Speciale, Immacolata (2017) Structural insight into new glycosylation patterns of microbial (bacterial and viral) origin. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Structural insight into new glycosylation patterns of microbial (bacterial and viral) origin
Creators:
CreatorsEmail
Speciale, Immacolataimmacolata.speciale@unina.it
Date: 10 April 2017
Number of Pages: 235
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
nomeemail
Paduano, Luigilpaduano@unina.it
Tutor:
nomeemail
De Castro, CristinaUNSPECIFIED
Date: 10 April 2017
Number of Pages: 235
Uncontrolled Keywords: Chlorella Viruses, NMR, Modelling
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/06 - Chimica organica
Date Deposited: 03 May 2017 17:22
Last Modified: 14 Mar 2018 10:06
URI: http://www.fedoa.unina.it/id/eprint/11744
DOI: 10.6093/UNINA/FEDOA/11744

Abstract

Sugars can be divided into two major subfamilies: the simple sugars (monosaccharides) and the complex sugars (oligosaccharides or polysaccharides depending on the unit of the monosaccharides). Complexity in structure arises from the elevated number of isomers that even two simple monosaccharides can generate, for instance, two glucose units can combine into 11 different disaccharides, while two identical aminoacids form only one dipeptide. Carbohydrates, simple or complex, can be further linked to other molecules, as proteins and fats to form glycoproteins and glycolipids (known as glycoconjugates). Sugars cover a wide range of fundamental roles, spanning from structural scaffolds to energetic intermediates and storage, they confer immunological protection, participate to cell-cell recognition processes and so on. In addition, their diversity in type and number guarantees to the organism the possibility to having a private and unique signature. In this regard, microorganisms display an extraordinary diversity that is confirmed by the continuous emergence of new glycosylation patterns: the studies of microorganisms disclose, indeed, the occurrence of atypical and rare sugars, which play distinctive roles in their interaction and adaptation with the environment, not known so far. Therefore, the structural analysis of the glycoforms produced by the microorganisms is crucial, it is the basis to understand the biosynthetic mechanism and the possible relationship between the structure and the biological activity. In this frame, my Ph.D. project focuses on the structural analysis of the glycosylation patterns of a special class of viruses, namely those having an autonomous glycosylation process. More in detail, I have studied the Chlorella viruses, belonging to the Giants Viruses class, deepening our understanding on the structures produced, their asset at the capsid and gaining clues on the biosynthetic process. Chlorella viruses (family Phycodnaviridae) infect certain unicellular, eukaryotic, symbiotic chlorella-like green algae. Their importance is related to their capability to encode most, if not all, of the component required to glycosylated their major capsid protein, unlike to the other viruses which use the host biosynthetic machinery. The prototype of this class of viruses is the Paramecium bursaria chlorella virus (PBCV-1), that infects Chlorella variabilis NC64A, which is a symbiont in the protozoan Paramecium bursaria. Previous studies established that it was constituted by a major capsid protein, called Vp54, that presents four glycosylation sites, which are unusual in several aspects: i) the glycans are not located in a typical Asn-X-(Thr/Ser) consensus site; ii) the glycans are attached to the protein by a beta-glucose linkage; iii) the oligosaccharides are highly branched; iv) each glycan have two rhamnose residues with opposite configurations. Therefore, the first aim of my project is to verify if the glycoform described by PBCV-1 is an isolated case or it is a structural motif shared by other Chlorella viruses. For this reason, I have extended the work to the other viral isolates that belong to the same genus of PBCV-1, but also with other host specificity, such as:NY2A, which infects Chlorella variabilis; MT325, CVM-1 and NeJV-1, that infect Micractinium conductrix; ATCV-1 and TN603, which infect Chlorella heliozoae and OSy-NE5, that infects Syngen 2-3 algae. This study discloses that the N-glycans of all chlorella viruses have a new structural core that do not resemble any other reported for bacteria, archea or eukarya, thus it can be considered a signature for this class of organisms. Moreover, the oligosaccharidic core can be decorated with different monosaccharides depending on the host specificities. The work was extended to the spontaneous mutants, also referred to as antigenic variants, of PBCV-1 in order to carry on the structure-to-function gene analysis of the prototype virus. Recent analysis about the PBCV-1 gene, discloses that it encodes, at least, six putative glycosyltransferases. One of them, the A064R protein, has drawn our attention because 18 of 21 mutants map to the related gene (a064r gene). This gene consists in three domains, whose function has been disclosed and in part confirmed during this PhD. On the basis of genetic analysis, the first domain encodes for glycosyltransferases, whereas the third domain seems to be a methyltransferase-like domain, while no clear information were deduced for the second domain. Therefore, the second goal of my PhD project consisted in two parts. First, I have undertaken the structural studies of the antigenic variants, to understand the function of the whole A064R gene by identifying the phenotype associated to the mutants. Antigenic variants are dived in six antigenic classes, depending on the reaction against antibodies raised against them. Variants