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The Emergence of "Nanospheres" as Basic Structural Components Adopted by AmelogeninCenter for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Room 103, Los Angeles, CA 90033, USA; joldak{at}usc.edu Martin Taubman, Editor
Key Words: amelogenesis • amelogenin nanospheres • biomineralization • enamel • macromolecules THE SCIENTIFIC EXPERIENCE "The field of biomineralization is poised to tackle the very difficult problems of understanding the functions of the matrix macromolecules in regulating crystal formation. This will require not only an understanding of the biochemistry of these macromolecules, but also a thorough understanding of the molecular organization of these matrix constitutes, their precise locations in the structure, their conformations, and the ways in which they interact with the mineral phase." (From a reprint given to the author in August, 1987, with a dedication: "Good way to start a PhD!...Steve") Under the supervision of Prof. Steve Weiner and Prof. Lia Addadi, I investigated the interactions of extracellular matrix proteins with calcium phosphate crystals and their biological significance during the formation of bone. My long-term goal was to apply fundamental chemical knowledge to the understanding of biological systems, namely, the formation of bones and teeth. FROM GROWING PRETTY CRYSTALS TO DISSECTING BABY MICE I met Dr. Harold Slavkin during the Fourth International Conference on the Chemistry and Biology of Mineralized Tissue (Feb., 1992) in San Diego. Dr. Slavkin, director of the Center for Craniofacial Molecular Biology (CCMB), introduced me to Dr. Alan Fincham, a research professor who was standing by his poster on "Amelogenin Biochemistry—Form and Function". I had read many papers by Dr. Fincham, but I did not know that colleagues knew him as the "Father of Amelogenin". I was also introduced to James Simmer, a postdoctoral Fellow at the CCMB, who presented "Characterization of Murine Amelogenin Isolated from E. coli". I was there to present "Acidic Macromolecule-Inorganic Calcium Phosphate Interactions". As a future post-doctoral Fellow, my responsibility would be to try to link these subjects and make my knowledge and experience useful for the future NIH-related research project. This was one step toward accomplishing my long-term scientific goal, to bridge the "artificial chemical" system, with which I was familiar, to the "real biological" world. In September, 1992, I joined Dr. Slavkin’s group as part of an NIH Program Project, and I was engaged in an exciting research field dealing with mineralized tissue formation, with a particular interest in tooth development and enamel biomineralization. My post-doctoral research was directed toward the study of enamel matrix processing during enamel maturation. My excitement was accompanied by disappointment and nervousness when I found out that I had to dissect neo-natal mouse mandibles for the extraction of developing molars. Apparently, that was the only way to get access to mouse enamel proteins and proteinases! Maggie Zeichner-David and Mary MacDougall, who had mastered the art of mandible dissection, enthusiastically taught me mouse dental anatomy, and with time I learned not to focus on the animal but on the proteins. Although the primary intention was to work on the mouse model, it became obvious that not enough material could be extracted from these animals, and I switched the focus to bovine and porcine material. INVESTIGATING THE "UNWANTED"—EMERGENCE OF THE "NANOSPHERES" "How far knowledge of the nature of the fetal enamel matrix will lead to an understanding of its biological role is an open question, but such are the unusual chemical and physical features of the matrix proteins so far ascertained, that it is to be hoped that by an understanding of the macromolecular chemistry of the structure we may more clearly discern its function." Alan used to talk about amelogenin with so much delight that one would think he "created" this molecule! He had mastered the history of enamel matrix biochemistry and the literature on amelogenin structure. He had the habit of citing statements made by investigators who worked on enamel, and went back as early as 1771 (John Hunter) to emphasize the progress in understanding enamel formation, and to define outstanding questions. Coming from a laboratory where the focus of my research was "acidic protein-mineral interactions", I personally had difficulty imagining how a generally hydrophobic protein with a transient nature like amelogenin, which was rapidly processed and eventually degraded, could have anything to do with controlling mineralization. It was clear that I needed to know more about amelogenin. It is now more than 40 years since Katz et al.(1965) first reported that the protein components of the enamel matrix appeared as polymerizing multi- or single components. Using embryonic bovine enamel proteins and a sedimentation equilibrium study, these investigators later showed that unusually high-molecular-weight complexes ranging from 1 to 4 million Daltons can be detected (Katz et al., 1969). It was around the same time when Nikiforuk and Simmons (1965) noticed the temperature-dependent reversible aggregation characteristics of developing bovine enamel proteins. Two decades later, Fearnhead and Kawasaki (1991) proposed that amelogenin might exhibit self-orienting properties characteristic of "liquid crystal mesophases". Clearly, amelogenin was capable of self-aggregation and some meso-structural organization. The focus on amelogenin structure and function reached its climax in our laboratory when we had enough pure amelogenin protein with which to experiment. Mouse recombinant amelogenin was available to us from James Simmer, who was involved in a robust study on amelogenin splicing and the recombinant expression of different isoforms. The ideal series of experiments on which I wanted to focus were functional studies, namely, the study of the effect of amelogenin on apatite crystal growth. However, before initiating such studies, we had to know much more about amelogenin biochemistry and its structure. One of the ideas was to crystallize the protein for elucidating its tertiary structure.
