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J Dent Res 86(2):153-157, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Processing of Ameloblastin by MMP-20

T. Iwata1,3, Y. Yamakoshi1, J.C.-C. Hu2, I. Ishikawa3, J.D. Bartlett4, P.H. Krebsbach1, and J.P. Simmer1,*

1 Department of Biologic and Materials Sciences and
2 Department of Orthodontics and Pediatric Dentistry, Dental Research Lab, University of Michigan School of Dentistry, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA;
3 Department of Hard Tissue Engineering, Tokyo Medical and Dental University, Tokyo, Japan; and
4 Department of Cytokine Biology, The Forsyth Institute Boston, MA, USA

* corresponding author, jsimmer{at}umich.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro2, Gln130, Gln139, Arg170, and Ala222. This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr223, as well as the 17-kDa (Val1-Arg170) and 15-kDa (Val1-Gln130) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.

KEY WORDS: enamelysin • ameloblastin • AMBN • MMP-20 • enamel • amelogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cleavage of enamel proteins by proteases is required for proper dental enamel formation (Bartlett and Simmer, 1999; Simmer and Hu, 2002). Two enzymes are involved: enamelysin (MMP-20) (Bartlett, 2004) and kallikrein 4 (KLK4) (Simmer, 2004). Enamelysin is the early enzyme, being expressed throughout the secretory stage and into earlier maturation, while KLK4 expression starts in the transition stage and continues throughout maturation (Hu et al., 2002; Simmer et al., 2004). Defects in MMP20 and KLK4 cause autosomal-recessive amelogenesis imperfecta (ARAI) in humans (Hart et al., 2004; Kim et al., 2005; Ozdemir et al., 2005). In vitro digestion of amelogenin by enamelysin yields the same cleavage products as those isolated from developing teeth (Ryu et al., 1999), while KLK4 digestion of amelogenin in vitro is more complete and does not yield the amelogenin peptides found in secretory enamel (Ryu et al., 2002).

Amelogenin (AMEL) is the most abundant protein in secretory-stage enamel, while ameloblastin (AMBN) is the most abundant non-amelogenin enamel protein (Fincham et al., 1999). Amelogenin is smaller and simpler than ameloblastin. Amelogenin is not glycosylated, and its only post-translational modification (other than proteolysis) is a single phosphorylation at Ser16 (Fincham et al., 1994). The major porcine amelogenin has 173 amino acids (P173) and is well-characterized, since it can be isolated from developing teeth (Yamakoshi et al., 2004) or expressed in bacteria (rP172). In contrast, the major porcine ameloblastin isoform has 395 amino acids (Hu et al., 1997), and contains diverse post-translational modifications, including sulfated O-linked glycosylations (Ser86 and Thr361) (Yamakoshi et al., 2001), hydroxylated prolines (Pro11 and Pro324), and phosphorylations (Ser17 and Thr251) (Hu et al., 2005). Uncleaved ameloblastin in developing teeth has never been isolated, but it has been identified in Western blots as a trace component (Murakami et al., 1997; Uchida et al., 1998). Recently, recombinant mouse ameloblastin has been expressed in eukaryotic cells and purified (Zeichner-David et al., 2006). For historical reasons concerning the independent cloning of AMBN cDNA homologues from the rat and pig, ameloblastin is variously described as ameloblastin, amelin, or sheathlin (Fincham et al., 1999).

Characterization of AMBN at the protein level has necessarily focused on its proteolytic cleavage products, which were first investigated in the porcine animal model (Fukae and Tanabe, 1987a,b; Shimizu, 1984). Antibodies against the porcine AMBN N-terminal region displayed a honeycomb pattern in immunohistochemical analyses of developing enamel (Uchida et al., 1991), indicating that it concentrated in the sheath space. Lectin-binding studies have suggested that the 13- to 17-kDa N-terminal AMBN cleavage products are O-glycosylated (Akita et al., 1992). In contrast to the relatively stable N-terminal cleavage products of ameloblastin, its C-terminal cleavage products are short-lived and are observed only in the superficial enamel among the crystals, and not in the sheath space (Murakami et al., 1997). Uncleaved rat ameloblastin is soluble in simulated enamel fluid, while its cleavage products are less soluble and more difficult to extract from the mineralized matrix (Brookes et al., 2001). The AMBN N-terminal region is basic, while the C-terminal region is acidic. Proteolysis disconnects these two regions, allowing them to segregate into different sub-compartments in the developing enamel and potentially serve different functions.

