….is a valuable source of knowledge in this area not only for graduate students but also for non-specialist readers.

Protein–Carbohydrate Interactions in Infectious Diseases

By Carole A. Bewley

The Royal Society of Chemistry

Copyright © 2006 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-802-1

Contents

Chapter 1 Atomic Basis of Protein–Carbohydrate Interactions: An Overview, 1,
Chapter 2 Mycobacterial Glycolipids and the Host: Role of Phenolic Glycolipid and Lipoarabinomannan G.J. Blaauw and B.J. Appelmelk, 6,
Chapter 3 Structures and Roles of Pseudomonas aeruginosa Lectins Anne Imberty, Michaela Wimmerová, Charles Sabin, and Edward P. Mitchell, 30,
Chapter 4 Protein–Carbohydrate Interactions in Enterobacterial Infections Nathan Sharon and Itzhak Ofek, 49,
Chapter 5 GM1 Glycomimetics and Bacterial Enterotoxins Anna Bernardi, Crtomir Podlipnik, and Jesús Jiménez-Barbero, 73,
Chapter 6 Retrocyclins: Miniature Lectins with Potent Antiviral Activity Robert I. Lehrer, 92,
Chapter 7 C-type Lectin Receptors that Regulate Pathogen Recognition Through the Recognition of Carbohydrates Sandra J. Van Vliet, Christian H. Grün and Yvette Van Kooyk, 106,
Chapter 8 Targeting Microbial Sialic Acid Metabolism for New Drug Development Eric R. Vimr and Susan M. Steenbergen, 125,
Chapter 9 Synthetic Carbohydrate-Based Antimalarial Vaccines and Glycobiology Alexandra Hölemann and Peter H. Seeberger, 151,
Chapter 10 Studies Toward a Rationally Designed Conjugate Vaccine for Cholera Using Synthetic Carbohydrate Antigens Pavol Kovác, 175,
Chapter 11 Carbohydrate Microarrays for High-Throughput Analysis of Carbohydrate–Protein Interactions Injae Shin, 221,
Subject Index, 247,

CHAPTER 1

Atomic Basis of Protein–Carbohydrate Interactions: An Overview

NATHAN SHARON

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

1 Introduction

Protein–carbohydrate interactions are the basis of numerous biological processes, both normal and pathological ones. They include the enzymatic synthesis and degradation of oligo- and polysaccharides, intracellular sorting of glycoconjugates, transport of carbohydrates into living cells and of their derivatives into subcellular organelles, the immunological response to carbohydrate antigens, and migration of leukocytes to sites of inflammation. These interactions also play a key role in a variety of cell adhesion phenomena, among them the attachment of parasites, fungi, bacteria, and viruses to host cells, the first step in the initiation of infection. The high selectivity required for this attachment, as well as the binding of a variety of microbial toxins to cells, is provided by a specific stereochemical fit between complementary molecules, one a carrier of biological information (such as complex carbohydrates) and the other capable of decoding such information (carbohydrate-binding proteins, belonging to the class of lee tins). This concept has its origins in the lock-and-key hypothesis, introduced by Emil Fisher at the end of the 19th century to explain the specificity of interactions between enzymes and their substrates, i.e., between molecules in solution; it was subsequently extended to describe the interactions of cells with soluble molecules and with other cells.

Complex carbohydrates are commonly found at the cell surface, where they are positioned to interact with suitable proteinaceous receptors, primarily lee tins, in solution or on the surfaces of other cells. These proteins, originally identified as sugar-specific hemagglutinins, are currently known as cell-recognition molecules. They are geared to distinguish between different oligosaccharides, whether as such or as part of glycoconjugates (primarily glycoproteins and glycolipids). Like other carbohydrate-binding proteins, whether enzymes, anticarbohydrate antibodies, or sugar transporters, lectins are structurally diverse, differing markedly in size, tertiary and quaternary structure, as well as the structure of their combining sites.

