Sugars And Splice: Glycobiology: The Next Frontier

Table of Carbohydrate Products Model of glycoprotein supplied by Seikagaku America Glycobiology. It has been called the "last frontier of pharmaceutical discovery." Hampered by a lack of economical and convenient tools, however, advances in glycobiology have been largely overshadowed by the rush to exploit PCR and the ready availability and comparative simplicity of the tools and enzymes for molecular biology. But that is changing. By all reports, the field of glycoprotein and carbohydrate analysis and synthesis is making a comeback and has a bright future. Fueled by recent Food and Drug Administration requirements for the characterization of the oligosaccharide portions of recombinant proteins with pharmaceutical applications, new findings concerning the roles carbohydrates play in biological systems, and the rise of proteomics, industry insiders report much optimism about the growth of the carbohydrate analysis market. The field of glycobiology has recently seen plenty of shuffling among the companies that manufacture and supply reagents for carbohydrate analysis. To illustrate, Genzyme Corp., Roche Molecular Biochemicals, and Oxford Glycosciences have left or are leaving the glycobiology market. In fact, Novato, Calif.-based Glyko Inc. has recently purchased Oxford Glycosciences' carbohydrate research reagent business. The purchase has made for exciting times at Glyko, the developer of the electrophoresis-based FACE® technology for carbohydrate analysis. "This move allows us to offer both electrophoretic and HPLC-based carbohydrate product lines to our customers," says Glyko's general manager, Brian Brandley. In addition, Glyko plans to release product lines for agricultural and food applications later this year and to distribute new catalogs and update its Web site in July. Meanwhile, other companies are expanding their glycobiology product lines while putting more effort into research and development. Calbiochem, for example, is carving out a strong niche in the reagent side of the carbohydrate analysis and synthesis market. Margaret Dentlinger, marketing director for Calbiochem, reports that the company plans to double its carbohydrate product line to nearly 800 items with the release of a new glycobiology catalog later this year. "We are very excited about our new catalog. It features good introductory articles by leading scientists, very useful comparative tables, and hundreds of new products including glycotransferases, lectins, and carbohydrate standards. It should be an excellent reference for researchers," she said in a telephone interview. She also said she sees much synergy developing between combinatorial chemistry and oligosaccharide synthesis, especially as new carbohydrate-related products become available. ProZyme Inc. of San Leandro, Calif., a manufacturer of specialty "PROteins" and "enZYMES" for diagnostics and research applications, is committing a significant amount of resources to research and development of new carbohydrate analysis products. In reference to glycobiology, ProZyme's Jo Wegstein states that "today, researchers need to understand the whole molecule. ProZyme is committed to supplying them with the best enzymes possible." ProZyme prides itself on the quality and characterization of its deglycosylation enzymes and relies heavily on an electronic catalog available through its Web page (www.prozyme.com). A quiet company known primarily within the glycobiology community for its quality products, Seikagaku America, a division of Associates of Cape Cod in Falmouth, Mass., markets and distributes a wide range of glycobiology products. Developed by Seikagaku Corp. of Japan, these products include enzymes, standards, reagents, and monoclonal antibodies to glycoconjugates. According to Seikagaku America's technical sales representative, Mary Beck, Seikagaku was the first to produce and market GAG degrading enzymes. She also explains that as "one of the first companies to foster industry/academic collaborations, Seikagaku has helped define what is known today as glycoscience." Indeed, the company backs its products with 52 years of research and experience. Glycobiology is recognized as the study of the biology of glycoconjugates in simple and complex systems.1 In this context, glycoconjugates are proteins and lipids with covalently attached oligosaccharide moieties. Glycoproteins, for instance, are proteins with carbohydrates attached covalently to asparagine or serine/threonine residues. N-linked oligosaccharides, or those attached to the asparagine residues, may contribute 3.5 kd or more per structure to the mass of a glycoprotein. The structures bound to the serine/threonine residues, called O-linked oligosaccharides, are less massive than the N-linked structures but may be more numerous. Together, both types of oligosaccharide structures perform critical biological functions in protein sorting, immune recognition, inflammation, and other processes. Proteoglycans are a class of glycoproteins with covalently linked sulfated glycosaminoglycans. This group includes proteins such as chondroitin sulfate, heparin, and keratan sulfate. Proteoglycans interact with important growth factors, extracellular