It was of interest to clone key genes involved in O-glycosylation with a view to using reverse genetics to establish their function. The Candida homolog of the S. cerevisiae MNT1 gene (Hausler and Robbins, 1992) was cloned by heterologous probing of a genomic DNA library. The CaMNT1 gene was found to be regulated differentially in response to the environment and exhibited a transitory increase in the level of transcription during early germ tube formation. Low stringency Southern analysis of C. albicans genomic DNA identified several CaMNT1 homologs suggesting CaMNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases. In order to demonstrate that specific glycosyl residues were actively involved in the host-fungus interaction, the CaMNT1 gene was disrupted in two strains using the ura-blaster technique. Disruption at the CaMNT1 locus led to a 90% reduction in -1,2-mannosyltransferase activity when -methyl mannoside was used as an acceptor, but had no obvious influence on viability, growth rate, germ tube formation or proteinase production. CaMnt1 appears to be involved in O-glycosylation since the Camnt1 null mutant strain accumulated intracellularly the O-glycosylated enzyme chitinase. Mannosyltransferase-deficient Camnt1 mutants were significantly reduced in their ability to adhere to human buccal epithelial cells in vitro and were attenuated in virulence in systemic models of candidosis. O-linked mannan may therefore be important for direct interactions with epithelial surfaces or for the stabilization and function of cell surface adhesins. The low virulence potential displayed by Camnt1 mutants clearly demonstrates the important role glycosylation plays in the virulence of C. albicans. Given that O-glycosylation differs significantly between yeast and man, this protein modification may constitute a novel target for antifungal agents.

CD46 is a ubiquitously expressed transmembrane protein in humans with a role in immune homeostasis. Originally identified as a complement regulator, CD46 has since been regarded a receptor for several pathogens and most recently, described as a T cell costimulatory molecule. Its coligation with CD3 and consequent cleavage from the T cell surface serves as a costimulatory stimulus for T cell activation. In addition, in the presence of IL-2, CD46 induces Tr1 cell differentiation which is characterised by low IFN-γ and high IL-10 secretion. CD3/CD46-induced Tr1 differentiation is defective in patients with MS, rheumatoid arthritis and asthma, highlighting the need to investigate the mechanisms involved in the regulation of the CD46 pathway. CD46 is a highly glycosylated protein with three N-glycosylation sites in the short consensus repeats and multiple O-glycosylation sites in the STP region. Previous data from the lab have shown that CD3 activation causes a change in CD46 glycosylation. Herein, I convey that this change is more pronounced in memory than naive CD4+ T cells and is mainly due to changes in CD46 O-glycosylation. Furthermore, these changes are required for the T cell responses triggered by CD46 costimulation including T cell activation and Tr1 differentiation. Interestingly, CD46 is recruited to the immunological synapse formed between a T cell and an antigen presenting cell and I illustrate that the STP region is needed for this also. These data suggest that the glycosylation status of CD46 regulates its function. In MS, vitamin D deficiency is considered to be a significant risk factor and many patients take vitamin D supplement to help manage their condition. Herein, I report that treatment of healthy and MS CD4+ T cells with vitamin D does not prevent T cell activation but it decreases adhesion molecule expression. Moreover, vitamin D supplementation in MS enhances CD46 cleavage. Therefore, vitamin D also plays a role in the regulation of the CD46 pathway and it would be interesting to investigate whether vitamin D affects CD46 glycosylation. During my MSc, I showed that a recombinant protein derived from adenovirus serotype 35 (which naturally binds CD46) known as Ad35K++ controls the CD46 pathway in CD4+ T cells. Lymphoma cells treated with Ad35K++ in combination with monoclonal antibody therapy rituximab have demonstrated increased sensitivity to rituximab and prove that virus-derived recombinant proteins that target CD46 have therapeutic potential. Considering the key role of CD46 as a T cell costimulatory molecule, I have investigated the effects of Ad35K++ on the CD46 pathway following its use in vivo and confirm CD46 is still cleaved from the cell surface and the cells still become activated. Overall these results provide insight into the mechanisms involved in the regulation of the CD46 pathway and highlight how it can be manipulated for therapeutic use.

