ProGlycProt: a repository of experimentally characterized prokaryotic glycoproteins

ProGlycProt (http://www.proglycprot.org/) is an open access, manually curated, comprehensive repository of bacterial and archaeal glycoproteins with at least one experimentally validated glycosite (glycosylated residue). To facilitate maximum information at one point, the database is arranged under two sections: (i) ProCGP—the main data section consisting of 95 entries with experimentally characterized glycosites and (ii) ProUGP—a supplementary data section containing 245 entries with experimentally identified glycosylation but uncharacterized glycosites. Every entry in the database is fully cross-referenced and enriched with available published information about source organism, coding gene, protein, glycosites, glycosylation type, attached glycan, associated oligosaccharyl/glycosyl transferases (OSTs/GTs), supporting references, and applicable additional information. Interestingly, ProGlycProt contains as many as 174 entries for which information is unavailable or the characterized glycosites are unannotated in Swiss-Prot release 2011_07. The website supports a dedicated structure gallery of homology models and crystal structures of characterized glycoproteins in addition to two new tools developed in view of emerging information about prokaryotic sequons (conserved sequences of amino acids around glycosites) that are never or rarely seen in eukaryotic glycoproteins. ProGlycProt provides an extensive compilation of experimentally identified glycosites (334) and glycoproteins (340) of prokaryotes that could serve as an information resource for research and technology applications in glycobiology

Methods in automated glycosaminoglycan tandem mass spectra analysis

Glycosylation is the process by which a glycan is enzymatically attached to a protein, and is one of the most common post-translational modifications in nature. One class of glycans is the glycosaminoglycans (GAGs), which are long, linear polysaccharides that are variably sulfated and make up the glycan portion of proteoglycans (PGs). PGs are located on the cellular surface and in the extracellular matrix (ECM), making them important molecules for cell signaling and ligand binding. The GAG sulfation sequence is a determining factor for the signaling capacity of binding complexes, so accurate determination of the sequence is critical. Historically, GAG sequencing using tandem mass spectrometry (MS2) has been a difficult, manual process; however, with the advent of faster computational techniques and higher-resolution MS2, high-throughput GAG sequencing is within reach. Two steps in the pipeline of biomolecule sequencing using MS2 are discovery and interpretation of spectral peaks. The discovery step traditionally is performed using methods that rely on the concept of averagine, or the average molecular building block for the analyte in question. These methods were developed for protein sequencing, but perform considerably worse on GAG sequences, due to the non-uniform distribution of sulfur atoms along the chain and the relatively high isotope abundance of 34S. The interpretation step traditionally is performed manually, which takes time and introduces potential user error. To combat these problems, I developed GAGfinder, the first GAG-specific MS2 peak finding and annotation software. GAGfinder is described in detail in chapter two. Another step in MS2 sequencing is the determination of the sequence using the found MS2 fragments. For a given GAG composition, there are many possible sequences, and peak finding algorithms such as GAGfinder return a list of the peaks in the MS2 mass spectrum. The many-to-many relationship between sequences and fragments can be represented using a bipartite network, and node-ranking techniques can be employed to generate likelihood scores for possible sequences. I developed a bipartite network-based sequencing tool, GAGrank, based on a bipartite network extension of Google’s PageRank algorithm for ranking websites. GAGrank is described in detail in chapter three

Understanding the innovative viral glycosylation machinery using a combination of chemical and structural methodologies