matrix proteins, extracellular enzymes, some path-ogens, and can also regulate growth, development and tissue architecture. Lastly, glycolipids consist of sugar and ceramide moieties and are ubiquitous components of vertebrate cell plasma membranes. They function as receptors for microorganisms and their toxins, organize cellular attachment to matrices, and modulate cell growth and differentiation. One good example of the biological significance of a carbohydrate epitope is a structure called alpha-Gal,2 found as part of the carbohydrate structures on cell-surface glycoproteins and glycolipids in nonprimate mammals and New World monkeys. Interestingly, this epitope is missing in humans, apes, and Old World monkeys, yet these organisms make antibodies against alpha-Gal because of antigenic stimulation from gastrointestinal bacteria that express structures related to alpha-Gal. The resulting interaction between alpha-Gal and the alpha-Gal antibodies causes almost immediate rejection when organs and tissues from pigs, for example, are transplanted into monkeys. This hyperacute rejection phenomenon has led to intensive research into alternative sources of organs for transplantation into humans. Alpha-Gal epitope, on the other hand, has potential applications for gene and recombinant protein therapy and use in tumor and viral vaccines. Incomplete processing of carbohydrates causes serious diseases in humans. Defective N-glycosylation results in a condition called carbohydrate-deficient glycoprotein syndrome, where patients develop multiple symptoms of neuropathy. Similarly, incomplete N-glycan processing causes congenic dyserythropoietic anemia. A major stumbling block to the growth of carbohydrate analysis has been the failure of technology to keep up with demand, unlike the explosive growth of tools available for molecular biology. Because working with carbohydrates is a much more complicated affair, glycobiology has been unable to keep pace. However, with the development of new technologies and the release of various kits for glycoprotein analysis, manufacturers are beginning to close the gap. In addition, better quality enzymes and a wide range of lectins, oligosaccharides, standards, and related reagents and antibodies are beginning to streamline the analysis of carbohydrates. Recent advances in glycoprotein analysis have appeared in the form of kits designed to determine if a protein is glycosylated and to provide structural and sequence data about the oligosaccharide moieties of those glycoproteins. For the most part, these kits are built around polyacrylamide gel electrophoresis. Glyko Inc. has developed and patented FACE technology, for instance, as a simple and reliable method of carbohydrate analysis based on polyacrylamide gel electrophoresis. FACE technology, or Fluorophore-Assisted Carbohydrate Electrophoresis, allows the quick profiling of carbohydrates released from various glycoconjugates through four steps. First, carbohydrates are released from glycoconjugates with chemical or enzymatic methods. Second, the released oligosaccharides are fluorescently labeled with ANTS by reductive amination. Third, the labeled oligosaccharides are separated on Glyko's optimized precast polyacrylamide gels. Finally, the FACE gels are imaged by photography on a long-wave UV light box or analyzed quantitatively with the FACE Imager. This device uses a CCD camera for high sensitivity and accurate band quantitation. Specific application kits provide the materials needed for N-linked profiling, N-linked sequencing, O-linked profiling, and the determination of monosaccharide composition. FACE technology has strong applications for the quality control of production-lot glycoproteins. In addition, the technology is suited for the determination of glycosylation type; the evaluation of purification procedures, culture conditions, and expression systems for the effects on glycosylation; and for the investigation of directed modification of glycosylation. The removal of all or a select class of oligosaccharides is often necessary to study the structure and function of a glycoprotein. Frequently, this approach allows the assignment of a specific biological function to the various components of a glycoprotein in many areas. These include the enhancement or reduction of blood clearance rates of glycoprotein therapeutics, the investigation of the role of carbohydrates in enzyme activity, and the simplification of protein sequencing. As an aid to these tasks, ProZyme manufactures the GlycoPro™ Deglycosylation Kit. Designed to deglycosylate up to 2 mg of glycoprotein, the kit includes PNGase F, Endo-O-Glycosidase™, Sialidase A™, and the other reagents necessary to remove all N- and simple O-linked (including polysialyated) carbohydrates from the glycoprotein in question. The extent of deglycosylation is monitored by observing mobility shifts of the treated protein with SDS PAGE. For proteins with more complex O-linked carbohydrates, ProZyme offers the prO-LINK Extender™ Kit for use in conjunction with the GlycoPro Deglycosylation Kit. The prO-LINK Extender contains ß-galactosidase and ß>-N-acetylglucosaminidase for the degradation of O-linked structures consisting of ß(1-4)-linked galactose