Lectins: Carbohydrate-specific Reagents and Biological Recognition Molecules

The occurrence in nature of proteins with hemagglutinating activity that in later years were shown to be sugar-specific and eventually named lectins has been known since the turn of the 19th century, but until about two decades ago they aroused little interest (for a historical survey, see Ref. 1Sharon N. Lis H. Lectins: from hemagglutinins to biological recognition molecules. A historical overview. Glycobiology. 2004; 14: 53R-62RCrossref PubMed Scopus (0) Google Scholar). My own involvement with these proteins began inadvertently and initially on a part-time basis in the early 1960s after my return to the Weizmann Institute from two and a half years of exciting and educational postdoctoral studies in the United States. During the first of these I worked in the laboratory of Fritz Lipmann at the Massachusetts General Hospital, Boston. Lipmann, one of the most influential biochemists of the last century, was then interested in the mechanism of protein biosynthesis. I was assigned to study the amino acid activation reaction (the first step in this process), work that resulted in two publications (2Sharon N. Lipmann F. Reactivity of analogs with pancreatic tryptophan-activating enzyme. Arch. Biochem. Biophys. 1957; 69: 219-227Crossref PubMed Google Scholar, 3Hoagland M. B. Zamecnik P. C. Sharon N. Lipmann F. Stulberg M. P. Boyer P. D. Oxygen transfer to AMP in the enzymatic synthesis of the hydroxamate of tryptophan. Biochim. Biophys. Acta. 1957; 26: 215-217Crossref PubMed Google Scholar). Concurrently, I greatly enriched my knowledge of biochemistry, mainly from my fellow postdoctoral students, and especially from the guest seminars in which ongoing biochemical discoveries and developments were reported. I spent the second postdoctoral year at the Massachusetts General Hospital with Roger Jeanloz, a leading carbohydrate chemist, where I got my training in the subject and also succeeded in isolating an unusual diamino sugar from a Bacillus polysaccharide I had brought with me from Rehovot (4Sharon N. Jeanloz R. W. The diaminohexose component of a polysaccharide isolated from Bacillus subtilis. J. Biol. Chem. 1960; 235: 1-5Abstract Full Text PDF PubMed Google Scholar) (see below) ; the remaining time I worked with Dan Koshland at Brookhaven National Laboratory on the mechanism of action of myosin ATPase (5Levy H. M. Sharon N. Koshland Jr. , D. E. Purified muscle proteins and the walking rate of ants. Proc. Natl. Acad. Sci. U. S. A. 1959; 45: 785-791Crossref PubMed Google Scholar, 6Levy H. M. Sharon N. Lindemann E. Koshland Jr. , D. E. Properties of the active site in myosin hydrolysis of adenosine triphosphate as indicated by the O18-exchange reaction. J. Biol. Chem. 1960; 235: 2628-2632Abstract Full Text PDF PubMed Google Scholar). Dan was then starting to make his mark on enzymology with his "induced fit" concept of enzyme action, originally greeted with much skepticism (7Koshland Jr. , D. E. Crazy, but correct. Nature. 2004; 432: 447Crossref PubMed Scopus (0) Google Scholar). Back at Rehovot my original aim was to establish the structure of that diamino sugar; I was fortunate to receive for this purpose my first National Institutes of Health (NIH) grant, a modest one of some 25, 000 for 3 years. (This would have been unheard of at the present time because nothing was known then about the function of the compound. ) The task took me (with a couple of graduate students) over a decade; eventually we were able to prove by degradation and synthesis that the compound in question, which we named bacillosamine, is 2, 4-diamino-2, 4, 6-trideoxy-d-glucose (8Zehavi U. Sharon N. Structural studies of 4-acetamido-2-amino-2, 4, 6-trideoxy-d-glucose (N-acetylbacillosamine), the N-acetyl diaminosugar of Bacillus licheniformis. J. Biol. Chem. 1973; 248: 433-438Abstract Full Text PDF PubMed Google Scholar, 9Liav A. Hildhesheim J. Zehavi U. Sharon N. Synthesis of 2-acetamido-2, 6-dideoxy-d-glucose (N-acetyl-d-quinovosamine), 2-acetamido-2, 6-dideoxy-d-galactose (N-acetyl-d-fucosamine) and 2, 4-acetamido-2, 4, 6-trideoxy-d-glucose from 2-acetamido-2-deoxy-d-glucose. Carbohydr. Res. 1974; 33: 217-227Crossref Scopus (36) Google Scholar). To my delight, the di-N-acetyl derivative of bacillosamine has recently been found attached glycosidically to the amide of asparagine or the hydroxyl of serine in the carbohydrate-peptide linkage region of several interesting glycoproteins of pathogenic bacteria (10Weerapana E. Imperiali B. Asn-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology. 