The epidermal growth factor receptor (EGFR) exists as a single, highly glycosylated subunit receptor on the plasma membrane of a cell. Upon ligand binding to its extracellular domain, the EGFR dimerizes with an adjacent receptor. This results in activation of the EGFR’s intracellular tyrosine kinase domain, and consequently, autophosphorylation of specific tyrosine residues on the receptor’s cytoplasmic tail. Adaptor proteins bind to these phosphorylated tyrosine residues and transduce the message internally, initiating a multitude of signalling cascades which stimulate cell growth, division, and movement. Despite all that has been elucidated regarding the activation and signalling pathways of the EGFR, the parameters controlling dimerization and activation remain unknown. Recently, Neu1 sialidase, an enzyme which cleaves α-2,3-linked sialic acids from glycosylated substrates, has been implicated as a critical mediator of TrkA receptor activation. Upon activation, the sialidase desialylates the external receptor glycosylation, removing a physical barrier which was formerly hindering receptor dimerization, and thus, receptor activation. Based on the known sialylation of EGFR glycosylation, as well as the demonstrated importance of receptor glycosylation in EGFR activation, we hypothesized that the EGFR may be activated by a similar mechanism. Here, we report an identical membrane signalling paradigm initiated by epidermal growth factor (EGF) binding to EGFR to rapidly induce Neu1 sialidase activity in live NIH3T3-EGFR cells but not in live Neu1-deficient human fibroblast cells. Furthermore, we report that Neu1 sialidase activity is required for EGFR activation, supported by the finding that tyrosine phosphorylation is inhibited in EGF-stimulated NIH3T3-hEGFR cells which have been pretreated with both broad-range (oseltamivir phosphate) and specific (anti-Neu1 neutralizing antibody) sialidase inhibitors. MMP-9 plays a role in the initiation of Neu1 sialidase post-ligand binding, as pre-treatment of NIH3T3-hEGFR cells with specific MMP-9 inhibitor prior to EGF stimulation blocks membrane sialidase activity as well as tyrosine phosphorylation. Of critical importance to this schematic is the finding that both Neu1 and MMP-9 co-immunoprecipitate with EGFR on the plasma membrane of both naïve and EGF-stimulated NIH3T3-hEGFR cells. Together, these findings reveal a novel EGFR activation mechanism in which cross-talk between Neu1 and MMP-9 plays a vital role in EGF-induced receptor activation. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-06-26 12:04:40.486

Reproduction de : Thèse de doctorat : Biochimie : Reims : 2004. / Titre provenant de l'écran titre. Bibliogr. f. 163-187.

The molecular mechanism(s) by which Toll-like receptors become activated are not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate NF-κB. However, the parameters controlling interactions between the receptors and their ligands still remain poorly defined. Previous reports have identified that neuraminidase-1 (NEU1) is an important intermediate in the initial process of TLR ligand induced receptor activation and subsequent cell function. What we do not yet understand is how NEU1 is activated following TLR ligand binding. In this thesis, the findings disclose a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via neuromedin-B (NMBR) Gα-proteins, matrix metalloproteinase-9 (MMP-9) activation, and the induction of Neu1 activation. Central to this process is that NEU1–MMP-9-NMBR complex is associated with TLR-4 receptors on the cell surface of naive primary macrophages and TLR-expressing cell lines. Ligand binding to the receptor initiate GPCR-signaling via GPCR Gα subunit proteins and MMP-9 activation to induce NEU1. Activated NEU1 targets and hydrolyzes sialyl α-2-3-linked to β-galactosyl residues at the ectodomain of TLRs, enabling the removal of steric hindrance to receptor association, activation of receptors and cellular signaling. Furthermore, a novel glycosylation model is uncovered for the activation of nucleic acid sensing intracellular TLR-7 and TLR-9 receptors. It discloses an identical signaling paradigm as described for the cell-surface TLRs. NEU1 and MMP9 cross-talk in alliance with neuromedin-B receptors tethered to TLR-7 and -9 receptors at the ectodomain is essential for ligand activation of the TLRs and pro-inflammatory responses. However, the mechanism(s) behind this GPCR and TLR cross-talk has not been fully defined. Here, GPCR agonists mediate GPCR-signaling via membrane Gα subunit proteins to induce NEU1 and MMP-9 cross-talk at the TLR ectodomain on the cell surface. This molecular organizational GPCR signaling platform is proposed to be an initial processing stage for GPCR agonist-induced transactivation of TLRs and subsequent cellular signaling. Collectively, these novel findings radically redefine the current dogma(s) governing the mechanism(s) of the interaction of TLRs and their ligands, which may provide important pioneering approaches to disease intervention strategies. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-04-30 12:23:42.429