Although protein crystallization was not my area of expertise, it was a possible project in which I considered becoming involved. Alan recommended that I participate with him in a hands-on one-day Workshop on Protein Crystallization, organized in the Laboratory of Structural Biology and Molecular Medicine, directed by David Eisenberg at UCLA. Part of this workshop included the demonstration of the tools and equipment used by protein crystallographers, among which the DynaPro 801 dynamic light-scattering unit attracted the attention of both of us. This equipment was recommended to protein crystallographers to screen for unwanted protein aggregations prior to crystallization attempts. Each of us had a chance to look at the instrument and understand its potential to detect protein "aggregates". At that time, I had already learned the lesson that amelogenin had a great tendency to aggregate. After some conversation during the break, we both agreed that the DP-801 would be a good tool for the study of amelogenin "aggregates", and we invited Dan Snyder, director of sales for Protein Solutions, to give us a demonstration in our laboratory at CCMB. Amelogenin aggregation has been a major obstacle for many investigators in the study of its structure and functions. It was exciting for me to know that we would start focusing on investigating the "unwanted" (namely, amelogenin aggregation). I prepared our recombinant amelogenin rM179 in 0.1% TFA and Tris-HCl buffer for the demonstration session, and the readings obtained in the buffer solution even surprised our sales representative! (Fig. 1
Dr. Slavkin, a leader who always looked at the "big picture", used to encourage me to implement the innovative technique of Atomic Force Microscopy for the study of apatite crystal growth. I was not yet ready to perform amelogenin-apatite interaction studies and needed to know more about this protein. I thought, "What if I tried to look at amelogenin aggregates using AFM?" Parallel to the DLS studies, we initiated a collaboration with Barney Drake (Imaging Services, Digital Instruments) for imaging amelogenin protein. Alan Fincham generated a two-dimensional computer image of amelogenin adsorbed on mica and zoomed for measurement purposes (Fig. 2  ). This was the first time that amelogenin "nanospheres" were imaged. On our way back from Santa Barbara to Los Angeles, both fascinated by those little yellow "spheres", we started to plan the manuscript. Following Alan’s recommendation, I continued to analyze the shapes of these "nanospheres" by transmission electron microscopy, using different staining techniques, while Alan focused on the size exclusion chromatography to further analyze the size distributions of the assemblies in different buffers.
The 4 different independent techniques (DLS, AFM, TEM, and HPLC) confirmed that amelogenin molecules do not just "aggregate", but rather assemble and self-organize to form supramolecular structures (Fincham et al. 1994; Moradian-Oldak et al., 1994). The term "nanospheres"—first used in the conclusion section of a biopolymer manuscript—attracted some criticism by colleagues because it sounded too "commercial"! Indeed, I adopted this term while reading an advertisement about silica nanospheres or polystyrene beads that were used as standards for molecular size determination by dynamic light-scattering. The story on amelogenin "aggregation" was first presented at the 1994 IADR meeting, and later at the Fifth International Conference on Tooth Morphogenesis and Differentiation in Rolduc, The Netherlands. At around the same time, and independent of our studies on recombinant amelogenin, Nagasaka (1994) reported the formation of "molecular aggregates" and polycrystalline deposits by a purified 21–20 kDa enamel protein with size and morphology similar to those of the "stippled material" described by Watson (1960). ARE THEY (THE SPHERES) RELEVANT? THE BREAKTHROUGH Enamel "stippled material" was originally described by Watson (1960) as fine, granular material between the distal end of the secretory ameloblasts and the mineralization front. In an attempt to provide evidence that enamel formation is an extracellular event, Watson noted that: "Between the cell membrane and the dentine can be seen globular masses of finely stippled material as well as occasional areas of calcification which are recognizable as enamel by virtue of the length of the dense profiles of inorganic material..." — Watson, 1960 Independent of Watson, Fearnhead (1960) had also described similar material as a granular "precursor substance". Other investigators have observed the "stippled material" that occasionally was found in vesicles in the Tomes’ processes (Reith, 1967; Kallenbach, 1973; Slavkin et al., 1976). During the Third International Symposium on Tooth Enamel (1979), controversial views about the nature and function of this material were expressed, and the possible artifactual nature of stippled material was suggested. Later, Nanci and Warshawsky (1984) proposed that the stippled material could be the breakdown product resulting from poor fixation. Lyaruu et al.