Reflections on Glycobiology (2001)

glycosaminoglycan 3′-phosphoadenosine-5′-phosphosulfate multiglycosyltransferase glycosyltransferase sialyltransferase Glycobiology has become a “hot” subject, 2A recent issue of Science features glycobiology (1Science. 2001; 291: 2263-2502Google Scholar), and the April, 2001 meeting of the Carbohydrate Division of the American Chemical Society emphasizes glycobiology as a major subject; their prestigious C. S. Hudson Award was presented to a well known glycobiologist, Y. C. Lee. How times have changed! In the 1950s, glycobiology was not a popular subject. There were a few interested biochemists at the meetings, and we had an annual lunch (Karl Meyer, Al Dorfman, Dick Winzler, Roger Jeanloz, Ward Pigman and a few others). After lunch, one might as well go home. My papers (glucosamine metabolism) were invariably scheduled as either last or next to last on Friday afternoon at the Federation Meetings in Atlantic City. The most hilarious incident was when my paper (next to last) was announced at one of these sessions. When I reached the platform, the chairman of the session apologized because he had to leave to make a train. My audience consisted of the next speaker and the slide projectionist. I stayed for the last paper, but unfortunately I never asked the projectionist how he liked the presentations. I had the same experience at the American Chemical Society meetings. Starch chemistry was a big thing for members of the Carbohydrate Division, and they walked out on papers devoted to the glycoconjugates or hexosamines. At one such meeting, my audience consisted of other members of the laboratory waiting to drive back to Ann Arbor with me. It was, however, a great time to do this kind of research. There was virtually no pressure. The handful of us in this country who worked in the field were supported by the National Institutes of Health. I can capture a little of the intellectual flavor of the times by my experience when I submitted my first independent application. It stated that I would work on the enzymatic synthesis of one of the monosaccharides in the glycoconjugates, but I did not know which to choose. I then listed about four monosaccharides (glucosamine, fucose, glucuronic acid, and galactosamine) and possible preliminary experiments for each; I would work on whichever problem appeared most fruitful. I was funded, and a short time later I met one of the members of the Study Section (Ef Racker) who told me that it was the best application he had read. What would happen today with an application that was so “unfocused” and with such nonspecific aims? Equally important to the National Institutes of Health support, we received unsparing help from a number of farsighted physicians such as Walter Bauer at Massachusetts General Hospital, who not only created a high caliber research unit (Roger Jeanloz, Jerome Gross, Karl Schmid, Morris Soodak, and others) but was also instrumental in the Helen Hay Whitney Foundation. In my case, it was William Robinson and Ivan Duff at the Rackham Arthritis Unit at the University of Michigan. Only once (when I was first interviewed) did I have to explain to Bill Robinson how work on Escherichia coli might relate to arthritis. Thus, we had the luxury of following our noses and serendipity wherever the work took us. We started with studies on the intermediary metabolism of glucosamine, which led in turn to the structure of the sialic acids and the discovery of N-acetylmannosamine, to the intermediary metabolism of these compounds, to CMP-sialic acid and its enzymatic synthesis, to the glycosyltransferases, and finally to the phosphotransferase system for sugar uptake by bacteria (reviewed in Refs. 2Roseman S. Chem. Phys. Lipids. 1970; 5: 270-297Crossref PubMed Scopus (799) Google Scholar and 3Roseman S. FEMS Microbiol. Rev. 1989; 63: 3-12Google Scholar). In recent years, the complex process of chitin catabolism by marine bacteria has become a major project (4Keyhani N.O. Roseman S. Biochim. Biophys. Acta. 1999; 1473: 108-122Crossref PubMed Scopus (240) Google Scholar).2A recent issue of Science features glycobiology (1Science. 