Receptor glycosylation is critical in receptor-ligand interactions in immune cells, but the exact role of glycosylation in receptor activation upon ligand binding has not been elucidated. In neuronal cells, we have shown that when neurotrophic factors bind their respective Trk tyrosine kinase receptors, receptor activation and subsequent neurotrophin-mediated signaling is dependent upon the induction and activity of an endogenous sialidase enzyme. In this thesis, we report that toll-like receptor (TLR) activation upon ligand binding is similarly dependent on the induction of a cellular sialidase, which we have identified as Neu1 sialidase, which specifically targets and hydrolyses alpha-2,3-linked sialic acid residues on the receptor. Blocking Neu1 sialidase activity with specific inhibitor Tamiflu detrimentally impacts ligand-induced TLR4/MyD88 interaction, NFkappaB activation and TLR-mediated effector responses like nitric oxide and pro-inflammatory cytokine production. Diminished cytokine production is also seen in vivo in Neu1-deficient mice. We propose a mechanism for the induction of Neu1 sialidase, upon ligand binding to TLR, that involves the activation of heterotrimeric G-alpha protein-dependent G-protein coupled receptor (GPCR) signaling to activate a matrix metalloproteinase (MMP) enzyme, likely MMP-9. It is suggested that MMP-(9) targets the cell surface elastin receptor complex of Neu1/protective protein cathepsinA/elastin binding protein (EBP), which potentially catalytically activates Neu1. In addition, we report an association between Neu1 and TLR2, TLR3 and TLR4 on the plasma membrane that has not previously been described. The idea that the multiple functionality and diversity of TLRs and TLR-mediated signaling may be an immunologic paradigm capable of explaining all human disease is provocative but plausible. Certainly, the structural integrity of TLRs, their ligand interactions and activation are essential for immunological protection. Thus, understanding the molecular mechanism of Neu1 sialidase regulation of TLR activation will provide important opportunities for disease control through TLR manipulation. The future directions of this research will also open a new area of glycobiology research (the glycomics of innate immune responses) and will widen the scope for the development of novel therapeutic drugs to combat infections and inflammatory diseases. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2009-01-26 12:33:32.743

The signaling pathways of tyrosine kinase Trk receptors and their downstream biological effects are well known, but the parameters controlling the interactions between the receptors and their natural ligands still remain to be defined. Recent published reports from our laboratory indicate that nerve growth factor (NGF)-induced TrkA receptor activation is dependent on a membrane cellular sialidase. This sialidase activity specifically targets and hydrolyzes sialyl α-2, 3-linked β-galactosyl residues resulting in the desialylation and activation of the receptor. These findings support a novel hypothesis that places mammalian sialidase(s) in a cycle of activation of these receptors by their natural ligand. Taken together, they also predict a prerequisite desialylation of Trk receptors caused by a sialidase on the cell surface enabling the removal of a steric hindrance to receptor dimerization. Until now, the sialidase associated with neurotrophin-treated live Trk-expressing cells has not been identified. The molecular mechanism(s) of sialidase activation by neurotrophin factors binding to their receptors also remains unknown. In this thesis, the novel role of Neu1 sialidase in the activation of ligand-induced TrkA and TrkB receptors has been identified. It has been reported for the first time that Neu1 is already in complex with naïve and ligand-induced Trk receptors. In addition, a membrane sialidase mechanism initiated by NGF binding to TrkA has been indentified. It suggests a potentiation of GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live TrkA- and TrkB-expressing cells and primary neurons. These results establish a unique mode of regulation of Trk receptors by their natural ligand and define a new function for Neu1 sialidase. Preliminary data indicate that members of the family of tyrosine kinase receptors like epidermal growth factor receptor (EGFR) and insulin receptor are also under the same regulatory control of Neu1 sialidase. Recent reports from the laboratory have indicated that ligand-induced activation of the highly glycosylated Toll-like receptors, TLR-2,-3 and -4 is also dependent on Neu1 sialidase on the cell surface. Taken all together, the findings in this thesis uncover a Neu1 and MMP-9 cross-talk on the cell surface which is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and neuron function. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2010-04-26 11:44:51.418

