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DISCOVERY! |
Department of Developmental Biology, Harvard School of Dental Medicine, and Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA; JBartlett{at}forsyth.org
Martin A. Taubman, Associate Editor
KEY WORDS: cloning • enamel • matrix metalloproteinase
In this issue of the Journal of Dental Research, Ozdemir et al. report only the second case in which a mutation in the human enamelysin gene has been demonstrated to cause amelogenesis imperfecta (AI). This is an exciting development, especially for those involved in the original cloning of the enamelysin mRNA. This companion "Discovery!" article is for those curious about the means by which enamelysin was originally discovered. The following highlights are what occurred along the enamelysin discovery path, starting with a brief preliminary overview of the circumstances leading to my entry into dental research and employment at The Forsyth Institute, proceeding to and finishing with the cloning and characterization of the enamelysin mRNA. Also mentioned are the key individuals whose contributions were essential to the discovery of enamelysin.
As is common for many young scientists starting a career, my path was far more serpentine than straight. I was awarded a PhD in Cell and Molecular Biology for DNA repair research from the University of Vermont, my first Post-doctoral research position was in endoplasmic reticulum stress responses at the National Institute on Aging, while my second Post-doctoral research position was in MMP expression in wound repair at Massachusetts General Hospital. My first job was at a small biotechnology company focused on identifying new enzyme inhibitors. My supervisor at this last position was a former British police officer who had a firmly defined sense of right and wrong that I consistently failed to interpret properly. Then the day came when a vendor called to tell me that my recent order would not be shipped, because my company was refusing to pay its bills. From this, I judged it a proper time to initiate a job search! When a telephone call accidentally came from The Forsyth Institute to my place of work (I had listed the wrong home ‘phone number on my CV) to inquire about a job interview, the first question I asked was, "Is The Forsyth a place where I’ll have to write grants?" I had seen first-hand the suffering that can occur by the loss or rejection of a grant application, and I had no wish to bank my career on writing grants. The answer was "Yes", but I reluctantly took the job anyway, because my corporate employment situation was so tenuous.
Dr. Edgard C. Moreno became my supervisor at The Forsyth Dental Center, now The Forsyth Institute. He was bright, intelligent, and animated, and I had more respect for him than I ever let be known. Less than a month after I joined The Forsyth, Dr. Moreno sent me to visit Dr. James P. Simmer, who was then at the University of Texas, San Antonio, now at the University of Michigan, with instructions for making recombinant amelogenin for future crystal growth experiments. Jim was a dynamo of activity and talked with high running energy, and he took it upon himself to help me find a grant-worthy line of independent research. I was very willing to listen to Jim, since prior to entering The Forsyth, I had never previously cracked a dental journal, and I did not even know what an enamel organ was. Jim informed me that a metalloproteinase(s) was secreted into the forming enamel, and that, because of my previous MMP research experience, I should consider cloning an enamel resident proteinase mRNA.
So, in October of 1994, Jim Simmer and I went to the library and made copies of virtually every paper published on enamel proteinases. The papers filled just one large, green, ring-binder that still sits on a shelf in my office. We took the photocopies to his office, and I recall papers strewn haphazardly about his floor as we both read about enamel proteinases. The reading was difficult for me, and Jim was mostly unaware of that fact. I was introduced to confusing terms such as LRAP (leucine-rich amelogenin peptide), TRAP (tyrosine-rich amelogenin peptide), CAP (collagenase activator protein), rose diagrams (a means to quantify amino acid content), and unit cell (the smallest repeating unit of crystal). Furthermore, I had not met or even seen a single author from any of these papers. What was apparent, and a bit alarming, was that with a few notable exceptions, most authors were listed on one or two enamel proteinase papers and were never again published in this area. I would have been lost if it were not for Jim’s running commentary on the merits and implications of the papers he was reading.
The defining moment that set the stage for the discovery of enamelysin occurred in Jim Simmer’s office when he read aloud a passage from a publication by scientists who had performed a zymography enzyme analysis on extracted enamel proteins (Overall and Limeback, 1988). The passage stated: "However, the purification of the enamel serine proteinases is likely to be a difficult undertaking, given the extremely small amounts of these enzymes that were present in the maturation enamel...." Jim noted that this spoke against the traditional cloning methods of the time, where purified proteins were sequenced to identify the non-degenerate nucleotide positions of a codon within an mRNA sequence. Jim then looked at me and suggested that I design PCR primers to conserved regions of the MMP family to clone the intervening mRNA fragment, and then use the fragment to clone the full-length mRNA. I knew that this technique was feasible, since MMPs have conserved cysteine switch- and zinc-binding domains. I later learned that the proper term for this technique was "PCR-based homology cloning".
Prior to flying back to Boston, I spent Halloween night with Jim and his wife, Dr. Jan Hu, helping them dole out candy to passing goblins and witches. I later boarded the plane with the satisfaction of having established two new friendships and with the reassurance that I now had a very clear research focus.