2006; 16: 91R-101RCrossref PubMed Scopus (0) Google Scholar). By a strange twist of fate, most of these glycoproteins were originally isolated in 2002 by Martin Young and his colleagues at the National Research Laboratories, Ottawa, from Campylobacter jejuni by affinity chromatography on immobilized soybean agglutinin (SBA) (11Young N. M. Brisson J. R. Kelly J. Watson D. C. et al. Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 2002; 277: 45230-45239Abstract Full Text Full Text PDF Scopus (321) Google Scholar), the first lectin I got involved with 40 years earlier. My studies of SBA began together with Halina Lis with whom it has been my good fortune to collaborate to this very day. It aroused our curiosity not because of its ability to bind sugars specifically and reversibly and to agglutinate cells, the hallmarks of proteins of this class, but because of other reasons that I shall presently mention. We did not have the slightest idea that lectins would become extremely useful carbohydrate-specific reagents, that they would be found to function as mediators of cell recognition, or that they would make a major contribution to glycobiology (12Taylor M. E. Drickamer K. Introduction to Glycobology. 2nd Ed. Oxford University Press, Oxford, UK2006Google Scholar). In fact, for a time we were not even aware of the term lectin, which was originally proposed in 1956 by William C. Boyd from Boston University for blood type-specific hemagglutinins. Because SBA, like the majority of the hemagglutinins, is not blood group-specific, we began referring to it as a lectin only in 1970, when it occurred to us that the original definition should be broadened to include all cell-agglutinating and sugar-specific proteins (13Sharon N. Lis H. Lectins: cell agglutinating and sugar-specific proteins. Science. 1972; 177: 949-959Crossref PubMed Google Scholar). Our interest in SBA developed in the course of investigations on soybean proteins carried out within the framework of a generous and long term grant from the United States Department of Agriculture that I received in 1961 jointly with Katchalski-Katzir (14Katchalski-Katzir E. My contributions to science and society. J. Biol. Chem. 2005; 280: 16529-16541Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Katchalski was the founding Head of the Department of Biophysics at the fledgling Weizmann Institute, which was officially inaugurated in 1949. I came to the department in 1954 after having received my Ph. D. degree from the Hebrew University, Jerusalem; Halina, with a Ph. D. degree from Uppsala University, joined the department 5 years later. The purpose of the above grant was to carry out a fundamental study of the soy proteins with the aim of providing information for their improved utilization for human nutrition. Katchalski and I were persuaded to embark on this project by Tim (M. L. ) Anson and Aaron Altschul, close friends, noted protein chemists, and enthusiastic believers in these proteins as the best solution to world hunger. After some time, Katchalski became immersed in his pioneering studies of polyamino acids as protein models and on enzyme immobilization and turned over the whole project to me, for which I am extremely grateful. Halina and I set out by trying to obtain pure proteins from soybeans by chromatographic techniques, but this proved to be a difficult task as most of them lack biological activity, are poorly soluble, and undergo complex association-dissociation reactions. We therefore chose to focus on SBA, originally isolated and characterized in the 1950s by Irvin E. Liener at the University of Minnesota, St. Paul. The main reason for our choice was the evidence presented by Liener that it contained glucosamine, raising the likelihood that it may be a glycoprotein (15Wada S. Pallansch M. J. Liener I. E. Chemical composition and end groups of soybean hemagglutinin. J. Biol. Chem. 1958; 233: 395-400Abstract Full Text PDF PubMed Google Scholar). In those days, research on glycoproteins was in its infancy, but I became intrigued by these compounds because of my interest in carbohydrates, as described elsewhere (16Sharon N. Half a century between carbohydrates and proteins. Comprehensive Biochemistry. in: A History of Biochemistry. Vol. 41. Elsevier Publishing Co. , Amsterdam2000: 391-448Google Scholar). Working on SBA, Halina and I soon found that it contains not only glucosamine but also mannose. We then isolated from a proteolytic digest of SBA an asparaginyloligosaccharide that contained all the N-acetylglucosamine and mannose of the lectin (17Lis H. Sharon N. Katchalski E. Soybean hemagglutinin, a plant glycoprotein. I. Isolation of a glycopeptide. J. Biol. Chem. 1966; 241: 684-689Abstract Full Text PDF PubMed Google Scholar). Eventually we also isolated from the lectin N-acetylglucosaminylasparagine (18Lis