Le coronavirus humain 229E (HCoV-229E) est responsable de rhumes mais peut entraîner de graves complications respiratoires chez les personnes âgées ou atteintes d’une maladie Chronique. Les coronavirus sont des virus enveloppés avec un génome à ARN positif simple brin. Trois protéines virales sont ancrées dans l'enveloppe virale : la protéine spike (S), la protéine de membrane (M) et la protéine d’enveloppe (E). Les protéines M et E sont impliquées dans l'assemblage viral et la sécrétion. La protéine S s'assemble en trimères à la surface des virions et joue un rôle-clé dans l’entrée du virus dans sa cellule-cible. Elle est constituée de deux domaines, le domaine S1 responsable de la liaison du virus à son récepteur et le domaine S2 responsable de la fusion de l’enveloppe virale avec une membrane cellulaire. La fusion est activée par des protéases cellulaires par clivage de la protéine S. Dans un premier temps nous avons caractérisé ce mécanisme. Pour cela, nous avons d'abord cloné la protéine S d’un isolat circulant de HCoV-229E. Nous avons analysé le clivage protéolytique de la protéine S par des sérine-protéases de type trypsine conduisant au processus de fusion à l’aide de particules pseudotypées rétrovirales. Les Résidus arginine, sites potentiels de reconnaissance par les protéases et présents au niveau de la jonction S1/S2 ou de la région S2’ ont été mutés individuellement (R565N, R679N, R683N ou R687N) afin d’étudier leur rôle lors de l'activation de la fusion. Contrairement à d'autres coronavirus, l'activation permettant la fusion de HCoV-229E semble être un processus en une seule étape. En effet, seule la mutation R683N inhibe l’infection médiée par des sérine-protéases et le clivage à l'interface S1/S2 ne semble pas être un pré-requis. Les protéines S de coronavirus sont fortement N-glycosylées et constituent la principale cible des anticorps neutralisants. Nous avons analysé le rôle de la N-glycosylation du domaine S1 dans les mécanismes d'entrée et dans la neutralisation par des anticorps. L'analyse de la séquence de la protéine clonée montre la présence de 33 sites potentiels de N-glycosylation, dont 18 dans le domaine S1 qui ont été numérotés de N1 à N18. Ces 18 sites de N-glycosylation ont été abolis individuellement par mutagenèse dirigée. L’effet des mutations sur l'infectiosité virale a été évalué en utilisant des particules pseudotypées rétrovirales. L'infectiosité des mutants N6, N7 ou N9 est diminuée tandis que deux mutants N12 et N15 montrent une augmentation de l'infectiosité. Nous n'avons détecté aucune différence d'interaction de ces mutants avec une forme soluble du récepteur, l'aminopeptidase N (APN). Des expériences d’activation de la fusion virale à la surface cellulaire par la trypsine suggèrent que les glycanes présents aux positions 6, 7 et 9 sont impliquées dans la fusion virale, cependant nous n’avons détecté aucune différence de clivage de ces mutants par la trypsine. Pour le mutant N17 uniquement, la diminution partielle de l'infectiosité pourrait s'expliquer par une diminution de l'incorporation de la protéine S dans les pseudoparticules, due au mauvais repliement de la protéine, comme le montre le profil du mutant en western blot en conditions réductrices ou non.Nous avons ensuite évalué si les N-glycanes pouvaient moduler la reconnaissance de la protéine S par des anticorps neutralisants. Des pseudoparticules contenant les différents mutants ont été produites et utilisées pour infecter des cellules en présence d'anticorps neutralisants. Nos données montrent que les mutants N4, N10, N11, N12, N15, N16, N17, N18 réduisent la sensibilité des pseudoparticules à la neutralisation des anticorps. Dans ensemble, nos résultats suggèrent que les N-glycanes de la protéine S jouent un