2001; 291: 2263-2502Google Scholar), and the April, 2001 meeting of the Carbohydrate Division of the American Chemical Society emphasizes glycobiology as a major subject; their prestigious C. S. Hudson Award was presented to a well known glycobiologist, Y. C. Lee. How times have changed! In the 1950s, glycobiology was not a popular subject. There were a few interested biochemists at the meetings, and we had an annual lunch (Karl Meyer, Al Dorfman, Dick Winzler, Roger Jeanloz, Ward Pigman and a few others). After lunch, one might as well go home. My papers (glucosamine metabolism) were invariably scheduled as either last or next to last on Friday afternoon at the Federation Meetings in Atlantic City. The most hilarious incident was when my paper (next to last) was announced at one of these sessions. When I reached the platform, the chairman of the session apologized because he had to leave to make a train. My audience consisted of the next speaker and the slide projectionist. I stayed for the last paper, but unfortunately I never asked the projectionist how he liked the presentations. I had the same experience at the American Chemical Society meetings. Starch chemistry was a big thing for members of the Carbohydrate Division, and they walked out on papers devoted to the glycoconjugates or hexosamines. At one such meeting, my audience consisted of other members of the laboratory waiting to drive back to Ann Arbor with me. It was, however, a great time to do this kind of research. There was virtually no pressure. The handful of us in this country who worked in the field were supported by the National Institutes of Health. I can capture a little of the intellectual flavor of the times by my experience when I submitted my first independent application. It stated that I would work on the enzymatic synthesis of one of the monosaccharides in the glycoconjugates, but I did not know which to choose. I then listed about four monosaccharides (glucosamine, fucose, glucuronic acid, and galactosamine) and possible preliminary experiments for each; I would work on whichever problem appeared most fruitful. I was funded, and a short time later I met one of the members of the Study Section (Ef Racker) who told me that it was the best application he had read. What would happen today with an application that was so “unfocused” and with such nonspecific aims? Equally important to the National Institutes of Health support, we received unsparing help from a number of farsighted physicians such as Walter Bauer at Massachusetts General Hospital, who not only created a high caliber research unit (Roger Jeanloz, Jerome Gross, Karl Schmid, Morris Soodak, and others) but was also instrumental in the Helen Hay Whitney Foundation. In my case, it was William Robinson and Ivan Duff at the Rackham Arthritis Unit at the University of Michigan. Only once (when I was first interviewed) did I have to explain to Bill Robinson how work on Escherichia coli might relate to arthritis. Thus, we had the luxury of following our noses and serendipity wherever the work took us. We started with studies on the intermediary metabolism of glucosamine, which led in turn to the structure of the sialic acids and the discovery of N-acetylmannosamine, to the intermediary metabolism of these compounds, to CMP-sialic acid and its enzymatic synthesis, to the glycosyltransferases, and finally to the phosphotransferase system for sugar uptake by bacteria (reviewed in Refs. 2Roseman S. Chem. Phys. Lipids. 1970; 5: 270-297Crossref PubMed Scopus (799) Google Scholar and 3Roseman S. FEMS Microbiol. Rev. 1989; 63: 3-12Google Scholar). In recent years, the complex process of chitin catabolism by marine bacteria has become a major project (4Keyhani N.O. Roseman S. Biochim. Biophys. Acta. 1999; 1473: 108-122Crossref PubMed Scopus (240) Google Scholar). a timely one for “Reflections.” The primary reason, I think, is illustrated in Fig.1, which shows the surface of an erythrocyte in cross-section. Just outside the plasma membrane of this and nearly all cells is a coat of fuzzy material called the glycocalyx, consisting of a myriad of carbohydrate-rich molecules, polysaccharides, proteoglycans, glycoproteins, and glycolipids. If the cell shown here was a fibroblast or an intestinal epithelial cell that secretes polysaccharides or mucins, it would be difficult to determine the location of the cell boundary; the polymers begin on the cytoplasmic face of the lipid bilayer, within it, or on its periphery, but it is not clear where they end. These extensive, complex structures must serve essential roles in cell surface phenomena, but we are only beginning to understand what some of these functions are. I believe that the glycoconjugates or glycans can serve as important informational macromolecules. In this remarkable age of genomics, proteomics, and functional proteomics, I am often asked by my colleagues why glycobiology has apparently lagged so far behind the other fields. The simple answer is that glycoconjugates are much more complex, variegated, and difficult to study than proteins or nucleic acids. To understand where we are and to appreciate what it has taken to get here requires some background, so this article will briefly survey the history of glycobiology from early studies on fermentation to the beginning of the contemporary era. Although glycobiology antedates biochemistry by many millenniums, their histories are inextricably linked. The principal foundations of both fields lie in the development of organic chemistry during the 19th century and in studies on the process of fermentation of glucose and sucrose. Fermentation was known to the cave man and has been the subject of intense study ever since. The Old Testament has many references to wine and libations, the first being to Noah (Genesis, 9:20–21): “Noah, the husbandman, began and planted a vineyard. And he drank of the wine and was drunk.” 