Nuclear Magnetic Resonance spectroscopy studies of proteins-glycoconjugates interactions
Creators: name: Marchetti, Roberta
Publisher: name: Università degli Studi di Napoli Federico II
Year: 2013

description: This PhD thesis work has been focused on the analysis of the structural requisites for recognition and binding between proteins and glycoconjugates, essential for the comprehension of mechanisms of paramount importance in chemistry, biology and biomedicine.
A large variety of techniques, such as crystallographic analysis, titration microcalorimetry (ITC), surface plasmon resonance (SPR) and fluorescence spectroscopy, allows the elucidation of molecular recognition events. In the last years, NMR-based methods have become among the most powerful and versatile techniques for detection and characterization of binding processes between a ligand and its receptor. In detail, Saturation transfer difference NMR (STD NMR) is a technique that allows to demonstrate the interaction between species and to detect ligand regions more involved in the interaction process; in parallel, important information on the bioactive conformation of the substrate in the bound state can be obtained by tr-NOESY experiments.
 Cell wall microbial glycoconjugates are very important players in the dynamic host-guest recognition that, in case of pathogens, is followed by the immune response and, in case of symbiosis, by its suppression. This PhD project proposes to use NMR techniques to achieve a deeper comprehension of the role of bacterial glycoconjugates in eukaryotic-pathogen and microbe-microbe interactions.
 For what concern eukaryotic host and pathogen interactions, we have studied the binding between a monoclonal antibody, designated as 5D8, and the O-polysaccharide chain isolated from the lipopolysaccharide of a Gram negative bacterium, Burkolderia anthina, an opportunistic pathogen of cystic fibrosis (CF) patients. Lipopolysaccharides (LPS) are endotoxins intercalated into the outer membrane of Gram negative organisms, essential for bacterial survival and defined as one of the most important virulence factor of bacterial infection in cystic fibrosis (CF). Among the LPS components, the O-chain is responsible of the activation of the adaptative immune response, that operates in the latter stage of the infection, providing a specific response and creating an immunological memory. Once activated by the innate immunity, leukocytes and B cells expose on their surfaces bacterial antigenic targets  leading the production of effector T cells and specific antibodies, which are able to recognize, as non-self, bacterial components. The above interaction system, mAb 5D8 - O-chain, was analyzed in detail to go in-depth, at molecular level, into the mechanism of infection,  key step toward the possible vaccines development. Thus, tr-NOESY and STD NMR experiments have been performed on the complex mAb 5D8 - O-chain from of the LPS of Burkolderia anthina, isolated from a CF patient from UK. Furthermore, in order to obtain more accurate information on structural requirements fundamental in the recognition process, we have also used, as carbohydrate ligands, the ad hoc synthesized trisaccharide and hexasaccharide O-antigen repeating units. The combined use of molecular dynamics simulations and NMR spectroscopy allowed to determine the binding epitope and the contribution to the binding of the sugar units composing the O-chain.
 As mentioned above, innate immunity represents the first line of defence against invading microorganisms in vertebrates and the only line of defence in invertebrates and plants. This ancient system is exemplified by C-type lectins, which have developed a sophisticated ability to differentiate between infectious agents and self components. Lectins achieve this by recognizing invariant exposed structures adorning pathogens, so called Pathogen Associated Molecular Patterns (PAMPs), such as carbohydrates and acetylated compounds, among others. The precise dynamics of binding between these human pattern recognition receptors (PRRs) and their ligands are poorly understood, and so, among the mechanism of interaction analyzed in this PhD project, we undertook a study to elucidate the physical and biochemical binding properties of the prototypic lectin, mannose-binding lectin (MBL). Previous studies have revealed that this protein modulates immune response and is able to mediate the phagocytosis, representing a potential clinical application against glycosylated enveloped viruses, such Ebola and Marburg. Unsuitable for industrial-scale production, due to the costs and the difficulties inherent in its extremely complex quaternary structure, its potential for use in successful anti-infective therapy is limited; to produce better, less expensive therapeutic agents, less complex chimeric fusion proteins, such as L-FCN/MBL76, with similar ligand recognition and enhanced effector functions, have been developed. Performing a large variety of NMR experiments, we have studied the binding activity of both, recombinant human, rhMBL, and its chimer achieving information, at a molecular level, on the mechanism of the interaction useful for the design and optimization of new therapeutic proteins.