Characterization of enamel matrix extracts has now identified proteolytic cleavage sites in porcine ameloblastin following Asn31, Gln130, Arg170, Ala222, Gly300 Arg319, and Ala342 (Uchida et al., 1995; Hu et al., 1997; Fukae et al., 2006; Yamakoshi et al., 2006). This incomplete but adequate knowledge of the in vivo pattern of AMBN cleavages allows us to test the hypothesis that MMP-20 is the enzyme that processes ameloblastin during the secretory stage of amelogenesis.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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.

AMBN Expression Construct
Porcine ameloblastin cDNA was amplified from enamel epithelial tissue cDNA with the oligonucleotide primers 5'-atcaccaggccctgagagca and 5'-gggctcttggaaacgccacg. The amplification products were cloned into pEF6/V5-His-TOPO (Invitrogen, Carlsbad, CA, USA). Recombinant pig ameloblastin was secreted as a 440-amino-acid protein with a 45-amino-acid combined V5-epitope and poly-histidine tag at the C-terminus (KGNSADIQHSGGRSSLEGPRFEGKPIPNPLLGLDSTRTGHH HHHH). The DNA sequences of the PCR insert and the adjacent vector at the junctions were confirmed by DNA sequence analyses.

Transient Transfection
The FreestyleTM 293F cell line, derived from a human kidney cell line (Invitrogen), was used for transient expression. The cell line has been adapted to grow in suspension in FreestyleTM Expression Medium (Invitrogen). The expression plasmid was introduced with the use of a cationic lipid-based transfection reagent (293fectinTM; Invitrogen). For each transfection reaction, 30 µg of purified plasmid and 40 µL of the transfection reagent (293fectinTM) were mixed with 1 mL of Opti-MEM® (Invitrogen), respectively, and incubated for 5 min at room temperature before being combined and allowed to form complexes for 20 min at room temperature. The DNA-293fectin complexes were added to 30 x 106 cells seeded in a 125-mL Erlenmeyer flask and incubated on the orbital shaker at 130 rpm at 37°C, 8% CO2. In the final procedure, the supernatant was harvested 48 hrs post-transfection by centrifugation (3000 g, 15 min at 4°C).

Purification of rpAMBN
The rpAMBN was purified from supernatants of transfected cells by immobilized metal ion-affinity chromatography on Co2+-charged resin (TalonTM Resin; BD Biosciences/Clontech, Palo Alto, CA, USA). The supernatant containing rAmeloblastin-V5-His was collected and bound to resin for 20 min at room temperature. The column was washed 3x with 50 mM sodium phosphate, 300 mM NaCl, pH 7.0, and bound proteins were eluted with 50 mM sodium phosphate, 300 mM NaCl, 150 mM imidazole, pH 7.0. Fractions of 0.5 mL were collected and analyzed by SDS-PAGE and Western blotting. The elutant was dialyzed and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC), with a Poros column (4.6 x 10 cm; PerSeptive Biosystems, Hertford, UK). The column was equilibrated with 0.05% trifluoroacetic acid (TFA) and eluted with a linear acetonitrile gradient (0–80%) containing 0.05% TFA at a flow rate of 1.0 mL/min at room temperature.

Expression and Purification of Recombinant Porcine MMP-20
Recombinant pig enamelysin (rpMMP-20) was expressed from the pPROEX-1 vector (Life Technologies, Carlsbad, CA, USA) in E. coli strain XL1-Blue (Stratagene, La Jolla, CA) (Bartlett et al., 1998). The rpMMP-20 with an N-terminal histidine tag was purified in the TALON purification system. The rpMMP-20 was dialyzed against de-salting buffer (50 mM Tris-HCl, pH 7.4) and activated by incubation for 24 hrs in de-salting buffer containing 5 mM CaCl2 at 37°C. The supernatant was concentrated by means of a Centriprep 10 spun filter (Amicon, Beverly, MA, USA) and stored at –20°C.

