Carbohydrate Chemistry Chemical and Biological Approaches Volume 38

A Review of the Literature Published between January 2011 and February 2012

By A. Pilar Rauter, Thisbe K. Lindhorst

The Royal Society of Chemistry

Copyright © 2012 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-439-4

Contents

Preface Amélia Pilar Rauter and Thisbe K. Lindhorst, vii,
Applications of glycobiology: biological and immunological effects of a chemically modified amylose-derivative Ghislain Opdenakker, Sandra Li, Nele Berghmans and Jo Van Damme, 1,
Lipopolysaccharide structure and biological activity from the cystic fibrosis pathogens Burkholderia cepacia complex Anthony De Soyza, Flaviana Di Lorenzo, Alba Silipo, Rosa Lanzetta and Antonio Molinaro, 13,
Synthesis of bacterial carbohydrate surface structures containing Kdo and glycero-D-manno-heptose linkages Stefan Oscarson, 40,
Synthetic glycopeptides in vaccine development and antibody epitope mapping Ulrika Westerlind, 61,
Posttranslational sialylation and its impact on leukocyte recruitment during inflammation Karin Bodewits and Markus Sperandio, 75,
Glycoengineering of protein-based therapeutics Sandrine Donadio-Andréi, Chloé Iss, Nassima El Maï, Valérie Calabro and Catherine Ronin, 94,
Congenital Disorders of Glycosylation (CDG): from glycoproteins to patient care Vanessa Ferreira, Paz Briones and Maria-Antonia Vilaseca, 124,
Bladder cancer–glycosylation insights Paulo F. Severino, Mariana Silva, Mylène A. Carrascal, Fernando Calais, Fabio Dall’Olio and Paula A. Videira, 156,
Levansucrases of Pseudomonas bacteria: novel approaches for protein expression, assay of enzymes, fructooligosaccharides and heterooligofructans Tiina Alamäe, Triinu Visnapuu, Karin Mardo, Andres Mäe and Alina D. Zamfir, 176,
Recent advances on the application of NMR methods to study the conformation and recognition properties of carbohydrates Ana Ardá, M. Álvaro Berbís, Pilar Blasco, Angeles Canales, F. Javier Cañada, Ma Carmen Fernández-Alonso, Filipa Marcelo and Jesús Jiménez-Barbero, 192,
Glycosidase inhibitors: versatile tools in glycobiology Oscar López, Penélope Merino-Montiel, Sergio Martos and Alejandro González-Benjumea, 215,
An overview of key routes for the transformation of sugars into carbasugars and related compounds Raquel G. Soengas, José M. Otero, Amalia M. Estévez, Amélia P. Rauter, Vasco Cachatra, Juan C. Estévez and Ramón J. Estévez, 263,
Multivalent glycoconjugates in medicinal chemistry José G. Fernández-Bolaños, Inés Maya and Ana Oliete, 303,
Glycotransporters for gene delivery Carmen Ortiz Mellet, José M. García Fernández and Juan M. Benito, 338,
Furanose-based templates in the chemoselective generation of molecular diversity Ana M. Gómez, Clara Uriel and J. Cristóbal López, 376,
Synthesis of carbohydrate-based artificial siderophores and their biological applications Marta M. Andrade and Amélia P. Rauter, 398,
Smart biomaterials: the contribution of glycoscience Laura Cipolla, Laura Russo, Francesca Taraballi, Cristina Lupo, Davide Bini, Luca Gabrielli, Alice Capitoli and Francesco Nicotra, 416,

CHAPTER 1

Applications of glycobiology: biological and immunological effects of a chemically modified amylose-derivative

