Enamel Protein Biochemistry - Avenues for Future Success V. C. HASCALL National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. J Dent Res 58(B):825-828, March 1979 I would like to thank Dr. Termine for the sulfate was injected into animals. Some rather fabulous title for this talk, but I am label was subsequently found by radioautonot so sure I want to thank him for the graphy localized in enamel tissue where it rather unenviable task of trying to sum- was later resorbed. The second was a poster marize the many, and occasionally conflict- abstract by Goldberg, et al., who used Alcian ing, results we have heard the past two days blue and bismuth nitrate to demonstrate the concerning the organic matrix of enamel. presence of acid polysaccharides in the Perhaps such tasks are an occupational interprismatic spaces. Someone commented hazard of working at NIDR; thus I have yesterday that he is an unashamed morphorequested combat pay from our Director, logist. Since I am a recidivist biochemist, Dr. Scott, for the duration of the confer- I will argue that a biochemical study is needed to support these initial morphoence. First, I want to compliment all the logical investigations. The enamel proteins appear to be susspeakers for their lucid and informative presentations. They have taught me a lot ceptible to endogenous tissue proteases. about a complex and difficult subject with It is encouraging that efforts are being made which I am not familiar. I also admire and by several laboratories (Termine, Slavkin) envy Jean-Paul Revel for doing such a good to prevent these enzymes from partially job of summarizing the morphology ses- degrading the enamel proteins during extracsions yesterday; his is a tough act to follow. tion and subsequent purification steps. He did ask one question which perhaps we Similar problems have been encountered in can clarify today. He asked why the amelo- attempting to isolate proteoglycans from blasts are such good soldiers and remain in cartilages and other connective tissues. place even in the absence of a recognizable Now most laboratories working on proteobasal lamina. The excellent histology slides glycans are very aware that procedures must shown by Dr. Smith seem to provide an be used which minimize breakdown during answer. If you look closely you will notice tissue preparation, extraction steps and that the feet of the cells are, in fact, embed- subsequent manipulation. The fact that ded in one of nature's forms of concrete, proteases are present in enamel tissue the inorganic enamel, and the ameloblasts suggests that they may have a critical role illustrate very nicely the Mafia maxim in modifying the organic matrix for the that bodies always go where the concrete ensuing deposition of the mineral phase. Indeed, this concept appears to be a central goes. I would also like to thank Dr. Slavkin working hypothesis and should continue to for mentioning proteoglycans to make me be a stimulus for the interesting initial feel more at home in this conference and studies described in this conference for Dr. Butler for suggesting that they may isolating and characterizing such enzymes play a role in regulating mineralization, at (Sasaki; Shimizu; Moe and Birkedal-Hansen). least in dentin. It is disappointing, however, It was stated earlier that proteolytic enzymes that there is so little being done to identify floating around outside the cells do not and characterize proteoglycans in either seem to be a very exquisite way to regulate enamel or dentin. While this will be a difficult matrix formation and modification. I would task, it would be a worthwhile objective for urge that we keep an open mind about this study, particularly in some of the model since nature is going to do things her own culture systems described in the past two way independent of how elegant we may days. There were two points brought out think it is. Certainly, the idea of proteolytic indicating that sulfated polysaccharides, and enzymes acting extracellularly to modify hence proteoglycans, are probably a com- a matrix has been a major element of models ponent of enamel. One was discussed by Dr. developed to explain endochondral mineraliReith who described a paper where 35 S- zation of ossification of osteoid. It has been 825
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HASCALL
recognized in such cases that proteoglycans are removed from the matrix just before mineralization and it is thought that the proteases known to be present in cartilage may regulate this process. How such regulation could be achieved either in the case of mineralizing cartilage or enamel matrix is a question for future studies, but I think it is entirely possible that the enzymes discussed the past two days may have a critical extracellular role in preparing an organic matrix for mineralization. There are some problems where the biochemist with his present limitations cannot provide much help. Dr. Smith illustrated the dilemma best. From morphological considerations he proposed that the early ameloblasts are secretory whereas the later ameloblasts are resorptive. This suggestion was heatedly discussed, and it could in principle be answered with biochemical approaches. The biochemist is in awe of the capability