and ß>-linked N-acetylglucosamine, such as polylactosamine. For added convenience, both kits are sold together in a combination pack for tackling the deglycosylation of most proteins. In a similar vein, Calbiochem sells the Glycoprotein Deglycosylation Kit. This kit contains the reagents and enzymes necessary to remove all N-linked, all simple O-linked, and nearly all complex O-linked oligosaccharides from glycoproteins in a single reaction at neutral pH. As with most of these kits, SDS PAGE is again used to monitor the extent of deglycosylation. The kit contains enough reagents to deglycosylate up to 1 mg of protein. Bio-Rad Laboratories' Glyco Analysis System handles the analysis of a glycoprotein using kits designed for each step of the analysis process. These steps include glycoprotein detection, glycoprotein deglycosylation, oligosaccharide purification, and oligosaccharide structural analysis. To begin with, Bio-Rad's Immun-Blot Kit for Glycoprotein Detection detects glycoproteins using SDS-PAGE and Western blotting technology. The Enzymatic Deglycosylation Kit uses three glycosidases to remove N-linked and sialic acid-substituted Gal( ß1-3)GalNAc(alpha1) O-linked oligosaccharides from glycoproteins. The enzymatic deglycosylation performed by this kit prepares intact deglycosylated proteins suitable for other analytical procedures. The Monosaccharide Compositional Analysis Kit uses standard hydrolysis techniques, fluorescent labeling, and polyacrylamide gel electrophoresis to determine the monosaccharide composition of glycoproteins and purified oligosaccharides. Two Oligosaccharide Profiling Kits provide simple and familiar methods for isolating and profiling N- and O-linked oligosaccharides through fluorescent labeling and PAGE. Finally, the N-linked Oligosaccharide Sequencing Kit uses exoglycosidases and PAGE to determine the sequence of N-linked oligosaccharides. In all, Bio-Rad provides several methods for the analysis of the carbohydrate moieties of glycoproteins that involve the familiar techniques of electrophoresis and chromatography. In support of these products, Bio-Rad sells precast carbohydrate PAGE gels and the Gel Doc 1000 imaging system for image analysis and documentation. Amersham Pharmacia Biotech sells the ECL Glycoprotein Detection System. This kit identifies the presence or absence of carbohydrates or, preferentially, sialic acid on a protein of interest blotted onto nitrocellulose or PVDF membranes. The kit works through biotinylation of the carbohydrate moieties on the protein followed by labeling with a streptavidin-horseradish peroxidase conjugate. The biotinylated proteins are then detected with the luminol-based ECL detection reagents. The Glycoprotein Carbohydrate Estimation Kit from Pierce Chemical Co. has been designed for the quick and easy identification of an unknown protein as a glycoprotein and is complementary to the electrophoretic and ELISA methods used for glycoprotein detection. The protein under study is reacted with Pierce's Glycoprotein Detection Reagent in a 96-well plate. The resulting colored complex is measured with a plate reader, and the approximate carbohydrate percentage in the glycoprotein is estimated by comparison to glycoprotein standards. Pierce has just introduced its new GelCode® Glycoprotein Stain for the detection of glycoproteins in SDS-PAGE gels and on Western blotting membranes. Designed to stain 10 mini gels, the kit reveals glycoproteins as magenta bands against a light pink or colorless background. Pierce plans to introduce new glycoprotein-related products in the coming months. Since their discovery in the late 1880s, lectins, a group of nonenzymatic proteins with high affinities for mono- and oligosaccharides, have been developed into powerful tools for the purification of glycoproteins and the analysis of carbohydrate structures. Lectins have been found in animals, plants, microorganisms, and viruses; in many cases, their sugar specificity has been determined. The exploitation of this specificity provides the basis for the use of lectins as tools for the separation and structural analysis of glycoproteins and oligosaccharides. To illustrate, lectin affinity chromatography using lectins immobilized on agarose beads has been used to isolate and purify soluble glycoproteins, hormones, oncofetal antigens, and many other compounds. Lectins are available from several sources and come in many forms. In addition to crude and purified preparations, lectins conjugated to enzymatic and fluorescent labels, colloidal gold, or biotin immobilized for chromatographic purposes are sold. Vector Laboratories of Burlingame, Calif., purified the first line of lectins in 1976 and has grown to become the world's leading lectin supplier. In addition to its unconjugated, fluorescein-labeled, rhodamine-labeled, biotinylated, agarose-bound, and peroxidase-conjugated lectins, Vector offers antibodies to lectins and biotinylated antilectin antibodies. Vector also sells three lectin-screening kits. Each kit provides researchers with a panel of seven lectins or lectin conjugates. Finally, the company Web site (www.vectorlabs.com) features descriptions of the uses and properties of each lectin, as well as pertinent references and