Antibody Production
Two 14-amino-acid segments (M63RPREHETQQYEYS and Q381QPQIKRDAWRFQE) from the porcine AMBN-deduced amino acid sequence were selected based upon their overall antigenic index, favorable secondary structure, peptide location, and cross-species reactivity. The peptides were conjugated to the carrier protein KLH. Antibodies were generated in rabbits according to a protocol that included 3 immunizations, 1 test bleed, a fourth immunization, and a final bleed. Specific anti-peptide antibodies were purified from the final bleed in an affinity column containing the immobilized unconjugated AMBN peptide, and ELISA-tested before being used for Western blot analyses. The antibodies were designated AMBN-63 and AMBN-381, respectively.

SDS-PAGE and Western Blotting
Samples were applied to pre-cast bis-(2-hydroxylethyl) aminotris (hydroxymethyl) methane (bis-tris) NuPAGE® gels (Invitrogen) run with MES buffer, then stained with Coomassie brilliant blue (CBB), stains-all (Sigma, St. Louis, MO, USA), or Pro-Q® Emerald 300 Glycoprotein Gel Stain Kit (Invitrogen). After electrophoretic transfer of the proteins onto a nitrocellulose membrane (0.4 µm, HybondTM-ECL; GE Healthcare Biosciences: Little Chalfont, UK), blocking was performed with 5% nonfat dry milk for 1 hr, followed by incubation with a 1:1000 dilution of AMBN-63 antibody or a 1:5000 dilution of AMBN-381 antibody in Tween Tris-buffered saline (TTBS) containing 5% milk for 1 hr. The blots were washed 3x for 20 min each in TTBS and incubated with anti-rabbit IgG secondary antibody (BioRad, Hercules, CA, USA) at a dilution of 1:10,000. Immunoreactive proteins were detected by enhanced chemiluminescence (ECL Plus; GE Healthcare Biosciences).

Proteolysis of AMBN by MMP-20 in vitro
The rpAMBN was incubated with rpMMP-20 at an enzyme-to-substrate ratio of 1:100 for various time intervals in 10 mM Tris-HCl with 10 mM Ca2+, pH 7.4. Proteolytic activity was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) with CBB or silver staining, and by Western blotting with the 2 AMBN-specific antibodies (AMBN-63 and AMBN-381). Digested AMBN samples were analyzed by Edman degradation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Following purification by affinity chromatography and RP-HPLC, recombinant porcine ameloblastin was characterized on 4–12% NuPage gels stained with Coomassie brilliant blue (CBB), stains-all, a glycoprotein staining kit, and immunostained in Western blot analyses with 2 AMBN-specific anti-peptide antibodies and a monoclonal antibody specific for the V5 epitope fused to the AMBN C-terminus (Fig. 1AGo). The rpAMBN expressed in 293F mammalian cells was CBB- and stains-all-positive, glycosylated, and migrated as a wide band at 65 kDa. The rpAMBN was immunodetected by both AMBN anti-peptide antibodies and the V5 antibody, while Edman sequencing of the purified rpAMBN yielded the porcine AMBN N-terminal sequence (V1XAFPXQ), confirming that the recombinant protein was full-length. These results showed that rpAMBN was sufficiently pure, intact, and modified following translation, to serve as a practical substitute for native AMBN, and could be used as a meaningful substrate for digestion by enamelysin. The porcine MMP-20 catalytic domain was prepared as described previously and corresponded to a 25-kDa band on a casein zymogram (Fig. 1BGo) (Bartlett et al., 1998; Ryu et al., 1999).


