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Splicing Determines the Glycosylation State of Ameloblastin
K. Kobayashi1,3,
Y. Yamakoshi1,
J.C.-C. Hu2,
K. Gomi3,
T. Arai3,
M. Fukae4,
P.H. Krebsbach1 and
J.P. Simmer1,*
1 Department of Biologic and Materials Sciences, Dental Research Lab, 1210 Eisenhower Place, Ann Arbor, MI 48108, and
2 Department of Orthodontics and Pediatric Dentistry, University of Michigan School of Dentistry, 1011 N. University, Ann Arbor, MI 48109-1078, USA; and
3 Department of Periodontics and Endodontics and
4 Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
Correspondence: * corresponding author, jsimmer{at}umich.edu
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ABSTRACT
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In developing porcine enamel, the space between enamel rods selectively binds lectins and ameloblastin (Ambn) N-terminal antibodies. We tested the hypothesis that ameloblastin N-terminal cleavage products are glycosylated. Assorted Ambn cleavage products showed positive lectin staining by peanut agglutinin (PNA), Maclura pomifera agglutinin (MPA), and Limulus polyphemus agglutinin (LPA), suggesting the presence of an O-linked glycosylation containing galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic acid. Edman sequencing of the lectin-positive bands gave the Ambn N-terminal sequence: VPAFPRQPGTXGVASLXLE. The blank cycles for Pro11 and Ser17 confirmed that these residues are hydroxylated and phosphorylated, respectively. The O-glycosylation site was determined by Edman sequencing of pronase-digested Ambn, which gave HPPPLPXQPS, indicating that Ser86 is the site of the O-linked glycosylation. This modification is within the 15-amino-acid segment (73-YEYSLPVHPPPLPSQ-87) deleted by splicing in the mRNA encoding the 380-amino-acid Ambn isoform. We conclude that only the N-terminal Ambn products derived from the 395-Ambn isoform are glycosylated.
Key Words: ameloblastin • Ambn • sheathlin • enamel • tooth • porcine
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INTRODUCTION
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A meloblastin is expressed predominantly by ameloblasts during amelogenesis (Torres-Quintana et al., 2005) and is critical for proper tooth enamel formation (Fukumoto et al., 2004). Ameloblastin was first described as a member of the non-amelogenin family of proteins in porcine enamel extracts that migrated between 13 and 17 kDa on SDS-PAGE (Fukae and Tanabe, 1985, 1987a). Antibodies raised against these enamel proteins produced a honeycomb pattern on histological sections of developing porcine (Uchida et al., 1991) and bovine (Fukae et al., 1993) teeth, suggesting that the protein concentrates in the sheath space separating enamel rods. Immunohistochemistry also demonstrated that various lectins selectively recognize components of the sheath space (Akita et al., 1992). When the N-terminal sequence of the 13- to 17-kDa non-amelogenins was first reported, these proteins were proposed to represent a new "class" of enamel proteins: the sheath proteins (Uchida et al., 1995).
Two groups independently cloned and characterized the first cDNAs encoding the rat homologue of the sheath protein, and named it ’ameloblastin’ (Krebsbach et al., 1996) and ’amelin’ (Cerny et al., 1996). The porcine cDNA was cloned and named ’sheathlin’ (Hu et al., 1997). The official gene name was designated ’ameloblastin’ (Ambn), which has become the term most commonly used for the gene and protein it encodes. Ambn cDNA sequences have permitted the complete primary structure of the protein to be deduced and led to the discovery that the 27- and 29-kDa calcium-binding proteins previously isolated from porcine enamel (Fukae and Tanabe, 1987b) are cleavage products from the Ambn C-terminus (Murakami et al., 1997). Antibodies raised against the ameloblastin C-terminal region only immunostained the superficial enamel, and did not localize to the sheath space (Uchida et al., 1997, 1998; Nanci, 1999). The C-terminal cleavage products of ameloblastin have at least one sulphated, O-linked glycosylation (Yamakoshi et al., 2001). The distinctly different localization patterns of the Ambn N- and C-terminal cleavage products suggested that enamel proteins might serve multiple functions, and that proteolysis might be necessary to separate the different functional components (Bartlett and Simmer, 1999; Simmer and Hu, 2002). Two hypothetical functions of ameloblastin are in ameloblast cell attachment (Fukumoto et al., 2004) and in the maintenance of rod/interrod boundaries (Iwata et al., 2007).