H. Sharon N. Katchalski E. Identification of the carbohydrate-peptide linking group in soybean agglutinin. Biochim. Biophys. Acta. 1969; 192: 364-366Crossref PubMed Google Scholar), the carbohydrate-peptide linking group, that was identical with the one originally obtained in 1963 by Albert Neuberger, the founding father of modern glycoprotein research, in his pioneering studies of ovalbumin. As pointed out recently by Liener (19Liener I. E. A trail of research revisited. J. Agric. Food Chem. 2002; 50: 6580-6582Crossref PubMed Scopus (3) Google Scholar), "The fact that SBA was shown to be a glycoprotein may not be particularly surprising to the modern day biochemists, but at the time the finding of a sugar moiety in a plant protein was accepted with reservation. It was thought that glycoproteins were strictly of animal origin and that the finding of a sugar with a plant protein was most likely because of noncovalent contamination. " In 1981, jointly with Hans (J. F. G. ) Vliegenthart from the University of Utrecht, the complete structure of the carbohydrate of SBA was established by NMR as the branched oligomannoside Man9 (GlcNAc) 2, found in animal glycoproteins too, demonstrating that protein N-glycosylation is a process conserved in plants and animals (20Dorland L. van Halbeek H. Vliegenthart J. F. G. Lis H. Sharon N. Primary structure of the carbohydrate chain of soybean hemagglutinin. A reinvestigation by high resolution 1H NMR spectroscopy. J. Biol. Chem. 1981; 256: 7708-7711Abstract Full Text PDF PubMed Google Scholar). A unique feature of SBA is that all its molecules carry the same oligosaccharide (21Ashford D. A. Dwek R. A. Rademacher T. W. Lis H. Sharon N. The glycosylation of glycoprotein lectins. Intra- and inter-genus variation in N-linked oligosaccharide expression. Carbohydr. Res. 1991; 213: 215-227Crossref PubMed Scopus (0) Google Scholar) in contrast to essentially all other glycoproteins, which bear a variety of glycans at each attachment site, i. e. consist of mixtures of distinct glycoforms. SBA serves therefore as an excellent source of this oligosaccharide (for an example, see Ref. 22Deras I. L. Kawasaki N. Lee Y. C. Quantitative recovery of Man9GlcNAc2Asn from concanavalin A. Carbohydr. Res. 1998; 306: 469-471Crossref PubMed Scopus (0) Google Scholar). Our few 1960s publications on SBA attracted little attention, and we sometimes felt like wanderers in a desert. Although the studies of lectins were in their eighth decade and several hundreds of these proteins (almost all from plants) had already been identified, the handful of other scientists active in the field at the time did not fare better. Irwin J. Goldstein from the University of Michigan at Ann Arbor, still a leading lectin researcher, tells that when he sent a note in 1963 to Biochemical and Biophysical Research Communications describing the purification of concanavalin A by affinity chromatography, it was rejected forthright because "this represents a modest advance in an obscure area. " The note was eventually published in the Biochemical Journal (23Agrawal B. B. L. Goldstein I. J. Specific binding of concanavalin A to cross-linked dextrans. Biochem. J. 1965; 96: 23c-25cCrossref Scopus (0) Google Scholar), and affinity chromatography soon became the method of choice for lectin isolation. However, as the 1960s were folding, the attitude toward lectins began to change, and a number of leading biochemists and immunologists, among them Gerald Edelman at Rockefeller University, Mel Greaves at London University, Elvin Kabat at Columbia University, Jerker Porath at Uppsala, and Jon Singer at University of California, San Diego, became involved with them. The reasons for this change in attitude were summarized by Kabat, who had become intrigued with lectins primarily because their combining sites seemed similar to those of antibodies and who in 1977 stated: "During the past 10 years there has been an extraordinary burst of activity in the study of plant and animal lectins, stimulated largely by the findings that they have specific receptor sites for carbohydrates and react with glycoproteins in solution or on cell membranes. . . " (24Kabat E. A. Dimensions and specificities of recognition sites on lectins and antibodies. J. Supramol. Struct. 1978; 8: 79-88Crossref PubMed Google Scholar). In 1970, affinity chromatography of glycoproteins on immobilized lectins was introduced (among others) by Donnely and Goldstein (25Donnely E. H. Goldstein I. J. Glutaraldehyde-insolubilized concanavalin A: an adsorbent for the specific isolation of polysaccharides and glycoproteins. Biochem. J. 1970; 118: 697-698Google Scholar). It became a must at one step or another for the isolation of membrane proteins, all of which are glycosylated, a classical case being that of the insulin receptor with the aid of wheat germ agglutinin (WGA) (26Jacobs S. Schechter Y. Bissell K. Cuatrecasas P. Purification and properties of insulin receptors from rat liver membranes. Biochem. Biophys. Res. Commun. 