Ghislain Opdenankker, Sandra Li, Nele Berghmans and Jo Van Damme

Carbohydrate chemistry, oligosaccharide sequencing, synthesis technologies and glyco-engineering have helped to establish glycobiology alongside molecular biology. However, examples of therapeutic implications of glycobiology are limited to oligosaccharides. These include engineered anti-bodies containing sialic acids, inhibition of leukocyte rolling effect in the interactions between mucins and selectins and antiviral imino sugars. The best example of a successful glycodrug is oseltamivir, Tamiflu®, as an inhibitor of the influenza virus neuraminidase. Polysaccharides, although at first sight structurally less complex and biologically less challenging, are interesting molecules with unexplored possibilities in biology and medicine. Polysaccharides may form protease-resistant scaffolds for tissue engineering and chemically modified polysaccharides (CMPs) have potent immunomodulating activities. This is illustrated here with chlorite-oxidized oxyamylose (COAM), a broad-spectrum antiviral agent. COAM interacts with the chemokine system and thus can be used to modulate leukocyte compartmentalization in vivo. This knowledge has been used to alter the clinical course of acute viral infections, cancer and experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The latter example is proof-of-concept that CMPs constitute important probes to study immune functions and are novel drugs applicable for specific disease entities.

1 Introduction

From semantic viewpoint the term molecular biology is a pleonastic expression and does not correspond with the present day interpretations. Indeed, all biological processes are driven by molecules. What is meant by molecular biology are processes driven by nucleic acids. Thus, molecular biologists are stricto sensu researchers using DNA and RNA tools for sequencing and synthesis, for practical uses in genetic engineering and for therapy with recombinant drugs. Glycobiology (the biology of glycans) is a more strict term in that it defines, by its name, the molecules under study, namely carbohydrates. Why does the field of glycobiology then still need promotion? Why do the DNA/RNA biologists generate more appreciation and the glycobiologists less recognition? This is certainly not because DNA and RNA are sugar-phosphate polymers. Maybe it is because of discovery of the beauty of the linear connection between codons and amino acids.

Maybe it is also because functional aspects of a branched glycan tree are not yet understood. As long as the interconnections between RNA/DNA biology with glycobiology are not yet hold by a solid stem, scientists need to invest time and resources in finding important examples in glycobiology. This is simply to state that the real basis of glycobiology, for instance relating structures to functions, is a barrier for many scientists and needs further promotion. The original concepts that glycoforms and glycotypes confer different functions to proteins must be seen as eye-openers. These concepts, well elaborated for immunoglobulin-G (IgG) and tissue-type plasminogen activator (tPA), are becoming apparent for many other glycoproteins. Aside effects of glycosylation on the specific activity of t-PA and functional aspects of the glycans on the Fc part of IgG (vide infra), glycoprotein folding, trafficking, recognition and involvement in the immune system demonstrate that oligosaccharides are endowed with multiple functions.

The successes in technology progresses in nucleic acid and protein sequencing and synthesis are evident and their impacts on medicine, industry and society are obvious. The field of RNA/DNA biology has the intrinsic advantage of a limited number of building blocks, with limited natural chemical modifications of the five bases. Protein biology, based on about 20 building blocks, is already more complex and subject to many more natural chemical modifications: proteolytic processing, phosphorylation and dephosphorylation, citrullination, isoprenylation, acetylation/deacetylation, to name a few. Glycosylation is nowadays recognized as the most diverse and complex posttranslational modification of proteins.

Glycobiology forms the next, and perhaps the most challenging level. Intrinsically, carbohydrate biochemistry is also based on a limited number of natural monosaccharides, but their chemical modifications are multifold. In addition, the glycosidic bonding of monosaccharides is subject to various linkages and the involved carbon atoms form stereochemical centers. The branching of oligo- and polysaccharides into arborized structures often supersedes our imagination capacity, which is too much trained for linear DNA, RNA and protein sequences. However, in practice in mammalian systems a limited set of monosaccharides are used. Even so the linkage variation creates an extraordinary diversity of structures.

2 Historical breakthroughs and examples

If glycobiology is to succeed as the science of the 21st century, then one needs to define new breakthroughs. Happily one can walk in the footsteps of those who prepared and enhanced the field with exoglycosidase oligosaccharide sequencing, with mass spectrometry and NMR analysis of sugars, with lectin studies and with glyco-engineering. The discovery of congenital disorders of glycosylation not only points to future therapeutic applications, but also has yielded a bridging function between glycobiology and nucleic acid biology.