rôle important dans l'entrée virale et modulent la reconnaissance de la protéine par des anticorps neutralisants. / The human coronavirus 229E (HCoV-229E) is a causative agent of common colds and can lead to severe respiratory complications in elderly persons and those with underlying disease. Coronavirus are enveloped viruses with a single stranded, positive-sense RNA genome. Three viral proteins are anchored in the viral enveloppe : the spike (S) protein, the membrane (M) protein and the enveloppe (E) protein. The M and E proteins are involved in viral assembly and secretion. The spike proteins assemble into trimers at the surface of the virions and play a key role in the early steps of viral infection. The spike protein comprised two domains, the S1 domain responsible for receptor binding and the S2 domain responsible for fusion of the viral enveloppe with the host cell membrane. Coronavirus fusion is activated by the proteolytic processing of the spike protein. First, we charaterized the proteolytic processing of the HCoV-229E spike protein by trypsin-like serine-proteases. To do so, we first cloned the spike protein of a circulating isolate of HCoV-229E. To investigate the role of the S1/S2 junction and the specific role of the 3 arginine residues located in the S2’ region in the proteolytic activation of HCoV-229E spike protein, the arginine residues present at these positions were mutated individually (R565N, R679N, R683N or R687N). Our results show that unlike other coronaviruses, HCoV-229E fusion activation appears to be a one step process. Indeed, the cleavage of the S1/S2 interface does not seem to be a pre-requisite, and the fusion activation strongly relies on the S2’ region, with R683 acting as the cleavage site.The spike protein is highly N-glycosylated and is the main target of neutralizing antibodies. We analysed the role of S1 domain N-glycosylation in the entry functions of the S protein and in neutralization by antibodies. Analysis of the sequence of the cloned protein shows the presence of 33 potential N-glycosylation sites, 18 being located in the S1 domain (numbered from N1 to N18). We mutated the 18 N-glycosylation sites of S1 individually by site-directed mutagenesis and studied the effect of the mutations using retroviral pseudotyped particles. Infectivity of the spike proteins with mutation either at the N6, N7 or N9 glycosylation site was strongly impaired. We did not detect any difference of interaction of these mutants with the soluble form of the receptor, the aminopeptidase N (APN). Results obtained by inducing the fusion of pseudoparticles at the cell surface with trypsin suggest that N-glycans located at the position N6, N7 and N9 are involved in viral fusion. However, the proteolytic processing of the protein required for fusion activation does not seem to be affected. Two mutants N12 and N15 show an increase of infectivity. Mutation of the N-glycosylation site N17 induces a partial decrease in infectivity. Indeed a decrease of spike protein incorporation into pseudoparticles was observed likely due to misfolding of the protein as shown by the profile of the mutant in western blot under reducing and non-reducing conditions. We next assessed if N-glycans can modulate the recognition of the spike protein by neutralizing antibodies. Pseudoparticles harbouring the different mutants were produced and used to infect cells in presence or absence of neutralizing antibodies. Our data demonstrate that mutants N4, N10, N11, N12, N15, N16, N17, N18 reduce the sensitivity of pseudoparticules to antibody neutralization. Taken together our results suggest that N-glycans of the S protein play an important role in viral entry and modulate the recognition of the protein by neutralizing antibodies.

