| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
DISCOVERY! |
Department of Cell and Molecular Biology, Feinberg Medical School, Northwestern University, Chicago, IL 60611; aveis{at}nwu.edu
KEY WORDS: Collagen • dentin • phosphoproteins • cross-linking
It is difficult to predict the directions, twists, and turns of one’s career, or at least it seems so to me. My PhD Dissertation research, completed in late 1950, was in physical chemistry and dealt with the thermodynamics of polyelectrolyte solutions. I was very much taken with the work of Ephraim and Aaron Katchalsky on the determination of the electrostatic free energy of polyanions, and with the works of Paul Flory and Maurice Huggins on the statistical mechanical approach to the entropy of polymer solutions. My thesis advisor at Northwestern, Irving Klotz, suggested a problem related to the effects of various micro-ions on the properties of polymeric anions, and I gladly took it on. But he had a large group of students studying the binding of ligands to proteins, and it turned out that he was only peripherally interested in my problem, so I was essentially left to fend for myself.
As my thesis work drew to a close, I knew that I wanted an academic career but felt the need to broaden my base in physical chemistry. Thus, I took a post-doctoral position with Lloyd Swearingen in the University of Oklahoma Chemistry Department to study the kinetics of free radical reactions in potential "high energy fuels" for jet and rocket propulsion. It was interesting work, but Career Disaster Number One. Shortly after I arrived at OU, Swearingen became Vice President for Research and had little time to devote to the study, so I was left on my own again. Further, as I was writing up my results, the Air Force, the sponsor of the study, decided that the work should be classified, and I could not have any open publication. All was not totally lost, however. I was offered a position on the Chemistry faculty and began teaching Swearingen’s graduate courses in classic and statistical thermodynamics. My second year at OU was an exhilarating but exhausting experience.
For my research I wanted something publishable that I could "get into" quickly. I turned back to the behavior of polyelectrolyte solutions but wanted to study polyampholytes. In 1951, the synthesis of polyampholytes was not at all routine. This led to another fateful decision. The polyampholyte of choice for physical biochemists at the time was gelatin—denatured, degraded soluble collagen. Funding sources were sparse, and NIH had just begun, but I happened on an Armour and Company advertisement in Chemical & Engineering News about the research they were trying to do with gelatins in their newly created Central Research Laboratory. I put together a research proposal and sent it off to them. To make a complex story very short, the upshot was that I was offered a position at Armour in Chicago to carry out the research that I had proposed in their Physical Chemistry Department.
It was an offer I could not refuse, so I moved there in September, 1952. Two wonderful people, Drs. Jules D. Porsche and James Harwood, Director and Associate Director of Central Research, respectively, allowed me to pursue my research as I saw fit. They gave me advice and unstinting support, but never once in my 8 years in Central Research did they "direct" my activities. I was allowed to publish as I believed appropriate, with the one proviso that I discuss each paper with the patent attorneys and submit applications as requested. Some 13 patents later, I believe that Armour received a good return on its modest investment. I should add that most of my work at Armour was carried out with the aid of two superb biochemists, Jerome Cohen and Joan Anesey, both of whom became life-long friends.
Back to the science. It did not take me long to discover that the commercially available gelatins were all polydisperse mixtures blended to give a particular set of properties. If I were to hope for any real insight, I had to make my own from the parent material, collagen. The ready availability of fresh bovine skin at Armour did influence my choice of mature skin collagen as the source. In contrast to the leather and gelatin-glue chemists, who were interested in cross-linking or degrading the collagen, and the biologists such as F.O. Schmitt and Jerome Gross, who began the study of soluble collagen about the same time, I approached the problem as one of the depolymerization of a cross-linked polymer network. A series of papers, capped by Veis and Cohen (1956), showed that two independent systems of cross-linkages of different placement and stability were present. The paper described a theoretical analysis of the molecular-weight distributions that should be obtained depending on the relative rates of disruption of the cross-linkages and the backbone peptide bond hydrolysis.
