JDR Woodhead Publishing
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, L.
Right arrow Articles by Moradian-Oldak, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, L.
Right arrow Articles by Moradian-Oldak, J.
J Dent Res 81(10): 664-667, 2002
© 2002 International and American Associations for Dental Research

RAPID COMMUNICATION
Biological

Assessment of Enamelysin (MMP-20) Selectivity to Three Peptide Bonds on Amelogenin Sequence

L. Wang, and J. Moradian-Oldak*

Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar Street, Los Angeles, CA 90033, USA;

* corresponding author, joldak{at}usc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have highlighted the potential role of the metalloproteinase enamelysin (MMP-20) in controlling some of the most critical stages during enamel development. This study was aimed to assess the selectivity of enamelysin to the three most abundant cleavage sites on the amelogenin sequence, and to gain insight into the factors that control the pattern of amelogenin processing during enamel mineralization. Three deca-peptides with sequences based on pig amelogenin and including the proteolytic cleavage sites W/L, S/M, and P/A were synthesized as substrates. Statistical analysis revealed no significant differences in the rates of cleavage among the three peptides, indicating comparable selectivity of enamelysin for these peptide bonds. Considering the selective appearance of amelogenin proteolytic products, we suggest that amelogenin folding and assembly are the primary factors in controlling the pattern of its proteolysis during the secretory stage of enamel development.

KEY WORDS: enamel • amelogenin • enamelysin (MMP-20) • metalloproteinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two different types of proteinases, which have been identified and cloned, have been reported to become active at different developmental stages during enamel mineralization (Bartlett and Simmer, 1999). The metalloproteinase enamelysin (MMP-20) has maximum expression during the secretory and early transition stages, where stepwise processing of enamel proteins takes place (Bartlett et al., 1998). Among numerous extracellular matrix proteins studied to date — namely, aggrecan (Stracke et al., 2000), ameloblastin, enamelin, dentin sialophosphoproteins (Bègue-Kirn et al., 1998), and amelogenins—the latter is the most clearly defined substrate of enamelysin for which the proteolysis pattern and cleavage sites have been systematically studied (Ryu et al., 1999; Moradian-Oldak et al., 2001). In vitro proteolysis experiments with recombinant pig amelogenin and enamelysin have shown not only that enamelysin cleaves amelogenin at its carboxy-terminal region, creating the pig "20K" and "23K" products, but also that the proteinase cleaves amelogenin at the amino-terminal region, generating the tyrosine-rich amelogenin polypeptide 45 residues (TRAP) (Fincham et al., 1981; Fincham and Moradian-Oldak, 1996; Ryu et al., 1999). In contrast, it has been shown that the most stable product formed during the early secretory stage of porcine amelogenesis is the "20K" amelogenin, which includes the N-terminal TRAP sequence (Yamakoshi et al., 1994). These observations indicate that cleavages at S148/M149 or P162/A163 (creating the "20K" and "23K", respectively) occur before the cleavage at W45/L46 (creating the TRAP polypeptide). Our recent in vitro controlled proteolysis experiments have demonstrated that the primary cleavage on recombinant pig amelogenin by recombinant enamelysin (rpMMP-20) occurs at the S148/M149 site. It is not known what factors control the selectivity of enamelysin in cleaving only certain regions of the amelogenin molecule. The objective of this study was to assess the selectivity of enamelysin in cleaving the three most abundant cleavage sites on the amelogenin sequence, and to define the factors that are responsible for the proteolytic pattern during the secretory and early transition stages of enamel development. We hypothesize that amelogenin folding and nanosphere assembly determine the pattern of proteolytic activitiy by enamelysin (Moradian-Oldak et al., 1994, 2001; Fincham et al., 1995). Three deca-peptides with sequences based on pig amelogenin and including the proteolytic cleavage sites at the amino and carboxy-terminals were synthesized. The use of polypeptides as substrates eliminated the factor of substrate folding and assembly and allowed for direct and comparative measurement of their hydrolysis by enamelysin. In the case of amelogenin, this substrate assembly results in the formation of nanospheres that may remarkably affect the action of proteinases by exposing certain domains, which will be more accessible to proteolytic activity (Moradian-Oldak et al., 2001).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Enamelysin Expression (rpMMP-20)
The recombinant enamelysin plasmid (provided by Dr. Jim Simmer) was transformed into competent E. coli XL1-Blue cells (Stratagene, La Jolla, CA, USA). Recombinant pig enamelysin (rpMMP-20) was expressed, and the active product of interest was purified with use of the His-TRAP chelating nickel affinity column (Pharmacia, Piscataway, NJ, USA) following the protocol described by Moradian-Oldak et al.(2001) and Ryu et al.(1999).