Le syndrome de Brugada (BrS) est une cardiopathie héréditaire à transmission autosomique dominante, se manifestant par une anomalie de l'ECG et un risque accru de mort subite. Les mutations retrouvées dans la sous-unité α du canal sodique cardiaque Nav1.5 chez certains patients entraînent un défaut d'adressage membranaire de ces canaux. Ceux-ci restent alors séquestrés dans des compartiments intracellulaires. L'étude de ces mutants se réduisant souvent à l'utilisation de traitements correcteurs, les mécanismes de rétention impliqués restent encore méconnus. L'objectif de ce travail est d'étudier des mutants Nav1.5 présentant un défaut d'adressage en tenant compte non seulement de l'hétérozygotie des patients BrS mais également de la présence de la sous unité régulatrice β1 prédominante dans le cœur. Des études fonctionnelles et biochimiques mettent en évidence un effet dominant négatif exercé par les mutants R1432G, L325R et S910L sur la densité de courant INa sauvage (WT). Cet effet nécessite la présence de la sous-unité β1 et passe par l'altération de l'adressage membranaire des formes WT. Ceci est la conséquence d'une interaction physique entre des sous-unités α mutantes et WT. D'autre part, les mutants étudiés présentent un profil de maturation lié aux N-glycosylations qui différent de celui des canaux WT. Nos données suggèrent que ces canaux peuvent emprunter (i) la voie classique d'adressage dans leur forme mature (ii) la voie dite non conventionnelle lorsqu'ils sont partiellement glycosylés. En conclusion, ces travaux mettent en évidence le rôle de la sous-unité β1 ainsi que l'implication des N-glycosylations dans la modulation de l'adressage des canaux Nav1.5 / Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy characterized by abnormal ECG pattern and an increased risk of sudden cardiac death. Several mutations on the cardiac sodium channel Nav1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane and are instead retained in intracellular compartments. Although pharmacological rescue is commonly used to characterize misfolded mutants, underlying cellular retention mechanisms remain unclear. The aim of this work is to investigate trafficking defective Nav1.5 mutants considering BrS patient heterozygosity and the presence of the regulatory β1-subunit which is largely expressed in cardiac tissue. By combining electrophysiology and biochemical approaches, we show that three distinct mutants, R1432G, L325R and S910L, exert a strong dominant negative effect upon wild-type (WT) sodium current density. Our data indicate that this effect requires the presence of the β1-subunit and is mediated by disruption of membrane trafficking of WT channels. Co-immunoprecipitation experiments demonstrate a physical interaction between mutant and WT α-subunits occurring only when the β1-subunit was present. Furthermore, we investigate the maturation pattern of Na channels. Our data show distinct N-glycosylated states between WT and mutant channels, suggesting that Nav1.5 α-subunits traffic (i) via unconventional secretion pathway as a partially glycosylated product, (ii) through the classical secretory pathway for mature fully-glycosylated form. This work highlights that β1-subunit and N-linked glycosylation process play key roles in modulating Nav1.5 trafficki

N-linked glycosylation is the most common post-translational modification (PTM) of proteins that exist in nature. N-glycosylation and change in cells serve as a criterion to monitor the activity of developmental stages and diseases severity. Currently, there is an increasing application of mass spectrometry on glycoprotein for malicious, chronic or acute diseases, such as cancers, rheumatoid arthritis (RA) or influenza. In this dissertation, several mass spectrometric assays have been utilized to, quantitatively and qualitatively, characterize protein N-glycosylation at the glycan, glycopeptide and peptide levels. The goals are to identify serum-based RA biomarker (Chapter 2), or to determine possible glycan structures from monoclonal antibody (Chapter 3), or comprehensively to study one influenza glycoprotein, hemagglutinin (Chapter 4). In Chapter 2, LC-MS/MS with CID as MS 2 is the primary technique that is applied to collect raw data for RA biomarker screening; western blot is the verification method for newfound biomarkers. This mass spectrometry based comparative analysis of N-glycoprotein in RA and healthy patients’ sera reveal 41 potential biomarkers for RA that can be applied in clinical research. Chapter 3 describes another LC-MS/MS based method developed for the structural analysis of N-glycan released from the monoclonal antibody, immunoglobin G. Higher-energy collision dissociation (HCD) was the surprior technique utilized to identify glycopeptide fragments. The results show that 19 and 23 N-glycan structures were determined from standard and modified mAb samples respectively by using SimGlycan software, while 38 and 35 glycan structures were recognized by manually mapping respectively. 13 N-glycoforms, out of 26 overlapped glycan structures, were identified with significant alterations by comparing standard sample (sample A) and modified mAb (sample B) utilizing our method. In Chapter 4, we comprehensively studied hemagglutinin by using LC-MS/MS and MALDI from both proteomic perspective and glycomics prospective. After confirmed and verified protein sequence and glycosylation sites, galactose-specific quantitation was performed with exoglycosidase digestion combined HPLC with fluorescence detection. The MALDI-MS/MS based method was utilized to confirm glycan structures. The results in this dissertation provide insights into the significance of protein glycosylation alterations as RA biomarkers, and these quantitative methods can be reapplied to any other disease biomarkers screening for clinical researchers.