Ameloblastin cDNA clones also revealed that ameloblastin transcripts undergo alternative splicing (Hu et al., 1997; Simmons et al., 1998). Two porcine ameloblastin isoforms are expressed from alternatively spliced RNA transcripts. Their translation products (excluding the signal peptides) have 380 and 395 amino acids, and, in the absence of post-translational modifications, have predicted molecular masses of 40 and 42 kDa, respectively. The smaller Ambn isoform differs from the larger by the absence of 15 amino acids encoded at the 5' end of exon 5 (73-YEYSLPVHPPPLPSQ-87), which is part of the Ambn N-terminal region. Although this peptide is highly conserved among species, its contribution to ameloblastin function is unknown. To gain insight into the structural properties of the porcine ameloblastin N-terminal region, we isolated ameloblastin N-terminal cleavage products and characterized their lectin-binding properties and post-translational modifications.
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MATERIALS & METHODS
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All experimental procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Program at the University of Michigan.
Preparation of Immature Enamel Extracts
Tooth germs of permanent second molars were surgically extracted from the maxillae and mandibles of 6-month-old pigs at the Michigan State University Meat Laboratory (East Lansing, MI, USA). The enamel organ epithelia and dental pulp tissue were removed with tissue forceps. The soft, cheese-like enamel was separated from the crowns and homogenized in Sörensen buffer (pH 7.4), made by mixing Na2HPO4 and KH2PO4 to achieve a final phosphate concentration of 50 mM and a pH of 7.4. The soluble material (supernatant) was the neutral (N) extract. Material not soluble in the neutral extract was obtained by centrifugation. The pellet was re-suspended in carbonate buffer, made by mixing bicarbonate NaHCO3 and carbonate Na2CO3 to a final concentration of 50 mM, and a pH of 10.8, and homogenized. The supernatant was designated the alkaline (AL) extract. The N and AL extracts included proteinase inhibitors (5 mM 1,10 phenanthroline, Sigma, St. Louis, MO, USA; and Protease Inhibitor Cocktail Set III, Novagen, Madison, WI, USA). Material not soluble in the alkaline extract was pelleted by centrifugation.
Purification of N-terminal Ameloblastin
The alkaline extract [AL] was applied to a Q-Sepharose Fast Flow column (1.6 cm x 20 cm, Amersham Pharmacia, Uppsala, Sweden) and was eluted with a linear gradient (from 0 to 0.2 M) of buffer B. Buffer A was 50 mM Tris-HCl/6 M urea; buffer B was buffer A/0.5 M NaCl. The fraction eluted in the first peak (Q1a) was desalted with a YM-3 membrane (Amicon, Houston, TX, USA), lyophilized, and dissolved in 1 mL of 0.01% acetic acid. For selective precipitation of amelogenins, a 25-mL quantity of Tris-buffered saline (pH 7.2)/0.5 mM CaCl2 (TBS-Ca) was added. The sample was stirred overnight at room temperature and centrifuged for 15 min at 5000 rpm. The supernatant (containing N-terminal ameloblastin) was mixed with PNA-immobilized lectin gel (EY Laboratories, Inc., San Mateo, CA, USA), equilibrated with TBS-Ca, and rotated overnight at room temperature. PNA has a specific affinity for proteins containing galactose. Unbound protein was collected by centrifugation for 1 min at 1000 rpm. The PNA-lectin gel was washed with 10 vol of TBS-Ca solution. Bound protein was eluted with 3 vol of Tris-buffered saline/0.5 mM CaCl2/0.1 M lactose solution. The unbound (UnB) and eluted (E) samples were dialyzed against 0.01% acetic acid, lyophilized, dissolved in 0.1% trifluoroacetic acid (TFA), and fractionated by non-porous (NPS) RP-HPLC in a PF2D-HPRP column (4.6 x 30 mm) (Beckman Coulter, Fullerton, CA, USA). The column was equilibrated with 0.1% TFA and eluted with a linear acetonitrile gradient (0–100%/30 min) containing 0.08% TFA at a flow rate of 0.75 mL/min at 50°C.