Tay-Sachs disease and its related disorders (GM2 gangliosidoses) are neurodegenerative diseases caused by the excessive accumulation of ganglioside GM2, an otherwise non-toxic plasma membrane component, in the lysosomes of cells of the central nervous system. The accumulation of ganglioside GM2 is the result of a defect in the gene encoding the α-subunit of β-hexosaminidase A (Hex A), an acid hydrolase responsible for the metabolism of gangloside GM2 in the lysosome. Though a debilitating disease in humans, Tay-Sachs model mice (𝘏𝘦𝘹𝘢-/-) escape symptoms by the action of lysosomal sialidase, which is expressed in mice at levels sufficient to metabolize ganglioside GM2 and effectively "bypass" Hex A deficiency. In an attempt to understand why a lysosomal sialidase-mediated bypass of Hex A deficiency is not observed in humans, we cloned ~ 2.9 kb of the human lysosomal sialdiase promoter and began characterization of the regulatory machinery that determines its activity. The transcription factor CDP (CCAAT -Displacement Protein) and truncations thereof were found to have a clear and consistent effect on promoter activity 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰, with the truncation CDP⁸³¹⁻¹⁵⁰⁵ resulting in a near 50-fold increase in activity. Adenovirus-mediated gene transfer of CDP⁸³¹⁻¹⁵⁰⁵ into CRB/TSD cells, a human Tay-Sachs neuroglia cell line, resulted in elevated lysosomal sialidase activity and a decrease in ganglioside GM2 stores. These results suggest that the regulatory machinery responsible for lysosomal sialidase expression may be manipulated in such a way as to "activate" a sialidase-mediated bypass of Hex A deficiency in human Tay-Sachs cells. Thus, induction of lysosomal sialidase may have a potential therapeutic benefit in human Tay-Sachs disease and other Hex A-deficient GM2 gangliosidoses. / Thesis / Master of Science (MS)

The upper respiratory tract (URT), including the nasal and oral cavities, is a reservoir for pathogenic and commensal microbial species, collectively known as the microbiota. Microbial colonization of the URT occurs right after birth, and URT microbial composition has been linked to development of respiratory infections, allergy, and asthma, though few direct mechanisms have been uncovered. Thus, I set out to establish animal models for characterizing the URT microbiota, and its role in infections. I found that nasal washes, a predominant method for measuring URT bacterial colonization, were insufficient for completely extracting the URT microbiota. The age and source of mice greatly affected the composition of the microbiota, which could be transferred to germ-free mice via cohousing. I also established that mice colonized with the Altered Schaedler’s Flora in the gut microbiota have no cultivable URT microbiota. To test the function of the URT microbiota, I colonized mice with Streptococcus pneumoniae, the leading cause of bacterial pneumonia worldwide. I show that the presence of a nasal microbiota increases permissiveness to pneumococcal infection in murine models. Addition of a single URT isolate, Actinomyces naeslundii, increased pneumococcal adherence to human respiratory epithelial cells in vitro and increased pneumococcal dissemination in vivo in a sialidase-dependent manner. The microbiota affects expression of several host genes throughout the respiratory tract involved in pneumococcal pathogenesis. Together, this work establishes new models for assessing the URT microbiota, and highlights the contribution of the URT microbiota to pneumococcal pathogenesis and identifies druggable targets to prevent and treat infections. / Dissertation / Doctor of Philosophy (PhD) / Bacteria living in the gut have been shown to benefit our health, but the role of bacteria living in our respiratory tract is relatively unknown. I describe the methods for characterizing the bacteria in the nose of a mouse as a model of the human nose. I found that pockets of the mouse nose are colonized by different bacteria. I also characterized a mouse model that had bacteria in the gut without nasal bacteria. I used this mouse model to understand infections with Streptococcus pneumoniae, the worldwide leading cause of bacterial pneumonia. The mice without nasal bacteria were protected from infections, which was due to a nasal bacteria helping S. pneumoniae escape from the nasal tissue. This work established new models for understanding how bacteria affect respiratory health, and identified new targets for protecting against infections.