2 Combining Sites

A major source of information about the combining sites of lectins is the X-ray crystallography study of their complexes with ligands. By now, the structures of close to 200 lectins, and over 300 of their complexes with carbohydrates, have been solved largely by this method (www.cermav.cnrs.fr/lectines/); most of those from bacterial or viral sources are listed in Table 1. Other inputs include binding experiments with sugars and their derivatives, site-directed mutagenesis, and, to a limited extent, also NMR experiments and molecular modeling. Such studies have shown that like the lectins themselves, the sites are diverse, even when their specificity is the same, although within a given lectin family the sites may be similar. The sites appear to be preformed, since few conformational changes occur upon ligand binding. They are mostly in the form of shallow depressions on the surface of the protein, where typically only one or two edges or faces of the ligand are bound, and are thus similar to those of anticarbohydrate antibodies or glycosidases. In a few lectins, the combining sites are in the form of deep clefts.

The participation of a particular amino acid of a lectin and of a specific group of the carbohydrate ligand in the interaction between the two can be assessed by different methods. In the case of the amino acids, it is done mainly by site-directed mutagenesis, as mentioned earlier. This technique, combined with ligand-binding experiments, also provides information on the relative contribution of individual residues to the protein-carbohydrate interaction. As to the carbohydrate ligand, its hydrogen bonding pattern in the complex can be mapped by studies with deoxy-, methoxy-, and deoxyfluoro sugar analogs. It is only X-ray crystallography, however, that provides detailed information at the atomic level on the interplay between the two molecules. Still, it should be kept in mind that a crystal represents a frozen constellation often obtained from a concentrated solution in a nonphysiological milieu, without any indication on the status of the involved molecules prior to complex formation. The use of nonphysiological crystallization conditions may induce conformational changes in the protein and alter the mode of binding. Therefore, although the value of X-ray crystallography is not disputable, it is essential to complement the data obtained by this method with information from other sources (e.g., NMR) on the solution structure of the lectin-ligand complex.

3 Lectin–Carbohydrate Bonds

The bonds involved in the formation of lectin–carbohydrate complexes are in principle not different from those involved in the formation of the corresponding complexes of other carbohydrate-binding proteins. Lectins combine with their ligands primarily by a network of hydrogen bonds and hydrophobic interactions; in rare cases, electrostatic interactions (or ion pairing) and coordination with metal ions also play a role. Bonding is sometimes mediated by one or more water molecules (explained later). Although in a single lectin a limited set of residues contribute to the interactions with the ligand, on the whole almost all kinds of amino acids participate in ligand binding.

3.1 Hydrogen Bonds

Hydrogen bonds that are directional are heavily involved in conferring specificity to protein–carbohydrate interactions, as well as contributing to their affinity. They depend largely on interactions between the hydroxyls of the carbohydrate and the amino acid side chains of the protein, most frequently of aspartic acid, aparagine, glutamic acid, glutamine, arginine, and serine residues. Main chain NH and CO groups also contribute to hydrogen bonding, but to a lesser extent.

A sugar hydroxyl has the capacity to interact with a protein both as a hydrogen bond donor and as an acceptor, by way of the lone electron pairs. Moreover, as donor, the hydroxyl possesses the added advantage of having rotational freedom about the C–OH torsional angle, thus enabling it to attain the best possible linear bond with an acceptor group, which is important in imparting specificity. When each of two adjacent hydroxyls of a monosaccharide interacts with different atoms of the same amino acid (e.g. the two oxygens of the carboxylate of glutamic or aspartic acid), they form bidentate hydrogen bonds. Another kind of hydrogen bond characteristic of protein–carbohydrate complexes is the cooperative bond, in which the hydroxyl group acts simultaneously as donor and acceptor.

