phosphate while with other hydrolysis techniques (e.g. sealed hydrolysis tubes), small and variable amounts of phosphoserine peaks were still seen on the amino acid chromatograms.

Acknowledgments-We gratefully acknowledge the technical assistance of Craig Cothran and Charles W. Braswell and the artistic and secretarial contributions of Brenda A. Neighbors. We thank Dr. Peter V. Hauschka for the y-carboxyglutamic acid analyses. We are also indebted to the American Scandinavian Foundation for some financial assistance to Dr. Linde during his stay in the United States.

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