Synthesis of Carbohydrates through Biotechnology - ACS Publications

Chapter 4

Probing the Antigenic Diversity of Sugar Chains

Downloaded by CORNELL UNIV on September 29, 2016 | http://pubs.acs.org Publication Date: January 9, 2004 | doi: 10.1021/bk-2004-0873.ch004

Ruby Wang, Brian J. Trummer, Ellen Gluzman, Chao Deng, and Denong Wang* Division of Functional Genomics, Columbia Genome Center, College of Physicians and Surgeons, Columbia University, 1150 St Nicholas Avenue, New York, NY 10032 *Corresponding author: [email protected]

Sugar chains of living organisms are structurally diverse and rich in biological information. The cellular functions of a specific sugar chain depend on its own structural characteristics, as well as on its interaction with other biological molecules that are able to recognize its structure. To facilitate investigations of carbohydrate-mediated molecular recognition, we have established a carbohydrate-based microarray technology. This biochip platform allows a single protein, such as an antibody, a lectin, or other cellular protein, to interact with thousands of carbohydrate molecules of distinct structures in a single assay and thus allows us to study the specificity and cross-reactivity of carbohydrate-protein interactions in a way that was previously impossible. A promising medical application is the detection and characterization of the anticarbohydrate antibodies that are elicited by a microbial infection or are associated with human diseases with abnormal expression of complex carbohydrates. Such technology is also a powerful means of studying the relationship between structural characteristics of carbohydrates and their antigenic properties. This article discusses the presence of different categories of carbohydrate-based antigenic determinants and the need for microarray technologies to preserve sugar chain conformations on chip.

© 2004 American Chemical Society

Wang and Ichikawa; Synthesis of Carbohydrates through Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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40 Carbohydrate-containing macromolecules of biological origin have been recently designated the term "glycomers" (/, 2), reflecting the influence of a new scientific discipline, glycomics. Polysaccharides and glycoconjugates of multiple structural configurations, such as glycoproteins, glycolipids, glycosphingolipids, glycosaminoglycans and proteoglycans, are all covered by this term. Cellular glycoconjugates are involved in many biological processes. Documented examples include molecular recognition at fertilization (5, 4\ cell-cell recognition, adhesion, and cell activation throughout the development and maturation of organisms (/, 5-7). Abnormalities in the expression of complex carbohydrates are seen in cancer (8, 9), retroviral infections (10, / / ) , and other diseases with a genetic or somatic defect in biosynthesis or metabolism of cellular glycans (12). Many carbohydrate molecules serve as the "signatures" of a specific species or strain of microorganism (See Ref (13, 14) for reviews). Such carbohydrate structures are important molecular targets for vaccine development and/or diagnosis of infectious diseases (15-19). Glycomics extends proteomics to include carbohydratecontaining biological molecules. This scientific discipline seeks to probe the diversity, decipher the information content, explore the biomedical applications, and examine the biosynthesis of glycomers. Glycomics also strives to facilitate efficient genetic and chemical engineering of these carbohydrate-containing macromolecules and seeks to firmly establish technologies for a large-scale production of such biological molecules. Our current research focuses on developing a biochip-based high throughput technology to explore the antigenic diversity of glycomers and to "decode" the sugar chain-based biological "codes." Additionally, we are developing a technology that can enable the simultaneous detection and immunological characterization of a wide range of microbial infections (2). Rapid progress of the genome-sequencing projects has led to the development of a generation of high throughput technologies for biological and medical research that include nucleic acid- (20-23), protein- (24-26), and carbohydrate-based microarrays (2, 27). These approaches are designed to monitor the expression of genes, proteins, and glycomers on a large scale, and to identify the characteristic patterns of



41 their differential expression in an organism at given conditions. In principle, c D N A microarray and oligonucleotide biochips (23) are interchangeable for the purpose of expression analysis, as the detection specificity of these biochips is determined solely by the A::T and C::G base pairing. A n on-chip denaturation process is prerequisite in order to allow D N A : D N A or D N A : R N A hybridization and their specific base pairing to take place. This is strikingly different from the protein-based microarrays, in which preservation of protein structure, especially its 3D conformation on chip, is necessary and poses a technical challenge to current protein microarray techgnology. Whether a carbohydrate microarray requires preservation of the conformational properties of carbohydrate molecules on chip is a more complicated issue and will be further explored in the following discussion. Structural diversity of carbohydrates While the primary structures of both proteins and carbohydrates are determined by the sequence of their respective monomers, the structure of carbohydrates is further characterized by the variety of their linkages. Unlike proteins that are connected solely by a peptide bond, carbohydrates utilize many possible glycosidic linkages so as to extensively diversify their structures. For example, whereas two amino acids can produce only one possible dipeptide, two molecules of glucose are able to generate eleven different disaccharides. Thus, while nucleic acids and proteins have a single type of covalent bond between their monomers, the structure of a sugar chain is determined by the composition and sequence of its sugar residues, as well as by their glycosidic linkages, and can therefore generate a variety of structures with identical residue compositions and sequences. Conformational flexibility, or plasticity, is an intrinsic property of sugar chains (14). Oligosaccharides and polysaccharides may exist in many different conformations wherein the lower energy forms predominate. Carbohydrates are further diversified by microheterogeneity. Unlike the translation of m R N A messages into proteins, which is precisely conducted by a protein biosynthesis pathway, the synthesis of sugar chains requires multiple enzymes and a complex pathway of biosynthesis. Carbohydrates with microheterogeneity are thus unavoidably generated during their biosynthesis.


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Biosignals of carbohydrates There are a number of biological systems that are able to specifically recognize carbohydrate structures. For example, the enzymatic recognition of dextran and glycogen by proteins of either microbial or mammalian origin is glycosidic linkage-specific. Dextran and glycogen are polymers composed entirely of glucose. Dextrans are produced by microbes, while glycogen is one of the forms of energy storage for mammals. Mammals can degrade a(l,4)-linked glycogen. However, they are unable to break down dextran, which has predominantly a(l,6)-linkages. oc(l,6)dextran of relatively low molecular weights can thus be used as a blood expander to maintain normal blood volume and blood pressure in the human body (28). The host's immune system can recognize dextrans and mount specific antibody responses to react with dextrans produced by the microbes (29-31). Differing from glycogen, a(l,6)dextrans with molecular weights of over 90,000 are antigenic in human and mouse (3733). Dextran molecules derived from different strains may differ significantly in their glycosidic linkage compositions (34). While some dextran preparations are predominantly or solely a(l,6)linked, forming molecules with predominantly linear chain structures; others are composed of multiple glycosidic linkages, including

Synthesis of Carbohydrates through Biotechnology - ACS Publications

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