The amino acid (2S, 4R)-4-hydroxyproline (Hyp) plays a critical role in animal kingdom as structural protein collagen. It is ubiquitous in plant cell walls performing various functions such as structural assembly, plant hormones, plant growth, defense against pathogens, etc. Glycosylation of Hyp is often seen in plant cell walls with L-Arabinofuranose and D-Galactopyranose and not in animal kingdom. Glycosylation is a post-translational modification, which affects characteristics of proteins and peptides. The main objective of this thesis is to synthesize various L-arabinofuranosylated hydroxyproline model amides and investigate their thermodynamic and kinetic properties of cis/trans amide isomerization. These results are compared with the previous research of D-galactopyranosylated hydroxyproline model amides, which may provide an insight to structural implications for their stability and conformations of peptides and specificity in plants. Both - and -L-arabinosylation of Hyp resulted in the stabilization of trans rotameric state at room temperature while the α-anomer leads to cis rotamer stabilization at higher temperature. Similarly, both unnatural 4S-hydroxyproline (hyp) building blocks resulted in stabilization of trans rotamer but α-anomer shows exo configuration instead of endo. This result shows a reverse trend when compared to galactosylated hydroxyproline building blocks as previous research results in our group. Our results may provide further insight to the role of glycosylation on protein structure and stability in plants.

Neisseria meningitidis is the causative agent of potentially life-threatening meningitis and septicaemia. According to W.H.O., meningococcal disease causes at least 500,000 cases and results in 50,000 deaths worldwide each year (W.H.O., 2008). Neisseria gonorrhoeae is causing the second most common sexually transmitted bacterial infection, with a global incidence of 62 million cases per year. Previous studies have shown surface expressed proteins like pilin, the subunit protein that forms pili (Type IV Fimbriae), in N. meningitidis and N. gonorrhoeae are post-translationally modified by O-glycosylation. This modification has been proposed to be of importance in the pathogenesis of these species. Although the exact function of these post-translational modifications are not fully understood, it is suggested that these modification have a role for immune evasion in the host. In this thesis, an additional outer membrane glycoprotein was identified in pathogenic Neisseria, the nitrite reductase AniA. Mass spectrometry analysis showed that AniA is glycosylated in its C-terminal imperfect (AASAP) repeat region by the pilin glycosylation pathway. This is the first report of a general O-glycosylation pathway in a prokaryote. It was shown AniA is surface exposed. To investigate whether AniA is subject to immune selection, a large collection of N. meningitidis and N. meningitidis clinical isolates were sequence analysed and evaluated. Analysis of published AniA 3D structure revealed that AniA displayed polymorphisms in residues that map to the surface of the protein. This suggests that AniA is under immune selection, and that glycosylation may facilitate immune evasion. Sequencing analyses revealed a frame shift mutation that abolished AniA expression in 34% of N. meningitidis strains surveyed. However, all N. gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology. In summary, the data presented here suggested that the protein may be under immune selective pressure. The addition of a phase variable glycan to this surface protein may serve as an additional immune evasion strategy. Immune selection on surface proteins in these host-adapted pathogens may have been the driving force for the evolution of this general O-glycosylation pathway. Therefore, the discovery that AniA is a glycoprotein has given insights into the pathogenesis and the host-pathogen interactions of these organisms.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19633号 / 医科博第71号 / 新制||医科||5(附属図書館) / 32669 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 斎藤 通紀, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Les fonctions endocriniennes des os sont médiées par au moins deux hormones, l’ostéocalcine et le facteur de croissance fibroblastique 23 « Fibroblast growth factor 23 » (FGF23), ces derniers sont secrétés par les cellules osseuses, les ostéoblastes et les ostéocytes. L’ostéocalcine est produite par les ostéoblastes et régule le métabolisme du glucose et énergétique. Elle améliore ainsi la tolérance au glucose et la sensibilité à l’insuline. Également, elle favorise la sécrétion d’insuline et la prolifération des