Proteomics Analysis of Human Dentin Reveals Distinct Protein

Proteomics Analysis of Human Dentin Reveals Distinct Protein Expression Profiles Eun-Sung Park,† Hye-Sim Cho,† Tae-Geon Kwon,§ Sin-Nam Jang,§ Sang-Han Lee,§ Chang-Hyeon An,| Hong-In Shin,‡ Jae-Young Kim,† and Je-Yoel Cho*,† Department of Biochemistry & BK 21, Department of Oral & Maxillofacial Surgery, Department of Oral & Maxillofacial Radiology, and Department of Oral Pathology & IHBR, School of Dentistry, Kyungpook National University, Daegu, South Korea Received September 4, 2008

The human tooth is the hardest organ of the body, and is composed of enamel, dentin, and dental pulp. Dentin provides the basis of the tooth shape by lining the inner parts of the root and crown. Odontoblasts deposit dentin, an organic matrix that contains collagen, noncollagenous proteins, phospholipids, and growth factors. In this study, we sought to reveal the proteins in human dentin by using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic approaches. Human third molar dentins were cut, isolated, and demineralized, and the extracted proteins were separated on SDS-PAGE. In-gel digested peptides were analyzed using reverse-phase LC-MS/MS. We identified 233 total and 68 common proteins from 3 individuals with high confidence, including a variety of collagenous and noncollagenous proteins such as DSPP, biglycan, osteoglycin, osteopontin, and osteocalcin. In addition to known proteins, we also identified various matrix and serum proteins deposited in the dentin, including asporin, lumican, mimecan, and SOD3. This study provides the first list of proteomes that are detected in human dentin. This proteome list is useful in that it defines the organic matrix of dentin and helps to characterize odontoblasts. Keywords: dentin • proteomics • tooth • odontoblasts

Introduction Global interest in bio-organs has substantially increased in the past decade. Thus, useful basic data for a variety of target bio-organs is greatly needed. Dentin forms the bulk of the tooth and is composed of a central chamber filled with soft pulp tissue. Dentin is first composed with the help of odontoblasts, which form the main structure of the tooth on which enamel and cementum are deposited. It has been hypothesized that dentin mineralization is initiated on the basis of the proteins deposited. To date, the organic matrix of dentin has been reported to contain collagen, noncollagenous proteins (proteoglycans, phosphophoryns, and glycoproteins), phospholipids, and growth factors.1 Odontoblasts are almost indistinguishable, in terms of matrix formation activity, from osteoblasts in that they both express almost all kinds of osteoblast marker genes, such as alkaline phosphatase, bone sialoprotein, osteopontin, and ostoecalcin. The nature of dentin formed by * To whom correspondence should be addressed. Je-Yoel Cho, Ph.D., Assistant Professor, Department of Biochemistry, School of Dentistry, Kyungpook National University, Dongin-dong 2Ga 101, Daegu, 700-422, Korea.Phone,+82-53-420-4997;fax,+82-53-421-1417;e-mail,[email protected]. † Department of Biochemistry, School of Dentistry, Kyungpook National University. § Department of Oral & Maxillofacial Surgery, School of Dentistry, Kyungpook National University. | Department of Oral & Maxillofacial Radiology, School of Dentistry, Kyungpook National University. ‡ Department of Oral Pathology, School of Dentistry, Kyungpook National University.

