Dissertations / Theses on the topic 'Glycans'

La eritropoyetina o EPO es una hormona glicoproteica. En los seres humanos adultos, se produce principalmente en el riñón, en respuesta a la reducción de oxígeno en los tejidos (hipoxia tisular). La EPO estimula la eritropoyesis, es decir, estimula a las células madre de la médula ósea para que aumenten la producción de eritrocitos. La molécula de EPO está formada por una cadena peptídica de 165 aminoácidos que contiene dos puentes disulfuro, un O-glicano (Ser-126), y tres N-glicanos (Asn-24, 38, 83). En conjunto, la glicosilación de esta proteína representa aproximadamente el 40% del peso total (29.4 kDa). La EPO sintetizada con tecnología recombinante (rEPO) se administra como fármaco, desde el año 1989, para el tratamiento de anemias, insuficiencia renal, cancer etc. También se ha observado la utilización de rEPO en deportistas con el fin de aumentar el suministro de oxígeno a los tejidos y así incrementar el rendimiento en deportes de resistencia. En el año 2001, se comercializó una "nueva proteína estimuladora de la eritropoyesis" llamada NESP. Esta proteína es un análogo hiperglicosilado de la EPO que posee dos N-glicanos adicionales (Asn-30, 88). El número y composición de los N-glicanos es muy importante para el metabolismo de estas glicoproteínas, ya que el contenido de carbohidratos (número de ácidos siálicos) determina su vida media. Los métodos que se utilizan actualmente para diferenciar la eritropoyetina endógena urinaria (uEPO) de sus análogos recombinantes (rEPO, NESP) están basados en las diferencias que existen entre sus perfiles isoelectroforéticos (IEF). Se cree que estas diferencias provienen de las células y/o especies en las que se expresan estas glicoproteínas. En este estudio, se llevo a cabo la caracterización estructural de diferentes preparaciones de EPO recombinante. Para ello, se utilizaron las técnicas más comunes en el campo de la glicoproteómica además de otras nuevas, desarrolladas para poder analizar los glicanos de muestras procedentes de geles 2-DE. Los perfiles de glicosilación total de cada una de estas glicoproteínas mostraron características estructurales que pueden facilitar la detección de rEPO y NESP en atletas que abusan de estas sustancias. Un ejemplo es la presencia de Neu5Gc únicamente en fármacos que han sido expresados en células CHO. La metodología desarrollada en este trabajo, podría emplearse también, para el control de calidad de estos fármacos y para el diagnóstico de ciertas patologías.
Erythropoietin (EPO) is a glycoprotein hormone secreted primarily by adult kidneys in response to tissue hypoxia. It is involved in the maturation and ultimately regulation of the level of red blood cells. The EPO molecule comprises a single polypeptide chain of 165 aminoacids with two disulfide bonds, 1 O-linked (Ser-126), and 3 N-linked (Asn-24, 38, 83) glycans representing about 40% of the total mass (29.4 kDa). The recombinant analogue (rEPO), available since 1989 has found widespread use in the treatment of anaemia, renal failure, cancer etc. Besides, rEPO is illicitly used by athletes to boost the delivery of oxygen to the tissue and enhance performance in endurance sports. In 2001, a novel erythropoiesis-stimulating protein (NESP) was also marketed. NESP possesses two additional N-glycans (Asn-30, 88). The number and composition of the N-glycans is very important in the metabolism of this glycoprotein because the carbohydrate content (sialylation degree) determines its half-life time. Current tests to differentiate between urinary endogenous (uEPO) and its recombinant analogues (rEPO, NESP) are based on differences in their iso-electric focussing (IEF) profiles. Those differences are believed to stem from the cells/species in which they are expressed. In this study, the structural characterisation of different rEPO preparations was conducted using standard techniques in the field and developing new ones to address glycans from 2-DE sample preparations. Overall glycosylation profiling of each glycoprotein revealed structural features that may pave the way to the unambiguous detection of rEPOs and NESP abuse, such as the presence of Neu5Gc in CHO cell derived drugs only, and the developed methodology may be also employed for maintaining pharmaceutical quality control and for diagnosing pathologic conditions.

Thesis (Ph.D.)--Tufts University, 2000.
Adviser: Marc d'Alarcao. Submitted to the Dept. of Chemistry. Includes bibliographical references (leaves 262-271). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Glycosylation is an abundant post translational modification of proteins, which occurs in the endoplasmic reticulum and Golgi. Nearly every secreted and plasma membrane localised protein is glycosylated. Mesenchymal stromal/stem cells (MSCs) are adult stem cells that can self-renew and differentiate into multiple cell types including: osteoblasts, adipocytes and chondrocytes. Due to their tri-lineage capacity and their immunoregulatory functions, MSCs are attractive tools for the treatment of multiple diseases and conditions. Key to their use in regenerative medicine is knowledge about what influences a MSC to differentiate into a particular cell type. Despite previous studies describing distinct glycan profiles of cells at different stages of development, whether glycans play a functional role in directing MSC differentiation is currently unknown. Here, utilizing an immortalized primary human MSC line (hTERT-MSCs), the N-glycans from MSCs and osteoblasts were harvested using the Filter aided N-glycan Separation (FANGS) method. Following permethylation, N-glycans were analysed by mass spectrometry (MALDI-TOF/TOF). This method allowed the reliable, quantitative, relative abundance of different glycan structures to be compared between the two cell types for the first time. Complex N-glycans were significantly more abundant in osteoblasts compared to MSCs. The N-glycan profiles of different hTERT-MSC lines were not significantly different, despite variations in differentiation potential. The glycosylation pathway was genetically disrupted by targeting a subunit of the Conserved Oligomeric Complex (COG) with a shRNA. As predicted, since COG is responsible for the tethering of vesicles carrying Golgi resident enzymes, Cog4 knock-down hTERT-MSCs had disrupted N- and O-glycan synthesis. Interestingly, Cog4 knock-down hTERT-MSCs showed reduced osteogenic capacity with reduced levels of mineralised extracellular matrix (ECM). Surprisingly, the chemical inhibition of complex N-glycan synthesis increased ECM mineralisation, whilst inhibition of O-glycan synthesis or proteoglycan sulfation mimicked the Cog4 knock-down cells with reduced mineralisation. These results showed a novel role of both N-and O-glycans in osteogenesis.