of a morphological study for predicting such elegant models but he is also frustrated since he would have to work with a relatively pure population of secretory ameloblasts to compare their synthetic and catabolic properties with those of a relatively pure population of resorptive ameloblasts in order to contribute support for or against the hypothesis. In this field, then, progress will be made biochemically as the morphologists and cell biologists begin to develop model systems in vivo and in vitro to answer some aspects of these problems. Can the cell biologist, as Jean-Paul Revel asked yesterday, isolate a cell population at an early, immature stage and find conditions which allow it to differentiate and mature? If so the biochemist can, in fact, work in concert to help unravel the sequence of events leading to mature enamel matrix. I am always amazed and impressed with Dr. Slavkin's quixotic attempts to endow chickens with teeth. It came as somewhat a shock to hear Dr. Eastoe mention last night that John Hunter had attempted and perhaps even succeeded in doing the same thing 250 years ago. Nevertheless, the transplantation of embryonic enamel organs onto chick chorioallantoic membranes is an excellent model system for studying some aspects of tooth morphogenesis. Like all model systems it has its limitations and even if Dr. Slavkin succeeds in endowing a chick with teeth, getting them into the
JDent Res Special Issue B, 1979
beak rather than under a wing will pose problems; even then there wil undoubtedly be severe cases of malocclusion which will need attention. The point is that all of us, morphologist, cell biologist, biochemist or whatever, are forced to work with model systems, to isolate and focus on variable parameters. The analogy in my own area of research interest is the use of stage 23-24 chick limb bud mesenchyme cells to study chondrogenesis and myogenesis in vitro. When isolated as a suspension, these mesenchyme cells can be plated onto petri dishes and provided with an environment which allows them to differentiate and express either chondrogenesis, where they elaborate cartilage nodules with type II collagen and cartilage proteoglycans, or myogenesis, where they fuse and form myotubes with actin and myosin. Whereas in the developing limb the spatial and temporal aspects of chondrogenesis and myogenesis are controlled to help form a morphologically distinct limb, in culture while regions of cartilage nodules or muscle may be indistinguishable from counterpart regions in the limb, no one expects the cultures to give rise to anything remotely resembling a limb. Thus, the cultures are valuable for studying biosynthesis of cartilage proteoglycans, for example, or even for studying how type II collagen and proteoglycan organize an extracellular cartilage matrix once you have spent the required time and energy to show that the cultures are making good facsimiles of the same macromolecules found in vivo. At this stage, these cultures would not be a useful model for studying endochondral ossification because investigators have not figured out a way to make the cartilage nodule in culture mineralize and be replaced by bone, nor, as mentioned above, can they be used to study limb formation. Nature can still do these things, however, since a suspension of stage 23-24 cells can be packed back into a stage 24 limb bud ectodermal jacket and give rise to a normal limb when grafted back into the embryo. It is just that we have not figured out how to do such elegant things in culture. We have heard descriptions of several promising model systems for studying biosynthesis of enamel and dentin organic matrices. Each of these will require the type of investigation discussed by Dr. Butler to demonstrate the validity of the system for making biochemical products
Vol. 58(B)
whiph
are characteristic of that particular tissue, and one must be cautious when one finds that it makes products in culture, such as the type I trimer which may or may not be present in vivo. The model system described by Sasaki and his collaborators shows promise, in this respect, for looking at biosynthesis of the organic matrix of enamel. A comparison of a protein component of 25,000 MW synthesized in culture was made with a sample of similar size isolated directly from enamel. This component may well give rise through programmed degradation steps to the smaller peptides which several investigators have discussed. Nevertheless, many problems need to be addressed. Have optimal conditions for culture been chosen? What is the effect of adding serum and/or embryo extract to the system? What are the kinetics of synthesis of the protein components over the time for which the cultures are maintained? Are there larger precursors which give rise to the 25,000 MW component which could be detected in pulse-chase experiments? Nevertheless, I think that this system, along with the ones developed in other laboratories, are going to produce new insights which will undoubtedly provide a major basis for the next. We now come to the knotty problem of protein chemistry. Dr. Eastoe said last night that compared with other fields of protein chemistry, problems of characterizing the organic matrix of enamel are unexpectedly difficult and beset with paradoxes, sometimes almost to the point of utter confusion. It should be noted that about 400 years ago, Francis Bacon considered that "truth emerges more readily from error than from confusion." So it is probably a good idea even with the rather limited knowledge at hand in this field to postulate some models which are consistent with most available data, take the chance that they resemble in some small way the truth of the matter, and begin to use them to serve as a guide for further research. It is reasonable to assume that the early secretory ameloblast is responsible for synthesizing and elaborating a series of building molecules-phosphoproteins, glycoproteins, proteoglycans, etc.