1,720,983 research outputs found
Structure of repeating unit of the capsular polysaccharide from Acinetobacter baumannii D78 and assignment of the K4 gene cluster
No full textThe structure of the K4 capsular polysaccharide (CPS) from a multiply antibiotic resistant Acinetobacter baumannii isolate D78 was elucidated by chemical and spectroscopical analysis. The K4 repeating unit is composed entirely by aminosugars and the trisaccharide repeating unit of the backbone polysaccharide is branched with a terminal N-acetyl-galactosamine capped with pyruvate as cyclic acetal. Each residue is in the pyranose form, a configured at the anomeric center, and has the D absolute configuration. The genes responsible for the synthesis of the K4 repeat unit and its polymerization and export are found in the capsule locus KL4 present in D78. The pyruvate acetal addition to galactosamine is formed by Ptr1, a novel pyruvate transferase, encoded at this locus
Dissecting Lipopolysaccharide Composition and Structure by GC-MS and MALDI Spectrometry
No full text: Lipopolysaccharides (LPSs) are the main components of the external leaflet of the outer membrane of Gram-negative bacteria. They exert multiple functions, starting from conferring stability to the bacterial membrane to mediating the interaction of the microbe with the external environment. The composition and the structure of LPSs present tremendous diversity even within bacteria of the same species, and for this reason, the determination of the structure of these molecules is crucial because it can provide information on the motifs key for the virulence of a pathogen or that are associated to a bacterium of the commensal or beneficial microbiota. In addition, structural data disclose the effects triggered from a mutation or from the use of an antibiotic, or they can be used as tools to check the quality of adjuvants and/or medications, as vaccines, that make use of LPS.The structural study of LPSs is complex, and it can be achieved with the right combination of different techniques. In this frame, this chapter focuses on the two MS-based approaches, the gas chromatography-mass spectrometry (GC-MS) and the matrix-assisted laser desorption/ionization (MALDI)
N-glycans from Paramecium bursaria chlorella virus MA-1D: Re-evaluation of the oligosaccharide common core structure
No full text: Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic (NMR) spectroscopy and MALDI-TOF mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the non-stoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses
Structure of the chlorovirus PBCV-1 major capsid glycoprotein determined by combining crystallographic and carbohydrate molecular modeling approaches
No full textThe glycans of the major capsid protein (Vp54) of Paramecium bursaria chlorella virus (PBCV-1) were recently described and found to be unusual. This prompted a reexamination of the previously reported Vp54 X-ray structure. A detailed description of the complete glycoprotein was achieved by combining crystallographic data with molecular modeling. The crystallographic data identified most of the monosaccharides located close to the protein backbone, but failed to detect those further from the glycosylation sites. Molecular modeling complemented this model by adding the missing monosaccharides and examined the conformational preference of the whole molecule, alone or within the crystallographic environment. Thus, combining X-ray crystallography with carbohydrate molecular modeling resulted in determining the complete glycosylated structure of a glycoprotein. In this case, it is the chlorovirus PBCV-1 major capsid protein
Structure and Conformation Study of the O-Antigen from the Lipopolysaccharide of Cupriavidus Metallidurans CH34
No full textCupriavidus metallidurans is a Gram-negative bacterium that has attracted the attention of the scientific community since its discovery back in 1976. It was initially studied as a model organism for bioremediation processes due to its ability to survive in heavy metal-rich environments. However, in recent years, there is evidence that this bacterium can be a potential pathogen for humans. How C. metallidurans can survive in such different environments is unknown and prompted the following work. Its great adaptability could be explained by the structural and conformational studies of the O-antigen portion of the lipopolysaccharide, the main constituent of the outer membrane of Gram-negative bacteria, which is the one in direct contact with the external environment. Therefore, a combination of chemical and spectroscopic analyses was used to define the O-antigen structure, disclosing that it is a polysaccharide constituted of a linear tetrasaccharide repeating unit that does not resemble other structures already reported for bacteria: [4)-α-d-GalNAc-(1→3)-α-d-Qui2NAc4NHBA-(1→3)-α-l-Rha-(1→3)-α-l-Rha-(1→]. Interestingly, the molecular dynamics studies revealed that the three-dimensional structure of the O-antigen is highly flexible: it might adopt three different right-handed helix conformations described by a two, three, or four-fold symmetry. This conformational behavior could represent the reason behind the survival of C. metallidurans in different environments
Rhamnolipid Self-Aggregation in Aqueous Media: A Long Journey toward the Definition of Structure–Property Relationships
No full textThe need to protect human and environmental health and avoid the widespread use of substances obtained from nonrenewable sources is steering research toward the discovery and development of new molecules characterized by high biocompatibility and biodegradability. Due to their very widespread use, a class of substances for which this need is particularly urgent is that of surfactants. In this respect, an attractive and promising alternative to commonly used synthetic surfactants is represented by so-called biosurfactants, amphiphiles naturally derived from microorganisms. One of the best-known families of biosurfactants is that of rhamnolipids, which are glycolipids with a headgroup formed by one or two rhamnose units. Great scientific and technological effort has been devoted to optimization of their production processes, as well as their physicochemical characterization. However, a conclusive structure–function relationship is far from being defined. In this review, we aim to move a step forward in this direction, by presenting a comprehensive and unified discussion of physicochemical properties of rhamnolipids as a function of solution conditions and rhamnolipid structure. We also discuss still unresolved issues that deserve further investigation in the future, to allow the replacement of conventional surfactants with rhamnolipids
Structural insight into new glycosylation patterns of microbial (bacterial and viral) origin
Get PDFSugars 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
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans
Get PDFThe structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of ge- netic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum–Golgi glycosyla- tion pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes
- …