Peptide Design, Synthesis, and Purification
Three deca-peptides with sequences based on pig amelogenin and including the proteolytic cleavage sites producing the Tyrosine Rich Amelogenin Polypeptide (TRAP), the P148 (20 kDa), and the P162 (23 kDa) were synthesized and used as substrates for examination of the comparative rates of their hydrolysis by recombinant enamelysin (rpMMP-20) (Fincham et al., 1981; Yamakoshi et al., 1994). The peptides were synthesized at the University of Southern California Microchemical Core Laboratory by means of an applied Biosystems Model 430A one-column peptide synthesizer with the modified Merrifield procedure (Merrifield, 1986), then purified by reverse-phase HPLC [Vydac C18 column (218TP52)] and characterized by electrospray ionization mass spectroscopy at the Mass Spectroscopy Facility at the University of California at San Francisco as described previously (Moradian-Oldak et al., 2001). The three synthetic deca-peptides PMGGWLHHQI (GWL), PLEAWPATDK (WPA), and LPPMFSMQSL (FSM) were characterized by comparison of the measured and theoretical mass values (Table 1Go). The amino acid sequence of pig amelogenin, with the peptides highlighted, is shown in the footnote to Table 1Go.


View this table:
[in this window]
[in a new window]
  		Table 1. Computed (Comp) and Measured (Meas) Masses of the Three Synthetic Deca-peptidesa Used in This Study
 
Peptide Hydrolysis by rpMMP-20
Series of experiments with different enzyme-to-substrate ratios have been performed for determination of optimal conditions so that digestion can be completed in a reasonable period of time. To test the activity of enamelysin on 3 peptides, we conducted proteolysis experiments by incubating each peptide (1 mg/mL) with enamelysin at a 1:200 (W/W) enzyme-to-substrate ratio, but for different time intervals (1-24 hrs). All experiments were performed at 37°C, and the following buffer was used: 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 150 mM NaCl, and 50 pM ZnCl2 (Li et al., 2001). Peptide digestion was terminated by rapid freezing of the incubated samples and storage at -20°C.

Comparative Analysis of Digest Experiments with Reverse-phase HPLC
Proteolytically digested peptide samples were analyzed by reverse-phase HPLC (RP-HPLC) on a Vydac C18 column (218TP52). Peptides were eluted by means of a linear gradient of 10-80% B (60% acetonitrile in 0.1% trifluoroacetic acid over 60 min. Elution was monitored at 220 nm (Fig. 1Go). We compared the rates of hydrolysis of the 3 peptides by presenting the ratio of substrate at time t to substrate at time zero (St/S0) as a function of time. These were evaluated based on the absorbance peaks corresponding to the substrate. Hydrolysis experiments were repeated at least 10 times for each polypeptide, and most of the data points in Fig. 2Go are presented as means of 3-8 samples + standard deviation. Regression analysis of the data was performed by Microsoft Excel. Statistical analysis for the evaluation of differences between peptides was performed with a two-way ANOVA with cells that had unequal sample sizes (n) that ranged from 3 to 8. The ratio data were arcsin-transformed prior to analysis so that the data distributions would satisfy the underlying assumptions of ANOVA, namely, normality and variance homogeneity (Sokal and Rohlf, 1981).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. Typical reverse-phase HPLC profiles of hydrolysis of the 3 synthetic deca-peptides WPA, GWL, and FSM by enamelysin, from zero to 24 hrs.