When I presented the enamel proteinase research project to Ed Moreno, his reception of it was not overly enthusiastic. He made the valid point that cloning an MMP mRNA from the enamel organ did not necessarily mean that the cloned MMP was secreted into the enamel matrix. Although we did have some difficulties in communicating research ideas, because my molecular biology background sometimes clashed with his physical chemistry background, Ed never prevented me from pursuing my goal of cloning enamel proteinases. This was all the more notable, considering that he had paid for my trip to Texas so I could learn how to express recombinant amelogenin for crystal growth experiments—which was something that I never did! I owe Ed Moreno a debt of gratitude for his patience. I doubt that he or his closest colleagues ever realized how fearful I was at the time, and still am, of the grant-writing process. My graduate student advisor had lost his grants, and subsequently went into industry. I had made up my mind early on that I would not write grants for my and my family’s livelihood, which is why I began my career at a biotechnology company. If I was going to succeed at The Forsyth Dental Center, then every effort was going to be toward the goal of obtaining grant funding. I therefore pursued the cloning of enamel proteinases with a will. To the dismay of few people at The Forsyth, almost everything else was set aside.
Jim and I decided that the pig would be the animal of choice for the identification of proteinases, because unerupted porcine teeth have large enamel organs from which we could extract significant quantities of RNA. One of the first difficulties I had was that I did not know the location of the unerupted teeth within the porcine mandible. Dr. Ziedonis Skobe suggested that I get a pig jaw and have it x-rayed by our chief dental hygienist. I drove to a slaughterhouse, and, instead of coming away with just the mandible, I brought an entire pig head back to The Forsyth. The chief hygienist was somewhat taken aback by the pig head sitting on the lab bench, and she went upstairs to tell the other hygienists that she would be x-raying a real pig. The other hygienists immediately demanded to know to whom she was referring! Afterward, I brought back only mandibles.
When I brought the pig jaw back with me to The Forsyth, my fine unerupted molar dissection technique consisted of delicately placing a chisel on the lingual surface of the mandible and then slamming the chisel with a mini-sledgehammer. The vibration was such that everything else on the bench appeared to jump into the air with glee. I usually attracted a crowd. Dr. Christian Caron, who is a dentist and was a post-doctoral researcher in my laboratory, would later show me how to use a handpiece so that I would no longer chance crushing a valuable tooth bud.
Others also contributed during the early years prior to the discovery of enamelysin. Proper design of the degenerate PCR primers necessary for the first step in the PCR-based homology cloning procedures was essential to the success of the project. Dr. Floyd E. Dewhirst provided valuable information that led to successful primer design. Jim Simmer and personnel from Dr. Philip P. Stashenko’s laboratory also helped me along the way. Jim and I split the cost of making a porcine enamel organ-specific cDNA library, and Jim was consulted when technical difficulties arose. Phil’s group taught us non-radioactive cloning methodologies. As luck would have it, the first RT-PCR procedure I performed generated a band of the appropriate size for our GAPDH positive control; the second RT-PCR I performed yielded a porcine cDNA fragment of 401 bp with the highest identity (56%) to a portion of the chicken gelatinase A cDNA. My laboratory assistant, Jun Xue, used the porcine cDNA fragment to clone the full-length mRNA. This mRNA would eventually be named enamelysin, and Dr. J. Frederick Woessner would later officially designate enamelysin as matrix metalloproteinase-20 (MMP-20).
It was the spring of 1995, and we were in the process of cloning the full-length enamelysin cDNA. During this time, I attended the San Antonio AADR meeting. Both Ed Moreno and Jim Simmer took the time to introduce me to many of their colleagues. However, there was one scientist about whom I had a great concern and to whom I had not yet been introduced. This scientist was Dr. Pamela K. DenBesten. I had come from the secret and sometimes combative world of industrial science, and I had not yet learned how welcoming those in dental research could be. I was very much concerned that I had impinged on Dr. DenBesten’s area of research expertise, and that she would be upset with me when we announced the discovery of enamelysin. After all, she was a pioneer in the realm of enamel metalloproteinases. Pam was at the well-attended Mineralized Tissue Group dinner with her husband, Dr. John D. Featherstone. When the dinner ended, the wait for a cab to the hotels was substantial. I had an opportunity to catch an earlier cab, but I held back because I thought that, if I timed it right, I could get a cab with John and Pam. This is exactly what happened. Both Pam and John were very open and courteous, and by the time my leg of the ride ended, I knew that my concerns were unfounded. (Neither one would take payment from me for my ride prior to continuing to their hotel.) Years later, I recounted this experience with Pam, who did not remember the cab ride, but she did admit to being a bit surprised when I told her the next year, at the IADR meeting in San Francisco, that a newly discovered MMP was soon to be published. Pam DenBesten remains a trusted friend.