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Master of Science / Division of Biology / Stella Y Lee / Neuronal Ceroid Lipofuscinoses (NCLs) are a group of lysosomal storage diseases that are characterized by accumulating autofluorescent lipopigments in cells. NCLs are a form of progressive neurodegenerative diseases with symptoms ranging from blindness, loss of speech and motor activities to ataxia and seizures. Patients do not live to adulthood in most cases, making it prevalent in children. Among the many genes that cause NCL, CLN5 leads to different forms of NCL (infantile, late infantile, juvenile, and adult). CLN5 protein resides in the lysosomes but its function has not been established. It is predicted to contain eight N-glycosylation sites, but the role of N-glycosylation on its function and trafficking has not been assessed. We analyzed the role of N-glycosylation on the transport and stability of human CLN5. We created N-glycosylation mutants of each site by changing the Asn to Gln and our analysis of these mutants show that all the eight N-glycosylation sites are used in vivo. We also report effects of abolishing individual N-glycosylation sites on the trafficking of CLN5. While the lack of glycosylation at some sites results in CLN5 being retained in the ER or Golgi, others do not affect CLN5 trafficking. Cycloheximide chase experiments show that one of the mutants (N401Q) in CLN5 leads to lower protein levels in cell pellets with an increased secretion compared to CLN5 wild type, while other mutations show differential stability in cell pellets. These results demonstrate that each N-glycosylation site plays a different role(s) in the stability, transport and/or function of CLN5.

Oral squamous cell carcinoma (OSCC) accounts for majority of head and neck cancers and ranks as the sixth most common cancer in the world. OSCC belongs to the most understudied cancers and little is known about molecular mechanisms underlying its etiology and progression to metastasis. A hallmark of cancer is the enhanced posttranslational modification of cell surface proteins with complex N-glycans. Our studies have shown that induced protein N-glycosylation via activation of the core N-glycosylation-regulating gene, DPAGT1, is associated with reduced E-cadherin adhesion, as well as deregulation of several oncogenic signaling pathways, including Wnt/β-catenin and Hippo. Modest increases in DPAGT1 expression are associated with dramatic amplification of Wnt/β-catenin activity and increased expression and nuclear localization of the Hippo pathway effectors TAZ /YAP. The goal of this study was to align the expression and localization of DPAGT1, complex N-glycans, β-catenin, and TAZ/YAP with the progression of oral cancer in vivo from dysplasia to OSCC. Human oral tissues from different stages of OSCC pathogenesis were characterized for DPAGT1/β-catenin/α-catenin/YAP/TAZ expression and localization and correlated with cell surface expression of complex N-glycans by PHA lectin staining and with expression of primitive cell surface markers, CD44, CD24 and CD29. Results showed that high DPAGT1 expression and nuclear TAZ became increasingly associated with disorganized E-cadherin junctions as oral epithelium progressed from mild to severe dysplasia to OSCC. This correlated with increasing expression of cell surface complex N-glycans and CD44. These studies suggest that DPAGT1/β-catenin/TAZ and high PHA staining represent novel signatures for OSCC pathogenesis.

The proper orchestration of voltage-gated ion channel gating is vital to maintaining normal heart rhythms throughout an animal's lifespan. Voltage-gated sodium channels, Nav, are responsible for the initiation of the cardiac action potential, which leads to cardiac systole. Comparison of neonatal ventricular and atrial myocyte Nav gating with adult indicated that the neonatal ventricular Nav gated following a ~10 mV greater depolarization than did atrial or adult ventricular Nav. In this study I questioned whether development- and/or chamber-dependent changes in Nav-associated functional sialic acids could account for these differences. When desialylated with neuraminidase, all gating characteristics for the lower voltage activated atrial and adult ventricular Nav shifted significantly to more depolarized potentials. However, desialylation of the higher voltage activated neonatal ventricular Nav had no effect on channel gating. Furthermore, channels were stripped of their N-glycosylation via PNGase-F in an attempt to separate the potential effects of the remaining glycosylation structure on Nav gating. Following treatment, neonatal ventricular Nav gating remained unchanged while atrial and adult ventricular Nav gating again shifted to depolarized potentials nearly identical to those of the neonatal ventricular channel. Immunoblot analyses indicated that atrial and adult ventricular Nav α subunits are more heavily sialylated than the neonatal ventricular a subunit, with approximately 15 more sialic acid residues. The data indicate that differential sialylation of myocyte Nav α subunits is responsible for much of the developmental and chamber-specific remodeling of Nav gating observed here. In addition, the Nav1.5 α subunit can associate with β subunits, also believed to be sialylated. The potential for functional β1 trans sialic acids to further modulate Nav1.5 gating was tested via co-transfection of β1 with the Nav1.5 α subunit into the Pro5/Lec2 mammalian expression system. Co-transfection revealed that the additional b1 trans sialic acids caused a hyperpolarizing shift in all tested gating parameters. When transfected into neonatal ventricular myocytes, β1 expression revealed no effect, implying that β1 expression alone is not responsible. Together, the myocyte and expression system studies describe a novel mechanism by which Nav gating, and subsequently cardiac excitability, are modulated by the regulated change in channel-associated functional sialic acids.