cellules β, elle augmente la dépense énergétique et réduit l’accumulation de graisse. L'ostéocalcine est gamma-carboxylée au niveau de trois résidus d'acide glutamique (Glu), un processus qui inhibe sa fonction endocrinienne chez la souris et l'humain. Le pH acide de la lacune de résorption décarboxyle l'ostéocalcine et libère sa forme non carboxylée (ucOCN), la forme active de cette hormone. Nos connaissances sur la régulation des fonctions endocriniennes d’ostéocalcine sont encore limitées à sa gamma-carboxylation. Puisque cette hormone est secrétée par les ostéoblastes et les ostéocytes, des cellules endocriniennes non classique, nous avons émis l’hypothèse que l'ostéocalcine pourrait être soumise à d'autres modifications post-traductionnelles (PTMs) au niveau de la voie de sécrétion contrôlant ses fonctions endocriniennes. Dans la première partie de cette thèse, nous avons montré que le propeptide de l'ostéocalcine pouvait être clivé dans son extrémité C-terminale au niveau du motif de base « RLRR » par la pro-protéine convertase furine, un processus qui se produit indépendamment de la gamma-carboxylation de l'ostéocalcine. L’inactivation du gène codant pour la furine, spécifiquement dans les ostéoblastes et les ostéocytes chez la souris, abolit totalement le clivage de la pro-ostéocalcine et altère son activation et sa libération lors de la résorption osseuse. Par conséquent, ces souris sont caractérisées par un niveau bas d'ucOCN dans le sérum, ce qui entraîne une altération de la tolérance au glucose, une diminution de la sécrétion d'insuline et de la dépense énergétique ainsi qu’une augmentation de l'accumulation de graisses. De plus, ces souris ont une perte d'appétit indépendamment de l'ostéocalcine. La restriction de la nourriture pour les souris contrôles ou « pair feeding » rend le phénotype des souris déficientes en furine plus apparent. Il apparait à un plus jeune âge avec une résistance à l'insuline. Dans la deuxième partie de cette thèse, nous avons découvert que l'ostéocalcine de souris est O-glycosylée au niveau de la sérine 8, un processus qui se produit indépendamment de sa gamma-carboxylation et de son clivage. Cette modification, qui n'est pas présente chez l'ostéocalcine humaine, augmente la demi-vie de l'ostéocalcine de souris dans le plasma ex vivo et in vivo. Il est intéressant de noter que la tyrosine 12 dans l'ostéocalcine humaine correspond à la sérine 8 dans la séquence de la souris, tandis que la mutation Tyr12Ser est suffisante pour générer une ostéocalcine humaine O-glycosylée et lui conférer une demi-vie plus longue dans le plasma de la souris comparativement à la forme native. FGF23 est une hormone secrétée par les ostéoblastes et les ostéocytes. Elle régule la réabsorption de phosphate et la production de vitamine D dans le tubule proximal du rein. Sa fonction endocrine est inhibée par un clivage endoprotéolytique qui libère ses fragments N- et C-terminaux. La mutation du motif « RHTR », un site de clivage consensus pour les proprotéines convertases PC(s), a été identifié chez les patients atteints du rachitisme hypophosphatémique génétiquement déterminés ou « Autosomal dominant hypophosphatemic rickets » (ADHR). Ces patients se caractérisent par une augmentation du taux de FGF23 intact, une hypophosphatémie et une ostéomalacie. Malgré l’importance de FGF23 dans plusieurs maladies, l’identité de l’enzyme responsable du clivage de FGF23 n’est pas encore connue, même si la furine et la proprotéine convertase subtilisine/kexine type 5 (PC5) peuvent cliver FGF23 in vitro. Dans la troisième partie de cette thèse, nous tentons de répondre à cette question en utilisant des souris déficientes en furine et/ou PC5 spécifiquement dans les ostéoblastes et les ostéocytes. Sous des conditions physiologiques, l’inactivation du gène de furine dans les ostéoblastes et les ostéocytes augmente le niveau du FGF23 intact par 25%. Malgré cette augmentation ces souris maintiennent une phosphatémie normale et elles ne montrent pas de signe d’ostéomalacie. On a aussi montré qu’une déficience en fer, une condition qui augmente la production de FGF23 au niveau de l’ARN messager et protéique, le FGF23 est totalement en forme intact dans les souris déficientes en furine, montrant que le clivage de FGF23 est totalement inhibé dans cette condition. En revanche, l’injection d’érythropoïétine ou d’interleukine 1-β, des conditions qui augmentent la production de FGF23, induit une augmentation significative du taux de FGF23 total dans le sérum des souris déficientes en furine et/ou PC5 dans les ostéoblastes et les ostéocytes, tandis que le niveau du FGF23 intact n’a pas augmenté de la même façon, suggérant que la FGF23 est