1977; 77: 981-988Crossref PubMed Google Scholar). Lectins proved also to be useful for the separation of purified glycoproteins into their glycoforms, i. e. differently glycosylated forms of the same protein. A very recent telling example is of different glycoforms of IgG with different degrees of sialylation, obtained by fractionation on the sialic acid-specific Sambucus nigra agglutinin and shown to differ in their anti-inflammatory activity (27Kaneko Y. Nimmerjahn F. Ravetch J. V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science. 2006; 313: 670-674Crossref PubMed Scopus (1201) Google Scholar). Interest in lectins intensified with the realization that they are extremely valuable reagents for the investigation of cell surface sugars, for the assessment of the role of the latter in cell growth and differentiation, in interactions of cells with their environment, and also in a variety of pathological processes. In this connection it is instructive to refer to two classical studies with lectins that provided very early evidence for the presence of sugars on cell surfaces and their potential role as cell identity markers, a common theme in modern glycobiology. One came from the laboratory of James Sumner at Cornell University, Ithaca, who in 1919 isolated concanavalin A in crystalline form but only in 1936, together with Howell, reported that it agglutinates cells such as erythrocytes and yeasts and that this agglutination is inhibited by sucrose, thus demonstrating for the first time the sugar specificity of lectins (28Sumner J. B. Howell S. F. Identification of concanavalin A with the hemagglutinin of jack bean. J. Bacteriol. 1936; 32: 227-237Crossref PubMed Google Scholar). Moreover, with much foresight they suggested that the hemagglutination induced by the lectin might be a consequence of its reaction with carbohydrates on the surface of the red cells. The other study was by Walter Morgan and Winifred Watkins at the Lister Institute, London, who in the early 1950s used blood type-specific hemagglutinins to show that the blood type A immunodeterminant is α-linked N-acetylgalactosamine and that the H (O) determinant is α-l-fucose (reviewed in Ref. 29Morgan W. T. J. Watkins W. M. Unraveling the biochemical basis of blood group ABO and Lewis antigenic specificity. Glycoconjugate J. 2000; 17: 501-530Crossref PubMed Scopus (0) Google Scholar). This was the first demonstration that cell surface carbohydrates can serve as carriers of biological information. Much excitement was created in the following decade by the reports of Joseph C. Aub from the Massachusetts General Hospital (30Aub J. C. Sanford B. H. Wang L. H. Reactions of normal and leukemic cell surfaces to a wheat germ agglutinin. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 400-402Crossref PubMed Google Scholar) and Max Burger from Princeton University (31Burger M. Goldberg A. R. Identification of a tumor-specific determinant of neoplastic cell surfaces. Proc. Natl. Acad. Sci. U. S. A. 1967; PubMed Google Scholar), who were with for N-acetylglucosamine and and of with from the Department of of our Institute, who used concanavalin A for mannose and M. L. of the protein concanavalin A with normal and Natl. Acad. Sci. U. S. A. 1969; PubMed Google Scholar), that these lectins cells but not their normal cells. The reports provided evidence that might be with a change in cell surface sugars, an idea that only a few years had been In with and we found soon that SBA for and also the ability to between normal and cells Lis H. Sharon N. L. of sites at the surface membrane of normal and Biol. 1970; PubMed Scopus (0) Google Scholar). studies have that high to agglutination by lectins is a by not cells. of were or their with the aid of lectins. concanavalin A and as an and Jon Singer at University of California, San Diego, found that the lectin bind specifically to the surface of the human and membrane and that the of the membrane of eukaryotic cells are Singer plant as specific for to to cell Natl. Acad. Sci. U. S. A. PubMed Google Scholar). for such was obtained by at University, who used and that the major of the human is that its is to the the other of the same are in the or into the T. W. Chemical and surface of the major glycoprotein of the human Natl. Acad. Sci. U. S. A. 1972; 69: PubMed Google Scholar). studies with lectins provided some of the most evidence for the membrane of Singer and to which the