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
SDS-PAGE was performed with Novex 18% or 4–20% Tris-Glycine Gels (Invitrogen, Carlsbad, CA, USA). Samples were dissolved in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA), and electrophoresis was carried out with a current of 30 mA for 70 min. The gels were stained with Simply BlueTM Safe Stain (Invitrogen). The apparent molecular weights of protein bands were estimated by comparison with SeeBlue® Plus2 Pre-Stained Standard (Invitrogen).
Western Blots and Lectin Staining
After electrophoretic transfer (Fukae et al., 1998) of proteins from SDS-PAGE gels to nitrocellulose membranes (0.4 µm, HybondTM-ECL; GE Healthcare Biosciences, Little Chalfont, UK), blocking was performed with 5% non-fat milk, followed by incubation with a 1:2000 dilution of Ambn-63 anti-peptide antibody (Iwata et al., 2007), a 1:5000 dilution of recombinant porcine amelogenin antibody (Ryu et al., 1999) in TTBS containing 5% milk, and stained with 7 horseradish peroxidase (HRP) conjugates: peanut agglutinin (PNA), Maclura pomifera agglutinin (MPA), and Limulus polyphemus agglutinin (LPA), concanavalin A (Con A), Ulex europens agglutinin I (UEA I), Griffonia simplicifolia lectin II (GS-II), and wheat germ agglutinin (WGA) (EY Laboratories) at dilutions of 1:500. The Western blots were washed 3x in TTBS and incubated with anti-rabbit IgG secondary antibody (BioRad, Hercules, CA, USA) at a dilution of 1:10,000. Immunostaining was by chemiluminescent detection with the ECL AdvanceTM Western Blotting Detection Kit (GE Healthcare Bioscience) or by colorimetric detection with diaminobenzidine (DAB, Sigma).
Pronase Digestion
Ambn was dissolved in 5 mL of 50 mM Tris-HCl buffer (pH 8.0) and digested at 37°C for 40 hrs with pronase (Wako Pure Chemical Industries Ltd, Osaka, Japan) at an enzyme/substrate ratio of 1/50. At the end of the incubation period, trichloroacetic acid (TCA) was added to a final concentration of 10%, and centrifuged for 10 min at 10,000 x g. The supernatant was applied to a Sephadex G-25 column (1.6 x 100 cm) equilibrated with 0.05 M pyridine/acetic acid (pH 5.0), and then continuously monitored at 280 nm.
Phenol-Sulfuric Acid Assay
Aliquots in each peak fractionated from the column of Sephadex G-25 or TSK-gel ODS-120T were evaporated, and 0.3 mL of water/0.3 mL of 5% phenol/1.5 mL of concentrated sulfuric acid were added. The peak containing oligosaccharides was identified by colorimetric determination at 490 nm (Masuko et al., 2005).
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RESULTS
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The ameloblastin N-terminal region appeared to be highly antigenic and was easily detected on Western blots with the Ambn-63 anti-peptide antibody. Ease of detection gave us the impression of abundance, but the ameloblastin N-terminal cleavage products were not abundant and proved difficult to isolate. The alkaline extract of porcine secretory-stage enamel was initially fractionated by ion exchange chromatography. N-terminal Ambn cleavage products eluted in the first chromatographic peak (Fig. 1A ). Next, we took advantage of the low solubility of many amelogenin cleavage products (Tan et al., 1998) by selectively precipitating them (Fig. 1B, left). The supernatant, enriched Ambn N-terminal cleavage products, was divided into proteins that bound to the lectin PNA and those that did not (Fig. 1B, right). PNA shows specific binding affinity for proteins containing galactose, so we used this step to separate glycosylated from unglycosylated N-terminal ameloblastin cleavage products. The unbound (Fig. 1C) and eluted (Fig. 1D) fractions from the PNA affinity purification were further fractionated by NPS RP-HPLC and analyzed by SDS-PAGE and Western blot analyses.