belonging to class B (EPA and EPA2) have a domain one affected (there is a point mutation in the first domain, or it was truncated), and in both cases the N-glycans present only six monosaccharides: b-rhamnose unit is missing. On the contrary, variant of class A and F, has this domain intact and -rhamnose elongates the previous oligosaccharide. This lead to hypothesize that the first domain encodes for a b-(1,4)-L-rhamnosyl transferase. CME6, the only variant of class F, has both domain 1 and 2 and the glycan displays an additional a-rhamnose unit. Thus, we suppose that the second domain encodes an a-(1,2)-L-rhamnosyl transferase. Furthermore, we belief that domain 3 methylates O-2 and O-3 of a-rhamnose. In support of this hypothesis are the minor forms of the glycan from P91 and CME6. These two variants have domain 3 in its full length or truncated, respectively, and their glycans are methylated to a low extent. We believe that P91 methylation is incomplete because the methyltransferase domain does not find its ideal substrate (there is b- and not a-rhamnose). In CME6, the right substrate acceptor is present, but domain 3 of A064R is truncated and impaired in its activity. Regarding the function of the first domain, its crystallographic data are available and literature reports that it encodes for a glycosyltransferase, for which the best ligand was a UDP-glucose. Considering our structural data, the best ligand for this glycosyltransferase is UDP-b-L-rhamnose. Therefore, additional experiments were performed and our hypothesis proved (manuscript in preparation). The enzyme encoded by the first domain (A064R-D1) was expressed in E. coli and its activity investigated by biochemical assays and by analyzing the product formed giving UDP-b-L-rhamnose as donor and a synthetic substrate resembling EPA1 glycan (hyperbranched fucose, substituted with a galactose, xylose and rhamnose residues, that presents a small lipophilic tail, at the reducing end), as acceptor. The biochemical experiments were performed by using the bioluminescent assay developed from Promega (https://ita.promega.com/products/cell-signaling/glycosylation/udp_glo-glycosyltransferase-assay/) and fully confirmed our initial hypothesis about the function of the first domain of a064r, but also broadens our knowledge about it. We have proved that this enzyme is a b-rhamnosyl transferase, manganese-dependent, and able to transfer the beta-rhamnose also onto free xylose monosaccharide, indicating that its specificity for the acceptor is rather broad. In addition, we have excluded that the enzyme can work using Mg as coordinating cation and also that it does not recognizes UDP-a-Glc as instead hypothesized in the literature. It is our idea to continue the experiments on other two domains encoded by the a064r gene (using biochemical assays and docking analysis) to address the role of the whole gene. Another important question regards the structure of the Vp54 major capsid protein. X-ray data combined with cryo-electron microscopy disclosed that the capsid has an icosahedral shape constructed with two higher order elements, the trysimmetron and the pentasymmetron, each in turn constituted by several capsomer unit.Each capsomer is a trimer of the capsid protein, Vp54, which consists of a 437amino acid, organized into two consecutive jelly-roll domains: D1 (residues 27-212) and D2 (residues 225-437; N-glycosylated at Asn 280, 302, 399 and 406), that are related by a 53° rotation approximately about the central threefold axis of the trimer giving to the capsomer a pseudo hexagonal symmetry. To gain insight into the function of these glycans, the original data of the Vp54 were re-examined to correct the inconsistencies reported in the first publication related to the unknown structure of the N-glycans. Upon revising the original X-ray data by using the correct sugar templates, the overall level of information increased. Indeed, the new structure contained the first aminoacids that were originally undetected, no sugar densities related to O-linked sugar could be fitted, while, more importantly, almost all the residues of the N-glycans were placed. Information from this new structure was further implemented through a Molecular Modelling approach that fixed some faults still existing in the X-ray structure. Actually, X-ray fitting produced some residues in the wrong ring conformation, while some few, especially those located far from the polypeptide backbone, were completely absent. The computation protocol integrated a systematic conformational search (Metropolis MonteCarlo) with Molecular Dynamic simulation that yielded in the end to determine the complete three-dimensional description of the chlorovirus PBCV-1 glycosylated major capsid protein. This study has given a first insight into the interactions existing between the carbohydrate and the protein part (paper under revision) and are at the basis to understand which role N-glycans play in the capsid packaging, or at what extent stabilize the whole capsid. Preliminary studies on this last aspect were performed using again adopting the molecular modelling approach. This work is still in progress, but preliminary simulations suggest that N-glycans not are involved in the protein folding, but probably they play an important role into the capsid packaging. This idea arises by the analysis of the normal mode of the glycoprotein: when the glycans are present, the two domains are maintained close, and the only permitted movement is the twisting mode; on the contrary, when the glycans are absent the two domains move away. This hypothesis needs further investigation, and will be the target of future work.

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