The aim of this thesis is the study of the innovative glycosylation machinery of the Mimiviridae family, using Mimivirus, Moumouvirus australensis and Megavirus chilensis as prototypes of lineages A, B and C, respectively. In 2003 the discovery of Mimivirus, the first giant DNA virus infecting amoeba, challenged the traditional view of viruses. Mimiviruses are giant viruses due to the size of their virions, easily visible by light microscopy, with a diameter of 700 nm against 200 nm for “traditional virus”. Their genomes encode 1000 proteins and count up to 1.2 Mbp, so they are as complex as the smallest free-living bacteria. Mimiviruses exhibit heavily glycosylated fibrils surrounding their capsid that differ in length depending on the lineages. Surprisingly, it was evidenced that they encode the proteins involved in their fibrils glycosylation. The glycosylation of the fibrils was confirmed by the analysis of their sugar content, revealing that the major saccharide components were rhamnose, N-acetylglucosamine, and viosamine for Mimivirus and N-acetylglucosamine and N-acetylrhamnosamine for Megavirus chilensis. Until now, we lack information on the sugar composition of fibrils from members of the B lineage. In this thesis, the innovative glycosylation machinery of these giant DNA viruses was investigated combining three different strategies: carbohydrate chemistry, bioinformatic and biochemical methodologies. The carbohydrate chemistry methodologies allowed to elucidate the structures/composition of the glycans associated to the giant DNA viruses fibrils. Mimivirus fibrils are decorated with two distinct polysaccharides, called poly_1 and poly_2. Poly_1 is characterized by a linear disaccharide repeating unit made of 3)--L-Rha-(1→3)--D-GlcNAc-(1→, with a pyruvic acid branched at position 4,6 of GlcNAc. Poly_2 has a branched repeating unit with the sequence 2)--L-Rha-(1→3)--D-GlcNAc-(1→ in the linear backbone and rhamnose further branched at position 3 by viosamine methylated at position 2 and acetylated at position 4. Regarding the novelty of the identified structures, they have no equivalent in eukaryotes, while some components were reported in bacteria. Megavirus chilensis has a different sugar composition of its shorter fibrils, with N-acetylglucosamine, N-acetylrhamnosamine and N-acetylquinovosamine as major components. Purification results suggested that Megavirus fibrils were decorated by more than one polysaccharides/oligosaccharide species, one having this trisaccharide: -L-4OMe-RhaNAc-(1→3)--L-RhaNAc-(1→3)--L-RhaNAc-(1→. A preliminary analysis revealed that Moumouvirus australensis fibrils were decorated with glucosamine and quinovosamine in addition to the rare sugar, bacillosamine. Starting from this experimental data, it was possible to identify new genes involved in glycosylation. As a result, the published nine-gene cluster of Mimivirus was extended to thirteen genes. A different cluster of fourteen genes was identified in Moumouvirus australensis, representing the first glycosylation gene cluster identified for the B lineage. A comparison of the glycosylation genes in the Mimiviridae family reinforced our finding that fibrils glycosylation was lineage specific. However, Moumouvirus australensis is an exception as it exhibits a cluster of glycosylation genes that is missing in other member of the B lineage. Among the genes with the glycosylation cluster, the function of L142 was investigated in vitro, demonstrating that it is a N-acetyltransferase that acetylates the 4 amino group of viosamine. N-L142 represents the first virally encoded N-acetyltransferase. To conclude, the fibrils of Mimiviridae are heavily glycosylated and the type of sugars and their organization depends on their lineage. The majority of the genes responsible for sugar production, sugar modification and glycosyltransferases were identified, strongly suggesting that Mimiviridae are autonomous for their fibrils glycosylation

Characterisation of the lipopolysaccharide and peptidoglycan and their structural determinants when bound to major proteins involved in its transport across the periplasm