Thesis (M. Eng. and S.B.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2004.
Includes bibliographical references (leaves 75-76).
Recent advances in biology have afforded scientists with the knowledge that polysaccharides play an active role in modulating cellular activities. Glycosaminoglycans (GAGs) are one such family of polysaccharides that play a very important role in regulating the functions of numerous important signaling molecules and enzymes in the cell. Developing bioinformatics tools has been integral to advancing genomics and proteomics. While these tools have been well-developed to store and process sequence and structure information for proteins and DNA, they are very poorly developed for polysaccharides. Glycan structures pose special problems because of their tremendous information density per fundamental unit, their often-branched structures, and the complicated nature of their building blocks. The GlycoBank, an online database of known GAG structures and functions, has been developed to overcome many of these difficulties by developing a common notation for researchers to describe GAG sequences, a common repository to view known structure-function relationships, and the complex tools and searches needed to facilitate their work. This thesis focuses on the development of GlycoBank. In addition, a large, NIGMS-funded consortium, the Consortium for Functional Glycomics, is a larger database that also aims to store polysaccharide structure-function information of a broader collection of polysaccharides. The ideas and concepts implemented in developing GlycoBank were instrumental in developing databases and bioinformatics tools for the Consortium for Functional Glycomics.
by Eric Zachary Berry.
M.Eng.and S.B.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 236-275).
Glycans are an important class of biological molecules that regulate a variety of physiological processes such as signal transduction, tissue development and microbial pathogenesis. However, due to the structural complexity of glycans and the unique intricacies of glycan-protein interactions, elucidating glycan structure-function relationships is challenging. Thus, uncovering the biological function of glycans requires an integrated approach, incorporating structural analysis of glycans, and glycan-proteins interactions with functional analysis. In this thesis, I develop new tools and implement integrated approaches to study glycans and glycan-binding proteins (GBPs). I apply these approaches to study glycans and GBPs in two areas: i) the role of hemagglutinin-glycan receptor specificity in human adaptation and pathogenesis of influenza and ii) the function of glycan regulation of cell-microenvironment interaction in cancer progression. Section 1: Influenza poses a significant public health threat and there is a constant looming threat of a pandemic. Pandemic viruses emerge when avian viruses acquire mutations that enable human adaptation, leading to infection of an antigenically naive host. Influenza Hemagglutinin (HA), and HA-glycan receptor interactions, play a central role in host tropism, transmissibility, and immune recognition. In section one, I develop and apply an integrated approach comprised of structural modeling, inter-amino acid network analysis, biochemical assays, and bioinformatics tools to study the hemagglutinin-glycan interaction and, in some cases, HA's antigenic properties. Using this approach, we i) identify the structural determinants required, and potential mutational paths, for H5N1 to quantitatively switch it's binding specificity to human glycans receptors, ii) identify the mutations that enable the 2013 outbreak H7N9 HA to improve binding to human glycan receptors in the upper respiratory tract, iii) uncover H3N2 strains that are currently circulating in birds and swine that possess features of a virus that could potentially re-emerge and cause a pandemic, and iv) characterize the glycan binding specificity of a novel 2011 Seal H3N8 HA. The approaches implemented here and the findings of these studies provide a framework for improved surveillance of influenza viruses circulating in non-human hosts that pose a pandemic threat. Section 2: Glycans are abundant on the cell surface, and at the cell-ECM interface where they mediate interactions between cells and their microenvironment. Despite this, the function of glycans in cancer progression remains largely understudied. Here, I develop an integrated approach to characterize the cell surface glycome, including N-linked, 0-linked glycans, and HSGAGs. This approach integrates glycogene expression data, analytical tools, and glycan binding protein reagents. I demonstrate that this platform enables rapid and efficient characterization of the N- and 0-linked glycome in a model cell system, representing metastatic versus non-metastatic cancer cells. Next, I apply this integrated approach to uncover new roles of glycans. I study the role that HSGAGs play in regulating cancer stem cell (CSC) activity in breast cancer. Here, we report that SULF1, an HSGAG modifying enzyme, is required for efficient tumor initiation, growth and metastasis of CSCs. Furthermore, we identify a putative mechanism by which SULF1 regulates interactions between CSCs and their microenvironment. The approaches implemented here and the finding of these studies Overall, this thesis provides important tools, approaches and insights to enable and improve the study of glycans and glycan binding proteins. Together the work here provides a framework for decoding structure-function relationship of glycans.
by Nathan Wilson Stebbins.
Ph. D.

Glycans are major components of every cell surface. In addition to their importance in many physiological processes, glycans play a key role during infection of many pathogens. The identification and characterisation of glycan-pathogen interactions at a molecular and atomic level is therefore a crucial step towards the design of novel antimicrobial drugs and vaccines. Protein functions related to glycan interactions include glycan biosynthesis (glycosyltransferases), glycan recognition (lectins) and glycan degradation (glycosylhydrolases). This thesis investigates structure-function relationships of four glycan binding proteins that are important for the infectivity of three major human pathogens: the polysialyltransferase (polyST) of Neisseria meningitidis serogroup B (NmB), the viral protein 8* (VP8*) of rotavirus, and the hemagglutinin (HA) and the neuraminidase (NA) of influenza A virus (IAV).
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
Full Text

Bacteria are coated with repeating units of oligosaccharides that exhibit remarkable diversity. Often, glycan units of three or even two sugars are sufficient to identify a species of bacteria. Such specificity makes bacterial surface glycans attractive vaccine targets. However, efforts to create effective vaccines against carbohydrates have been hampered by poor vaccine design as well as the human immune tendency to respond to glycan antigens with non-specific, T-cell independent mechanisms. As a result, carbohydrate vaccines have historically produced only adequate memory responses in healthy individuals and poor responses in the elderly or immunocompromised. To circumvent these issues, a novel conjugate vaccine was developed that utilizes theQβ virus-like particle carrier that displays both a carbohydrate antigen as well as a Natural Killer T cell adjuvant. This unique vaccine has been reported to stimulate the production of high affinity (nanomolar) antibodies against carbohydrate antigens. To further conjugate vaccine research, the present work synthesizes two bacterial surface antigens: a trisaccharide from Streptococcus pneumoniae serotype 23F (Sp23F), and a pentasaccharide from Ruminococcus gnavus (Rg). Sp23F has been characterized as one of the more virulent and disease-causing strains of S. pneumoniae. Rg secretes highly immunostimulatory proteins and is associated with irritable bowel syndrome. The Sp23F antigen is synthesized with an alkyne at the reducing end of the sugar to facilitate coupling to Qβ. A selection reagent for Sp23F is also synthesized to enable the extraction of antibodies and B cells that bind the antigen. In conjunction with providing a conjugate vaccine antigen, the Rg pentasaccharide will be examined as a TLR4 ligand and was therefore synthesized without an alkyne. The Rg conjugate vaccine shows promise in treating irritable bowel syndrome as well as facilitating research into the role Rg plays in the human microbiome.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The epithelial cell-extracellular matrix interface primarily comprises of complex glycans and glycoconjugates. The widespread distribution of these glycans on the epithelial cell surface makes them ideal targets for interaction with microbial pathogens. In this thesis, a framework of integrated approaches was developed to characterize the structure-function relationships of host cell surface glycans and examine their role in mediating hostpathogen interactions. The first part of the thesis involves a study of the effect of secreted bacterial sphingomyelinases on the epithelial cell surface proteoglycan (a large glycan- protein conjugate), syndecan-1 and on epithelial tight junctions. The findings presented in this work suggest mechanisms by which sphingomyelinases could enhance bacterial virulence by regulating epithelial cell function. The second part of the thesis investigates the glycan binding requirements that govern the human adaptation and transmission of influenza A viruses by characterizing the molecular interactions between sialylated glycan-receptors and viral hemagglutinin (HA). The study puts forth the concept that the topology or shape (going beyond the chemical c2-3 versus a2-6 sialic acid linkage) adopted by the sialylated glycans is the critical determinant for efficient human adaptation of these viruses. In conclusion, this thesis provides insights into the molecular mechanisms of host-pathogen interactions and enables development of improved strategies for targeted antimicrobial therapies.
by Aarthi Chandrasekaran.
Ph.D.