Epithelial cells represent the ability to polarize with an apical and basolateral domains which differ markedly in proteins, lipid composition and therefore in function. This asymmetry reflects the ability of epithelial cells to sort newly synthesized proteins and lipid to either cell surface. While the signals responsible for basolateral targeting of the proteins have been clearly understood, the situation regarding the apical sorting of proteins is more obscure. We have previously shown that differently from basolateral GPI-APs oligomerization in the Golgi apparatus is necessary for apical sorting of Glycosylphosphatidylinositol- anchored proteins (GPI-APs). Interestingly this mechanism is conserved in two different kinds of epithelial cells, MDCK and FRT cells, which exhibits a difference in the sorting of GPI-APs. However the precise mechanism leading to this event is not understood. Our previous data demonstrated that simple addition of cholesterol to MDCK cells is necessary and sufficient to induce the oligomerization and apical sorting of a basolateral GPI-AP. Whereas, in this present study in FRT cells we showed that in contrast with MDCK cells cholesterol is not an active player in the regulation of GPI- APs apical sorting. In addition, we also showed that apical and basolateral GPI-APs are not segregated in the Golgi on the bases of the cholesterol content of the surrounding membrane environment. Furthermore, we demonstrated that N- glycosylation of the protein ectodomain is critical for oligomerization and apical sorting of GPI-APs. Our data indicates that at least two mechanisms depending either on cholesterol or on N-glycosylation exist to determine oligomerization in the Golgi and sorting to the apical membrane of GPI-APs.

The chloroplast is the organelle within a plant cell where photosynthesis takes place. This organelle originates from a cyanobacterium that was engulfed by a eukaryotic cell. During the transition from endosymbiont to organelle most of the cyanobacterial genes were transferred to the nuclear genome of the host cell, resulting in a chloroplast with a much reduced genome that requires massive import of gene products (proteins) back to the organelle. The majority of these proteins are translated in the cytosol as pre-proteins containing targeting information that directs them to a translocon complex in the chloroplast envelope, the Toc-Tic system, through which these proteins are transported. We have identified a protein in the model plant Arabidopsis thaliana, CAH1, that is trafficked via the endomembrane system (ER/Golgi apparatus) to the chloroplast instead of using the Toc-Tic machinery. This transport is partly mediated by canonical vesicle trafficking elements involved in ER to Golgi transport, such as Sar1 and RabD GTPases. Analysis of point mutated variants of CAH1 showed that both N-linked glycans and an intra-molecular disulphide bridge are required for correct folding, trafficking and function of the protein. Since chloroplasts lack N-glycosylation machinery, we propose that a route for chloroplast proteins that require endomembrane-specific post-translational modifications for their functionality exists as a complement to the Toc-Tic system. We also show that mutant plants with disrupted CAH1 gene expression have reduced rates of CO2 uptake and accumulate lower amounts of starch compared to wild-type plants, indicating an important function of the CAH1 protein for the photosynthetic capacity of Arabidopsis. Further study of CAH1 will not only be important to reveal its role in photosynthesis, but characterization of this novel protein pathway to the chloroplast can also shed light on how the plant cell evolved and clarify the purpose of keeping several chloroplast import pathways working in parallel. In addition, knowledge about this pathway could increase the opportunities for using plants as bio-factories for production of recombinant glycoproteins, which make up the vast majority of the bio-pharmaceutical molecules. / Kloroplasten är den organell i växtcellen där fotosyntesen sker. Denna organell härstammar från en cyanobakterie som togs upp av en eukaryot