Figure 1
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  		Figure 1. Purified rpAMBN expressed in 293F mammalian cells and activated rpMMP-20. (A) The rpAMBN migrated as a smeared band at 65 kDa on 4–12% NuPage gels. The rAMBN reacted with Coomassie brilliant blue (CBB), stains-all (SA), and a glycoprotein gel stain (Pro-Q). The rpAMBN reacted with 3 antibodies: 2 AMBN anti-peptide antibodies (AMBN-63 and AMBN-381) and a monoclonal antibody specific for the V5 epitope (V5) fused to the AMBN C-terminus. (B) The 25-kDa rpMMP-20 catalytic domain used to digest rpAMBN is indicated by an arrowhead on a casein zymogram (Zym).

 
The rpAMBN was digested by rpMMP-20 for up to 27 hrs. A time-course for the digestion was analyzed by SDS-PAGE stained with CBB and silver (Fig. 2AGo), and by Western blot analyses with the AMBN anti-peptide antibodies (Fig. 2BGo). The 65-kDa starting material was eliminated during the time-course. The Western blot analyses showed that rpMMP-20 cleaved intact rpAMBN nearer to its N-terminus, generating 3 distinct lower-molecular-weight N-terminal products migrating at approximately 19, 18, and 17 kDa, and a group of larger C-terminal products ranging from 40 to 50 kDa. The pattern of AMBN cleavage products generated in the in vitro digestion was similar to that observed in Western blot analyses of AMBN cleavage products in porcine secretory-stage enamel extracts (Uchida et al., 1998).


Figure 2
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  		Figure 2. Time-course for the digestion of rpAMBN by rpMMP-20. (A) 4–12% NuPage gels stained with CBB (left) and silver (right). (B) Western blots of a similar time-course immunostained with the AMBN N-terminal (AMBN-63) and C-terminal (AMBN-381) anti-peptide antibodies. The lanes are marked according to the times (in hrs) of the digestions. An asterisk marks negative controls with no rpMMP-20 added. The rpAMBN incubated without rpMMP-20 was stable and was undigested for the duration of the time-course, indicating that rpMMP-20 was solely responsible for the AMBN cleavages in the experimental conditions. Western blot analyses with region-specific anti-peptide antibodies showed that the smeared band at 48 kDa did not contain the AMBN N-terminus, while the smaller bands at 16, 18, and 19 kDa did.

 
For precise definition of the AMBN sites cleaved by rpMMP-20, the 0.5- and four-hour AMBN digestions were separated by SDS-PAGE and transblotted to a membrane, and selected bands were excised for N-terminal sequence analyses (Fig. 3Go). Five AMBN cleavage sites catalyzed by MMP-20 in vitro were identified: after Pro2, Gln130, Gln139, Arg170, and Ala222. Three of these sites are known to occur in vivo: after Gln130, Arg170, and Ala222. In addition, the amino acid context of the Gln139 site (Q/QV) was identical to the Gln130 site, suggesting that this site might also be cleaved in vivo, but the in vivo cleavage products starting at this site have not been identified. A summary of AMBN cleavages identified by the characterization of AMBN-derived polypeptides in porcine enamel extracts and in the MMP-20 digestion in vitro is shown in Fig. 4Go.


Figure 3
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  		Figure 3. N-terminal sequence characterization of rpAMBN polypeptides generated by rpMMP-20 digestion. The rpAMBN digestions after 0.5 and 4 hrs were separated on 4–12% NuPage gels, transblotted to a membrane, and lightly stained with CBB; 7 strips of membrane were analyzed by N-terminal sequencing. CBB-stained replica gels of the ones used for this analysis are shown here. Arrowheads indicate the positions of the bands excised for analysis. The band labels (i.e., S65, etc.) indicate the apparent molecular weight of the excised protein. The numbers at the start of the protein sequences indicate the number of the first amino acid in the 365-amino-acid porcine AMBN sequence. When more than one sequence was obtained for a single strip of membrane, the separate sequences are indicated by a, b, or c.