-which organize an organic lattice which must serve both as a template onto which crystals of hydroxyapatite can be laid down and as a filler to temporarily occupy the space which the inorganic component will even-
827
tually fill. Some of these extracellular components then must serve, for want of better terms, as nucleation and crystal organizing functions. They may be modified during the process or end up embedded in or around the prisms. Other extracellular space-filling components would have to be removed in a systematic way as crystalfilling components would have to be removed in a systematic way as crystallization proceeds. Many, if not most, of the organic molecules used in the early scaffolding ot the extracellular matrix may be extensively modified during the maturation process quite likely by highly specific proteases. Throughout, cellular regulation in secretory and later resorptive phases may be a controlling factor. Many of the data presented the past two days can be considered within this framework. The high molecular weight phosphoproteins discussed by Dr. Termine are particularly intriguing. Since they were only solubilized when EDTA was added to the 4 M guanidine HC1 extractant it is likely that they are tightly associated with the mineral phase and may be critical for organizing the crystals. What is its relationship to the high molecular weight component described by Dr. Chrispens? Does this phosphoprotein remain intact during enamel maturation or is it degraded to generate the intermediate sized proteins described by the Japanese workers and/or the smaller phosphoproteins described by Dr. Glimcher and Dr. Fincham? It is quite possible that the small phosphoproteins, the J fragments discussed by Dr. Shimizu and the E3, E4 complexes described by Dr. Glimcher are secreted as larger, soluble precursor molecules which must be enzymatically cleaved to release a soluble fragment which is lost from the tissue (perhaps the C fragment discussed by Dr. Shimizu) leaving behind the small, insoluble, proline-rich phosphoprotein to serve perhaps as a nucleation site for initial mineral formation. The immunological approaches being developed by Drs. Schonfeld and Christner may help sort out which of these many fragments are immunologically similar. Certainly the different high MW phosphoproteins can be treated with the enzymes which are being characterized to see if smaller fragments generated are similar to those which have been isolated from the enamel matrix. In other words, test the possibility that the C and J type proteins do indeed
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come from these larger macromolecules. A final comment with relation to the elegant sequence studies presented by Glimcher, et al.: The E3 and E4 components had a very large degree of sequence homology including identity through the first 5 residues. Are these two components, then, products of different, very similar polypeptides or are they part of a common polypeptide precursor? If they are in the same polypeptide, then there would have to be a great deal of internal repetitive sequence. Such repetition could be dictated by the function of the molecules in a manner similar to collagen, or it could reflect the type of sequence homology found in immunoglobulins. In any event, the eloquent protein chemistry will undoubtedly raise new and important questions about the structure and function of these fascinating polypeptides. I will conclude by saying that I have appreciated the science discussed in the past two days, and I have learned a lot
J Dent Res Special Issue B, 1979
about a complex and interesting biological system. I also hope that any naive errors I have made in attempting to understand the presentations will be excused. Some of the presentations have presented data almost in art forms which do not need to be understood to be appreciated. The polarized light microscopy and ground enamel sections presented by Dr. Fejerskov and the color analogues used by Dr. Suga for converting density changes into displays of brilliant colors were particularly beautiful examples of art in science. In many ways this conference represents a sunrise in which the first glimmers of light begin to reveal the models and mechanisms which will provide the basis for the debates at the next conference. It may take a long time for the sun to rise above the horizon, but there will be constant progress in this area and like any scientific endeavor, there will be those novel flashes of insight to speed the course of our work.