 


View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Comparative cleavage rates of the 3 deca-peptides — FSM {square}, GWL •, and WPA {blacktriangleup} — by recombinant pig enamelysin (rpMMP-20). The rates, which are represented as the ratio of substrate at time t to substrate at time zero (St/S0) as a function of time, are exponential. The data presented in the curve are the means and standard deviations of from 3 to 8 determinations (except 5 hrs of FSM, 8 hrs of GWL, and 12 hrs of FSM, GWL, and WPA, each of which represents one determination). Statistical analysis (two-way ANOVA) revealed no significant difference among the rates of hydrolysis of the 3 peptide bonds by rpMMP-20. Cells had unequal sample sizes (n = 3-8). F = 2.27 (P > 0.10) for the differences among the peptides regardless of time, and F = 84.86 (P < 0.001) for the differences among times regardless of peptide.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
rpMMP-20 Hydrolyzed Each Deca-peptide into Two Truncated Fragments
Fig. 1Go represents typical HPLC profiles of the hydrolysis of the deca-peptides by rpMMP-20 from 0 to 24 hrs, demonstrating the progress of the hydrolysis reactions. In the case of GWL, 2 truncated fragments with masses corresponding to the sequences PMGGW and LHHQI were detected, indicating that cleavage occurred at the W/L peptide bond (Fig. 1Go, Table 2Go). In the case of WPA, the mass of one product corresponding to the sequence PLEAWP was detected, indicating cleavage at the P/A site (Table 2Go). We presume that the peptide ATDK was too small and hydrophilic to bind to the column and could be lost at the very beginning of sample injection (Fig. 1Go). Two truncated products with masses corresponding to the sequences LPPMFS and MQSL were detected in the case of the FSM deca-peptide, indicating cleavage at the S/M peptide bond (Fig. 1Go, Table 2Go). The quantities of these products increased and remained stable until 24 hrs, when the quantity of the substrate decreased drastically and almost disappeared (Fig. 1Go).


View this table:
[in this window]
[in a new window]
  		Table 2. Measured (Meas) and Computed (Comp) Mass Analysis of the Hydrolysis Products of the Synthetic Peptides GWL, WPA, and FSM, Indicating that, under the Experimental Conditions Used in this Study, Cleavages by rpMMP-20 Occur at W/L, P/A, and S/M, Respectively
 
No Significant Differences were Found in the Rates of Cleavage among the Three Peptide Bonds by rpMMP-20
We evaluated the rates of hydrolysis of the 3 deca-peptides by enamelysin and compared them by plotting St/S0 as a function of time (Fig. 2Go). As seen in Fig. 2Go, in all 3 cases, the trend for substrate hydrolysis was exponential, which was found to be a good fit as judged by the R-square values. The half-life values (t1/2, where St/S0 is 0.5) calculated based on the exponential functions were 2.4 hrs for FSM, 3.1 hrs for GWL, and 4.6 hrs for WPA (Fig. 2Go). Although the rate of cleavage at P/A appears to be slightly slower, statistical analysis revealed no significant difference among the 3 peptides regarding degradation (F = 2.27; P > 0.10). There is, as expected, a highly significant difference with time, indicating the progress of degradation (F = 84.86; P < 0.001). There was no interaction between peptide and time with respect to degradation. That is, the degradation with increasing time is the same for all 3 peptides.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The localization of enamelysin (MMP-20) in the inner enamel layer (Fukae et al., 1998), its maximum mRNA expression at the secretory stage of enamel development (Bartlett et al., 1998), and the abnormal enamel formation in the recent transgenic enamelysin-deficient mice studies (Bartlett JD, personal communication)—all support the hypothesis that enamelysin is essential for normal enamel formation. In addition, the presence of enamelysin in odontoblasts and in various tumors (Bègue-Kirn et al., 1998) suggests that the enzyme may have important functions in dentin development and possibly in the pathogenesis of odontogenic tumors (Takata et al., 2000).