trans-Sialidase de Trypanosoma cruzi (TcTS) pertence à família de glicoproteínas de superfície do parasita e constitui um dos poucos exemplos naturais de glicosiltransferases superficiais encontradas em eucariotes. T. cruzi é incapaz de sintetizar ácido siálico e utiliza esta enzima para retirar este monossacarídeo de glicoconjugados do hospedeiro para sialilar moléculas aceptoras, como mucina-GPI (glicosilfosfatidilinositol), presentes na sua membrana plasmática. Esta enzima é específica em catalisar, preferencialmente, a transferência de ácido siálico para moléculas de mucina, originando ligações -2,3 com moléculas de galactose aceptoras na superfície do parasita. Considerando a heterogeneidade das moléculas de mucina de T. cruzi, é necessário que novas moléculas sejam sintetizadas a fim de que estas atuem como substratos glicopeptídicos, os quais podem levar ao melhor entendimento das interações entre enzima e substratos e permitir o planejamento racional de inibidores seletivos. Por isso, o trabalho foi divido em três rotas sintéticas: (i) preparação do doador de galactose, (ii) preparação dos aceptores-doadores e (iii) acoplamento dos dissacarídeos com aminoácidos aceptores para obtenção dos blocos de construção. Apesar dos objetivos propostos inicialmente não terem sido totalmente alcançados, o trabalho desenvolvido durante esse período permitiu a síntese do doador de galactose (3) em três etapas, aceptor de galactose (6) em cinco etapas, dissacarídeo (11) na glicosilação de 6 com 3, aminoácidos aceptores (13 e 14) e também dos blocos de construção (17 e 18) decorrente do acoplamento de 11 com os aminoácidos aceptores. Não obstante, é importante ressaltar que apesar da extensa rota planejada, porém necessária, a síntese dos blocos de construção é inédita. Portanto, pode-se concluir que o trabalho trouxe relevante contribuição no que diz respeito à química de carboidratos e à disponibilização de dados espectrométricos de compostos orgânicos para a literatura. / Trypanosoma cruzi trans-sialidase (TcTS) belongs to the family of glycoproteins expressed on the surface of the parasite and constitutes one of the few examples of natural surface glycosyltransferases found in eucariotes. T. cruzi can not synthesize sialic acid itself and uses this enzyme to scavenge this monosaccharide from host glycoconjugates to sialylate acceptors molecules, such as GPI (glycosylphosphatidylinositol) mucins, that are present in parasite plasma membrane. This enzyme is specific to catalyze, preferentially, the transference of sialic acid to mucin glycoproteins, generating -2,3-linkages with acceptor galactose molecules in the parasite surface. Considering the heterogeneity of T. cruzi mucin molecules, its necessary to synthesize new compounds that can act as glycopeptide substrates, leading to a better understanding concerning the enzyme and substrates and allow the rational design of some selective inhibitors. Thus, this work was developed in three synthetic routes: (i) the synthesis of galactose donor, (ii) synthesis of donor-acceptors and (iii) coupling between disaccharides and acceptors amino acids in order to obtain building blocks. Despite of some objectives initially proposed had not been accomplished, the developed work during this period allow the synthesis of the galactose donor (3) in three steps, donor-acceptor (6) in five steps, disaccharide (11), acceptors amino acids (13 and 14) and also the building blocks (17 and 18). However, its important highlight that the synthesis of the building blocks by this necessary, but extensive, synthetic route is unpublished. Therefore, it can be concluded that the present work brought rich contribution concerning the carbohydrate chemistry and the availability of spectrometric data of organic compounds to the literature.