3.2 Hydrophobic Interactions

Even though carbohydrates are highly polar molecules, the steric disposition of the hydroxyl groups creates hydrophobic patches on their surfaces, which can form contacts with hydrophobic side chains of the protein. One widely occurring interaction of this kind is the stacking of a monosaccharide on a side chain of an aromatic amino acid. It is the result of the presence of partial positive charges on the aliphatic protons on one face of a hexopyranose ring and a partial negative charge from the π-electrons of the aromatic system. Such stacking is found in the sugar complexes of almost all legume lectins, of the galectins and some C-type lectins, as well as of bacterial toxins (e.g. E. coli lytic toxin and tetanus toxin). In addition, the methyl moiety of the acetamide of acetamido sugars often interacts with aromatic residues of the lectins (e.g. WGA and influenza virus hemagglutinin that are specific for such sugars), as does the methyl group of fucose. Hydrophobic contacts also occur with side chains of aliphatic amino acids, such as valine or leucine.

3.3 Other Interactions

Most saccharides are uncharged, and therefore ionic, i.e., charge–charge, interactions do not commonly participate in the formation of protein–carbohydrate complexes; such interactions, however, occur in complexes with sialic acids, e.g., influenza virus hemagglutinin, and are common in complexes with glycoamino-glycans. An unusual type of bond, found only in the C-type lectins, is between the protein-bound Ca2+ and certain hydroxyls of the ligand.

4 Role of Water

As mentioned, contacts between the protein and its ligands are sometimes mediated by water bridges. Water acts as a molecular mortar, its small size and ability to serve as both a hydrogen bond donor and acceptor endowing it with ideal properties for this purpose. Such bridges, which consist of a single water molecule or chains of several water molecules, may be important for ligand recognition.

Both the protein and the ligand in aqueous solutions are normally bonded to water molecules by hydrogen bonding. Therefore, the overall process of binding between the two involves the meeting of a solvated polyhydroxylated glycan and a solvated protein combining site. In the process of complexation, the protein–water and ligand–water hydrogen bonds are replaced by protein-ligand bonds and the released water returns to the bulk solvent. When the complex finally forms, it presents a new surface to the surrounding medium, which is also hydrated. Solvation–desolvation energies are very large, due to entropy and cannot be reliably calculated for hydrophilic compounds such as sugars. Thus, even though the energetic contributions of van der Waals and hydrogen bonding interactions in the combining site can be estimated, errors in the estimation of the attendant solvation energy changes can be much larger, making the overall calculations of binding energy difficult. Comparison of the structures of unligated lectins from the same family with those of their complexes with the same or different sugars has shown that certain ordered water molecules are conserved in all such structures.

CHAPTER 2

Mycobacterial Glycolipids and the Host: Role of Phenolic Glycolipid and Lipoarabinomannan

GJ. BLAAUW AND BJ. APPELMELK

Department of Medical Microbiology, VUmc Vrije Universiteit Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

1 Introduction

Among mycobacterial infectious diseases, tuberculosis and leprosy are the best known. Their causative agents are Mycobacterium tuberculosis and Mycobacterium leprae, respectively. They have in common the staining characteristic referred to as acid fastness, which is due to the presence of a highly characteristic cell wall that includes the presence of very large covalently attached lipids known as mycolic acids. Leprosy, also referred to as Hansen’s disease, is a chronic infection of the skin and peripheral nerves. It is not very contagious and is transmitted via droplets from the nose and mouth during close and frequent contact. It is a mutilating disease because of the progressive and permanent damage to the skin, nerves, limbs and eyes. It mainly occurs in third world countries such as India and Brazil. It is estimated that 2 million people worldwide are disabled because of leprosy and between 500,000 and 1,000,000 cases are detected each year. Leprosy in fact presents itself by a spectrum of manifestations: on one end the tuberculoid type, which is a non-progressive disease, mainly affecting the peripheral nerves, resulting in loss of sensation and muscle atrophy, whereas on the other end the lepromatous type, which is the progressive form of leprosy, mainly affects the skin, mutilating the face, fingers, and toes. Progression in the latter type is due to the inability of the host immune response to inhibit bacillary multiplication. Leprosy can be treated and should be detected as early as possible, because of the irreversible damage it causes.