826
HASCALL
recognized in such cases that proteoglycans are removed from the matrix just before mineralization and it is thought that the proteases known to be present in cartilage may regulate this process. How such regulation could be achieved either in the case of mineralizing cartilage or enamel matrix is a question for future studies, but I think it is entirely possible that the enzymes discussed the past two days may have a critical extracellular role in preparing an organic matrix for mineralization. There are some problems where the biochemist with his present limitations cannot provide much help. Dr. Smith illustrated the dilemma best. From morphological considerations he proposed that the early ameloblasts are secretory whereas the later ameloblasts are resorptive. This suggestion was heatedly discussed, and it could in principle be answered with biochemical approaches. The biochemist is in awe of the capability of a morphological study for predicting such elegant models but he is also frustrated since he would have to work with a relatively pure population of secretory ameloblasts to compare their synthetic and catabolic properties with those of a relatively pure population of resorptive ameloblasts in order to contribute support for or against the hypothesis. In this field, then, progress will be made biochemically as the morphologists and cell biologists begin to develop model systems in vivo and in vitro to answer some aspects of these problems. Can the cell biologist, as Jean-Paul Revel asked yesterday, isolate a cell population at an early, immature stage and find conditions which allow it to differentiate and mature? If so the biochemist can, in fact, work in concert to help unravel the sequence of events leading to mature enamel matrix. I am always amazed and impressed with Dr. Slavkin's quixotic attempts to endow chickens with teeth. It came as somewhat a shock to hear Dr. Eastoe mention last night that John Hunter had attempted and perhaps even succeeded in doing the same thing 250 years ago. Nevertheless, the transplantation of embryonic enamel organs onto chick chorioallantoic membranes is an excellent model system for studying some aspects of tooth morphogenesis. Like all model systems it has its limitations and even if Dr. Slavkin succeeds in endowing a chick with teeth, getting them into the
JDent Res Special Issue B, 1979
beak rather than under a wing will pose problems; even then there wil undoubtedly be severe cases of malocclusion which will need attention. The point is that all of us, morphologist, cell biologist, biochemist or whatever, are forced to work with model systems, to isolate and focus on variable parameters. The analogy in my own area of research interest is the use of stage 23-24 chick limb bud mesenchyme cells to study chondrogenesis and myogenesis in vitro. When isolated as a suspension, these mesenchyme cells can be plated onto petri dishes and provided with an environment which allows them to differentiate and express either chondrogenesis, where they elaborate cartilage nodules with type II collagen and cartilage proteoglycans, or myogenesis, where they fuse and form myotubes with actin and myosin. Whereas in the developing limb the spatial and temporal aspects of chondrogenesis and myogenesis are controlled to help form a morphologically distinct limb, in culture while regions of cartilage nodules or muscle may be indistinguishable from counterpart regions in the limb, no one expects the cultures to give rise to anything remotely resembling a limb. Thus, the cultures are valuable for studying biosynthesis of cartilage proteoglycans, for example, or even for studying how type II collagen and proteoglycan organize an extracellular cartilage matrix once you have spent the required time and energy to show that the cultures are making good facsimiles of the same macromolecules found in vivo. At this stage, these cultures would not be a useful model for studying endochondral ossification because investigators have not figured out a way to make the cartilage nodule in culture mineralize and be replaced by bone, nor, as mentioned above, can they be used to study limb formation. Nature can still do these things, however, since a suspension of stage 23-24 cells can be packed back into a stage 24 limb bud ectodermal jacket and give rise to a normal limb when grafted back into the embryo. It is just that we have not figured out how to do such elegant things in culture. We have heard descriptions of several promising model systems for studying biosynthesis of enamel and dentin organic matrices. Each of these will require the type of investigation discussed by Dr. Butler to demonstrate the validity of the system for making biochemical products
Vol. 58(B)
whiph
are characteristic of that particular tissue, and one must be cautious when one finds that it makes products in culture, such as the type I trimer which may or may not be present in vivo. The model system described by Sasaki and his collaborators shows promise, in this respect, for looking at biosynthesis of the organic matrix of enamel. A comparison of a protein component of 25,000 MW synthesized in culture was made with a sample of similar size isolated directly from enamel. This component may well give rise through programmed degradation steps to the smaller peptides which several investigators have discussed. Nevertheless, many problems need to be addressed. Have optimal conditions for culture been chosen? What is the effect of adding serum and/or embryo extract to the system? What are the kinetics of synthesis of the protein components over the time for which the cultures are maintained? Are there larger precursors which give rise