Includes bibliographical references.
The structures of some non-cellulosic β D-Glycans from three plant sources have been investigated and each was found to be characterised by linked D-pyranosyl a main chain consisting of β -(1-44)- sugars. The polysaccharides were, however, different in structural features in a manner apparently related to their respective locations within the organs of the plants concerned. The polysaccharides were isolated and purified using standard fractionation methods including chromatographic techniques and selective precipitation methods. Structural information was obtained by employing techniques such as methylation analysis (involving use of gas liquid chromatography mass spectrometry), optical rotation measurements, mass spectrometry and n.m.r. spectroscopy on the original polysaccharides and on degraded products obtained by methods such as acid- or enzyme-catalysed hydrolysis and Smith degradation.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February, 2021
Cataloged from the official PDF of thesis.
Includes bibliographical references.
The human body is colonized by trillions of microbes, many of which reside in a layer of mucus that covers all wet epithelia in the body. In this way, mucus serves as the first line of defense to the host, simultaneously protecting against pathogens while providing a habitat where commensal microbes thrive. It has long been known that defects in mucus production or biochemistry are associated with opportunistic infections; however, few studies have focused on how components of the intact mucus barrier interact with resident microbes to promote health. In this thesis, I fill this gap using a clinically relevant 3-dimensional model of the mucus environment based on mucin glycoproteins, the major structural component of mucus. This in vitro culturing system mimics the natural mucus environment, where mucin polymer domains interact and entangle into a flexible hydrogel, as opposed to 2-dimensional surface coatings, which can create artificially concentrated amounts of surface mucins. I apply this system to answer three major conceptual questions, separated into three projects. In the first project, I study the ability of mucin and their attached glycans to regulate interactions between a clinically-important opportunistic pathogen, Pseudomonas aeruginosa, and its host. I then investigate the underlying genetic mechanisms that enable P. aeruginosa to sense and respond to the mucus environment, and explore how mucin glycan-sensing in turn impacts microbe-microbe interactions in the mucosal niche. I end by investigating how mucin glycan-mediated microbial regulation modulates the composition of complex microbial communities isolated from the human oral cavity. Collectively, the work presented in this thesis lays the framework for characterizing the therapeutic nature of mucin and how specific mucin glycan moieties modulate the behavior, pathogenicity, and competitive interactions of host-associated microbes.
by Kelsey M. Wheeler.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2019
Cataloged from PDF version of thesis. Page 184 blank.
Includes bibliographical references (pages 151-183).
Despite the importance of carbohydrates in cellular processes, there are few tools for their study in the context of a cell. The finding that non-natural monosaccharides could be internalized, processed, and displayed in cellular glycans in the early 1990s led to the development of metabolic incorporation probes for mammalian and microbial organisms. Taking advantage of the development of rapid, bioorthogonal chemistries these probes have provided valuable insight into intermolecular interactions, biosynthetic and metabolic pathways, and intercellular interactions. The successful application of metabolic incorporation probes to bacteria has been hampered by their unfastidious use of monosaccharides for energy production. In this work, we describe an alternative approach to metabolic incorporation, termed biosynthetic incorporation, using synthetic sugar donors that do not require intracellular processing prior to glycosyl transfer.
We evaluated our approach in cells of the suborder Corynebacterianeae, for which no useful probes had been described. Within Corynebacterianeae exist important human pathogens, including Mycobacterium tuberculosis. These bacteria utilize a host of lipid-linked sugar donors to construct polysaccharides implicated in immune avoidance and intrinsic antibiotic resistance. We produced a library of sugar donor analogs that were assessed for processing in cells. The most promising analog was used to evaluate incorporation in Corynebacterium glutamicum and Mycobacterium smegmatis, two widely used models of M. tuberculosis. We found that the sugar donor analog could work within the cell's traditional workflow, so analogs bearing azido-groups were synthesized. Incorporation of the azido-analogs labels nascent cell wall as determined by fluorescence microscopy. We have also begun synthesizing and evaluating probes targeting other polysaccharides within Corynebacterianeae.
These findings establish biosynthetic incorporation as a novel mode of polysaccharide structure modification. Furthermore, Biosynthetic incorporation probes offer advantages over metabolic incorporation, including the lack of requisite intracellular processing and the ability to target glycans that were previously recalcitrant to current methods.
by Phillip Joseph Calabretta.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemistry

In the biopharmaceutical industry, identification of glycans in a glycoprotein is a regulatory requirement and is a part of the characterization of the protein. Glycans are constructed of several monosaccharides linked together. N-linked glycans, which have been studied in this project, are attached to the nitrogen atom in asparagine. A method for separating N-linked glycans by high performance anion exchange chromatography had already been developed at the department. To develop a method for identification of the N-glycans by mass spectrometry, a desalting method on porous graphitic carbon (PGC) columns was used and optimized resulting in the eluents A (0,05% TFA in ACN:water 5:95 v/v) and B (0,05% TFA in ACN:water 50:50 v/v). Also the sample introduction on the mass spectrometer was optimized and resulted in a sensitive on-line liquid chromatography mass spectrometry (LC-MS) approach which gave mass spectrometric peaks with high signal to noise ratios and with high mass accuracy. The developed procedure was then successfully used on glycans cleaved from a glycoprotein separated by high performance anion exchange chromatography with pulsed amperometric detector.

Mestrado em Bioquímica Clínica
Uma das alterações fenótipicas mais comuns nos tumores são modificações no padrão de O-glicosilação na superfície da célula e nas glicoproteínas secretadas. Como consequência têm implicações nas suas funções biológicas. Em particular, tem sido descrito que algumas células tumorais sobreexpressam ou expressam de novo antigénios associados ao antigénio Thomsen-Friedenreich (TF), ou seja, sialil-Tn, sialil-T e disialyl-T. Estes epítopos resultam da paragem prematura do processo de O-glicosilação em proteínas e têm um impacto direto sobre a biologia do tumor. Assim sendo, a identificação destas modificações póstranslacionais anormais em proteínas é essencial para determinar as relações estrutura-função e descobrir novos alvos terapêuticos. Além disso, as proteínas que transportam estas alterações podem ser secretadas na corrente sanguínea, urina, e em outros fluidos corporais e, portanto, são explorados como biomarcadores em testes não invasivos. Atualmente a detecção de antigénios associados ao antigénio TF é baseado em métodos imunohistoquímicos em que, embora úteis para uma investigação de rotina, não conseguem descrever totalmente o padrão de glicosilação de uma dada proteína. Sendo assim, neste trabalho apresentamos uma abordagem analítica para determinar estes glicanos em quantidades minimas de glicoproteínas (picomole) isoladas a partir de géis SDS-PAGE. Resumidamente, as glicoproteínas são de-Oglicosiladas no gel por beta-eliminação redutiva, permetiladas e analisadas por nanoLC-MALDI-TOF/TOF. De seguida, os dados provenientes são sujeitos a uma seleção melhorada dos sinais analíticos relevantes, utilizando ferramentas de bioinformática. Esta abordagem foi, em seguida, aplicada com sucesso na validação do western blotting quanto à expressão de sialil-Tn numa glicoproteína isolada a partir da urina de ratos com tumores na bexiga induzidos quimicamente e no plasminogénio isolado a partir do soro de pacientes com lesões precursoras do cancro gástrico.
A common phenotypic change in tumors comprises alterations in the O-glycosylation of cell-surface and secreted glycoproteins with implications in their biological functions. In particular, it has been described that some tumor cells overexpress or de novo express Thomsen-Friedenreich (TF)-related antigens, namely sialyl-Tn, sialyl-T and disialyl-T. These epitopes result from a premature stop in protein Oglycosylation and have direct impact on tumor biology. As a result, the identification of these abnormal post-translational modifications of proteins is essential to determine structure-function relationships and designs novel therapeutics. Moreover, the proteins carrying these alterations can ultimately be shed into the blood stream, urine and other body fluids and thus be explored as biomarkers in non invasive tests. Currently the detection of TF-related antigens relies on immuno-based methods that, even though useful in a routine basis, often fail to fully highlight the glycosylation pattern of a given protein. Herein, we have systematized a target-driven analytical approach to determine these glycans in minute amounts of glycoproteins (picomole) isolated from SDS-PAGE gels. Briefly, the glycoproteins are to be de-O-glycosylated in-gel by reductive beta-elimination, permethylated and analyzed by nanoLC-MALDI-TOF/TOF with enhanced selection of the relevant analytical signals using bioinformatics tools. This approach was then successfully applied to validate western blotting assignments regarding the expression of sialyl-Tn in a glycoprotein isolated from the urine of rats with chemically-induced bladder tumors and in plasminogen isolated from the serum of patients with gastric cancer precursor lesions.