Tuberculosis, also referred to as Koch’s disease, is mainly a pulmonary disease, but other sites of infection are also possible, owing to early bacteremia after initial infection. It is caused by M. tuberculosis or, less frequently, by other mycobacterial species within the M. tuberculosis complex, such as M. bovis, M. africanum, and M. microti. Tuberculosis is currently one of the leading causes of death worldwide, responsible for 50 million new infections and claiming 2–3 million lives per year. Infection is acquired through inhalation of aerosols that contain infectious bacilli. However, only 10% of those who are infected actually get the disease. The other 90% is able to contain the infection with the help of an adequate immune response, resulting in the formation of granulomas in which (infected) macrophages and lymphocytes are predominant. The tubercle bacilli enter into a state of dormancy in which they do not replicate or disseminate. Dormancy reflects a state of equilibrium, which is beneficial to both the host and the infectious agent. The former is able to contain the infection, while the latter is able to survive. The hypoxic milieu within granulomas seems to be a major factor in induction of this state of dormancy. It is estimated that nearly one-third of the world population is in this way latently infected. The moment this equilibrium is disturbed, the bacilli can be reactivated to cause disease. Therefore host factors and especially the state of the immune system, such as the number of CD4 T-cells, are crucial determinants for disease progress after initial infection or, alternatively, for containment or even elimination. This chapter will focus on two glycolipid components of these pathogenic mycobacteria, lipoarabinomannan (LAM) and phenolic glycolipids (PGLs). These glycolipids play, through interactions with host receptors such as lee tins, a crucial role in the patho-genesis of tuberculosis and leprosy. A brief history of these compounds, their structure, biosynthesis and role in pathogenicity and immune modulation will be discussed in this chapter.

2 Phenolic Glycolipids

2.1 Phenolic Glycolipids History

Characterization by infrared (IR) spectroscopy of antigen fractions from mycobacteria signified the beginning of phenolic glycolipids (PGLs) history. These fractions contained lipids, characteristic for a given mycobacterial species. In 1960 these lipids were named mycosides and were defined as “glycolipids or glycolipid peptides limited in distribution to a single species of mycobacteria.” Not long afterwards, the presence of mycosides A, B, and G in lipid extracts of M. kansasii, M. bovis and M. marinun was also demonstrated. They were classified as members of the PGL family, because IR spectra indicated the presence of an aromatic group, differentiating them from other mycobacterial cell wall glycolipids. Analytical tools, such as gas-liquid chromatography, NMR- and mass spectrometry, made it possible to elucidate the chemical structure of these different PGLs. The structures of PGLs derived from different mycobacterial species are principally the same in their lipid core and are attached to mycocerosic acids (Figure 1), but they vary in the composition of oligosaccharide side chain (Figure 2) attached to the phenol moiety.

After a period of negligence, interest in PGLs was revived when a M. leprae- specific antigen (PGL-I) (Figure 3) was isolated from M. leprae-infected liver tissue of armadillos. This antigen was also classified as a member of the PGL family. Owing to its high antigenicity, PGL-I could be used in serodiagnosis of leprosy. With respect to the diagnosis of leprosy, the synthesis of neoglycoproteins of PGL-I, the major PGL of M. leprae, has been a major leap forward. Antibodies against PGL-I can now be detected in the sera of leprosy patients with high sensitivity and specificity.

In addition to its high antigenicity and the use for serodiagnosis, the role of PGL-I in pathogenicity of M. leprae and immune modulation of the host was investigated. In analogy with M. leprae, a PGL (PGL-Tb 1) (Figure 3B) was extracted from M. tuberculosis (strain Canetti). In contrast to leprosy, PGL of M. tuberculosis cannot be used for serodiagnosis of tuberculosis: a too large variation is observed in the anti-PGL antibody responses in the sera of tuberculosis patients. Nevertheless, the production of PGLs by specific strains of M. tuberculosis has recently been recognized as a virulence factor of major importance and we foresee major future interest in studies on the interaction of PGL with host receptors such as immune cell-surface lectins and signaling pathways.

(Continues…)Excerpted from Protein–Carbohydrate Interactions in Infectious Diseases by Carole A. Bewley. Copyright © 2006 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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