correctement clivée chez ces souris. D’une façon intéressante et malgré les défauts développementaux et le retard dans la minéralisation osseuse observée dans les souris complètement déficientes en PC5, la suppression conditionnelle de PC5 dans les ostéoblastes et les ostéocytes chez la souris n'a entraîné aucun défaut osseux. Cependant, l’inactivation du gène codant pour la furine dans les ostéoblastes et les ostéocytes chez la souris a augmenté les paramètres osseux trabéculaires et a diminué l'épaisseur de l’os cortical. De plus, ces souris ont eu une diminution de la densité minérale et la rigidité des os reflétant une mauvaise qualité osseuse. En résumé, nous avons décrit pour la première fois que la furine est un régulateur multifonctionnel de la fonction des ostéoblastes et des ostéocytes in vivo. Elle régule le métabolisme du glucose en assurant le clivage de la pro-ostéocalcine, qui est nécessaire à la maturation et à la bio-activité de l'ostéocalcine, et en régulant l'appétit indépendamment de l'ostéocalcine. Ces résultats suggèrent la présence d'ostéokines supplémentaires régulant l'appétit et contrôlées par la furine. De plus, dans les ostéoblastes, la furine régule partiellement le clivage de FGF23 en assurant une phosphatémie normale, suggérant que la régulation de l'accumulation de masse osseuse par la furine est indépendante du FGF23. En outre, nous avons découvert que l'ostéocalcine de souris est soumise à l’O-glycosylation, une modification qui n'est pas conservée chez l'humain, ni chez d’autres espèces, et qui augmente la demi-vie de l'ostéocalcine de souris. La glycosylation artificielle confère à l'ostéocalcine humaine une demi-vie plus longue, offrant ainsi une approche permettant d'augmenter potentiellement la bio-activité de l'ostéocalcine humaine dans les futures applications thérapeutiques de l'ostéocalcine dans les maladies humaines. / Bone endocrine functions are mediated by at least two hormones, osteocalcin and fibroblast growth factor 23 (FGF23) which are secreted by the bone cells, osteoblasts and osteocytes. Osteocalcin is an osteoblast-derived hormone regulating glucose and energy metabolism. It improves glucose tolerance and insulin sensitivity, promotes insulin secretion and β-cell proliferation, increases energy expenditure and reduces fat accumulation. Osteocalcin is gamma-carboxylated on three of its glutamic acid residues (Glu), a process that inhibits its endocrine function in mice and humans. It is the acidic pH in the resorption lacuna which decarboxylates osteocalcin releasing the uncarboxylated osteocalcin (ucOCN), the active form of this hormone. Our knowledge on osteocalcin regulation by post-translational modifications is limited to its gamma-carboxylation. Since osteocalcin is secreted by differentiated osteoblasts, a non-classical endocrine cell, we hypothesized that osteocalcin may be subjected to additional post translational modifications (PTMs) in the secretory pathway that regulates its endocrine functions. In the first part of the thesis we showed that osteocalcin’s putative pro-peptide is cleaved in its C-terminus at the basic motif «RLRR», by the proprotein convertase furin. This process occurs independently of osteocalcin gamma-carboxylation. Furin inactivation specifically in osteoblasts in mice totally abolishes osteocalcin processing and impairs its activation and release during bone resorption. Consequently, these mice have decreased serum level of ucOCN resulting in impaired glucose tolerance, reduced insulin secretion and energy expenditure, and increased fat accumulation. Moreover, these mice have a decrease in the appetite independently of osteocalcin. Pair feeding of control mice resulted in more apparent phenotype in furin deficient mice, as it appears at younger age alongside with insulin resistance. In the second part of this thesis, we discovered that mouse osteocalcin is O-glycosylated on serine 8, a process that occurs independently of its gamma-carboxylation and processing. This modification is not conserved in human or any other species and it increases mouse osteocalcin half-life in plasma ex vivo and in vivo. Interestingly, tyrosine 12 in human osteocalcin corresponds to the serine 8 in the mouse sequence. Tyr12Ser mutation was sufficient to O-glycosylate human osteocalcin and to confer this hormone a longer half-life in mouse plasma compared to the native form. FGF23 is a hormone secreted by osteoblasts and osteocytes which regulates phosphate reabsorption and vitamin D production in the kidney proximal tubule. Its endocrine function is inhibited by endoproteolytic cleavage which releases its N-terminal and C-terminal fragments. Mutations in the «RHTR» motif, a consensus cleavage site for proprotein convertases (PCs), were found in patients with autosomal dominant hypophosphatemic rickets (ADHR). These patients are characterized by an increased intact FGF23 levels, hypophosphatemia and osteomalacia. Despite the importance of FGF23 in the pathology of multiple diseases, the identity of the enzyme(s) involved in FGF23 cleavage is yet unclear, even though furin and the proprotein convertase subtilisin/kexin type 5 (PC5) were shown to cleave FGF23 in vitro. In the third part of the thesis, we addressed this question using mice model deficient in furin and/or PC5 in osteoblasts and osteocytes in mice. Under physiological conditions, furin inactivation resulted in a 25% increase in intact FGF23; however, these mice maintained normal phosphate level and did not shown any sign of osteomalacia. We also showed that under iron restriction, a condition that induce FGF23 expression at the mRNA and protein level, FGF23 processing is totally impaired in furin deficient mice. However, the injection of erythropoietin or interleukin 1-β, two conditions that increase FGF23 production, induce FGF23 serum level while it is still properly processed in mice deficient in furin and/or PC5 in osteoblasts and osteocytes. Interestingly, despite the patterning defects observed in global inactivation of PC5, conditional inactivation of PC5 in osteoblasts and osteocytes in mice did not result in any bone defect. However, furin inactivation in osteoblasts and osteocytes in mice increases trabecular bone parameters and decreases cortical thickness. Moreover, these mice have decreased bone mineral density and bone strength reflecting a poor bone quality. In summary, we described for the first time that furin is a pleotropic regulator of osteoblast and osteocyte function in vivo. It regulates glucose and energy metabolism by mediating pro-osteocalcin processing which is required for osteocalcin maturation and bioactivity, and by regulating appetite independently of osteocalcin. These findings suggest the presence of additional osteokines controlling appetite and which are regulated by furin. Moreover, furin partially regulates FGF23 processing while maintaining normal phosphate homeostasis, suggesting that the regulation of bone mass accrual by furin occurs independently of FGF23. Additionally, we discovered that mouse osteocalcin is subjected to O-glycosylation, a species-specific modification that is not conserved in humans or any other species and increases mouse osteocalcin half-life. Artificial O-glycosylation confer human osteocalcin a longer half-life, thus providing an approach to increase human osteocalcin bioactivity in future therapeutic applications of osteocalcin in human diseases.

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<p>In the recent years considerable progress has been made to understand how the protein Bcl-2 regulates apoptosis at the mitochondria. By comparison, the cell death mechanisms at the endoplasmic reticulum remain unclear. In response to the agents that cause endoplasmic reticulum stress in breast cancer cells, the cell-cell adhesion molecule E-cadherin is modified by two independent modifications comprising pro-region retention and O-glycosylation. Both the modifications on E-cadherin inhibit its cell surface transport and the resultant loss of E-cadherin on the plasma membrane sensitizes cells to apoptosis. During this process binding of E-cadherin to type I gamma phosphatidylinositol phosphate kinase (PIPKIγ), a protein required for E-cadherin trafficking to the plasma membrane is prevented by O-glycosylation. E-cadherin deletion mutants that cannot be O-GlcNAcylated continue to bind PIPKIγ, traffick to the cell surface and delay apoptosis, confirming the biological significance of the modifications and PIPKIγ binding in the cell death regulation. These results also led me to determine whether there is a cell death pathway in which commitment to cell death is mediated by proteins primarily located at the endoplasmic reticulum. The studies show that the growth of estrogen receptor-positive breast cancer cells in charcoal stripped bovine serum leads to a form of programmed cell death which is protected by Bcl-2 exclusively localized at the endoplasmic reticulum instead of the mitochondria. Interestingly, the BH3 mimetic ABT-737 can abolish the protection mediated by Bcl-2 localized at the endoplasmic reticulum. Taken together, these studies suggest the novel role of the endoplasmic reticulum in programmed cell death through the identification and elucidation of the mechanisms that regulate the cell death pathway at this organelle.</p> / Doctor of Philosophy (PhD)