3This reference was kindly called to my attention by Dr. Michael Edidin.Treatises and philosophical discourses were published on the process during and after the Middle Ages. 4One that struck a chord was a 74-page treatise by John Richardson (1790) entitled “Theoretic Hints on an Improved Practice of Brewing Malt-liquors … ”. He defines fermentation as: “A spontaneous internal motion of constituent parts, which occasions a spontaneous separation and removal from their former order of combination, and a remarkable alteration in the subject, by a new arrangement and re-union.” Not a bad definition of intermediary metabolism and the thermodynamics of glycolysis. Fermentation was not confined to making alcohol but has been used for thousands of years to make cheese, soy sauce, etc. The first chapter of the biochemical classic Alcoholic Fermentation by Arthur Harden (1st edition, 1911) reviews the history. (a) The most important early study was that of Lavoisier (1789) who quantitatively established the stoichiometry of the process and concluded that the sugar was split into two parts, one of which was oxidized (carbonic acid) at the expense of the other (alcohol). Furthermore, “if it were possible to recombine these two substances, sugar would result.” The methodology was insufficient to permit him to see an increase in the weight of the yeast or of other products that were formed. (b) Yeast at the time was regarded as a catalyst, much like alumina or diatomaceous earth, and during the first 60 years of the 19th century, this was the prevailing view of the leading chemists and journal editors of the time (Liebig, Berzelius, Wohler). This was despite the fact that in 1837 three independent investigators, Cagniard-Latour, Schwann, and Kutzing, presented evidence that yeast was a living organism, an idea that was ridiculed by the establishment. (c) In 1860, the pivotal experiments of Louis Pasteur finally laid this ghost to rest (5Conant J.B. Pasteur's Study of Fermentation. Harvard University Press, Cambridge, MA1952Google Scholar), and he showed unequivocally that yeast was a living organism. He also did careful stoichiometry. The balance sheet showed that about 95% of the C,H,O of the sugar was converted to CO2 and ethanol. The remainder, from 1.6 to 5%, consisted of substances that the “yeast had taken from the sugar.” The result of this and subsequent work by Pasteur led to his famous dictum, “no fermentation without life.” In an extension of this work, he came to the conclusion (1875) that fermentation was the result of life without oxygen, the cells being able under anaerobic conditions to avail themselves of the energy liberated by the decomposition of substances containing combined oxygen (i.e. anaerobic glycolysis). (d) Enzymes (called ferments), generally hydrolases, were known during the 19th century; indeed, invertase (i.e. sucrase) had been extracted from yeast. In 1858, Traube proposed that fermentation resulted from the action of ferments secreted by cells on sugar. Many attempts were made to extract yeast cells and obtain cell-free fermentation of sugar but without success. Finally, while attempting to preserve yeast extracts for therapeutic purposes, Eduard Buchner succeeded in 1897. The preservative was sugar, and he noted that carbon dioxide was formed. This fortunate and serendipitous result marks the beginning of biochemistry as we know it today. It is interesting to note that the Journal of Biological Chemistry was founded only a few years later by Christian Herter. The story would not be complete without summarizing what was learned between Buchner’s landmark result in 1897 and the publication of Harden’s monograph in 1911. Kinetic experiments were conducted using yeast extracts and glucose, and the rate of fermentation was followed by measuring the rate of CO2 evolution. The following results, especially by Harden and Young, were obtained. (i) Fructose and mannose were fermented as well as glucose, but the yeast had to be “trained” (i.e. adapted) for the extract to ferment galactose. They speculated that different ferments were required for galactose utilization. (ii) Inorganic phosphate was required. (iii) A hexose diphosphate was isolated, characterized as fructose-di-P, and was shown to be an intermediate in the process. (iv) The extract was pressure filtered through a gelatin film, giving a dialysate and a “residue.” Neither alone supported fermentation, but it was restored by mixing the two. The residue contained the heat-labile