1338 Journal of Proteome Research 2009, 8, 1338–1346 Published on Web 02/04/2009

odontoblasts, however, is different from bone in its hardness and its structure. Type I collagen is reportedly the major protein of the dentin matrix, which also contains lesser amounts of types III, V, and VI collagens.1 Fibronectin has also been found in association with collagen fibrils in the predentin. Interestingly, the tissue inhibitor of matrix metalloproteinase 1 (TIMP1), another secretory product of odontoblasts, is found in high concentrations in the predentin.2 In terms of noncollagenous proteins, biochemical and immunohistochemical studies indicate that there are specific differences in proteoglycan composition between the predentin and dentin.3,4 Decorin, a chondroitin-dermatan sulfate proteoglycan with binding affinity for type I collagen, is found in the dental pulp, odontoblasts, at the mineralization front, and along the mineralized walls of the dentinal tubules.5 Dentin phosphophoryn (DPP) is the major noncollagenous component of dentin. Immunocytochemical studies indicate that phosphophoryns are localized in small granules distinct from the larger collagen-containing secretory granules.6 Dentin matrix protein 1 (DMP1) reportedly localizes to mature odontoblasts, cementoblasts, and osteoblasts.7-9 Dentin sialoprotein (DSP) is a sialic acid-rich glycoprotein that is expressed early in tooth development, prior to basement membrane degradation.1 Osteocalcin, a glycoprotein rich in glutamic acid, is also reportedly found in odontoblasts and in the dentin matrix.10,11 In terms of growth factors, bone morphogenetic proteins 10.1021/pr801065s CCC: $40.75

 2009 American Chemical Society

Proteomics Analysis of Human Dentin

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Figure 2. X-ray confirmation of demineralization. All dentin samples were demineralized with 0.5 M EDTA (pH 8.0) under agitation at 4 °C for about 14-16 days. (A) Erupted third molar at day 0. (B) Third molar at the 10th day of decalcification, and (C) completely decalcified third molar after 14 days.

Figure 3. SDS-PAGE analysis of human dentin extract. Three different dentin samples and sample buffer were separated by 1-D electrophoresis and stained with Coomassie blue dye. Whole gel lanes were cut into 15 equally sized pieces along the black lines on the gel images.

Figure 1. Flowchart of the sequential extraction procedures for preparing human dentin proteins to be analyzed in LC-MS/MS.

(BMPs) and transforming growth factor β (TGF-β) have been isolated from demineralized dentin matrix.12 Up to date, there have been no proteome analyses of human dentin. Therefore, the present study was carried out to provide the useful proteome list data of human dentin for defining the organic matrix of dentin and helping to characterize odontoblasts.

Materials nad Methods Preparation of Human Dentin Samples. Healthy erupted permanent human third molars, extracted for valid clinical reasons, were obtained from the Department of Oral and Maxillofacial Surgery at Kyungpook National University Hospital. Third molars were taken from 3 patients (2 females and 1 male) with ages ranging from 20 to 33 years after acquiring from the patients their informed consent for tooth donation for research. Cementum and soft connective tissues adhering to the teeth were first carefully scraped off with a curet and a chisel. Under a continuous water spray, each tooth was cut into crown and root portions with a diamond disk. The pulp was removed from the pulp chamber of the root with endodontic files. The pure dentin samples taken from the roots were about 1.5 g in wet weight. Extraction of Dentin Proteins. A flow diagram of the sequential extraction procedure is presented in Figure 1. After demineralization with a solution of 0.5 M EDTA · 2Na (pH 8.0) and protease inhibitors for about 15 days with vigorous agitation in a cold room, the demineralization of the dentin samples was confirmed by X-ray (Figure 2). The completely demineralized dentin samples were then pulverized using a cold mortar and pestle after freezing in liquid nitrogen. RIPA buffer was added to the pulverized dentin sample in an