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Figure 1. Purification of N-terminal Ambn cleavage products from developing porcine molars. (A) Chromatogram showing absorbance at 280 nm of alkaline extract proteins eluting from an ion exchange column. The contents of each fraction are shown on 5–20% gradient SDS-PAGE. Fraction Q1a (boxed) was used in subsequent purification steps. (B, left) 18% Tris/glycine gel showing fractionation of Q1a by precipitation of amelogenins with TBS (pH 7.4). Western blot with Ambn-63 shows that ameloblastin N-terminal cleavage products are in the supernatant (Sup), but not the precipitate (Ppt). (B, right) 18% Tris/glycine gel and Western blots showing Q1a-Sup fractionation by PNA affinity binding: UnB, unbound; washes (W1, W2, W3), eluted (E), and PNA beads (B). (C) Chromatogram showing absorbance at 220 nm of fraction Q1a-Sup-Ub separated by NPS RP-HPLC and SDS-PAGE and Western blots of the 12 fractions. (D) Chromatogram showing absorbance at 220 nm of fraction Q1a-Sup-E separated by NPS RP-HPLC and SDS-PAGE and Western blots of the 4 fractions. Ambn-positive fractions (boxed) were selected for further analyses.
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To gain information about the types of glycosylation sites attached to the Ambn N-terminal region, we tested the lectin-binding properties of Ambn N-terminal cleavage products in Far-Western blot analyses. The Ambn N-terminal cleavage products were separated on SDS-PAGE, transferred to a membrane, incubated with a panel of HRP-conjugated lectins, and detected by chemiluminescent or colorimetric assays (Fig. 2). The lectins and their binding specificities were: PNA (galactose), MPA (N-acetyl-galactosamine), LPA (sialic acid), Con A (mannose), UEA-I (fucose), GS-II (N-acetyl-glucosamine), and WGA (N-acetyl-glucosamine). All of the unbound proteins (i.e., those that did not bind to the PNA beads) were negative for lectin binding, suggesting that these proteins are not glycosylated. Of note, an Ambn-positive band at 17 kDa did not bind the PNA beads and was negative for binding of all lectins. This finding suggests that not all Ambn N-terminal cleavage products are glycosylated. In contrast, two Ambn N-terminal cleavage products eluting from the PNA beads migrated at 15 and 17 kDa on SDS-PAGE and were positive for PNA, MPA, and LPA binding, indicating the presence of galactose, N-acetyl-galactosamine, and sialic acid. This pattern is consistent with a simple, unbranched, O-linked glycosylation containing one or two sialic acids (Dennis et al., 1999).
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Figure 2. Lectin staining of final fractions containing N-terminal Ambn cleavage products. RP-HPLC fractions 2, 5, and 7 that did not bind to the PNA affinity beads (UnB) and RP-HPLC fractions 2 and 3 eluted (E) from the beads were separated by SDS-PAGE, transferred to membranes, and tested for their ability to bind Ambn-63 antibody and 7 different lectins with various carbohydrate specificities. The lectins and their binding specificities were: PNA (galactose), MPA (N-acetyl-galactosamine), LPA (sialic acid), Con A (mannose), UEA-I (fucose), GS-II (N-acetyl-glucosamine), and WGA (N-acetyl-glucosamine). These results suggest an O-linked glycosylation having a structure like that shown on the lower right. Protein bands selected for N-terminal sequencing are labeled in the SDS-PAGE on the upper left. Bands a through f all gave the Ambn N-terminal sequence VPAFPRQP... . Bands marked with an asterisk gave the amelogenin N-terminal sequence MPLPPHPG... . This confirmed that the lectin-positive bands were ameloblastin N-terminal cleavage products. Based upon integration of the chromatographic peaks (2 and 3 for the PNA-bound and 5 for the PNA-unbound), the abundance of Ambn395 relative to Ambn380 is at least 2:1. We say a minimum of 2:1, because it is apparent that the PNS-bound material is almost all Ambn, while the unbound fractions still contain some amelogenin.
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Next we confirmed the identity of selected bands by transblotting them onto a membrane and determining their N-terminal sequences (Fig. 2 ). The major Ambn-immunopositive bands all gave the Ambn amino-terminal sequence starting with VPAFPRQP, including the lectin-positive bands. A quality N-terminal Ambn sequence was obtained through Glu19 (VPAFPRQPGTPGVASLSLE). Cycle 11 was hydroxyproline, with its peak in the normal position of histidine. Ser17 gave a blank cycle, consistent with its being phosphorylated. The phosphorylation of Ser17 is predicted by its context (SXE) and by analogy to other proteins in the secretory calcium-binding phosphoprotein (SSCP) family (Kawasaki and Weiss, 2003).