(1984) have proposed that "the appearance of stippled material was due to temperature dependent aggregation-deaggregation properties of amelogenin", and the authors concluded that "the stippled material is a fixation artifact". We have faced similar hesitations regarding the "nanosphere" concept. In fact, the conditions under which they were described were not physiological. This hesitation was also justified because of the spherical shapes of these assemblies. The first report of globular structures in unfixed enamel examined by a freeze-fracture technique was from Robinson et al.(1981), who reported that, "...in the youngest enamel globular structures of 300–500 Å in diameter seem to have occupied virtually the entire volume of the tissue". It was then suggested that these "globular structures" (which were proposed to consist of protein and mineral), arranged in rows, might be involved in the control of organized and elongated growth of enamel crystals. The next logical step for us was to compare those spherical structures formed by recombinant amelogenin with the granular structures ("stippled material") observed by TEM in sections of the extracellular matrix of developing enamel. Alan took the lead in discussing this issue with various researchers who had examined the enamel extracellular matrix ultrastructure. In our laboratory, Thomas Diekwisch, another post-doc working with Dr. Slavkin, had just published his work on the "Antisense inhibition of AMEL translation" (Diekwisch et al., 1993), and had many TEM images of enamel developing matrix. In collaboration with Diekwisch, D.M. Lyaruu, J.T. Wright, E. Bonucci, and P. Bringas, we accumulated evidence that "amelogenin nanospheres appear as beaded rows of electron-lucent structures aligned with, and separating, the enamel mineral crystallites" (Fincham et al., 1995). In the Sixth International Symposium on Tooth Enamel (1998), Fincham presented a paper on the DLS analysis of "intermediately sized" structures during nanosphere assembly, as a logical extension of our work on amelogenin, and Diekwisch presented a paper on the AFM analysis of "subunit compartments" of secretory stage enamel matrix. During one of the discussion sessions, Colin Robinson noted, "We published some freeze-etching pictures of enamel—some twenty-odd years ago—and sizes are about the same, but they stacked themselves into long very, very tidy rows and as you proceeded through towards transition they simply became crystals, you got no indication of any residue. And it fits very nicely with what has been said so far..." (referring to work by Tom, Janet, and Alan) (Robinson et al., 1981). It seemed that there was finally some consensus about the presence of spherical structures related to amelogenin nanospheres in developing enamel. Malcolm Snead was another creative scientist with whom I had many fruitful discussions, mostly in the hallway of our laboratory at CCMB, about protein folding and misfolding, assembly, aggregation, and disassembly. Malcolm, who had cloned mouse amelogenin (Snead et al., 1983), convinced me that we should not use the term "aggregates" but rather "assemblies". We continued the work on amelogenin "self-assembly" in solution and in gel matrices. In collaboration with Malcolm Snead and Michael Paine, who used a two-hybrid system to demonstrate protein-protein interactions for amelogenin self-assembly, we have studied the effects of mutations in amelogenin sequences on the process of self-assembly (Moradian-Oldak et al., 2000). Dr. Fincham retired in July, 2001, to live in a quiet home facing the Mediterranean. I have continued efforts for amelogenin crystallization with Dr. Guiseppe Falini from Bologna University. We did not succeed in obtaining protein crystals, even after trying almost 2000 conditions for crystallization. Instead, we obtained elongated "fettuccine-like" fibers (microribbons) consisting of amelogenin nanospheres assembled in linear arrays (Moradian-Oldak et al., 2006). These were probably related to the fibers previously reported by Travis and Glimcher (1964), or to other periodic structures observed earlier in vivo (Ronnhölm, 1962; Smales, 1975). The discovery that amelogenin assembles into linear arrays of nanospheres, which can further control elongated growth of mineral crystals, has great significance for defining the function of this "generally hydrophobic protein" during enamel biomineralization, as well as its potential for developing advanced and improved biomaterials. There is still much to learn. It will take major multidisciplinary efforts to integrate the physical chemistry, biochemistry, molecular biology, and biomaterial sciences to study one of the most intriguing mineralized tissues in the human body (enamel).
ACKNOWLEDGMENTS I thank the National Institute of Dental and Craniofacial Research for supporting my studies, Prof. Alan Fincham and Prof. Margarita Zeichner-David for reading the manuscript prior to its submission, and the anonymous reviewers whose comments and suggestions helped improve the manuscript. I thank all the students, post-docs, technicians, investigators, and faculty from whom I have learned and with whom I have interacted during these past 20 years. Finally, I am grateful to all the investigators in the field, without whose invaluable reports and discoveries, nanospheres would not emerge! Received for publication September 10, 2006. Revision received January 31, 2007. Accepted for publication February 6, 2007. REFERENCES
Journal of Dental Research, Vol. 86, No. 6,
487-490 (2007)
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