N-glycans of the microalga Chlorella vulgaris are of the oligomannosidic type but highly methylated

Microalgae of the genus Chlorella vulgaris are candidates for the production of lipids for biofuel production. Besides that, Chlorella vulgaris is marketed as protein and vitamin rich food additive. Its potential as a novel expression system for recombinant proteins inspired us to study its asparagine-linked oligosaccharides (N-glycans) by mass spectrometry, chromatography and gas chromatography. Oligomannosidic N-glycans with up to nine mannoses were the structures found in culture collection strains as well as several commercial products. These glycans co-eluted with plant N-glycans in the highly shape selective porous graphitic carbon chromatography. Thus, Chlorella vulgaris generates oligomannosidic N-glycans of the structural type known from land plants and animals. In fact, Man5 (Man5GlcNAc2) served as substrate for GlcNAc-transferase I and a trace of an endogenous structure with terminal GlcNAc was seen. The unusual more linear Man5 structure recently found on glycoproteins of Chlamydomonas reinhardtii occurred - if at all - in traces only. Notably, a majority of the oligomannosidic glycans was multiply O-methylated with 3-O-methyl and 3,6-di-O-methyl mannoses at the non-reducing termini. This modification has so far been neither found on plant nor vertebrate N-glycans. It’s possible immunogenicity raises concerns as to the use of C. vulgaris for production of pharmaceutical glycoproteins

Electron-transfer dissociation mass spectrometry for revealing protein O-glycosylation fine structure

Diese kumulative Doktorarbeit beschreibt die Implementierung einer Analysenmethode für dicht zusammenliegende Protein O-Glykosylierungen mittels Elektronen-Transfer Dissoziierungs Massenspektrometrie (ETD-MS/MS) und demonstriert ihre erfolgreiche Anwendung in drei Studien. Das anfängliche Bestreben dieser Arbeit war die tiefergehende Beschreibung der Rinderfetuin O-Glykosylierung in allen molekularen Details. Porös-graphitische Kohlenstoff Chromatographie gekoppelt an Massenspektrometrie konnte die O-Glykane aufklären, während ETD-MS/MS die Glykosylierungsstellen bestätigte und die Verteilung der O-Glykane an diesen Stellen zeigte. Dabei wurde festgestellt, dass die UDP-N-Acetylgalaktosamin:polypeptid-N-Acetylgalaktosaminyltransferasen (ppGalNAcTs) sequenziell an den verschiedenen Glykosylierungsstellen arbeiten. In einer zweiten Studie wurden bis jetzt kaum beachtete mehrfache O-Glykosylierungen im humanen Neuropilin-1 untersucht. Die massenspektrometrische Untersuchung des intakten Proteins zeigte ein bis vier, hauptsächlich komplett sialylierte, gebundene core-1 und core-2 Glykane. ETD-MS/MS stellte erneut eine hierarchische Ordnung der Anheftung von N-Acetylgalaktosamin (GalNAc) an die jeweiligen vier Aminosäuren fest. In einem dritten Projekt wurden die Substratpräferenzen der ersten rekombinanten ppGalNAcT aus der Klasse der Schnecken auf einem spezifisch gestaltetem Akzeptorpeptid getestet. Die Ergebnisse ermöglichten die Erstellung einer Rangordnung der jeweiligen Aminosäuren bezüglich ihres Glykosylierungspotenzials. Alles in allem geben die drei in dieser Arbeit präsentierten Publikationen Aufschluss über molekulare Details der untersuchten O-Glykoproteine und Peptide und ermöglichen dadurch weiterführende Studien über die Funktion dieser Glykosylierungen. Die Information über Substratpräferenzen von ppGalNAcTs bezüglich der verschiedenen Akzeptorstellen könnte Erkenntnisse über ihre physiologische Rolle mit sich bringen.This cumulative doctoral thesis describes the implementation of an analysis method for clustered protein O-glycosylation via Electron-transfer dissociation mass spectrometry (ETD-MS/MS) and demonstrates its successful application in three studies. The initial target of this work was the in-depth analysis of bovine fetuin O-glycosylation in all molecular details. Porous graphitic carbon chromatography coupled to mass spectrometry could reveal O-glycan species while ETD-MS/MS approved the glycosylation sites and characterized the O-glycans in a site-specific manner. UDP-N-acetylgalactosamine:polypeptide-N-acetylgalactosaminyltransferases (ppGalNAcTs) were thereby found to act sequentially on the specific sites. In a second study, yet hardly studied clustered O-glycosylation sites of human neuropilin-1 were investigated. Mass spectrometry on the intact protein elucidated the degree of glycosylation ranging from one to four mainly fully sialylated core-1 and core-2 O-glycans. ETD-MS/MS again showed a hierarchical order of the initial attachment of N-acetylgalactosamine (GalNAc) residues onto the four respective amino acids. In a third project, substrate preferences of the first recombinant ppGalNAcT of snail origin on a specifically designed peptide were investigated. The results allowed a ranking of the acceptor sites regarding their potential of getting modified. Altogether, the studies presented in this thesis shed light on the molecular details of respective O-glycoproteins/peptides and thus enable future work on the function of these O-glycosylations. The information on substrate preferences of ppGalNAcTs for the various acceptor sites may give insight into their physiological role.submitted by Markus WindwarderAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2015OeBB(VLID)193155