Oligosaccharide sequencing evolved over the last 30 years from an extremely labor-intensive discipline towards an efficient analytical method in the hands of experts. Pioneering work by the groups of Akira Kobata (University of Tokyo) and of Raymond Dwek (Oxford Glycobiology Institute) led to the understanding of the proinflammatory effects of agalactosyl IgGs in rheumatoid arthritis. This knowledge was complemented with the findings of anti-inflammatory sialic acids in IgGs. Together, these breakthroughs can be summarized in one sentence. Oligosaccharides attached to proteins fine-tune the biological activities of these molecules. This should be not compared with an on/off switch (as is often the case with protein phosphorylation), but rather with the selection of a specific program or tuning. Presently, oligosaccharide exoglycosidase sequencing has been perfected to such a level that broad-spectrum and high-throughput analysis and glycomics are within reach. One next breakthrough will come from coupling of this type of sophisticated analysis with proteomics, genomics and transcriptomics.

A second aspect of structural analysis is mass spectrometry and NMR analysis. Whereas mass spectrometry per se is nowadays routinely used in proteomic analysis, for oligosaccharide analysis the identification of structural details by mass spectrometry remained more complicated for the simple reason that, for example, the various hexoses maintain the same molecular mass. Nevertheless, sophisticated mass spectrometry technology for sugars has been developed, is presently the expertise of few specialists, but is becoming common practice, because the combination of high-performance liquid chromatography and mass spectrometry of oligosaccharides is very powerful and rapid. Considerable growth is expected in this area and basic mass spectrometry analysis is already becoming classical technology for structural analysis. Unfortunately and as is the case for other applications of mass spectrometry, high-end equipment is rather expensive and a limited number of specialized centers, homing dedicated specialists and equipped with such instruments, can answer easily any exotic demands for the analysis of complex glycoconjugates. Similar economic considerations may apply for NMR analysis, with the additional practical point that the amounts of purified glycoconjugates needed for NMR analysis supersede considerably those for mass spectrometry identification.

A third aspect is related to practical aspects, which we best place under the term glyco-engineering. Agalactosyl and sialyl IgGs have already been mentioned as disease promoting and disease-limiting factors, respectively, in inflammation. Such knowledge is successfully addressed by glyco-engineering companies making optimized recombinant glycoprotein preparations. Another example is related to the classical ABO blood group system. Nobel Prize laureate Karl Landsteiner was a glycobiologist avant-la-lettre. Present-day glyco-engineers have managed to modify the ABO blood group antigens, heralding novel possibilities in transfusion medicine. Because the immunological insights with blood transfusions formed the basis of organ transplantation medicine, it is not excluded that the glyco-engineering of the ABO blood group antigens forms the basis of new applications in xenotransplantation, where galactosylation is a major hinder in the application of pig donor organs.

A fourth aspect to be recognized as historical in glycobiology is the study of lectins as probes for sugars. Plant lectins, recognizing in defined ways oligosaccharides, have been widely used in medical diagnostics, often before their mechanism of action was known. Lymphoblast transformation with phytohaemagglutinin for karyotyping purposes and definition of chromosomal aberrations was used long before the cytokines that are induced in this process were identified as interleukin-2, interleukin-6 and others. Lectins are also used for disease diagnosis in histochemical analysis of cancer and, more recently, with the use of lectin microarrays. The literature on this topic is vast, exemplary reviews are available for detailed and general information. In contrast, the information about lectins as therapeutics is still rather sketchy. After the discovery of selectins, the mammalian lectins that regulate rolling of leukocytes at an early step in the inflammatory response, selectin knockout mice were developed to study immunophenotypes (how these mice differ from wildtype animals with reference to immunological parameters) and attempts were made to develop novel anti-inflammatory agents on the basis of these lectin-glycan interactions. A second example about glycotherapy relates to viral infections. Human immunodeficiency virus is enveloped in a glycocoat, limiting access to protein (peptide) epitopes. One way to flag this virus is with mannose-recognizing lectins, a strategy that is investigated with increasing intensity.