2004/2005
Se si considerano i carboidrati non solo per il loro più semplice ruolo di riserva energetica nel sostentamento de11a vita ma anche per que11o più intrigante di modulatori della comunicazione cellulare, entriamo nel campo della glicobiologia. Quest'aspetto non-triviale della funzione dei glicani deriva dalla loro inerente complessità strutturale (grande varietà di legami e possibilità di ramificazione) e dalla loro localizzazione ubiquitaria nella cellula e nella matrice extracellulare. La maggior parte dei glicani ricopre densamente la parte esterna della superficie cellulare ed è esposta ad un ambiente ricco di proteine (come i fattori di crescita, citochine, tossine, enzimi ed altro). Questa posizione preferenziale permette loro di mediare numerose interazioni tra cellule o tra cellula e matrice extracellulare e di fungere da "agenti del traffico" in molti processi cellulari (riconoscimento, adesione, proliferazione e differenziamento). Perciò, la comprensione della funzione biologica peculiare svolta dai carboidrati, non può prescindere dalla caratterizzazione strutturale dei saccaridi, considerati o come catene a se stanti o come parte integrante di glicoconiugati, e dei loro partners d'interazione proteici. Scopo. Lo scopo del presente lavoro è essenzialmente di tipo metodologico, ossia rivolto allo sviluppo di tecniche e procedure sperimentali per l'analisi (identificazione/rilevamento, caratterizzazione, sintesi) di determinate sequenze saccaridiche frequentemente riscontrate in natura, di glicoproteine e di proteine che legano carboidrati. l. Derivatizzazione di saccaridi per il rilevamento UV-visibile all'elettroforesi capillare (CE) Per quanto riguarda la determinazione della composizione in monosaccaridi, che spesso rappresenta il primo passo per la completa elucidazione strutturale di un campione zuccherino, abbiamo sviluppato ed ottimizzato delle metodologie sperimentali volte ad un migliore rilevamento UV-visibile di mono- ed oligosaccaridi presenti in glicoproteine di mammiferi e pianta. I carboidrati sono privi di gruppi cromofori e debbono essere opportunamente derivatizzati, cioè legati covalentemente ad un cromoforo, solitamente per amminazione riduttiva, prima di essere analizzati alla CE. Le rese di derivatizzazione, tuttavia, dipendono dalla natura del residuo saccaridico e gli zuccheri N-acetilati, come l'N-acetilgalattosammina (GalNAc) e l'Nacetilglucosammina (GlcNAc) che occupano, rispettivamente, il terminale riducente di 0- ed N-glicani, sono notoriamente difficili da derivatizzare. Si è quindi condotto uno studio comparativo finalizzato al miglioramento della procedura di derivatizzazione, aumentando il grado di rilevabilità degli zuccheri N-acetilati. I risultati hanno mostrato come, tra vari agenti derivatizzanti comunemente usati, l'acido 2-aminobenzoico sia il più efficiente, otirendo alta e pari sensibilità al detector UV-vis per tutti i saccaridi analizzati. Inoltre, scegliendo adeguate condizioni di corsa alla CE, una miscela di undici monosaccaridi è stata efficientemente separata in un intervallo di tempo assai ristretto. 2. Sintesi di N-acetillattosamina (LacNAc) con beta-galattosidasi immobilizata I risultati sopra menzionati, relativi ad una efficace procedura di derivatizzazione di zuccheri riducenti, sono stati applicati con successo al rilevamento di LacNAc da miscele di reazione grezze via CE. Questo disaccaride è solitamente situato nella porzione più esterna di oligosaccaridi complessi facenti parte della superficie cellulare ed è quindi direttamente coinvolto in processi di riconoscimento cellulare. Qui è stata provata la sintesi enzimatica in fase eterogenea di LacNAc. Lo scopo dello studio era quello di migliorare sia la resa del prodotto ma anche il recupero del biocatalizzatore attraverso la sua immobilizzazione su supporto solido (polimeri commerciali Eupergit® e Sepabeads) in confronto alla stessa sintesi effettuata con enzima libero in soluzione. La biosintesi è stata fatta per reazione di transglicosilazione con betagalattosidasi da B. circulans partendo da p-nitrofenil galattopiranoside (come glicosil donatore) e GlcNAc (come glicosil accettore). La reazione è stata seguita monitorando via CE la produzione di disaccaride in funzione del tempo. L'analisi cinetica ha rivelato che la procedura di immobilizzazione non sopprime l'attività catalitica ma, al contrario, incrementa l'efficienza di trasferimento di galattosio dell'enzima. I profili cinetici delle reazioni effettuate con Eupergit® o Sepabeads sono risultate essere molto diversi, suggerendo che le proprietà chimico-fisiche delle matrici di supporto influenzano il comportamento dell'enzima. La resa molare massima di LacNAc (64%) è stata ottenuta usando Eupergit® come carrier polimerico. 3. Analisi strutturale di glicani L'identificazione e l'analisi quantitative di porzioni oligosaccaridiche di glicoproteine di importanza biotecnologica, come la beta-glucosidasi, o terapeutica, come le proteine di membrana di linee cellulari epatiche, è stata qui affrontata seguendo due strategie di processamento glicoproteico alternative. (3a) Prima del rilascio sul mercato, beta-glucosidasi (GCasi) ricombinante prodotta da semi di tabacco transgenico, deve essere ampiamente caratterizzata al fine di verificare se lo schema di glicosilazione della glicoproteina espressa in pianta somiglia a quello della CGasi da placenta umana. Il trattamento con acido trifluoroacetico ha portato ad idrolisi esaustiva del campione saccaridico e sui monosaccaridi rilasciati si è svolta un'analisi CE. Dal momento che non sono stati ritrovati né fucosio né xilosio, unità monosaccaridiche frequenti in glicoproteine vegetali, si è potuta escludere una potenziale immunogenicità per la somministrazione all'uomo. (3b) La rilevanza scientifica dello studio della struttura glicanica di proteine di membrana, differentemente espresse in linee cellulari epatiche sane o cancerose, sta nella possibile diagnosi di markers tumorali saccaridici, presenti già nella fase precoce dell'insorgere del tumore. Al fine di sviluppare e ottimizzare delle procedure metodologliche, basate sulla deglicosilazione enzimatica in soluzione o in gel (con PNGasi F) con rilascio delle catene oligosaccahridiche intatte e analisi LC/MS dell'idrolizzato, da esportare poi ali' analisi del pattern di glicosilazione delle proteine di membrana, è stata qui utilizzata fetuina bovina commerciale come modello di una proteina pesantemente glicosilata. Dai dati ottenuti, una struttura biantennaria e due triantennarie sono state infine assegnate. 5. Biosintesi su ampia scala di un lisozima da H.pylori Un grande sforzo è stato dedicato allo studio di un Iisozima da H.pylori (Lys ), un enzima che sembra svolgere un ruolo chiave nell'autolisi del batterio durante la colonizzazione dell'epitelio gastrico dei primati. Al fine di tentare una caratterizzazione strutturale della proteina, sola ò in associazione con il suo substrato saccaridico (acido N-acetylmuramico), si è reso necessario lo "'scaling-up" dei processi di biosintesi e purificazione. Ciò è stato possibile dopo l'identificazione del gene codificante la sequenza del lisozima in H.pylori e clonaggio del frammento di DNA di interesse inun vettore d'espressione adeguato, promuovendo l'espressione ad alti livelli in E. coli. Due sistemi d'espressione sono stati provati: Il sistema pGEX ha prodotto il Lys come proteina di fusione con la GST, preferenzialmente prodotta in Eco/i come corpi di inclusione, specialmente quando sono stati usati grandi volumi di colture batteriche. La proteina è stata isolata dal precipitato cellulare insolubile, solubilizzata e successivamente ripiegata con successo. Dopo purificazione e taglio proteolitico con trombina per la rimDziDne della coda GST da Lys, le prove di attività litica sono risultate essere positive, nonostante la fallita separazione tra le due proteine. Studi di affinità su tali campioni condotti alla CE, non hanno portato nessuna evidenza a favore dell'affinità di Lys per un disaccaride (LacNAc) che mima il substrato naturale di Lys. Quindi il vettore pET è stato successivamente scelto per esplorare una stategia di clonaggio alternativa. Il sistema d'espressione ha fornito Lys con coda poliistidinica C-terminale. Anche in questo caso la proteina è stata recuperata quasi interamente nella frazione insolubile del lisato cellulare. Dopo processo di rinaturazione, direttamente effettuato in colonna d'affinità Ni-NTA agarosio, si è ottenuta proteina ad un elevato grado di purezza ma inattiva. L'intero lavoro è stato svolto all'Università di Trieste, nel dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole nel laboratorio del Prof. S.Paoletti sotto la supervisione della dott.ssa A.Gamini, dove ho avuto la possibilità di imparare i rudimenti della biochimica analitica. All'intemo del progetto lisozima da H.pylori, è stata di grande importanza la permanenza durata 4 mesi all'Università di Halle-Wittenberg all'Istituto di Biotecnologia (Germania) nel laboratorio del Prof R.Rudolph e sotto la supervisione del Dott. C.Lange che mi ha portato ad acquisire esperienza nel campo della chimica delle proteine.
lf carbohydrates are viewed not merely as energy suppliers for sustaining life but their intriguing biological role, chiefly as modulators of cell communication, is investigated, then we approach the field of glycobiology. This nontrivial task of glycans arises from their inherent structural complexity (great variety of linkage and branching occurrence) an d their ubiquitous location in the cell and extracellular matrix. Most glycans densely cover the outer cellular surface and are exposed to an environment of many proteins (such as growth factors, cytokines, toxins, enzymes and others). This particular position enables them to mediate several cell-cell or cell-matrix interactions and act as recognition determinants in a great variety of important cellular events ( adhesion, proliferation and differentiation). Therefore, the comprehension of the unique function of carbohydrates in biology calls for a structural characterization of saccharides, considered either as sugar chains or as integral part of glycoconjugates, and their interaction partners, such as carbohydratebinding proteins. The aim of the present work was essentially methodological in kind, i.e. directed towards the development of techniques and experimental procedures for the analysis (identification/detection, characterization, synthesis) of given carbohydrate sequences, frequently occurring in nature, glycoproteins and sugar-binding proteins. l. Derivatization of saccharides for UV -visible detection on capillary electrophoresis CE). Concerning the compositional monosaccharide determination of a hydrolyzed glycan pool, which is frequently the first step in oligosaccharide mapping, we developed and optimized experimental methods aimed to improve the UV -vis detection of monoand oligosaccharides present as widespread components in mammalian and plant glycoproteins. Saccharides inherently lack chromophores and must be suitably derivatized, i.e. covalently linked to a chromophore usualiy through reductive amination, prior to detection on CE. Derivatization yield, however, depends on the nature of the sugar residue and N-acetylamino sugars, such as N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), occupying the reducing end of respectively 0- and N-glycan sequences, are notoriously difficult to label. A comparative study aimed at the improvement of a labelling procedure, enhancing the detectability of N -acetylamino sugars, was therefore performed. The results showed that, among frequently used tagging dyes, 2-aminobenzoic acid turned out to be the most efficient, offering high and comparable sensitivity at the CE UV -vis detector for ali saccharides tested. Choosing adequate CE running conditions, a mixture of eleven monosaccharides was efficiently separated in a very short time frame. 2. Immobilized beta-galactosidase-based synthesis ofN-acetyllactosamine (LacNAc) The abovementioned findings of suitable derivatization procedure for the UV -vis detection of reducing sugars was successfully applied to the detection of LacNAc from crude reaction mixtures via CE. This disaccharide is very commonly located at the outermost portion of complex celi surface oligosaccharides of glycoconjugates and is therefore directly involved in recognition processes between cells. The enzymatic synthesis in heterogeneous phase of the LacNAc disaccharide was here attempted. The goal of the study was to improve both product yield and recovery of the biocatalyst through its immobilization on solid supports (commercial polymers Eupergit® and Sepabeads ), in comparison with the synthesis performed with the free enzyme in solution. The biosynthesis was performed through transglycosylation reaction with beta-galactosidase from B. circulans starting from p-nitrophenyl galactopyranoside as the glycosyl donor and GlcNAc as the acceptor. The reaction was followed monitoring via CE the disaccharide production in function of time. The kinetic analysis revealed that the immobilization procedure did not suppress catalytic activity, but, on the contrary, improved the galactose transfer efficiency of the enzyme. Kinetic profiles of the reactions performed with Eupergit® or Sepabeads were qui te different, suggesting that the physico-chemical properties of the supporting matrices infuence enzyme behaviour. Maximum LacNAc molar yield (64%) was obtained using Eupergit® as solid carrier. 3. Glycan structure analysis The identification and quantitative analysis of oligosaccharide portions of glycoproteins of biotechnological, like beta-glucosidase, or therapeutical interest, such as membrane proteins of hepatic celllines, was here carried out according to two alternative glycoprotein processing strategies. (3a) Prior to commercialization, recombinant beta-glucosidase (GCase) produced in transgenic tobacco seeds must be first thoroughly characterized in order to test whether the glycosylation pattem of the glycoprotein expressed in plant resembles that of the human placenta} GCase. Treatment with trifluoroacetic acid led to exhaustive hydrolysis of the glycan moiety and o n the released monosaccharides a CE analysis was carried out. Since neither fucose nor xilose, frequent sugar residues found in plant glycoproteins, could be detected, potential immunogenicity for delivery to humans was excluded. (3b) The scientific relevance of studying the glycan structure of membrane-bound proteins, differently expressed in a healthy and hepatoma cell line, relies on the possible detection of early-stage saccharidic tumoral markers. In order to develop and optimize a methodological procedure, based on the in-solution or in-gel enzymatic release (with PNGase F) of entire oligosaccharide chains and analysis through LC/MS of the hydrolizate, that could be exported to the glycosylation pattem analysis of membrane proteins, the commerciai bovine fetuin was here used as a model of a heavily glycosylated protein. From the obtained data, a biantennary and two triantennary oligosaccharide structures, pertaining to the N-glycosylation profile of Fet, could be finally assigned. 5. Large-scale biosynthesis of a Hpylori lysozyme Much effort has been devoted to the study of a Hpylori lysozyme (Lys), an enzyme that seems to play a key role in the autolysis of the bacterium during colonization of the gastric epithelium of primates. In order to attempt structural characterization of the protein, either alone or in complexed form with its saccharidic substrate (Nacetylmuramic acid), the scaling-up of the biosynthesis and the purification procedures were required. This could be performed after identification of the gene encoding the lysozyme sequence in Hpylori and cloning the DNA sequence of interest into a suitable expression vector and promoting high-level expression of the Lys in E. coli. Two expression systems were tested: the pGEX system yielded the Lys as fusion protein with GST, which was produced preferentially as inclusion body in E. coli host cells, especially when larger culture volumes were used. The protein was isolated from insoluble cell pellet, solubilized and successfully refolded. After purification and digestion with thrombin protease to remove the GST fusion tag from the Lys protein, tests for lytic activity were positive, even though separation could not be achieved. Binding studies performed through affinophoresis on these samples, unfortunately did not provide any evidence of the affinity of Lys fora disaccharide (LacNAc) mimicking the natural sugar substrate. The pET vector was therefore chosen for an alternative cloning strategy. The expression system gave the Lys with a C-terminai 6xHis-tag. Also in this case the protein was almost totally recovered in the insoluble fraction of the celllysate. After a refolding step, directly performed on Ni-NTA agarose affinity column, highly pure, but unactive protein was obtained. The whole work was carried out at the University of Trieste, department of Biochemistry, Biophysics and Macromolecular Chemistry in the laboratory of Prof. S.Paoletti under the supervision of Dr. A.Gamini, where I had the opportunity to learn the basic issues of analytical biochemistry. Within the Hpylori lysozyme project, a four-months stay at the Martin-Luther University of Halle-Wittenberg at the Institute of Biotechnology (Germany) in the laboratory of Prof. R.Rudolph and under the supervision of Dr. C.Lange was of great importance for getting expertise in the field of protein chemistry.
XVIII Ciclo
1974
Versione digitalizzata della tesi di dottorato cartacea.