Eppendorf tube, and the samples were incubated at 37 °C for 30 min with gentle tilting and rotation. After incubation, the samples were centrifuged for 20 min at 12 000 rpm and 4 °C. The concentration of the total proteins in the supernatants was assayed with a protein assay kit by using BSA as a standard. SDS-PAGE and In-Gel Digestion. SDS-PAGE and in-gel digestion were done as previously reported by our group.13 Briefly, protein bands were excised from Coomassie-stained gels (Figure 3) and destained by incubation in 75 mM ammonium bicarbonate/40% ethanol (v/v, 1:1). After destaining, for protein alkylation, the gel was incubated in a solution of 55 mM iodoacetoamide at room temperature for 30 min, and the gel pieces were dehydrated in 100% acetonitrile (ACN) and dried. Gel pieces were then swollen in 10 µL of 25 mM ammonium bicarbonate buffer containing 20 µg/mL modified sequencing grade trypsin (Roche Applied Science), and were incubated overnight at 37 °C. The tryptic peptide mixture was eluted from the gel with 0.1% formic acid. LC-ESI-MS/MS Analysis. LC-MS/MS analysis was also carried out as previously reported by our group.13 Briefly, LC-MS/ MS analysis was conducted using Thermo Finnigan’s ProteomeX workstation LTQ linear ion trap MS (Thermo Electron, San Jose, CA) equipped with NSI sources (San Jose, CA). Twelve microliters of the peptide mixture was injected and loaded onto a peptide trap cartridge (Agilent, Palo Alto, CA). Trapped peptides were eluted onto a 10-cm reverse-phase PicoFrit column packed in-house with 5 µm, 300 Å pore size C18, and then separated on an RP column by gradient elution. The solutions used as the mobile phases were H2O (A) and ACN (B), and both contained 0.1% (v/v) formic acid. The flow rate was maintained at 200 nL/min. The gradient was started at 2% B, reached 60% B in 50 min, 80% B in the next 5 min, and 100% A in the final 15 min. Data-dependent acquisition mode (m/z 300-1800) was enabled, and each survey MS scan was followed by five MS/MS scans with the 30 s dynamic exclusion option on. The spray voltage was 1.9 kV and the temperature of the Journal of Proteome Research • Vol. 8, No. 3, 2009 1339

research articles ion transfer tube was set at 195 °C. The normalized collision energy was set at 35%. Data Analysis. For database searching, tandem mass spectra were extracted by Sorcerer version 3.4 beta 2. Charge state deconvolution and deisotoping were not performed. All MS/ MS samples were analyzed using Sequest (ThermoFinnigan, San Jose, CA; version v.27, rev. 11), which was set up to search the ipiHUMAN3.29 database (unknown version, 68161 entries) assuming a nonspecific digestion enzyme. Sequest was searched with a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 1.5 Da. An iodoacetamide derivative of cysteine was specified in Sequest as a fixed modification. For improved false-positive statistics, the decoy option was selected during the data search process in the Sorcerer program, which improves the quality of the results by reducing the effects of noise. Oxidation of methionine was specified in Sequest as a variable modification. Scaffold (version Scaffold-01_07_00, Proteome Software, Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95.0% probability, as specified by the Peptide Prophet algorithm,14,15 and if they contained at least 2 identified peptides. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Western Blot Analysis. Western blot analysis was performed as previously reported.16 Briefly, after SDS-PAGE, the transferred nitrocellulose membranes (Whatman, Germany) were incubated with antibodies against PEDF (1:1000 dilution, BioProducts MD, Middletown, MD), DSP (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), asporin (1:1000, Abcam, Cambridge, U.K.), lumican (1:1000 dilution, R&D Systems, Minneapolis, MN), and mimecan (1:1000, R&D Systems, Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat or anti-rabbit IgG secondary antibodies (1:2000). Signals were developed with ECL-PLUS detection reagent (Amersham Biosciences, U.K.) and the membranes were exposed to X-ray film for an appropriate time and then developed. Immunohistochemistry (IHC). IHC was performed as previously described on wax sections using standard protocols.17 Briefly, demineralized dentin samples were fixed in 4% paraformaldehyde, embedded in paraffin wax, and sectioned at 7 µm. Sections were incubated with a goat polyclonal antibody against lumican (R&D Systems) and a rabbit polyclonal antibody against SOD3 (Abcam). Normal serum, instead of the primary antibody, was used as a negative control. Light microscopic observation was carried out with Leica DMRE (Leica Microsystems, Germany).