In vitro studies have shown that rpMMP-20 cleaves pig amelogenin at S/M, F/S, P/L, P/A, A/L, P/M, S/Q, and W/L sites (Ryu et al., 1999). The present study was aimed to investigate whether enamelysin showed any preference in cleaving certain sites over others in the amelogenin sequence. We compared the rates of hydrolysis of the 3 most abundant cleavage sites, W/L, P/A, and S/M. Based on the exponential curves in Fig. 2Go and statistical analysis (two-way ANOVA), no significant differences were found in the rates of hydrolysis of the 3 peptide bonds, indicating comparative selectivity of rpMMP-20 for cleaving these locations.

It appears that the situation in vivo is different when these 3 sites are cleaved at different rates. The cleavage at the c-terminal (S/M) results in the formation of the most abundant product in the enamel extracellular matrix ("20K"), when the N-terminal W/L results in the formation of the TRAP molecule, which is more abundant during the maturation stage of amelogenesis following c-terminal cleavage. A dosimetric analysis of the SDS-PAGE pattern of amelogenin extract from the developing pig enamel has revealed the presence of 50% of the "20K" (result of S/M cleavage), while only 10% of the extract is 23K (result of P/A cleavage) (Wen et al., 1999). This pattern can also be created in vitro with recombinant full-length pig amelogenin (rP172) (unpublished data). Based on analysis of the present data, we speculate that, during amelogenesis, substrate conformation and assembly are the primary factors that control the pattern of amelogenin processing. The idea that nanosphere assembly can control the pattern of proteolysis was supported by our recent controlled proteolysis experiments, which showed that, under limited proteolytic conditions, the FSM locus was the target of other proteinase activities, including chymotrypsin and thermolysin, suggesting that this region is exposed to the surfaces of amelogenin nanospheres (Moradian-Oldak et al., 2001).

Substrate specificity can be the second important factor, and some common features can be found among the cleavage sites studied. It is noteworthy to highlight the fact that W/L and S/M have proline residues in their P5 location, while P/A has proline in the P6 location (see amelogenin sequence in the footnote to Table 1Go). This may explain the slightly slower rate of hydolysis at the P/A location. Mutation of a proline at location 5 of the W/L site by methionine in a synthetic polypeptide has remarkably inhibited its rate of hydrolysis by enamelysin, indicating that the presence of this proline is critical for normal cleavage by enamelysin (Li et al., 2001). Similarly, the presence of proline in the P3 location was found to be necessary for the activity of gelatinase-3 (MMP-13) (Deng et al., 2000). It is of particular interest to note that, among 7 cleavage sites on pig amelogenin sequences identified to be targeted by rpMMP-20, 4 of them have proline on their P4 site, 2 have proline on their p3, and one on p3 (Ryu et al., 1999). Further systematic studies are required to determine the substrate specificity of MMP-20.


   ACKNOWLEDGMENTS
 
We thank Mr. David Maltby from the University of California at San Francisco Mass Spectrometry Facility for analyzing masses of the polypeptides. We are grateful to Dr. Jim Simmer for kindly providing the plasmid for the expression of recombinant rpMMP-20. We appreciate the useful advice of Prof. Michael Melnick in performing the statistical analyses. This research is supported by NIDCR-NIH grants DE12350 and DE13414 (JMO).