Glycosylation of proteins is ubiquitous and has the ability to significantly alter the biological and biophysical properties of proteins. The need to study structure-function relationships of glycans in a living organism requires the continuous development of rapid and sensitive technologies for the characterization of glycan components. In recent years, a broad range of technologies have evolved to provide new developments and emerging glycomics techniques. In this thesis we present the development of new methodologies based on capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the study of protein N-glycosylation in complex biological systems and therapeutic recombinant drugs. Our approach involves the use of a flow-through microvial, a novel technology that provides a robust and easy-to use strategy for interfacing CE separation with MS detection. In chapter 2, we report a simple and robust CE-ESI-MS methodology for comprehensive characterization of glycosylated proteins at the level of intact protein, enzymatically released glycopeptide and glycans. In chapter 3, we characterize a complex set of enzymatically released N-glycans from a recombinant therapeutic drug that revealed extensive glycan heterogeneity. The study demonstrated the potential of our approach to complement established techniques for glycan characterization of therapeutic glycoproteins in the pharmaceutical industry. Chapters 4 and 5 of this thesis are devoted study protein glycosylation of relevant biological systems. In chapter 4, O-acetylated N-glycans from fish serum were characterized in their native state and the structural variations of their isomeric species were investigated by tandem MS approaches. The developed CE-MS methods may be useful not only for the characterization of acetylation of complex glycans but also to study other types of glycan modifications in different contexts. In chapter 5, we present CE-MS methodologies for characterizing protein N-glycosylation in human serum associated with prostate cancer and asthma. Comparison of glycan compositions and relative abundances revealed abnormal glycosylation in prostate cancer and asthma serum. The capability of our CE-ESI-MS method to perform global glycan profiling of human serum demonstrates its potential for comprehensive glycan profiling in the context of malignancies and for the discovery of glycan disease markers with high selectivity and specificity.
Science, Faculty of
Chemistry, Department of
Graduate