Results and Discussion Identification of Dentin Proteins by LC-MS/MS. The human dentin proteome analysis procedure is summarized in Figure 1. Briefly, dentins were cut out by dental disk from a human third molar, demineralized in 0.5 M EDTA, and pulverized in liquid nitrogen. Demineralized condition of dentin was confirmed by X-ray photography (Figure 2). Extracted proteins from dentin samples were resolved by SDS-PAGE (Figure 3), and each sample was divided into 15 gel pieces, which were then subjected to in-gel digestion by trypsin. Each peptide mixture was eluted and analyzed by liquid chromatography with tandem mass spectroscopy (LC-MS/MS). 1340

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Park et al. The SEQUEST algorithm in the Sorcerer program was used to search the IPI human protein database using our MS/MS data. The identified data were validated by peptide and protein prophet TPP scores of 0.95 and peptide hits of 1 or more in the Scaffold program. From the data combined from 3 individual demineralized dentin samples, 233 proteins were identified. This was after 34 proteins, about half of which were keratins, were subtracted as background, since they also appeared in the sample buffer-only control lane. After removal of background keratins from the dentin proteome list, several keratins were still found in the dentin, especially the cuticular types Ha3, Ha6, Hb2, and Hb5. Sixty-eight of these 233 proteins appeared in all 3 samples, 55 in 2 samples, and 110 in 1 sample. The search results were arranged by occurrence and by the number of peptides that identified the proteins (Supporting Information Table 1). In the individual dentin samples, 165 (20F), 145 (29F), and 114 (33M) proteins were identified (Figure 4A). A list of all 233 human dentin proteins in the three samples is shown in Table 1 and Supporting Information Table 1. Cellular Localization and Functional Classification of the Identified Proteins. The cellular localizations of the proteins identified from all three individuals are shown in Figure 4B. The majority of proteins identified (35.2%) originated from the extracellular space. The next significant localizations were the cytoplasm (23.6%), nucleus (12.4%), endoplasmic reticulum (9.4%), mitochondrion (8.2%), membrane (7.7%), unknown (2.6%), and lysosome (0.9%). As shown in Figure 4C, the proteins had a variety of functions, including metabolic enzymes (28.8%) and cellular organization (18.5%), signal transduction (11.65%), growth/maintenance (11.2%), chaperone/ stress response (6.9%), transport (6.9%), immune response (3.4%), transcription factor activity (2.1%), and nucleic acid binding (0.9%). Proteins of unknown function corresponded to 9.9%. Most of the known noncollagenous matrix proteins of dentin or bone matrix proteins were identified in our proteome analysis of the dentin (Table 2). In addition to collagen and matrix proteins, significant numbers of cellular metabolic proteins were also detected in dentin in this study. It should be noted that odontoblasts extend odontoblastic processes into dentinal tubules up to the mantle dentin from the predentin.18,19 Secretory vesicles and intratubule proteins contain some odontoblast intracellular proteins including rich microtubule and microfilament proteins which were routinely detected in our study. Indeed, our protein list includes tubulin chains and actin proteins. Thus, a novel and interesting finding of the present work is that metabolic enzymes comprise the largest functional category of human dentin proteins (Figure 4C). This is different from what we had expected, and indicates that dentin, especially the dentinal tubule and peritubular dentin area, is not a fixed region, but a constantly active and physiologically dynamic one. Validation of Candidate Proteins. To validate the candidate proteins identified by MS/MS, we performed a Western blot analysis and immunohistochemistry for some of the proteins that were already known to be present in or were newly identified in human dentin. With the use of a Western blot analysis in which human dentin samples were compared with various cell lines and human mandibular bone (Figure 5), DSP, asporin, lumican, and mimecan were not detected in osteosarcoma (MG63), human breast cancer (MCF7), lung cancer, human umbilical vein endothelial cell lines, bone marrow stem cells and normal human serum. However, PEDF was intensely