membrane of proteins and glycoproteins in a (reviewed in Ref. of lectins with animal cell 1974; PubMed Google Scholar). among these was the finding of the and of the membrane receptors on and other of as for example by the with concanavalin A of rat or M. L. of sites on the surface in to of cell 1973; 32: PubMed Scopus (0) Google Scholar). of cell surface carbohydrates was later shown to be for of lectins on cells such as and of The for animals of plant lectins has been since the of lectin research, at the end of the 19th However, research on the action of lectins on cells only decades later with being to cell and to different lectins, primarily the and the and (reviewed in Ref. P. and the of Full Text PDF Google Scholar). the field was one cell isolated in by at University, at the National Institute for London, and at University, This the enzyme in the of complex and N-linked carbohydrate of other cell with different enzymatic glycosylation became proved extremely valuable for the investigation of the of glycoproteins and and of the function of their carbohydrates, especially those on the cell they also serve for the of useful glycoproteins such as In the of I at the Department of University of at for a year as My was with whom I at the of lectins to the on the of carbohydrates as information and recognition molecules. had been by from University S. The synthesis of complex carbohydrates by and their potential function in 1970; PubMed Scopus Google Scholar) and at the A. and of of in and of Press, Scholar). Although there a few and several on lectins, of them with their properties did they their potential as for biological Because Dan Koshland from the same department at was then a of the of I with the that I a on lectins for that This was accepted by then of was by me in the of that year in the laboratory of Albert at St. Hospital in London, where I for a few to study on which and I were at that time However, I by chromatography from wheat germ together with it into and that its specificity is similar to that of A. A. Sharon N. The purification and specificity of wheat germ J. 1973; PubMed Google Scholar), because it a affinity not only for from as originally by Burger and Goldberg (31Burger M. Goldberg A. R. Identification of a tumor-specific determinant of neoplastic cell surfaces. Proc. Natl. Acad. Sci. U. S. A. 1967; PubMed Google Scholar), but also of In we also proved to in the was not a glycoprotein. This work stimulated the interest of in lectins with which he to be for several years into his The was jointly with Halina my return to Rehovot early in (13Sharon N. Lis H. Lectins: cell agglutinating and sugar-specific proteins. Science. 1972; 177: 949-959Crossref PubMed Google Scholar). It summarized the of the research on lectins since their their specificity for and cells, and the properties of concanavalin A and the few other lectins that had been purified at the The that on cell surfaces as by lectins, were their was not and were by as to they are a of cells. of we that lectins, and a and useful for the study of the of cell we in with the on the role of lectins in about which nothing was known with on lectins was published by us in the following year in the of N. Lis H. The of plant lectins Biochem. 1973; PubMed Scopus Google Scholar) and a in the same in H. Sharon N. Lectins as molecules and Biochem. PubMed Google Scholar). In these we to to the our and for the In we a on lectins and in a second of the same N. Lis H. Google Ed. The Scholar), of which have been into years ago I a on lectins to which Halina and I several I. E. Sharon N. Goldstein I. J. The and in and Press, Scholar). A activity of was the in of a in which lectins are and where I my the specificity of is in of sugar not of amino acids or N. and Scholar). The was on that I for the graduate my course on the same subject and in for a long I still this the and The the of the study of the properties of lectins, a for a of their at the In concanavalin A became the first of these proteins for which the and have been the latter by This was to the of Gerald group at the Rockefeller University Jr. , M. J. Wang The and structure of concanavalin Natl. Acad. Sci. U. S. A. 1972; 69: PubMed Google Scholar) and of the of and F. at National of concanavalin A at 1972; PubMed Google Scholar). The first in this an of into two became known as the or lectin N. M. B. A of proteins with the lectin Biochem. Biol. Scholar). The of the concanavalin A structure was soon by the by at University of the structure of as as of its with even the complete amino acid of this lectin had become The structure of wheat germ agglutinin at Biol. 1977; PubMed Scopus (0) Google Scholar). It is that at present the of close to 10