 
Besides being involved in the immune response, as a result of their interactions with glycoproteins, glycolipids and oligosaccharides, lectins play a large variety of biological functions in all living organisms, from viruses to bacteria, fungi, plants and animals. In pathogenic micro-organims, lectins are mostly involved in host recognition and tissue adhesion. Indeed, soluble lectins, are often secreted as virulence factors from opportunistic pathogens, such as B. cenocepacia that produces, among the others, a lectin called BclA with specificity for fucosylated and mannosylated glycoconjugates. Since the biological role of that lectin is unknown, in order to ascertain a correlation between its structure and function, a combination of techniques, crystallography, micro-calorimetry, fluorescence, electron microscopy and NMR spectroscopy, have been used. In detail, we have studied by NMR the affinity of BclA for different ligands with a structure that remembers the glycoconjugates exposed on the external membrane of Gram negative organisms, such as the oligosaccharide portion (core) of the lipooligosaccharide of Burkolderia cenocepacia. By this approach, we have verified the hypothesis that this lectin could be involved in quorum sensing mechanisms defining its biological role.
 Moving to plant-pathogen interactions, a combination of NMR techniques was used even in the analysis of the binding between glycoside hydrolases and xyloglucan oligosaccharides. Xyloglucans are plant cell wall polysaccharides that can be degraded by glycoside hydrolases, ubiquitous among species, providing essential energy to organisms from microbes to higher animals. Thus, the study of this system of interaction is fundamental to diverse applications in medicine, food production, but in particular to biomass resource utilization. In the present project, we focused our attention on the main α-xylosidase, member of glycoside hydrolase family 31, Xyl31A,  from the Gram negative bacterium Cellvibrio japonicus. NMR techniques have been used to perform the epitope mapping on both the natural substrate and the product of the enzyme. Results obtained from STD NMR and tr-NOESY experiments, taken together with molecular dynamic simulations and docking, allowed a rationalization of previous kinetic data, underlying a peculiar architecture of enzyme active site. Indeed, we demonstrated that Xyl31A recognized the entire backbone of glycosidic units with four sites and also makes significant interactions, with other sub-sites, with branched xylose residues. This is due to the presence of an appended PA14 domain that plays a key role in the interaction and explains the strong affinity of the enzyme for long oligosaccharide substrates.
 Other important components of fungal, cell wall, on which we have focused our attention, are chitooligosaccharides,  potent elicitors of defense responses in a wide range of plant species. Their perception and transduction, that plays a fundamental role in the establishment of plant resistance to pathogens, thought to be mediated by a chitin elicitor binding protein CEBiP, that is a key receptor in rice cells. It contains a transmembrane domain, two extracellular domains (known as Lysin Motif domains), that bind to the chitoligosaccharides, but lacks any intracellular region, so it could require additional factors for signal transduction, such as a chitin elicitor signaling, denoted as CERK1. In order to study the mechanism of plant immune reaction to fungal oligosaccharides, we performed many STD NMR experiments on chitooligosaccharides of different length and the extracellular domain of CEBiP. Combining NMR results with molecular biology experiments, it has been possible to define the ligand regions more involved in the interaction and, at the same time, to detect the portion of the protein essential for the binding.
The above work is a part of a large project in which we are studying molecules possessing  a similar primary structure and immunity-stimulating activities, involved in nodulation process and in the establishment of plant resistance to pathogen. Between these molecules, the peptidoglycan stands out; it is a signaling molecule involved not only in the establishment of bacterial resistance in plant but even in the interaction between bacteria. Indeed, it is known that PGN fragments, named muropeptides, are produced during bacterial growth and represent a molecular signal that growing conditions are promising, playing a key role in the germination of dormant bacterial spores. They are indeed recognized by the extracellular domain of bacterial eukaryotic-like Ser/Thr membrane kinase that, in response to muropeptides, allows the germination of spores. By coupling protein mutagenesis and NMR techniques we succeeded in defining the structural requirements of both protein and ligand necessary for recognition and binding of muropeptides. In particular, an arginine residue belonging to the third PASTA (Penicillin and Serine Threonine Associated) domain played a key role in the interaction with meso-diaminopimelic moiety of PGN. It is worth to note that this aminoacid is a peculiar component of PGN of all Gram negative bacteria and of few Gram positive bacteria, such as bacilli; thus, our results explain also the specificity of the interaction between the protein and only DAP-type PGN.
 In conclusion, the results obtained have allowed the analysis, at a molecular level, of fundamental mechanisms of interaction, thus allowing to clarify the molecular determinants involved in recognition and binding events. Therefore, the obtained results represent an important platform pre-requisite that will permit a better comprehension of recognition events, key step for the design and optimization of new molecules with therapeutic aims.