Proteomics Analysis of Human Dentin

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Figure 4. (A) Venn diagram of overlapping proteins among the three individuals (20F, 29F, and 33M) and the number of individual proteins identified with LC-MS/MS. Cellular (B) and functional (C) distributions of the 233 identified human dentin proteins in three individuals. Cellular location and biological function analyses were carried out with assistance of the Human Protein Reference Database (www.hprd.org/protein).

detected in normal human serum. Compared with human mandibular bone, DSP, PEDF, and lumican were detected at a similar intensity as in human dentin. In contrast, asporin was not found and mimecan was only weakly detected in human mandibular bone. Dentin sialophosphoprotein (DSPP), which is an important nucleator for dentin mineralization, was identified at mid to low levels (average 7 peptides per sample). This peptide hit was not as large as that of other matrix proteins, such as biglycan, vitronectin, osteoglycin, and osteomodulin (Table 2). DSP and DPP (also known as phosphophoryn) are encoded by a single gene, Dspp.7,20-22 DSP and DPP are the two major noncollagenous components of dentin, being solely expressed by the ectomesenchymal-derived odontoblasts of the tooth. It has been suggested that a deficiency in DPP is a causative factor in dentinogenesis imperfecta (DI).23 In particular, it has been suggested that phosphophoryn is synthesized and secreted only by physiologically differentiated odontoblasts, and that the mineralization processes of mantle, secondary, reparative, and DI type II dentins may be different from that of circumpulpal orthodentin.24 Some reports have shown that DSPP is also found in the bone.25 In the current study, DSP was not detected in several cell lines analyzed, but was detected in human mandibular bone (Figure 5), which also supports the recent report that DSP is not a dentin-specific protein. Pigment epithelium-derived factor (PEDF) which is also known as serpin F1, among several serpin antiproteases found in our analysis including serpins A1, A3, C1, F1, and H1, showed

the highest spectral counts of the serpins observed in this study. These serpins are likely to be deposited from the blood, although some reports show that osteoblasts and osteoclasts express PEDF,26 and that human mesenchymal stem cells (MSCs) also secrete PEDF when they differentiate to osteoblasts.27 On the basis of our results, the PEDF detected in human dentin may have originated from the blood and dentin. This is supported by the observations that PEDF was also immunoreactive in human normal serum and dental pulp stem cells, and that odontoblasts can transcribe the pedf gene to synthesize PEDF. Class II small leucine-rich proteoglycans, such as lumican, prolargin (PRELP, proline arginine-rich and leucine-rich repeat protein), asporin, and fibromodulin, were detected in the dentin. Our data showed that asporin (ASPN or periodontal ligament-associated protein-1; PLAP-1) was only present in the dentin (Figure 5) and cartilage (data not shown), and not in various other cell lines analyzed or in human mandibular bone. Asporin belongs to a family of leucine-rich repeat proteins that comprise a major noncollagen component of the ECM and are especially associated with the cartilage matrix.28 The name asporin reflects its unique aspartate-rich N terminus, and the structure has an overall similarity to decorin.29 Clinically, asporin is abundantly expressed in osteoarthritis articular cartilage, and its expression increases with progressive cartilage degeneration.29,30 It was recently suggested that asporin is an important regulator of TGF-β in articular cartilage, and thus plays an essential role in cartilage homeostasis and osteoarJournal of Proteome Research • Vol. 8, No. 3, 2009 1341

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Park et al. a

Table 1. Complete List of Human Dentin Proteins Identified from Three Individuals

a

Human dentin proteins are abbreviated. Protein names are separated by semicolons.