A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

The cell surface of the oral pathogen Tannerella forsythia is heavily glycosylated with a unique, complex decasaccharide that is O-glycosidically linked to the bacterium’s abundant surface (S-) layer, as well as other proteins. The S-layer glycoproteins are virulence factors of T. forsythia and there is evidence that protein O-glycosylation underpins the bacterium’s pathogenicity. To elucidate the protein O-glycosylation pathway, genes suspected of encoding pathway components were first identified in the genome sequence of the ATCC 43037 type strain, revealing a 27-kb gene cluster that was shown to be polycistronic. Using a gene deletion approach targeted at predicted glycosyltransferases (Gtfs) and methyltransferases encoded in this gene cluster, in combination with mass spectrometry of the protein-released O-glycans, we show that the gene cluster encodes the species-specific part of the T. forsythia ATCC 43037 decasaccharide and that this is assembled step-wise on a pentasaccharide core. The core was previously proposed to be conserved within the Bacteroidetes phylum, to which T. forsythia is affiliated, and its biosynthesis is encoded elsewhere on the bacterial genome. Next, to assess the prevalence of protein O-glycosylation among Tannerella sp., the publicly available genome sequences of six T. forsythia strains were compared, revealing gene clusters of similar size and organization as found in the ATCC 43037 type strain. The corresponding region in the genome of a periodontal health-associated Tannerella isolate showed a different gene composition lacking most of the genes commonly found in the pathogenic strains. Finally, we investigated whether differential cell surface glycosylation impacts T. forsythia’s overall immunogenicity. Release of proinflammatory cytokines by dendritic cells (DCs) upon stimulation with defined Gtf-deficient mutants of the type strain was measured and their T cell-priming potential post-stimulation was explored. This revealed that the O-glycan is pivotal to modulating DC effector functions, with the T. forsythia-specific glycan portion suppressing and the pentasaccharide core activating a Th17 response. We conclude that complex protein O-glycosylation is a hallmark of pathogenic T. forsythia strains and propose it as a valuable target for the design of novel antimicrobials against periodontitis

UDP-sulfoquinovose formation by Sulfolobus acidocaldarius

The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-d-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD+-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-d-glucose is converted via UDP-4-keto-d-glucose to UDP-d-glucose-5,6-ene, the structure of which was determined by 1H and 13C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-d-glucose and NAD+ could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-d-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested

Genome Analysis and Characterisation of the Exopolysaccharide Produced by Bifidobacterium longum subsp. longum 35624™.

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide

¹H and ¹³C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from <i>B</i>. <i>longum</i> 35624.

<p>¹H and ¹³C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from <i>B</i>. <i>longum</i> 35624.</p

B. longum 35624 EPS characterization.

<p>(A) The 600 MHz ¹H NMR proton spectrum of the acid-treated <b>35624</b> EPS (D₂O, 338 K) is illustrated. A part of the high-field region is displayed in the insert. <b>(B)</b> Expansion plot of the 150 MHz 13C NMR spectrum of the acid-treated <b>35624</b> exopolysaccharide. The anomeric signals on the left confirmed the presence of a hexasaccharide repeat unit.</p