review articles. EY Laboratories of San Mateo, Calif., offers the widest selection of lectins available. Supplying lectins from organisms as diverse as potatoes (Solanum tuberosum) and garden slug (Limax flavus), this 23-year-old company carries lectins in crude and purified states, as well as conjugated and immobilized forms, affinity chromatography kits with selected lectins, carbohydrates, and a thorough line of biochemical reagents. Sigma and Calbiochem also sell a wide range of unconjugated, conjugated, and immobilized lectins. Sigma carries antisera to lectins and specially tested lectins used to induce mitogenic activity in cell cultures. Calbiochem, meanwhile, has added 24 new lectins and agarose-conjugated lectins to its inventory. United States Biological, ICN, and Amersham Pharmacia Biotech also sell lectins and related products. While too numerous to cover in detail, more enzymes and related reagents are becoming available to the life scientist. Presently, researchers have access to a battery of carbohydrate analysis enzymes. These enzymes fall into several categories. Glycosidases, the glycobiologist's equivalent of the restriction enzyme, cut oligosaccharides with a high degree of specificity. Both exo- (terminal cleavage) and endo- (internal cleavage) glycosidases are available. More than 500 have now been discovered and classified into 45 groups. Glycosidases are useful for the analysis of oligosaccharide structure and sequence and for removing oligosaccharides from proteins. Natural and cloned glycosidases are on the market. New England Biolabs (NEB) of Beverly, Mass., offers a wide array of high concentration endoglycosidases and novel prokaryotic exoglycosidases. In fact, as production is linked to basic research at NEB, the glycobiology group discovered a wide array of novel and unique exoglycosidases in the genus Zanthomonas.3 These well-characterized exoglycosidases have become powerful tools in the repertoire of the glycobiologist. The company Web site (www.neb.com) provides detailed property and specificity information for each enzyme. Next, glycosyltransferases are a group of enzymes that synthesize oligosaccharides in nature. These are useful for the in vitro synthesis of natural and unnatural oligosaccharides. Calbiochem offers a unique and proprietary line of recombinant glycosyltransferases. Last, glycosaminoglycan (GAG) degrading enzymes, or polysaccharide lyases, are a class of enzymes used for the analysis of glycosaminoglycans and the glycosaminoglycan portions of proteoglycans.4 This group includes enzymes such as Chondroitin ABC Lyase and Heparinase. Seikagaku Corp. in Japan was the first company to develop and sell GAG degrading enzymes beginning about 1965. Currently, Seikagaku America carries the broadest range of GAG degrading enzymes on the market, including some that are unavailable here. Sigma, ICN, and Calbiochem also carry selections of these enzymes. A wide selection of carbohydrate related mono- and polyclonal antibodies are offered as useful tools for the glycobiologist. EY Laboratories and Vector Labs, among other vendors, sell antilectin antibodies. Temecula, Calif.-based Chemicon International carries a line of monoclonal antibodies to proteoglycans and carbohydrates, and Seikagaku America has a wide range of monoclonals to many glycoconjugates. Of course, purified and conjugated forms of the antibodies are available. Other available products for carbohydrate analysis include inhibitors of glycoprotein processing, carbohydrate standards, glycolipids, glycoproteins, and mono-, oligo-, and polysaccharides. Overall, the companies supplying these types of enzymes and reagents include ProZyme, Seikagaku America, Calbiochem, ICN, Sigma Chemical Co., United States Biological, Glyko, Chemicon International, Upstate Biotechnology, and Vector Laboratories. For additional information about the field of glycobiology, visit the Glycoforum Web page (www.glycoforum.gr.jp) Designed by and for glycobiology researchers, the forum provides basic information, links to related Web sites, and a forum for researchers to exchange information and ideas. Another good site, Seikagaku America's Web page (www.seikagaku.com), offers plenty of resource and technical options. Specifically, the site features a "Tech to the Rescue" section with lists of protocols, hot reference papers, and direct E-mail connections to experts in the glycobiology field. The author, Michael Brush, can be contacted at mdbrush@compuserve.com. A. Varki et al., "Preparation and analysis of glycoconju- gates," Current Protocols in Molecular Biology, New York, John Wiley & Sons, 1999, page 17.0.5. U. Galili, "Biological significance of the carbohydrate epitope Gal (alpha)1,3Galß1,4GlcNAc-R," Calbiochem Glycobiology Catalog, 1999, page 95. S. T. Wong et al., "Purification and characterization of novel glycosidases from the bacterial genus Xanthomonas," Glycobiology, 5[1]:19-28, 1995. R.J. Linhardt, "Analysis of glycosaminoglycans with poly- saccharide lyases," Current Protocols in Molecular Biology, New York, John Wiley & Sons, 1999, page 17.13.13. For additional reading visit www.the-scientist.com. Table of Carbohydrate Products

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