Table 2. List of Selected Dentin Matrix Proteins no. of unique peptides IPI accession number

29F

33M

20F

total peptide

coverage

symbol

description

IPI00010790 IPI00007960 IPI00298971 IPI00025465 IPI00020990 IPI00014572 IPI00021000 IPI00003323 IPI00009802 IPI00022418 IPI00015315 IPI00292732 IPI00419916 IPI00294662 IPI00009028 IPI00023015 IPI00296099

83 40 66 18 14 10 19 14 4

78 40 54 18 13 5

194 150 101 38 36 26

355 230 221 74 63 41 19 17 17 11 9 8 6 5 4 3 2

42.4 27.8 19.3 24 16.3 11.1 7.6 2.2 1.1 1.8 15.2 6.7 9.2 3.2 10.4 6 1.4

BGN POSTN VTN OGN OMD SPARC SPP1 DSPP VCAN FN1 ECM2 FMOD ALPL IBSP CLEC3B PMF1 THBS1

Biglycan precursor Isoform 1 of periostin precursor Vitronectin precursor Osteoglycin (osteoinductive factor, mimecan) Osteomodulin precursor. Sparc precursor (Osteonectin) Isoform A of Osteopontin precursor dentin sialophosphoprotein preproprotein Isoform v0 of versican core protein precursor Isoform 1 of fibronectin precursor Extracellular matrix protein 2 precursor Fibromodulin precursor Alkaline phosphatase, tissue-nonspecific isozyme precursor. Bone sialoprotein 2 precursor Tetranectin precursor. Osteocalcin precursor Thrombospondin-1 precursor

3 4

1 2

13 7 8

6 6

5 4 1

2 2

thritis pathogenesis. Inhibition of asporin, then, should enhance cartilage regeneration by increasing TGF-β activity.31 Recently, asporin mRNA was reportedly detected in odontoblasts, and its levels were reduced by fluoride treatment.32 Asporin may also be involved in collagen fiber assembly and in chondrogenesis. Asporin has been shown to bind to BMP2, inhibiting the binding of BMP-2 to its receptor and blocking periodontal ligament mineralization.33,34 Interestingly, mimecan (osteoglycin) and lumican, which exist at high levels in the cartilage, were also detected at high levels in dentin. Western blot analysis confirmed that mimecan 1342

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was specifically expressed in dentin, and not or minimally expressed in other cell types including bone (Figure 5), although its expression level in dentin was lower than that in cartilage (data not shown). Mimecan/osteoglycin, a member of the small leucine-rich proteoglycan (SLRP) gene family, was initially isolated in a truncated form from bovine bone. It was subsequently characterized as one of the three major keratin sulfatecontaining proteoglycans in the cornea, along with lumican and keratocan.35,36 In mimecan-deficient mice, a lack of mimecan leads to increased collagen fibril diameters in the cornea and skin.37 A novel finding of the present study is that the

Proteomics Analysis of Human Dentin

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Figure 5. Validation of protein expression by Western blot analysis. Antibodies against DSP (dentin sialoprotein), PEDF (pigment epithelium derived factor), asporin, lumican, and mimecan were used for the cells and tissues as indicated. MG63, osteosarcoma cell line; MCF7, human breast cancer cell line; HUVEC, human umbilical vein endothelial cells.

Figure 6. Immunohistochemistry showing the localizations of lumican and SOD3 in human dentin tissues. (A) In the microphotogram of lumican, lumican was detected with a strong localization pattern in the predentin region. (B) Higher magnification of panel A. (C) Weaker localization of lumican was detected at the outer region of dentin-enamel (DEJ). (D) Higher magnification of panel C. (E) In the microphotogram of SOD3, broad localization patterns were seen in the dentin tissue. In particular, SOD3 localized along the dentinal tubules. (F) Higher magnification of panel E. (G) Similar SOD-3 localization patterns were examined in the outer region of dentin. (H) Higher magnification of panel G. (I-L) There was no immuno-positive signaling in the negative control specimens. Scale bars in A, C, E, G, I, K, 200 µm; and in B, D, F, H, J, L, 50 µm.