The first reporting of enamelysin, later to be named MMP-20, was in the journal Gene (Bartlett et al., 1996). The rest of the story is an interesting history that can be found in journal articles. It is appropriate, however, to highlight and briefly summarize some of the more notable MMP-20 discoveries. They are presented here in approximate order of occurrence. Dr. Carlos-Lopez-Otín’s group reported the cloning of the human enamelysin mRNA and tentatively named it MMP-20 (Llano et al., 1997). Drs. William T. Butler and Catherine Bègue-Kirn and, separately, Drs. Jan Hu and James P. Simmer characterized the temporal-spatial expression of enamelysin (Bègue-Kirn et al., 1998; Hu et al., 2002). They demonstrated, by in situ hybridization, that enamelysin was expressed in ameloblasts just prior to mineral formation, and that expression continued through the early maturation stage. Jan and Jim also demonstrated that odontoblasts express enamelysin. Drs. Makoto Fukae and Takako Tanabe definitively demonstrated that enamelysin activity caused the doublet bands of approximately 41–46 kDa observed on zymograms of extracted porcine enamel (Fukae et al., 1998). They did this by performing a Western blot with a zymogram gel and therefore proved conclusively that enamelysin was secreted into the enamel matrix. Dr. Ok-Hee Ryu, when she was a member of Drs. Simmer and Hu’s group, demonstrated that basically all of the secretory-stage amelogenin cleavage sites were attributable to enamelysin activity (Ryu et al., 1999). Drs. Takashi Takata, Hiromasa Nikai, and colleagues demonstrated that enamelysin was expressed in odontogenic tumors (Takata et al., 2000). Drs. Tuula Salo, Timo Sorsa, Leo Tjäderhane, and colleagues demonstrated that MT1-MMP activates proMMP-20 (Palosaari et al., 2002). Drs. Henning Birkedal-Hansen and John J. Caterina spent a lot of time and effort engineering an MMP-20 knockout mouse that, once achieved, provided important insight into enamelysin function (Caterina et al., 2002). Drs. Elia Beniash and Charles E. Smith assessed the mineral content in enamelysin null mice vs. controls (Bartlett et al., 2004). I am especially grateful to Charlie Smith for his time and labor-intensive analysis of enamel strip dissections from those tiny murine incisors. Charlie did all this work himself. Most recently, Jung-Wook Kim, while working in Drs. Simmer’s and Hu’s laboratory, discovered the first human mutation in MMP-20 that causes amelogenesis imperfecta (Kim et al., 2005). The report in this issue of the Journal of Dental Research is the second such discovery.
Finally, I am very appreciative of those who initially welcomed me into the field of dental enamel research, and who were subsequently responsible for our collaborative successes. Many friends and collaborators were not mentioned in this article because they were not directly involved in the discovery of enamelysin, but they are nevertheless appreciated. The past few years for me have been a time of substantial personal and scientific growth, and I credit those who have supported me along the way. If I could offer just one bit of advice to young aspiring researchers, it would be to find a project that inspires you (it is not an easy accomplishment, so a mentor can be of great help), pursue it with a will, and do not allow others to distract you from your goals (perhaps the toughest obstacle). I also enthusiastically thank The Forsyth Institute and the National Institute of Dental and Craniofacial Research for supporting our research pursuits (DE12098, DE13237 Project 4, DE14084).
Received July 26, 2005; Last revision September 14, 2005; Accepted September 15, 2005
REFERENCES
Bartlett JD, Simmer JP, Xue J, Margolis HC, Moreno EC (1996). Molecular cloning and mRNA tissue distribution of a novel matrix metalloproteinase isolated from porcine enamel organ. Gene 183:123–128.[ISI][Medline]
Bartlett JD, Beniash E, Lee DH, Smith CE (2004). Decreased mineral content in MMP-20 null mouse enamel is prominent during the maturation stage. J Dent Res 83:909–913.
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.[ISI][Medline]
Caterina JJ, Skobe Z, Shi J, Ding Y, Simmer JP, Birkedal-Hansen H, et al. (2002). Enamelysin (matrix metalloproteinase 20)-deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem 277:49598–49604.
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.
Hu JC, Sun X, Zhang C, Liu S, Bartlett JD, Simmer JP (2002). Enamelysin and kallikrein-4 mRNA expression in developing mouse molars. Eur J Oral Sci 110:307–315.[ISI][Medline]
Kim JW, Simmer JP, Hart TC, Hart PS, Ramaswami MD, Bartlett JD, et al. (2005). MMP-20 mutation in autosomal recessive pigmented hypomaturation amelogenesis imperfecta. J Med Genet 42:271–275.
Llano E, Pendas AM, Knauper V, Sorsa T, Salo T, Salido E, et al. (1997). Identification and structural and functional characterization of human enamelysin (MMP-20). Biochemistry 36:15101–15108.[Medline]
Overall CM, Limeback H (1988). Identification and characterization of enamel proteinases isolated from developing enamel. Amelogeninolytic serine proteinases are associated with enamel maturation in pig. Biochem J 256:965–972.[ISI][Medline]
Palosaari H, Ding Y, Larmas M, Sorsa T, Bartlett JD, Salo T, et al. (2002). Regulation and interactions of MT1-MMP and MMP-20 in human odontoblasts and pulp tissue in vitro. J Dent Res 81:354–359.
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.
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.
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