Gonadotrophic hormones are heterodimeric glycoproteins and their N-glycans attached to specific amino acid residues are currently thought to play important roles in hormonal biosynthesis, secretion and function. The studies reported in this thesis aimed at isolation and characterisation of structural properties of the N-glycans on ovine and human luteinizing hormones. Initially, chromatographic methods were developed using reverse-phase HPLC for the analytical separation of the three human pituitary glycoprotein hormones and their subunits. Separation of intact oLH and its subunits was also effected by a single HPLC step. A preparative procedure was developed for the efficient purification of hLH and hTSH from crude human pituitary extracts using hydrophobic chromatography which gave highly purified hormones in good yields and with high biological activities. This method did not significantly influence the hormones' extensive charge heterogeneity and it offered potential advantages in the characterisation of their carbohydrate structures. A preparative scheme was developed for the isolation of the N-linked oligosaccharides from each glycosylation site of o- and hLH. Charge heterogeneity of oligosaccharides, which were released by hydrazinolysis from subunits and glycopeptides, was characterised by anion-exchange HPLC. 1H-NMR analysis showed that the structures of all three N-glycans on hLH were highly heterogeneous but mainly diantennary complex-type, with site-specific patterns of terminal sialylation and sulphation as well as core-fucosylation. Sulphated/sialylated and/or disialylated oligosaccharides were the major components at each site. A set of new mono- and disialylated oligosaccharides with the terminal sequence NeuAcα2-6GalNAcβ1-4G1-cNAcβ1-2Manα1-3 was identified. This finding suggested unique site-specific terminal sialylation of oligosaccharides at Asn 78 (hLHα) by an unknown α2-6 sialyltransferase(s) in the human pituitary gonadotroph cell. Each glycosylation site in oLH had a distinct set of oligosaccharides ranging from mainly monosulphated hybrid-type at the two sites of oLHα to predominantly disulphated diantennary complex-type on oLHβ. Core-fucosylation also differed at each site. These results suggested that processing of the oligosaccharides of the α- and β-subunits by α-mannosidase II and α1-6 fucosyltransferase was differently regulated by protein structure in oLH. Whereas hCG, hLH and oLH share similar biological activities, no apparent relationship between their N-glycan structures was found, which suggested that specific branching and peripheral structures of N-glycans on LH and hCG may not be essential for biological function, although the N-glycan nearer the N-terminus of the α-subunit of hCG has been implicated in hormonal activity.

Metastasis, the process by which cancer spreads in the body, is a complex, multi-step cascade which is poorly understood and is the cause of death of most cancer patients. Aberrant glycosylation is an established characteristic of cancer cells and appears to have a role in metastatic mechanisms. The lectin from Helix pomatia, the Ron (Helix pomatia agglutinin, HPA), recognises aberrant glycans terminating in α-N-acetylgalactosamine (GalNAc). The presence of GalNAc-glycans on cancer cell surfaces, detected by HPA binding, is associated with metastasis and consequent poor survival.

Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed Aug. 19, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Glycans are carbohydrate structures coating the cell surface of virtually all nucleated cells of vertebrates. A crucial function of glycans is in cell interaction and recognition, which is constantly exploited by bacteria and viral pathogens as targets for toxins, and as a receptor to aid recognition of and adhesion to host cells. Sialic acids, a 9-carbon compound commonly added as a terminal sugar on glycoconjugates is crucial to both bacteria and viruses causing a successful infection. Bacterial pathogens utilize sialic acids as a component of LOS and capsule, both key virulence factors. The decoration of the bacterial cell surface with sialic acid glycoconjugates has two roles; first, to mimic host glycans to evade the host immune system, second, in reducing the efficiency of complement mediated lysis and opsonophagocytosis. Several key bacterial pathogens including Haemophilus influenzae and Pneumococcus also utilize sialic acids as a carbon source. In viruses, host sialic acids glycoconjugates act as a receptor for viral recognition and adherence to host cells. In the case of influenza A virus, a viral neuraminidase is required to cleave and release newly formed viral particles from infected host cell. This thesis investigates the role of sialic acid glycoconjugates in pathogenesis to increase the understanding of sialic acid biology in human host adapted pathogens. The role sialic acids play in pathogenicity is investigated in the major human pathogens; non-typeable Haemophilus influenzae (NTHi) and Neisseria meningitidis and in human influenza A viral strains.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
Full Text

Glycosyltransferases ST6GAL1 and B4GALNT2 (and their cognate antigens Sia6LacNAc and Sda, respectively) are associated with colorectal cancer (CRC) but it is not fully clear their biological and clinical significance. We explored the clinical relevance of both glycosyltransferases by interrogating The Cancer Genome Atlas (TCGA) database while the phenotypic/transcriptomic effects of ST6GAL1/B4GALNT2 overexpression were studied in genetically modified CRC cell lines. Transcriptomic data from CRC patients in TCGA database suggested a moderate impact of ST6GAL1 on CRC progression, although it was not possible to define a clear role for this glycosyltransferase. Transcriptomic analysis of ST6GAL1-transduced cell lines revealed a much deeper effect of ST6GAL1 on gene expression in SW948 than in SW48. The overexpression of ST6GAL1 induced opposite effects on soft agar growth and wound healing in both cell lines. These results indicate that the impact of a cancer-associated glycosyltransferase change on phenotype/transcriptome can be extremely variable, depending on the molecular context of the tumor cell. On the contrary, transcriptomic analysis of B4GALNT2-modified cell lines together with TCGA database survey demonstrated a strong impact of B4GALNT2 on the transcriptional activity of CRC cells, in particular its association with a better prognosis. We suggest an anti-tumoral role of B4GALNT2 in CRC. We also investigated the glycan changes related to ST6GAL1/B4GALNT2 expression in a small cohort of tissues/plasma as well as the N-glycomic profile of CRC, normal and polyp tissues. We found an increase of ST6GAL1 activity in CRC and inflammatory bowel disease plasma samples comparing with plasma from healthy donors. A different Sda protein carrier pattern was observed between healthy donors and CRC plasma samples. β-arrestin 1 is a possible candidate as Sda carrier protein in plasma samples although future validation studies are needed. The alterations found in the N-glycan pattern highlight the importance of N-glycome as a molecular signature in cancer.