to the 25,000 MW component which could be detected in pulse-chase experiments? Nevertheless, I think that this system, along with the ones developed in other laboratories, are going to produce new insights which will undoubtedly provide a major basis for the next. We now come to the knotty problem of protein chemistry. Dr. Eastoe said last night that compared with other fields of protein chemistry, problems of characterizing the organic matrix of enamel are unexpectedly difficult and beset with paradoxes, sometimes almost to the point of utter confusion. It should be noted that about 400 years ago, Francis Bacon considered that "truth emerges more readily from error than from confusion." So it is probably a good idea even with the rather limited knowledge at hand in this field to postulate some models which are consistent with most available data, take the chance that they resemble in some small way the truth of the matter, and begin to use them to serve as a guide for further research. It is reasonable to assume that the early secretory ameloblast is responsible for synthesizing and elaborating a series of building molecules-phosphoproteins, glycoproteins, proteoglycans, etc.-which organize an organic lattice which must serve both as a template onto which crystals of hydroxyapatite can be laid down and as a filler to temporarily occupy the space which the inorganic component will even-
827
tually fill. Some of these extracellular components then must serve, for want of better terms, as nucleation and crystal organizing functions. They may be modified during the process or end up embedded in or around the prisms. Other extracellular space-filling components would have to be removed in a systematic way as crystalfilling components would have to be removed in a systematic way as crystallization proceeds. Many, if not most, of the organic molecules used in the early scaffolding ot the extracellular matrix may be extensively modified during the maturation process quite likely by highly specific proteases. Throughout, cellular regulation in secretory and later resorptive phases may be a controlling factor. Many of the data presented the past two days can be considered within this framework. The high molecular weight phosphoproteins discussed by Dr. Termine are particularly intriguing. Since they were only solubilized when EDTA was added to the 4 M guanidine HC1 extractant it is likely that they are tightly associated with the mineral phase and may be critical for organizing the crystals. What is its relationship to the high molecular weight component described by Dr. Chrispens? Does this phosphoprotein remain intact during enamel maturation or is it degraded to generate the intermediate sized proteins described by the Japanese workers and/or the smaller phosphoproteins described by Dr. Glimcher and Dr. Fincham? It is quite possible that the small phosphoproteins, the J fragments discussed by Dr. Shimizu and the E3, E4 complexes described by Dr. Glimcher are secreted as larger, soluble precursor molecules which must be enzymatically cleaved to release a soluble fragment which is lost from the tissue (perhaps the C fragment discussed by Dr. Shimizu) leaving behind the small, insoluble, proline-rich phosphoprotein to serve perhaps as a nucleation site for initial mineral formation. The immunological approaches being developed by Drs. Schonfeld and Christner may help sort out which of these many fragments are immunologically similar. Certainly the different high MW phosphoproteins can be treated with the enzymes which are being characterized to see if smaller fragments generated are similar to those which have been isolated from the enamel matrix. In other words, test the possibility that the C and J type proteins do indeed
828
HASCALL
come from these larger macromolecules. A final comment with relation to the elegant sequence studies presented by Glimcher, et al.: The E3 and E4 components had a very large degree of sequence homology including identity through the first 5 residues. Are these two components, then, products of different, very similar polypeptides or are they part of a common polypeptide precursor? If they are in the same polypeptide, then there would have to be a great deal of internal repetitive sequence. Such repetition could be dictated by the function of the molecules in a manner similar to collagen, or it could reflect the type of sequence homology found in immunoglobulins. In any event, the eloquent protein chemistry will undoubtedly raise new and important questions about the structure and function of these fascinating polypeptides. I will conclude by saying that I have appreciated the science discussed in the past two days, and I have learned a lot
J Dent Res Special Issue B, 1979
about a complex and interesting biological system. I also hope that any naive errors I have made in attempting to understand the presentations will be excused. Some of the presentations have presented data almost in art forms which do not need to be understood to be appreciated. The polarized light microscopy and ground enamel sections presented by Dr. Fejerskov and the color analogues used by Dr. Suga for converting density changes into displays of brilliant colors were particularly beautiful examples of art in science. In many ways this conference represents a sunrise in which the first glimmers of light begin to reveal the models and mechanisms which will provide the basis for the debates at the next conference. It may take a long time for the sun to rise above the horizon, but there will be constant progress in this area and like any scientific endeavor, there will be those novel flashes of insight to speed the course of our work.