Contemporary work by Helga Boedtker and Paul Doty (1956) on carp swim bladder soluble collagen ("tropocollagen") showed that the collagen molecule was a semi-rigid rod-like structure composed of three separate polypeptide chains, each about 105 in molecular mass. These concepts were incorporated into our work to develop a refined model of the cross-linked fibrillar collagen network. Another line of thinking, obviously influenced by my work at Armour, was that it would be economically valuable if one could disperse and subsequently re-aggregate mature skin collagen to form uniform sheets and films and fibers. This could be done from dissolved tendon and skin, but the yields were too low. Such a process could be viable, I thought, if one could reconstitute or re-assemble the rod-like molecules from denatured gelatins of the correct high-molecular-weight cross-linked types. The conventional wisdom at the time was that denatured proteins could not be "renatured". But since we knew from viscosity, optical rotation, and infrared studies that gelatin gels re-formed a collagen-fold in part, we set out to renature collagen.
That work, although not received with much attention, was actually the first demonstration of the complete renaturation of any protein (Veis and Cohen, 1960). Critics had said that they would be convinced only by seeing the correct re-appearance of collagen fibrils at the EM level. Fortunately, Jerry Cohen was teaching a Biochemistry Laboratory course at the Evening Division of Northwestern University at the time. Dr. Leonard Fosdick, Chair of Biochemistry in the Dental School, was the course director. He had one of the few electron microscopes in Chicago. Jerry told him of our needs, and Dr. Fosdick was very generous in letting us use his EM, enabling us to complete the renaturation study successfully.
The interaction with Dr. Fosdick proved to be even more important in other ways—and leads finally to the subject of this Discovery! By the end of the 1950s, two events at Armour spelled doom for my career as an independent scientist in Central Research. First, as in many corporations, basic research had about run its course at Armour. The various Divisions, mostly developed from the research accomplished (Armour Pharmaceuticals, Armour Chemicals, etc.), needed and wanted their own dedicated applied research laboratories, and these were to be created out of Central Research. Second, we had been too successful: Our patents on the reconstitution and re-assembly of collagen into sheets and films (US Patent 2,838,363, Method of Preparing Filaments and Sheets from Procollagen, Arthur Veis and Jerome Cohen, June 10, 1958; US Patent 2,935,413, Procollagen Material, Arthur Veis and Jerome Cohen, May 3, 1960) (Fig.
) were cited against a process being developed by Jerome Gross and John Highberger with the United Shoe Machinery Corp. USMC had made perhaps a greater than 50-fold investment than Armour and had built a pilot production plant, but our patents covered—indeed exceeded—their process.
|
Dr. Fosdick knew that I had ultimately intended to seek an academic position, so when he learned of the developments at Armour, he asked if I would like to join his department at the Dental School. It was clear that I knew nothing about dental science or dental education; I was still a physical chemist edging my way into protein chemistry via my studies in the physical chemistry of collagen. Dr. Fosdick was undeterred and suggested that I think about a problem related to collagen that had always bothered him. Why did collagen in bone and dentin become mineralized, whereas the collagen in skin and tendon became mineralized only under pathological conditions? There was no open position in his small Biochemistry Department, so he asked me to submit an application to NIDR, which had just initiated the possibility of NIH Career Development Awards. My literature search was not very rewarding, but I concluded that there were perhaps two possibilities: one, that there might be more than one kind of collagen—different kinds in soft and hard tissues; and two, that there were special molecules in one tissue as compared with the other. These ideas were somewhat "far out", for at that time, there was only "collagen", not the 27 types known today, and little was known about the non-collagenous proteins of either matrix. Nevertheless, I prepared and submitted the Career Development Award application. Surprisingly, many months later, I received notice that the grant would indeed be funded.
In the intervening period, however, a group of angels was looking out for me, and an alternative possibility arose at Northwestern in the Medical School Department of Biochemistry. Henry Bull, a prominent physical biochemist of the day, and professor in that department, took the chair in Biochemistry at Iowa, creating a vacancy. Without my knowledge, Richard Winzler, chair of Biochemistry at the University of Illinois Medical School, and Professors J.W. Williams and John Ferry at the University of Wisconsin, and Irving Klotz at NU in Evanston all independently recommended me for that position. I had taught Winzler’s graduate Physical Biochemistry course in 1957 while he was on sabbatical. Williams and Ferry knew of my work through meetings and my papers on gelatin, and I had kept in touch with Irv Klotz. Smith Freeman, chair of Biochemistry in the Medical School, called and asked me to come see him about the faculty position. This happened before I had heard anything from NIDR. I felt that I was obligated to Dr. Fosdick and the Dental School, but Fosdick urged me to check out the Medical School position. He believed that I would receive a better offer and future than he or the Dental School could provide. That indeed was the case, and I joined the Medical School in September, 1960, as Associate Professor, on the tenure track. When I received word that my Career Development Award had been approved for funding, I wrote to NIDR about my changed plans. Their response was that the Study Section and Dental Council had been very positive about the proposed work, so, at their request, I reworked the budget, and the grant was awarded as an R01. Thus began my work on dentin and my association with NIDR/NIDCR that continues to this day, with the same grant now approved through year 45.