For determination of the position of the O-glycosylation, an Ambn- and lectin-positive fraction was digested with pronase. Pronase is a mixture of endo- and exo-peptidases that catalyze unspecific degradation of proteins down to individual amino acids, excepting glycosylated segments. Steric inhibition by attached glycosylations causes pronase to digest glycoproteins into a mixture of glycopeptides averaging about 8 amino acids. The pronase digest was fractionated by RP-HPLC, and each fraction was tested for glycosylation by a phenol-sulfuric acid assay. The fraction from the third chromatographic peak of the pronase digest was positive for glycosylation, and Edman sequencing gave the Ambn sequence as 80-HPPPLPXQPS-89 (Fig. 3). The blank cycle indicates that Ser86 is the site of the O-linked glycosylation. This amino acid is within the 15-amino-acid segment (73-YEYSLPVHPPPLPSQ-87) deleted by alternative splicing in the 380-amino-acid porcine Ambn isoform.
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Figure 3. (left) Chromatogram showing absorbance at 230 nm of Pronase-digested Ambn N-terminal cleavage products separated on a Sephadex G-25 column. The contents of peak 3 were positive for glycosylation with the phenol-sulfuric acid colorimetric assay, and gave the Edman sequence: HPPPLPXQPS. The blank cycle for Ser86 identifies this as the position of the O-linked glycosylation. (right) Deduced amino acid sequence of the 395-amino-acid ameloblastin. Protein sequences characterized in this study are boxed. Underlined amino acid positions are modified following translation. The 15-amino-acid sequence deleted by alternative splicing is in bold. Previously determined ameloblastin cleavage sites from in vivo sources are marked by arrowheads (Iwata et al., 2007).
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DISCUSSION
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The sheath space of developing porcine enamel can be selectively immunostained with antibodies specific for the N-terminal region of ameloblastin or by lectins having affinity for sugars commonly included in protein glycosylations. Based upon the apparent molecular weight of the slowest-migrating Ambn-positive band of enamel extracts on Western blots, uncleaved porcine ameloblastin has an apparent molecular weight of 62 kDa (Uchida et al., 1995). This is well above the deduced molecular weight of the unmodified protein (42 kDa), suggesting that ameloblastin is glycosylated. There are no predicted N-linked glycosylation sites in ameloblastin. Previously, it was shown that the C-terminal region of porcine ameloblastin has a sulphated O-linked glycosylation at Thr361 (Yamakoshi et al., 2001), and full-length recombinant Ambn transiently expressed in a kidney cell line was glycosylated (Iwata et al., 2007). This is the first demonstration that ameloblastin is glycosylated in its N-terminal region. Surprisingly, the glycosylation is at Ser86, which is present only on the 395-amino-acid isoform. Thus, alternative splicing plays a role in deciding if the N-terminal region of Ambn is glycosylated.
Although it is clear that Ambn is required for normal enamel formation, evidence for its functions at the molecular level is largely circumstantial. The specific localization of Ambn N-terminal cleavage products in the sheath space suggested that it plays a role in maintaining prism organization; however, other observations and considerations challenge this conclusion. A single-ameloblast Tomes’ process generates each enamel rod, and any disruption in the prism pattern implies that the Tomes’ process is faulty, and it is difficult to imagine how enamel rods, once formed, would be obliterated. Another problem is that in some mammals, such as in rodents, ameloblastin cleavage products accumulate throughout the enamel layer, rather than within the sheath space. Furthermore, ameloblastin is expressed by crocodiles (Shintani et al., 2002) and toads (Shintani et al., 2003), which lack enamel prismatic structure altogether. Ambn expression in the crocodile is tooth-specific, and Ambn antibodies that recognize different regions of the protein show dissimilar immunolocalization patterns in developing enamel (Shintani et al., 2006).
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ACKNOWLEDGMENTS
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We thank Mr. Tom Forton, Manager of the Michigan State University Meat Laboratory, and members of the Michigan State University Department of Animal Science. We thank Dr. Myron Crawford, director of the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University, and Nancy Williams for the protein sequencing. This investigation was supported by USPHS Research Grants DE12769 and DE15846 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 29892, USA.
Received for publication April 20, 2007.
Revision received May 29, 2007.
Accepted for publication June 5, 2007.
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Journal of Dental Research, Vol. 86, No. 10,
962-967 (2007)
DOI: 10.1177/154405910708601009
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ABSTRACT
INTRODUCTION