immunoreactivity of mimecan in human dentin was more intense than that in human mandibular bone. This indicates that mimecan is more abundant and active in dentin than in mandibular bone for its role in regulating collagen fibrillogenesis. Immunohistochemistry results showed the localizations of lumican and SOD3 in human dentin tissues (Figure 6). The photomicrogram of lumican showed that positive immunoreactivity of lumican was detected in peritubular dentin and in the predentin region (Figure 6A-D). Lumican, a small interstitial leucine-rich proteoglycan gene (SIPG) family member, is a keratan sulfate proteoglycan present in large quantities in the corneal stroma and the interstitial collagenous matrices of the heart, aorta, skeletal muscle, skin, and intervertebral discs.38 Other SIPG members include decorin, biglycan, and fibromodulin. Similar to decorin, lumican interacts with collagen

and limits the growth of fibrils in diameter.38 In comparison with its levels in various cell lines and in human mandibular bone, lumican was more abundantly localized in human dentin (Figure 5). Lumican exist in gradients across the predentin (Figure 6).39 Some proteoglycans are expressed at high levels in the predentin, and are degraded near the mineralization front, where proteoglycanases and metalloproteinases exist at high levels. This supports the idea that some proteoglycans may inhibit the mineralization of the dentin matrix.40,41 Thus, it is plausible that lumican, a soft tissue matrix protein, exists in predentin at high levels and thus may inhibit mineralization in predentin. Furthermore, the reports on the potential biological function of lumican in dentin formation have been obtained by using the lumican null mice. They have serious functional defects including cornreal opacity as well as skin and tendon Journal of Proteome Research • Vol. 8, No. 3, 2009 1343

research articles fragility associated with disorganized and loosely packed collagen fibers.38,42-44 Therefore, we think that lumican in dentin formation may control the collagen fibril assembly and maintain dentin homeostasis. Further studies are required for the roles of lumican during the dentinogenesis and for the elucidation whether it has inhibitory or excitatory roles in the mineralization of dentins. SOD3 was detected in all 3 cases (Supporting Information Table 1). In the photomicrogram of SOD3, broad localization patterns of SOD3 were shown in the dentin tissues, particularly along the dentinal tubules (Figure 6E-H). The SODs are antioxidant enzymes that catalyze the dismutation of two superoxide radicals into hydrogen peroxide and oxygen. The product of SOD is thought to protect the brain, lungs, and other tissues from oxidative stress.45 The protein is secreted into the extracellular space and forms a glycosylated homotetramer that is anchored to the ECM and cell surfaces through an interaction with heparan sulfate proteoglycan and collagen. SOD3 (also known as extracellular SOD) is found in plasma, lymph, and synovial fluids, as well as in tissues. Like SOD1, SOD3 is a CuZn SOD; however, it is distinct from SOD1 in its amino acid sequence, antigenic properties, and tissue distribution. SOD3, an endogenous antioxidant, may mediate scavenging in the dentinal tubules to protect dentin itself or after its secretion into the dentinal tubule space. The presence of SOD3 in the human dentin again suggests that the dentin or, at least, the dentinal tubule is in physiologically active and dynamic state. An odontoblast-specific knockout animal model will reveal the precise role of this protein in the dentin and dentinogenesis. Characteristics of Other Proteins Identified in Dentin. From our proteomic analysis, we identified most of the known collagenous and noncollagenous proteins in dentin reported in the literature.1,21 Types IR1, VIR3, XIR2, and XIIR1 collagens were identified in all 3 samples. Types VIR1 and R2 collagens were identified in 2 samples. Although type III collagens were not detected, our data did reveal the presence of types VR1 and IVR1 collagens. As the synthesis of type I collagen increases, the expression of type III collagen reportedly decreases in odontoblasts.46 A novel finding of our data is the presence of types XI and XII collagens in dentin. The quantities of type XII collagens were roughly a half to one-third those of the type I collagens. Type XI collagen levels were higher than those of type V collagens, as estimated by the peptide hits of the identified proteins. Until now, collagen types IX, X, and XI are known to be minor cartilage constituents, with type XI collagen belonging to the group of fibrillar collagens. It has been suggested that the expression of type XI collagen is not restricted to cartilage, as previously thought, since the cDNA libraries from which the clones were isolated originated from both cartilaginous and noncartilaginous tissues.47 Gordon et al. showed that types IX and XII collagen are 2 homologous members of a family of unique collagenous proteins that display tissue-specific expression patterns.48,49 This family of collagen appears to be distinct from fibrillar collagens. The abundance of the type XI and XII collagens imply that dentin structures require these collagens for their proper structural maintenance. Thus, it would be interesting to measure the level of these collagens secreted by odontoblasts when the cells are differentiated from dental pulp stem cells. Dentin is also known to contain collagenases and gelatinases. Our data showed the presence of the gelatinase MMP2, a type IV collagenase that degrades collagen IV and fibronectin,50 and 1344