Doutoramento em Bioquímica
Helicobacter pylori is a bacterial pathogen that affects more than half of the world’s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions. The major goals of this thesis were the structural analysis of LPSs from H. pylori strains isolated from gastric biopsies of symptomatic Portuguese patients and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins. LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex and Ley antigens and N-acetyllactosamine (LacNAc) sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a 6-glucan. Other structural domains, namely ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains. For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulosecoated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides. The H. pylori LPS microarray was probed for recognition with carbohydratebinding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydratebinding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray. Microarray analysis revealed the occurrence of type-2 Lex and Ley, but not type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The -fucose-specific lectin UEA-I, showed restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of H-type-2, as well Htype- 1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant. Microarray analysis also revealed that plant lectins known to bind 4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to 4GlcNAc tri- and pentasaccharides, differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of 4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS. H. pylori LPS microarray was also applied to analysis of two human sera. The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+ serum, than the control serum. A specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection. The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H. pylori LPS microarray constructed enabled the study of interactions with host proteins and showed promise as a tool in serological studies of H. pyloriinfected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.
O Helicobacter pylori é uma bactéria patogénica que afeta mais de metade da população mundial com doenças gastrointestinais e está associado ao cancro gástrico. A superfície celular do H. pylori é decorada com lipopolissacarídeos (LPSs), que são constituídos por três regiões distintas: uma região polissacarídica variável (“O-chain”), um “core” oligossacarídico estruturalmente conservado e o lípido A, que ancora o LPS à membrana celular. A “O-chain” apresenta estruturas oligossacarídicas únicas, tais como os antigénios de Lewis (Le), que são semelhantes às presentes na mucosa gástrica e que estão envolvidas na interação com o hospedeiro. Glucanas, heptoglicanas e ribanas também constituem o “core” dos LPSs do H. pylori. Glicanas do tipo amilose e mananas também são referidas como integrantes de estirpes de H. pylori, possivelmente co-expressas com os LPSs. A complexidade dos LPSs do H. pylori tem dificultado o estabelecimento de uma relação entre a estrutura e a função em interação com o hospedeiro, que é crucial para o desenvolvimento de vacinas. Os microarrays de carboidratos são ferramentas recentes no estudo de antigénios e de novos ligandos oligossacarídicos, e que têm revelado a sua função em interações patogéniohospedeiro. O trabalho desta tese teve como objetivos principais a análise estrutural de LPSs de estirpes do H. pylori isoladas de biópsias gástricas de Portugueses sintomáticos e a construção de um microarray de LPSs do H. pylori (H. pylori LPS microarray) para estudos funcionais de interação com proteínas. Os LPSs foram extraídos da superfície celular de cinco isolados clínicos do H. pylori e de 1 estirpe de referência (NCTC 26695) pelo método fenol/água, fracionados por cromatografia de exclusão molecular e analisados por cromatografia em fase gasosa acoplada à espetrometria de massa. Os oligossacarídeos resultantes da hidrólise ácida parcial do LPS foram analisados por espetrometria de massa de electrospray. Para além de estruturas existentes no “core”, a análise estrutural, revelou a presença de antigénios de Le do tipo-2, Lex e Ley, e de sequências de resíduos de N-acetillactosamina (LacNAc), tipicamente encontrados em H. pylori. A identificação de resíduos de glucose ligados na posição O-6 nos LPSs das estirpes 2191 e NCTC 26695, indicou a presença de 6-glucanas. Outros domínios, nomeadamente ribanas, compostos por resíduos de ribofuranose ligados em posição O-2, foram identificados nos LPSs da maioria dos isolados clínicos. A presença de uma galactana (O-3 galactose), recentemente identificada noutra estirpe do H. pylori, foi também identificada para a estirpe 14382. Nos LPSs das estirpes 2191 e CI-117, a elevada quantidade de resíduos de Nacetilglucosamina (GlcNAc) ligados na posição O-4, sugeriu a presença de glicosídeos semelhantes à quitina (4GlcNAc) que, ao nosso conhecimento, ainda não foram descritos para o H. pylori. Para a construção do novo microarray, os LPSs analisados e os neoglicolípidos (NGLs) derivados de frações oligossacarídicas dos LPSs foram imobilizados não-covalentemente em suportes de nitrocelulose, juntamente com NGLs com sequências oligossacarídicas definidas, LPSs de outras bactérias e outros polissacarídeos. O microarray foi avaliado com proteínas de especificidade conhecida, que incluíram anticorpos monoclonais (mAbs) específicos para os antigénios de Le e estruturas relacionadas, um módulo de ligação a carboidratos (CBM), lectinas de plantas e os recetores do sistema imunitário DC-SIGN e Dectina-1. A análise com estas proteínas forneceu informação complementar à análise estrutural, para além de ter sido útil para o controlo de qualidade do microarray. A análise do microarray revelou a ocorrência de antigénios de Le do tipo-2, Lex e Ley, mas não do tipo-1, Lea e Leb, o que está de acordo com os resultados obtidos na análise estrutural. Os LPSs do H. pylori foram reconhecidos pela DC-SIGN, que é uma lectina conhecida por interagir com esta bactéria através dos antigénios de Le expressos nos seus LPSs. A lectina UEA-I, específica para -fucose, mostrou ser restrita para determinantes do grupo sanguíneo H do tipo-2 e para os LPSs das estirpes CI-117 e 14382. A ocorrência de antigénios do tipo H-2 e do tipo H-1 nos LPSs destas estirpes foi corroborada utilizando mAbs específicos. Os antigénios do tipo H-1 já tinham sido identificados em H. pylori, mas os do tipo H-2 foram identificados pela primeira vez neste estudo. Os LPSs do H. pylori foram também reconhecidos por lectinas com especificidade para oligossacarídeos de quitina (4-linked GlcNAc). A STL, que mostrou uma ligação restrita aos tri- e pentassacarídeos, reconheceu distintivamente o LPS da estirpe CI-117, que poderão ser internas, dada a ausência de ligação detectada pela lectina WGA, com especificidade para terminais não redutores 4GlcNAc. A análise dos LPSs do H. pylori por SDS-PAGE e Western blot com a STL apontou para a presença destas estruturas na “O-chain” deste LPS. O microarray dos LPSs do H. pylori foi utilizado para a análise do soro de um indivíduo infetado com H. pylori (H. pylori+ CI-5), e mostrou um aumento na detecção de IgGs para os LPSs de H. pylori no soro H. pylori + quando comparado com o soro de um indivíduo não-infetado (H. pylori-). A resposta observada foi específica para o LPS do isolado da estirpe CI-5, causadora da infeção. O trabalho desenvolvido nesta tese contribuiu para a extensão do conhecimento estrutural dos LPSs dos isolados clínicos do H. pylori. A construção do microarray dos LPSs de H. pylori permitiu o estudo de interações com proteínas do hospedeiro, e mostrou ser útil na análise serológica de indivíduos infetados com esta bactéria. Deste modo, espera-se que o uso destas técnicas complementares possa contribuir para uma melhor compreensão da complexidade molecular dos LPSs e do seu papel na patogenicidade.