I have gone into this background story in more detail than perhaps I should have, to emphasize how important mentorship can be to a career. In my case, I never did have a true mentor, someone to offer advice, direction, or aid during the early development years. But the people mentioned above, especially Leonard Fosdick and Richard Winzler, who had no formal relationship with me, or any obligation to do so, filled that role at crucial moments. I have tried to honor their contribution by supporting my students and younger colleagues as much as I could.
My first NU PhD student, Robert Schlueter, began the dentin work with the simple objective of isolating and purifying the dentin collagen. Was it the same as skin collagen? We selected bovine collagen, because the skin and teeth were readily available, and the bovine teeth were large. One look at the worn, forage-contaminated teeth of a mature cow, however, dictated that we use unerupted teeth. The teeth were demineralized with EDTA, and the remaining insoluble collagen was collected. We simply discarded the extracted proteins and any other soluble components. The soft enamel of the unerupted teeth was scraped away, and the pulps were removed mechanically. Since the lab was being set up, we did not have an atomic absorption spectrophotometer available for routine calcium assay to follow completion of the demineralization, and elected to determine demineralization by residual phosphate. This was a fateful decision, because, try as we could, we were unable to remove a small amount of phosphate from the insoluble dentin residue, whereas there was no phosphate in comparably treated insoluble skin collagen. The phosphate-containing dentin residue was shown to be completely calcium-free. Bob Schlueter’s meticulous record-keeping left us no alternative but to go out on the trail of that phosphate—the overriding effort of the next few decades.
Another finding was that the dentin collagen did not swell appreciably in acid, again, a very different property from those of skin and tendon collagens. This led to the conclusions that the dentin collagen was either more highly cross-linked than the soft-tissue collagens, or that it cross-linked differently, possibly through the mediation of phosphate cross-linkages (Veis and Schlueter, 1963, 1964; Schleuter and Veis, 1964). These two themes—the source and nature of the dentin-matrix-related phosphate, and the nature of the dentin collagen and its cross-linking—were the focus of our research for the next few decades. We made slow but steady progress. There was not much initial interest in our papers, since dentin was not a very popular subject compared with bone, and there was some skepticism about the validity of the work as we described the amazingly high Asp- and P-Ser-containing polypeptides related to the dentin collagen matrix.
Nevertheless, we persisted, and a series of great post-doctoral fellows (Ann Perry, David Carmichael, Dino Volpin, Paul Scott, Yuzo Takagi, Ian Dickson, Adrian Shuttleworth, Joe Lechner) and PhD students (Alan Spector, Maureen Sharkey-Miamoto, Michael DiMuzio, Sandra Lee, Peter T.-Z. Tsay, Mohammed Rahima, William Stetler-Stevenson, Abu-Bakr Rabie, Chou Bing Wu, Charles Sfeir, Jerry Dillon) developed the details of the nature of the phosphorylated proteins and their relationship to the dentin collagen. Prof. Doug Maier spent two productive years with me while on sabbatical from Colby College in Maine. Boris Sabsay (MD, PhD), an émigré from Russia, joined the group in 1978 and became a highly valued colleague.
[Part II of this Discovery! (Phosphophoryn, the DMPs, and More) will follow next month (J Dent Res 83[1], 2004).]
Received July 25, 2003; Accepted September 3, 2003
 |
REFERENCES |
|---|
 TOP REFERENCES |
|---|
Schlueter RJ, Veis A (1964). The macromolecular organization of dentine matrix collagen. II. Periodate degradation and carbohydrate cross-Linking. Biochemistry 3:1657–1665.
Veis A, Cohen J (1956). A non-random disaggregation of intact skin collagen. J Am Chem Soc 78:6238–6244.
Veis A, Cohen J (1960). Reversible transformation of gelatin to collagen structure. Nature 186:720–721.[Medline]
Veis A, Schlueter R (1963). Presence of phosphate-mediated cross-linkages in hard tissue collagens. Nature 197:1204.
Veis A, Schlueter RJ (1964). The macromolecular organization of dentine matrix collagen. I. Characterization of dentine collagen. Biochemistry 3:1650–1656.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