 

Figure 4
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  		Figure 4. Ameloblastin cleavage sites in vivo and in vitro. Analysis of porcine ameloblastin cleavage products isolated from developing teeth show that ameloblastin is cleaved in vivo after Asn31 (a), Gln130 (b), Arg170 (c), Ala222 (d), Gly300 (e), Arg319 (f), and Ala342 (g). Three AMBN cleavage sites targeted by MMP-20 in vitro are identical to known in vivo cleavage sites (after Gln130, Arg170, and A222). Two sites that were cleaved in vitro have not been identified in vivo: Pro2 (h*) and Gln139 (i*). The context of the Gln139 site (Q:QV) is identical to the Gln130 site, suggesting that this site is likely to be cleaved in vivo, but in vivo cleavage products starting with this cleavage have not yet been described. Key: The in vivo and in vitro AMBN cleavage sites are marked by arrows. The arrows are boxed for the sites cleaved in vitro.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Enamelysin cleaved AMBN in vitro at sites identical to those sites used in vivo to generate AMBN cleavage products that had been isolated from secretory-stage enamel, providing strong evidence that MMP-20 processes AMBN during amelogenesis. Perhaps the most important finding was that MMP-20 catalyzed the cleavages (after Gln130 and Arg170) that generate the AMBN cleavage products that segregate into the sheath space. As yet, we have not produced evidence that MMP-20 is responsible for AMBN cleavages near the C-terminus. We believe that this is due to our failing to identify these cleavages, not to their being generated by a different enzyme in vivo.

The roles played by ameloblastin during enamel formation are still largely a matter of speculation. The best evidence that ameloblastin is critical for proper dental enamel formation comes from the Ambn–/– mice, which produced virtually no enamel layer (Fukumoto et al., 2004), while the Ambn+/– mice had no detectable dental phenotype. No disease-causing AMBN mutations have yet been described in humans (Kim et al., 2006), but the knock-out mice findings suggest that enamel phenotypes caused by AMBN mutations would probably be recessive, and hence rare. Ameloblasts in the Ambn–/– mice detach from the matrix surface at the secretory stage and lose cell polarity, suggesting that ameloblastin is a key adhesion molecule for enamel formation. However, the enamel surface to which the ameloblasts are proposed to attach is itself highly defective, which might interfere with attachment even if ameloblastin was not directly involved. In vitro assays of ameloblastin as a cell attachment protein are inconsistent, being highly dependent upon the cell type tested.

Because ameloblastin N-terminal cleavage products accumulate in the sheath space, it was originally proposed (before the first cDNA was cloned and the ameloblastin designation adopted) that this protein was part of a new family of proteins, designated as ’sheath proteins’ (Uchida et al., 1995). The sheath proteins were thought to form a barrier that maintained rod-interrod boundaries during the secretory stage (Hu et al., 1997). Consistent with this hypothesis is the finding that enamel rod integrity is disrupted by the inhibition of Ambn expression by ribozymes (Lyngstadaas, 2001).

One feature of the enamel phenotype in Mmp20–/– mice is that the decussating enamel rod pattern is absent (Caterina et al., 2002), indicating that matrix protein processing by enamelysin is necessary to establish or maintain prism definition. In this study, we demonstrated that MMP-20 generates the 17- and 15-kDa AMBN cleavage products that concentrate in the sheath space during the secretory stage of amelogenesis, thus linking independent observations that AMBN and MMP-20 are required for proper prism organization. Ameloblastin is a bipolar molecule. Cleavage by MMP-20 disconnects basic N-terminal AMBN fragments (that concentrate in the sheath space) from acidic C-terminal polypeptides, which are more rapidly degraded, but which can be shown to concentrate in the rods near the enamel surface. We concluded that one function of MMP-20 is to generate AMBN sheath proteins that maintain rod boundaries, presumably by forming a physical barrier to their fusion during the secretory stage of amelogenesis.


   ACKNOWLEDGMENTS
 
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.

Received July 19, 2006; Last revision September 12, 2006; Accepted October 31, 2006


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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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K. Kobayashi, Y. Yamakoshi, J.C.-C. Hu, K. Gomi, T. Arai, M. Fukae, P.H. Krebsbach, and J.P. Simmer
Splicing Determines the Glycosylation State of Ameloblastin
J. Dent. Res., October 1, 2007; 86(10): 962 - 967.
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