An important way to enhance the field of glycobiology is to define new medical applications. These may involve deficiencies, resulting from aberrant glycosylation, in need for substitution therapy or storage diseases with a pathogenic role of accumulated saccharides, in which reduction forms a treatment strategy. An important class of rare diseases is the family of congenital diseases of glycosylation (CDG). However, a critical aspect to recognize is the fact that CDGs create the link between nucleic acid and protein biology and glycobiology. Identified CDGs, resulting from the mathematics of evolution (combinations of random mutations that result in life offspring with clinical phenotypes), demonstrate that glycosylation is a biological process of vital importance. In addition, the creation of knockout mouse models of CDGs is an important step to understand better the biological importance of sugars, but may also form a solid basis to define treatments of these rare diseases, both in cases of deficiencies and of substrate reduction therapy in storage diseases, like Gaucher disease.

Finally, we come to the level of commonly used drugs. One viral target enzyme stands out as a glycobiological example: the influenza virus neuraminidase with its inhibitors oseltamivir (Tamiflu®) and zanamivir (Relenza®). The concept is simple, but the efforts done are considerable: definition of a microbial target enzyme (sufficiently different from sister enzymes in the host) and screening for inhibitors. It does not take much imagination that future antivirals and other antimicrobial agents may be developed on the basis of other microbial sugar modifying enzymes. In virology, such examples of interesting glycotarget enzymes may be restricted because of the limitation of the coding capacity of viral genomes and, therefore, the influenza neuraminidase will keep the lead for a while. However, in the world of bacteria, fungi and parasites an enormous potential exists to discover target enzymes, generate inhibitors and define new medicines.

Most of the examples mentioned so far relate to oligosaccharides. What is the status about polysaccharides?

3 Polysaccharides and derivatives

Although changes are emerging with the recognition of the importance of polysaccharide vaccines, in biomedical education the time spent to study polysaccharides is inversely correlated with their abundance in nature. Cellulose [polyβ(1-4)D-glucose], amylose [polya(1-4)D-glucose] and bran ched amylopectin in starch form most biomass and energy supply for most organisms. Intestinal resorption of starch glucose (from amylose and amylopectin) as monosaccharides and disaccharides and conversion into glycogen is basic medical knowledge that can be linked to rare glycogen storage diseases and to metabolic disease number one, i.e. diabetes. Deficiencies of amylase and disaccharidases lead to alterations of the gut microbiomes and to gastrointestinal disorders as is the case with specific polysaccharide derivatives. Indeed, dextran sodium sulphate (DSS) is commonly used to induce inflammatory bowel disease in mice (used as an animal model for Crohn’s disease or ulcerative colitis). In other words, polysaccharide derivatives may be harmful or not, depending on their degradability and their location.

4 An historical finding in virology?

After the discovery of interferon (IFN) in 1957, great hope was generated for its use as therapy for viral diseases and mass production lines were explored ending in the recombinant expression of IFN and use in the clinic. IFNs were not developed into first choice antivirals. Eventually, IFN-α from leukocytes and IFN-β from fibroblasts became first choice anti-inflammatory drugs for the treatment of multiple sclerosis. Recombinant IFN-γ, also dubbed immune interferon, because it is produced by lymphocytes and natural killer cells, found its way to the treatment of chronic granulomatous disease.

Piet De Somer (1917–1985), founder of the Rega Institute (Fig. 1) was an insightful man because he thought about and tried to solve the IFN production problem (before its cloning and expression) by finding ways to induce endogenous IFN. He admired and followed his scientist example, Karl Landsteiner, by trying to use simple but practical rules. Karl Landsteiner, who discovered the ABO blood group system, used simple serological tests for discovery.

(Continues…)Excerpted from Carbohydrate Chemistry Chemical and Biological Approaches Volume 38 by A. Pilar Rauter, Thisbe K. Lindhorst. Copyright © 2012 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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