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A transsialidase é uma glicoproteína de membrana pertencente a uma família de genes de cópia múltipla, envolvida no processo de invasão celular do Trypanosoma cruzi no hospedeiro vertebrado. Esta dissertação foi concebida com um amplo componente analítico que dependia de dados publicamente disponíveis, ou seja, as sequências oriundas do projeto genoma de T. cruzi e cDNAs de trans-sialidase depositadas no Genbank-dbEST. Este componente analítico necessitou ser complementado e ampliado com a obtenção experimental de novas sequências, a partir da metodologia baseada na transcrição reversa acoplada a PCR. Os fragmentos obtidos de cepas de T. cruzi Dm28c (T. cruzi I), Y (T. cruzi II), CL-Brener (T. cruzi II, cepa híbrida), INPA4167 (zimodema III), 3663 (zimodema III) e Colombiana (zimodema III) foram clonados, sequenciados e analisados composicionalmente. Essas sequências foram editadas e alinhadas usando-se o software CLUSTAL X. Em uma seção específica do Genbank, denominada dbEST, buscamos os cDNAs homólogos a trans-sialidase. Esta busca por similaridade foi realizada individualmente com os números de acesso referentes às seqüências supracitadas contra o dbEST utilizando o BLAST a fim de obtermos informações funcionais e evolutivas. Em seguida, desenvolvemos metodologias experimentais que nos permitiu avaliar segmentos da 5 UTR, tais como os sítios de trans-splicing adicionais ou múltiplos em TS e seus respectivos sinais (região rica em polipirimidina), variação composicional e tamanho da região das sequências entre diferentes linhagens de T. cruzi. O resultado dessa averiguação também nos mostrou a quantidade de cDNAs relacionados com a transsialidase no dbEST bem como a relação desses cDNAs com o mini-exon. As cepas do zimodema III apresentaram tamanho médio dos fragmentos de 312 bases, enquanto T. cruzi I e T. cruzi II apresentaram, respectivamente 209 e 218. Trans splicing adicional ou duplicações gênicas com mutações no sítio primário de trans splicing não parece ser um fenômeno exclusivo de algum grupo populacional, embora seja mais evidente em T. cruzi zimodema III.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A transsialidase é uma glicoproteína de membrana pertencente a uma família de genes de cópia múltipla, envolvida no processo de invasão celular do Trypanosoma cruzi no hospedeiro vertebrado. Esta dissertação foi concebida com um amplo componente analítico que dependia de dados publicamente disponíveis, ou seja, as sequências oriundas do projeto genoma de T. cruzi e cDNAs de trans-sialidase depositadas no Genbank-dbEST. Este componente analítico necessitou ser complementado e ampliado com a obtenção experimental de novas sequências, a partir da metodologia baseada na transcrição reversa acoplada a PCR. Os fragmentos obtidos de cepas de T. cruzi Dm28c (T. cruzi I), Y (T. cruzi II), CL-Brener (T. cruzi II, cepa híbrida), INPA4167 (zimodema III), 3663 (zimodema III) e Colombiana (zimodema III) foram clonados, sequenciados e analisados composicionalmente. Essas sequências foram editadas e alinhadas usando-se o software CLUSTAL X. Em uma seção específica do Genbank, denominada dbEST, buscamos os cDNAs homólogos a trans-sialidase. Esta busca por similaridade foi realizada individualmente com os números de acesso referentes às seqüências supracitadas contra o dbEST utilizando o BLAST a fim de obtermos informações funcionais e evolutivas. Em seguida, desenvolvemos metodologias experimentais que nos permitiu avaliar segmentos da 5 UTR, tais como os sítios de trans-splicing adicionais ou múltiplos em TS e seus respectivos sinais (região rica em polipirimidina), variação composicional e tamanho da região das sequências entre diferentes linhagens de T. cruzi. O resultado dessa averiguação também nos mostrou a quantidade de cDNAs relacionados com a transsialidase no dbEST bem como a relação desses cDNAs com o mini-exon. As cepas do zimodema III apresentaram tamanho médio dos fragmentos de 312 bases, enquanto T. cruzi I e T. cruzi II apresentaram, respectivamente 209 e 218. Trans splicing adicional ou duplicações gênicas com mutações no sítio primário de trans splicing não parece ser um fenômeno exclusivo de algum grupo populacional, embora seja mais evidente em T. cruzi zimodema III.