Received June 7, 2002; Last revision July 17, 2002; Accepted July 19, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bartlett JD, Simmer JP (1999). Proteinases in developing dental enamel. Crit Rev Oral Biol Med 10:425–441.[Abstract/Free Full Text]

Bartlett JD, Ryu OH, Xue J, Simmer JP, Margolis HC (1998). Enamelysin mRNA displays a developmentally defined pattern of expression and encodes a protein which degrades amelogenin. Connect Tissue Res 39:101–109.[Medline]

Bègue-Kirn C, Krebsbach PH, Bartlett JD, Butler WT (1998). Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth specific molecules that are distinctively expressed during murine dental differentiation. Eur J Oral Sci 106:963–970.[Medline]

Deng SJ, Bickett DM, Mitchell JL, Lambert MH, Blackburn RK, Carter HL 3rd, et al. (2000). Substrate specificity of human collagenase 3 assessed using a phase-displayed peptide library. J Biol Chem 275:31422–31427.[Abstract/Free Full Text]

Fincham AG, Moradian-Oldak J (1996). Comparative mass spectrometric analyses of enamel matrix proteins from five species suggest a common pathway of post-secretory proteolytic processing. Connect Tissue Res 35:151–156.[Medline]

Fincham AG, Belcourt AB, Termine JD, Butler WT, Cothran WC (1981). Dental enamel matrix: sequences of two amelogenin polypeptides. Biosci Rep 1:771–778.[Medline]

Fincham AG, Moradian-Oldak J, Diekwisch TGH, Lyaruu DM, Wright JT, Bringas P Jr, et al. (1995). Evidence for amelogenin "nanospheres" as functional components of secretory-stage enamel matrix. J Struct Biol 115:50–59.[Medline]

Fukae M, Tanabe T, Uchida T, Lee SK, Ryu OH, Murakami C, et al. (1998). Enamelysin (matrix metalloproteinase-20): localization in the developing tooth and effects of pH and calcium on amelogenin hydrolysis. J Dent Res 77:1580–1588.[Abstract/Free Full Text]

Hu CC, Bartlett JD, Zhang CH, Qian Q, Ryu OH, Simmer JP (1996). Cloning, cDNA sequence, and alternative splicing of porcine amelogenin mRNAs. J Dent Res 75:1735–1741.[Abstract/Free Full Text]

Li W, Gibson CW, Abrams WR, Andrews DW, DenBesten PK (2001). Reduced hydrolysis of amelogenin may result in x-linked amelogenesis imperfecta. Matrix Biol 19:755–760.[Medline]

Moradian-Oldak J, Simmer JP, Lau EC, Sarte PE, Slavkin HC, Fincham AG (1994). Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering. Biopolymers 34:1339–1347.[Medline]

Moradian-Oldak J, Jimenez I, Maltby D, Fincham AG (2001). Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions. Biopolymers 58:606–616.[Medline]

Ryu OH, Fincham AG, Hu CC, Zhang C, Qian Q, Bartlett JD, et al. (1999). Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins. J Dent Res 78:743–750.[Abstract/Free Full Text]

Sokal RR, Rohlf FJ (1981). Biometry. The principles and practice of statistics in biological research. 2nd ed. Chapter 11. New York: W.H. Freeman and Company.

Stracke JO, Fosang AJ, Last K, Mercuri FA, Pendas AM, Llano E, et al. (2000). Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP). FEBS Lett 478:52–56.[Medline]

Takata T, Zhao M, Uchida T, Wang T, Aoki T, Bartlett JD, et al. (2000). Immunohistochemical detection and distribution of enamelysin (MMP-20) in human odontogenic tumors. J Dent Res 79:1608–1613.[Abstract/Free Full Text]

Wen HB, Moradian-Oldak J, Leung W, Bringas P Jr, Fincham AG (1999). Microstructures of an amelogenin gel matrix. J Struct Biol 126:42–51.[Medline]

Yamakoshi Y, Tanabe T, Fukae M, Shimizu M (1994). Porcine amelogenins. Calcif Tissue Int 54:69–75.[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, L.
Right arrow Articles by Moradian-Oldak, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, L.
Right arrow Articles by Moradian-Oldak, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
IADR Journals Advances in Dental Research ®
Journal of Dental Research ® Critical Reviews (1990-2004)