Gram-negative bacteria possess an outer membrane (OM), a lipidic bilayer that surrounds a thin peptidoglycan (PG) layer and the cytoplasmic membrane (CM). The OM is an asymmetric bilayer whose outer leaflet is mainly composed by lipopolysaccharides (LPSs). LPS and PG have a direct role in antibacterial resistance and in the communication bacteria-host. There are many open questions on how the bacterial surface of pathogens and commensals interacts with the host in order to escape immune recognition and produce harmful or beneficial effects as well as regarding the details on how the bacterial envelope is built. Therefore, the main focus of this Ph.D. thesis is to contribute to the characterisation of the LPS and PG to increase the knowledge on the interaction of pathogen and commensal Gram-negative bacteria with the host and to deepen the understanding of their structural determinants when bound to major proteins involved in its transport across the periplasm. With that aim the composition, structure and immune activities of the LPS and PG of Akkermansia muciniphila and Fusobacterium nucleatum is disclosed and the trans-envelope machineries of the model bacterium Escherichia coli studied. A. muciniphila is one of the few bacteria that successfully inhabits the mucus layer of humans and other mammals' intestines. Not only its presence is associated with a healthy intestine, but also it seems to improve insulin sensitivity, increase the mucosal barrier function, regulate glycemia levels, and reduce fat accumulation, insulinemia, cholesterol, body weight gain and inflammation in the intestine and body. The lipooligosaccharide (LOS or rough LPS) of A. muciniphila MucT is very complex: it includes more than the two canonical units of Kdo, is rich in fucose units and most of the fatty of the lipid A are branched at the penultimate carbon. The LOS seems to be a mild activator of TLR4, while it is a relevant activator of TLR2 which may play a role in the development of the beneficial effects of the bacterium. The PG of A. muciniphila MucT contains muropeptides with de-N-acetylated glucosamine, being the first time, such structure is described in a Gram-negative bacterium. Moreover, this modification of the PG has been linked to the avoidance of recognition by NOD-1 immune receptors and therefore bacterial clearance. F. nucleatum is an oral commensal that plays a crucial role in the formation of biofilms, being also involved in extra-oral disorders such as intrauterine infections and colorectal cancer, in which the subspecies animalis is the most-commonly isolated. The LPS of F. nucleatum spp. animalis ATCC 51191 has a trisaccharide repeating unit rich in amino- and aminuronic-monosaccharides, and a lipid A similar to that of Burkholderia cenocepacia. In addition, F. nucleatum ssp. polymorphum ATCC 10953, F. nucleatum ssp. animalis ATCC 51191 and F. nucleatum ssp. nucleatum ATCC 25586 full cells, outer membrane vesicles (OVMs), and LPSs stimulate monocyte-derived dendritic cells leading to an increased production of TNFa, IL-8 and IL-6, while in monocyte-derived macrophages the stimulation leads to the production of IL-10, IL-6 and IL-8 and to low levels of TNFa. These effects are measured in the three strains and seem to be mediated by Siglec-7, a sialic acid receptor, even though the O-antigen of only two of the strains tested (ATCC 10953 and 25586) expose this monosaccharide or the sialic acid-like molecule fusaminic acid. The PG of F. nucleatum spp. animalis ATCC 51191 presents an alteration of the most common stem peptide by substitution of the L-meso-diaminopimelic acid by the sulfur-containing diamino acid lanthionine or another amino acid. This may be crucial to avoid the recognition by NOD-1 immune receptors potentiating colorectal cancer development. The trans-envelope machineries were studied on E. coli, because of its importance as antibiotic-resistant "priority pathogen" of WHO and due to the fair amount of existing literature. The T5SS that transports, folds and insets b-barrel proteins in the OM is comprised of Skp, SurA, DegP and the BAM complex (that consists of four lipoproteins BamB, BamC, BamD, BamE and one OMP named BamA). In order to determine the extent of their influence on the composition and structure of LPS, the E. coli mutants delta-surA, delta-skp, delta-degP, delta-bamB, delta-bamC, and delta-bamE were produced and the structure of their LPS determined. The results suggested that the alterations on the BAM machinery do not significantly alter the composition nor the structure of the LPS, providing an insight on the mechanism by which the alteration of the BAM machinery may alter the integrity of the OM. The LPS transportation machinery (Lpt) deploys seven LPS transport proteins named Lpt A-G that extracts the LPS from the external leaflet of the CM, transports it across the periplasm and the PG, flips it across the OM and locates the LPS in its external face. All Lpt proteins are essential, which makes them candidates as targets for new antibiotics. There are many open questions on the working of this machinery, for instance the details of the interaction sites of the hydrophobic pocket and how the periplasmic bridge is formed. During this Ph.D., the development of a semi-synthetic lipid A with active nuclei instead of acyl chains was attempted in order to study the details of the interaction between LPS-Lpt proteins by NMR. The introduction of a paramagnetic group on the fully de-acylated lipid A failed, but the introduction of fluoropropanoyl chloride seemed to be partially successful. However, the product presents a high level of contamination that prevented the reliable determination of the product as well as the interaction studies. In addition, it is disclosed that LptA does not act as an amidase regulator for AmiA, AmiB nor AmiC nor as a ligand for the amidase activators YgeR or NlpD. Leaving unanswered the question on how the hole on the PG is open for the Lpt bridge. In conclusion, the architecture of bacterial envelope is crucial for the interaction with the host and the knowledge of the structure of its components is a fundamental prerequisite to proceed with functional studies, and to dissect the role of each component. In this frame, the knowledge of the molecular determinants of the bacteria of the microbiota is preliminary. Through the characterisation of LPS and PG, this Ph.D. thesis demonstrates that they have unexpected structures and activities. Likewise, their transport across the periplasm, dissected on model organisms, still presents many gaps to be filled. Thus, our understanding of the cell envelope and of its metabolism is still to an early stage, but it is mature enough to devise alive bacteria and/or synthetic analogues of their surface structures for clinical applications

Accessing Pseudaminic Acid (Pse5Ac7Ac) containing Glycosides through the Characterisation of Pse5Ac7Ac Processing Enzymes

Cell-surface carbohydrate pseudaminic acid (Pse5Ac7Ac) is known to contribute to the virulence of several multi-drug resistant bacterial pathogens. Pse5Ac7Ac and its derivatives are not commercially available in appreciable quantities and chemical synthesis of these molecules has proved to be challenging. Access to Pse5Ac7Ac and activated CMP-Pse5Ac7Ac has been a hindrance in studies into the biological significance of Pse5Ac7Ac, including Pse5Ac7Ac-processing enzymes, which may be novel therapeutic targets. This project aimed to characterise enzymes which process pseudaminic acid and to chemoenzymatically synthesise glycosides which contain pseudaminic acid. Firstly, nucleotide-activated pseudaminic acid (CMP-Pse5Ac7Ac) was produced via a chemoenzymatic synthesis route. Six recombinant biosynthetic enzymes were purified for use in this reaction. With CMP-Pse5Ac7Ac in-hand, a library of bacterial glcosyltransferases were assayed for activity with CMP-Pse5Ac7Ac as donor. Success from this initial screen led to the synthesis of glycosides containing β-linked Pse5Ac7Ac, mediated by promiscuous glycosyltransferases. Finally, a putative pseudaminyltransferase was recombinantly produced, through the construction of a fusion protein. Activity studies revealed that the enzyme was able to utilise CMP-Pse5Ac7Ac and the product of the reaction was analysed, to confirm that the enzyme functions as a retaining pseudaminyltransferase. To our knowledge the work presented herein details the first examples of chemoenzymatic synthesis of glycosides containing Pse5Ac7Ac and the first in vivo study of a pseudaminyltransferases to provide unequivocal functional characterisation of this novel class of enzym