cell. Under omvandlingen från endosymbiont till organell har de flesta av den ursprungliga cyanobakteriens gener flyttats över till växtcellens eget kärngenom, vilket resulterat i en kloroplast som endast kan producera ett fåtal av de proteiner den behöver och som istället kräver att en mängd genprodukter (proteiner) transporteras tillbaka till organellen. De flesta av dessa proteiner syntetiseras i cytosolen som polypeptider innehållande en speciell signal för kloroplasten, och tranporteras över kloroplastens dubbelmembran (envelop) med hjälp av ett specifikt importsystem (Toc-Tic). Vi har identifierat ett protein i modellväxten Arabidopsis thaliana (CAH1) som istället för att använda Toc-Tic tranporteras via det endomembrana systemet (ER/Golgi). Transporten sker delvis med hjälp av faktorer involverade i normal vesikeltransport, t.ex. Sar1 och RabD GTPaser (mellan ER och Golgi). Genom att uttycka och analysera punktmuterade varianter av CAH1 har vi kunnat visa att både sockergrupper kopplade till proteinet, samt en intern svavelbrygga, är nödvändiga för korrekt veckning, transport och funktion av proteinet. Då kloroplasten saknar eget maskineri för att koppla sådana sockergrupper till proteiner så föreslår vi att anledningen till att denna rutt existerar, som ett komplement till Toc-Tic, är för att proteiner beroende av denna typ av modifiering ska kunna finnas i kloroplasten. Vi visar också att muterade växter som inte kan uttrycka genen som kodar för CAH1 uppvisar lägre upptag av CO2, samt ackumulerar mindre stärkelse än vildtypplantor, vilket antyder att CAH1 har en viktig funktion för den fotosyntetiska förmågan hos Arabidopsis. För att kunna fastställa den exakta funktionen för CAH1 kommer ytterliga studier att vara nödvändiga. En fördjupad karaktärisering av transportvägen som CAH1 följer till kloroplasten kan dessutom ge kunskap om hur växtcellen uppkom, samt besvara varför flera importvägar arbetar till synes parallellt med varandra. Kunskap om denna transportväg kan även bidra med användbar information i försöken att nyttja växter till att uttrycka rekombinanta N-glykosylerade proteiner, t. ex. antikroppar och vacciner.

The HIV-1 envelope glycoprotein trimer (Env) is covered by an extensive array of glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints that limit the actions of glycan processing enzymes, such that multiple under-processed structures remain on specific locations. These oligomannose-type glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, perhaps paradoxically, target it. In multiple studies, bNAbs have been shown to be capable of providing passive protection from viral challenge, making Env a focus of antibody-mediated vaccine design. Here, the development of a workflow for the semi-quantitative, site-specific N-glycosylation analysis of a soluble recombinant, native-like trimer mimic (BG505 SOSIP.664) is reported. The resulting data reveal a mosaic of dense clusters on the outer domain of Env and allow mapping the extremes of simplicity and diversity of glycan processing. Although individual sites usually minimally affect the global integrity of the glycan shield, examples are identified of how deleting certain glycans can subtly influence neutralization by bNAbs that bind at distant sites. Env is a trimer of heterodimers of gp120 and gp41, which is generated by cleavage of an endogenous protease. In this thesis, the detailed effect of protease cleavage on glycan processing is examined by comparing the site-specific N-glycosylation profiles of the native-like trimer mimic to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. Trimer-associated glycan remodeling forms a localized subdomain of the native mannose patch. Furthermore, the glycosylation analysis of further Env immunogens – a glycan-depleted trimer and a flexibly-linked, uncleaved trimer (both based on BG505 SOSIP.664) – provides important insights into the robustness of the HIV-1 glycan shield and the Env maturation pathway. Overall, this thesis reveals how structural constraints shape Env glycosylation and the network of bNAb-targeted glycans that should be preserved on recombinant vaccine candidates.