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Park et al. much higher levels of MMP20. MMP20 is known to degrade amelogenin, the major protein component of the enamel matrix. The presence of MMP20 in dentin was not surprising, as the enamel proteins are endocytosed in coated vesicles at the odontoblast cell surface.51,52 Therefore, MMP20 might be synthesized by odontoblasts and secreted into the predentin or dentin to degrade the amelogenin integrated in dentin. TGFβ1, a growth factor important for hard tissue formation, was also detected in the dentin. TGFβ1 binds to fibronectin and decorin, inhibits cell proliferation, and stimulates odontoblast differentiation. Odontoblasts express high levels of TGFβ and its receptor.53,54 Interestingly, the TGFβ-induced protein ig-h3 (Big-H3) was also detected at a high level in all 3 samples. Although we could not detect BMP or other cytokines, probably due to their low expression level and/or to their detachment during the EDTA demineralization process, other growth factorrelated molecules such as IGFBP-5 and β-catenin were detected. Other than those intrinsic proteins synthesized, stored in dentinal tubules, or secreted and deposited by odontoblasts listed above, dentin also has extrinsic proteins that are carried into the tubules from the dentinal fluid and those that are trapped in the lamina limitans or bound to the mineral phase of the dentin from the blood. These proteins are mainly synthesized by the liver and by hematopoietic cells, and include immunoglobulins and carrier proteins. Thus, dentinal tubules contain serum proteins, including fibrinogen, albumin, and immunoglobulins.55,56 Our dentin proteome list also showed a variety of extrinsic proteins, including albumin which was also present in bone at a higher concentration than in blood prothrombin, transferrin, complements, HMW kininogen-1, hemoglobin alpha, apolipoproteins, and others.

Concluding Remarks Our proteome results can be used as markers for the studies of the differentiation of dental pulp stem cells to odontoblasts which synthesize dentin. For this, the expression of these proteins first needs to be determined in the process of dental pulp stem cells (DPSC) differentiation process and matrix mineralization process by way of RT-PCR, Western blot, ELISA, immunohistochemistry, and so forth. Also, it is needed to study the patterns of these proteins or genes in the developmental process of tooth. Other applications of our proteome results are to the studies of dental formation-related diseases. It is known that DI is caused by the defects in the matrix proteins such as Dspp in dentin. Thus, our results provide valuable information for the investigation looking for new target genes or proteins involved in the malformation of tooth structure. Our matrix proteins identified can also be used as components for the biotooth regeneration studies. That is, for optimal tooth regeneration, it might be needed to focus on how to stimulate the odontoblasts to synthesize and produce the matrix proteins identified by our studies and how to provide these matrix proteins by artificial synthesis. We think that the results of proteins identified in human dentin can also be used as useful criteria for validating the similarity of the function and composition of a biotooth when it may be made and used clinically in the near future. In conclusion, we present for the first time a list of 233 proteins identified in human dentin by LC-MS/MS proteomics, including a variety of known proteins and many new proteins. We further confirmed several of these proteins by Western blot and immunohistochemical staining. Further study is needed

Proteomics Analysis of Human Dentin to clarify the physiological function of the newly found proteins and to confirm other candidate proteins discovered in this study. Abbreviations: DSPP, dentin sialophosphoprotein; PEDF, pigment epithelium derived factor; SOD3, superoxide dismutase 3.

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