Dendritic cells (DCs) are regulators of the immune system and express a class of pattern recognition receptors known as C-type lectin receptors (CLRs) to recognize and respond to carbohydrates (glycans). Dendritic cells are hypothesized to be key mediators in the immune response to implanted materials and ligation of CLRs has been shown to have diverse effects on DC phenotype ranging from tolerogenic to pro-inflammatory. Thus, designing future biomaterials and combination products that harness the potential of CLR ligation on DCs has great promise. Additionally, many of the proteins which adsorb to biomaterials when implanted are glycosylated and thus understanding this interaction would provide further insight into the host response to currently implanted materials. However, DC responses to glycans presented from non-phagocytosable surfaces has not been well characterized and optimal factors for DC phenotype modulation by surface presented glycans are unknown. Additionally, studies relating DC response to glycan structures from soluble and phagocytosable displays to that of non-phagocytosable display have not been performed. This is of critical importance to the field because of the extremely limited supply of complex glycan structures that are able to be obtained. Because of this limited supply of glycans the trend in glycomics has been toward creation of glycan microarrays to assess initial candidates of interest for further study. However, the assumption that cell response to these glycoconjugate microarrays is equivalent to soluble or phagocytosable conjugates has not been validated. Therefore, the purpose of this study was to 1) determine the optimal molecular contextual variables of glycoconjugate presentation from a non-phagocytosable surface, namely, charge, density, and glycan structure for modulating DC phenotype; and 2) determine if modality of glycoconjugate presentation, i.e. soluble, phagocytosable, or non-phagocytosable will modulate DC phenotype differentially. To determine the effect of the molecular contextual variables primary human immature DCs (iDCs) were exposed to a range of adsorbed glycoconjugates in a 384 well plate and their subsequent phenotype assessed via a novel in house produced high throughput (HTP) assay. Bovine serum albumin (BSA) was modified to have a range of glycan densities and isoelectric points to determine which of these were optimal for DC phenotype modulation. Next, several poly-mannose structures were presented to DCs to determine if DC response was structure specific. Finally, contextual variables were modeled in a multivariate general linear model to determine underlying trends in DC behavior and optimal factors for glycan presentation from non-phagocytosable surfaces. To determine the effect of the modality of glycoconjugate display on DCs, optimized glycoconjugates from 1) were adsorbed to the wells of a 384 flat well plate, delivered at varying soluble concentrations, or adsorbed to phagocytosable 1 µm beads and subsequent DC phenotype assessed via the HTP assay. The cell response to the glycoconjugates was then validated to be CLR mediated and the DC response to glycan modality was modeled in another general linear model. Results from these studies show that highly cationized high density glycoconjugates presented from non-phagocytosable flat well display modulate DC phenotype toward a pro-inflammatory phenotype to the greatest extent. Additionally, significant impacts on DC phenotype in response to adsorbed conjugates can be seen when grouping glycan structure by terminal glycan motif. Finally, DC response to glycoconjugates were found to be CLR mediated and that each modality of glycan display is significantly different, in terms of DC phenotype, from the others. These results provide indications for the future design of glycan microarray systems, biomaterials and combination products. Furthermore, this work indicates that different mechanisms are involved in binding and processing of surface bound versus soluble glycoconjugates. With further study these differences could be harnessed for use in the next generation of biomaterials.

The sujet de cette these portait sur la caractérisation de la machinerie originale utilisée par les Mimiviridae pour glycosyler les fibrilles entourant leurs capsides en travaillant sur les prototypes des 3 lignées connues, Mimivirus (A), Megavirus chilensis (B) et Moumouvirus australensis (C). Les fibrilles de Mimivirus sont décorées par 2 polysaccharides différents :l’un est caractérisé par la répétition d’un disaccaride linéaire fait de 3)-α-L-Rha-(1→3)-β-D-GlcNAc-(1→, avec un pyruvate branché en position 4,6 du GlcNAc ; l’autre présente une unité répétée branchée de séquence 2)-α-L-Rha-(1→3)-β-D-GlcNAc-(1→ pour le squelette linéaire et du rhamnose branché en position 3 par de 2OMeVioNAc. Nous avons suggéré que les fibrilles de Megavirus sont décorées par plus d'une espèce de polysaccharides/oligosaccharides, donc l’un ayant présentant un trisaccharide de RhaNAc:α-L-4OMe-RhaNAc-(1→3)-α-L-RhaNAc-(1→3)-α-L-RhaNAc-(1→. Les fibrilles de Moumouvirus sont décorées de glucosamine, quinovosamine et bacillosamine. A partir de ces données expérimentales il devenait possible de rechercher de nouveaux gènes responsables de ces glycosylations spécifiques. Le cluster de 9 gènes déjà publié de Mimivirus a pu être étendu à 13 gènes. Un cluster de 14 gènes a été d’autre part identifié dans le génome de Moumouvirus, le premier cluster de gènes de la glycosylation identifié dans la lignée B. Parmi les gènes de glycosylation, l’analyse fonctionnelle in vitro de la protéine L142 a permis de démontrer qu’il s’agit d’une N-acétyltransferase. En conclusion, les fibrilles des Mimiviridae sont lourdement glycosylées and le type de sucres et leur organisation dépend de la lignée considérée
The aim of this thesis is the study of the innovative glycosylation machinery used by the Mimiviridae family for the glycosylation of the fibrils sourrounding their capsid, using Mimivirus, Moumouvirus australensis and Megavirus chilensis as prototypes of lineages A, B and C, respectively. Mimivirus fibrils are decorated with two distinct polysaccharide: one is characterized by a linear disaccharide repeating unit made of 3)-α-L-Rha-(1→3)-α-D-GlcNAc-(1→, with a pyruvic acid branched at position 4,6 of GlcNAc.; the other has a branched repeating unit with the sequence 2)-α-L-Rha-(1→3)-β-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. We suggested that Megavirus chiliensis fibrils are decorated by more than one polysaccharides/oligosaccharide species, one having this trisaccharide: α-L-4OMe-RhaNAc-(1→3)-α-L-RhaNAc-(1→3)-α-L-RhaNAc-(1→. Moumouvirus australensis fibrils are 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.Among the glycosylation genes, the function of L142 was investigated in vitro, demonstrating that it is an N-acetyltransferase. To conclude, the fibrils of Mimiviridae are heavily glycosylated and the type of sugars and their organization depends on their lineage

Normal mammalian urothelium lines the majority of the urinary tract performing a barrier function against penetration of toxic substances and adventitious microorganisms from the urine. These protective functions of the urothelium are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification and complex assembly to regulate the basal to apical organisation of multiple cell layers, including the highly-differentiated lumen-facing superficial cell. As a poorly studied part of the urothelial differentiation programme, protein glycosylation is reported to contribute to the apical glycome, with possible roles in urothelial cancer and infection by uropathogenic E. coli. It is thus important to investigate the apical surface glycome of the normal superficial urothelial cell that is presumed to act in healthy urothelial function and disease. The aim of this study was to investigate the urothelial cell apical surface glycome using MALDI-MS to analyze permethylated glycans. The first step was to define the N-glycome of the porcine urothelial total cell lysate using the established filter-aided N-glycan separation approach; this represents the first normal mammalian urothelial N-glycome to be defined. Moreover, this thesis presents development of a method to differentially collect the urothelial cell apical surface N- and O-glycans. An in-house designed and built device for trypsinization of the luminal urothelial surface, with subsequent β-elimination and PNGase F digestion was used to release apical surface O- and N-glycans. The effect of trypsinization on superficial glycoproteins was post-monitored using immunolabelling of apically-expressed uroplakin 3a protein to optimize the period of trypsinization, without compromising the integrity of the superficial urothelial layer. 47 N- and 12 O-glycan species were observed on the apical surface of porcine urothelium. The approach developed for releasing apical cell surface N- and O-glycans allowed identification of apical surface glycans as candidates implicated in urothelial barrier function.

Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2008.
Title from PDF t.p. (viewed May 13, 2009). Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4721. Adviser: James P. Reilly.