zymase, and the dialysate contained the heat-stable coenzyme(s) or cozymase. Soon after, it was shown that yeast anaerobic glycolysis was closely connected to anaerobic glycolysis by muscle and muscle extracts. The cozymase, of course, was the source of ATP, NAD+, etc. The close connection between the development of organic chemistry and biochemistry in the 19th century is summarized in an exemplary, early textbook (6Fruton J.S. Simmonds S. General Biochemistry. John Wiley & Sons, Inc., New York1953Crossref Google Scholar). However, carbohydrate history goes back many centuries earlier. Cellulose in the form of cotton, for instance, was known from ancient times, and sucrose was one of the first organic substances to be crystallized (300 A.D., from the juice of sugar cane in India). Because the climate in Europe was not favorable for growing sugar cane, alternative sweetening agents were sought early in the 19th century, leading to the discovery of new sugars (glucose, fructose, mannose, galactose, etc.), all with the same elementary composition (CH2O)n. Clarifying the structural relationships between these compounds occupied carbohydrate chemists for most of the century. Finally, the structure of d-glucose was established by Emil Fischer in 1891, which marks the beginning of modern carbohydrate chemistry. Fischer's multitudinous and brilliant contributions were likewise in the fields of amino acid and purine/pyrimidine chemistry. It is worth reminding the reader that chromatography and electrophoresis were unknown at the time, and substances were purified by fractional crystallization and characterized by elemental analyses and their physical properties (melting point, optical rotation, solubility, etc.). 5My interest in carbohydrate chemistry began as a graduate student working in the laboratory of Karl Paul Link at the University of Wisconsin. He was both a carbohydrate and natural products chemist, with very high standards and an ability to inspire the best in us. The laboratory had isolated and characterized dicumarol as the hemorrhagic factor in spoiled sweet clover hay prior to my arrival (warfarin is a synthetic analogue). My project was to study the metabolism of its parent compound, 4-OH-coumarin, which was not toxic. Four large dogs used as subjects were fed the drug and maintained in very large metabolic cages so that their urine could be collected. (In those days, graduate students took complete and very good care of their animals, including feeding, exercising them, and cleaning their cages.) The metabolic product turned out to be 4-OH-coumarin β-d-glucuronide. However, this had to be established by synthesis and also by elemental analysis. I spent a very muggy, frustrating summer in Madison recording the swings on a microbalance and learning how to do microanalyses before the standards finally came out right. Somewhat later, I developed considerable experience with fractional crystallization, particularly of anomeric glycosides. They were being synthesized for Joshua Lederberg, a young faculty member in a neighboring department (genetics), who was using them for assaying the expression of glycosidases, such as β-galactosidase in E. coli. Fractional crystallization, like elemental analysis, is tedious work, but above all it is a real art and when it works, it is most gratifying. In doing this kind of work, we even invoked the help of the Lord. To this day my children remember that my wife Martha (who is not a scientist) concluded the evening prayers over the Sabbath candles with the following phrase: “and may Daddy have crystals.” It worked! In this age of electronics and the internet, one always thinks that science moves forward too slowly, but it is mind boggling to realize how far we have come since the 1890s (Fischer, Buchner). Although mucins from various sources were studied by organic chemists as early as 1846 (see reviews by Blix, Gottschalk, and Morgan (7Gottschalk A. Glycoproteins: Their Composition, Structure and Function. Elsevier Science Publishers B.V., Amsterdam1972Google Scholar)) and were thought to contain sugars, there was always an unresolved question of purity. In 1925, the distinguished chemist, P. A. Levene, who had made fundamental contributions to the structures of the nucleic acids, published a monograph entitled “Hexosamines and Mucoproteins.” Chondroitin sulfate had been isolated in 1884 from cartilage, but the nature of its monosaccharides and structure were controversial until Levene showed conclusively that the were acid, acid, and acid in He the structure as to and at on the might be from the methodology and on sugar there were major in the structural including the fact that it was a sulfate (i.e. acid) was as a containing but also He also substances such as were or such substances even In Chem. 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