Étude du rôle de la N-glycosylation dans la localisation et les fonctions de la nucléoline humaine

La nucléoline, protéine ubiquitaire, est impliquée dans la croissance et la prolifération cellulaires. Bien que nucléaire, elle est aussi exprimée à la surface cellulaire. Des formes atypiques de la nucléoline de surface, N- et O-glycosylées, ont été mises en évidence dans notre laboratoire. Nous avons émis l hypothèse que la N-glycosylation pourrait modifier son trafic, ses propriétés d interaction et ses fonctions. Premièrement, nous avons montré que la nucléoline de surface est exclusivement N-glycosylée, que la glycosylation est essentielle à son expression de surface, et qu elle possède une activité de signalisation intracellulaire utilisant le calcium comme messager secondaire. Dans un deuxième temps, nous avons examiné la capacité de N-glycoformes recombinantes de nucléoline à interagir avec elles-mêmes, mais aussi avec plusieurs des ligands naturels de la molécule. Nous avons mis en évidence la capacité de la nucléoline à interagir avec elle-même en fonction de son état de glycosylation, et un rôle de cette glycosylation dans la modulation de l interaction de la nucléoline avec certains ligands, et donc probablement dans ses fonctions. Enfin, nous avons étudié le rôle de la N-glycosylation de la nucléoline dans son trafic. Nous avons démontré que seules les cellules qui expriment une nucléoline recombinante dont la N-glycosylation est possible présentent la nucléoline à leur surface, et que la nucléoline transite par les endosomes précoces et le trans-Golgi.Nos travaux suggèrent que la N-glycosylation est une modification essentielle de la nucléoline, non seulement pour son expression et sa distribution à la surface cellulaire, mais également pour ses fonctions.Nucleolin, a ubiquitous protein, is involved in cell growth and proliferation. In addition to its nuclear localization, it is also expressed at the cell surface. Atypical N- and O-glycosylated forms of surface nucleolin have been previously evidenced in our laboratory. We postulated that N-glycosylation could change trafficking, interactions and functions of nucleolin. First, we have evidenced that the only N-glycosylated nucleolin isoform is present on the cell surface and that glycosylation is an absolute requirement for its expression on cells, and that nucleolin can induce signals in cells using calcium as a secondary messenger. Second, we have investigated the ability of recombinant nucleolin N-glycoforms to interact with themselves, but also with several nucleolin ligands. We have demonstrated that nucleolin is able to interact with itself and that glycosylation may modulate, or not, the interactions with the ligands. Hence, N-glycosylation may play an important role in the modulation of nucleolin functions. Lastly, we have studied the role of nucleolin N-glycosylation in its trafficking. We have demonstrated that only the cells expressing recombinant nucleolin which was not mutated in its N-glycosylation sites also expressed nucleolin on their surface, and that nucleolin passes through early endosomes and the trans-Golgi.Our work suggests that N-glycosylation is an essential post-translational modification of nucleolin, not only for its expression and distribution at the surface of cells, but also for its functions.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Glycosylation network mapping and site-specific glycan maturation in vivo

Glycoprotein processing along a complex highly compartmentalized pathway is a hallmark of eukaryotic cells. We followed the kinetics of intracellular, site-specific glycan processing of a model protein with five distinct N-glycosylation sites and deduced a mathematical model of the secretory pathway that describes a complex set of processing reactions localized in defined intracellular compartments such as the endoplasmic reticulum the Golgi, or the lysosome. The model was able to accommodate site-specific N-glycan processing and we identified phosphorylated glycan structures of the mannose-6-phosphate pathway responsible for the lysosomal sorting of the glycoprotein. Importantly, our model protein can take different routes of the cellular secretory pathway, resulting in an increased glycan complexity of the secreted protein.ISSN:2589-004

Mutations in STT3A and STT3B cause two congenital disorders of glycosylation

We describe two unreported types of congenital disorders of glycosylation (CDG) which are caused by mutations in different isoforms of the catalytic subunit of the oligosaccharyltransferase (OST). Each isoform is encoded by a different gene (STT3A or STT3B), resides in a different OST complex and has distinct donor and acceptor substrate specificities with partially overlapping functions in N-glycosylation. The two cases from unrelated consanguineous families both show neurologic abnormalities, hypotonia, intellectual disability, failure to thrive and feeding problems. A homozygous mutation (c.1877T \u3e C) in STT3A causes a p.Val626Ala change and a homozygous intronic mutation (c.1539 + 20G \u3e T) in STT3B causes the other disorder. Both mutations impair glycosylation of a GFP biomarker and are rescued with the corresponding cDNA. Glycosylation of STT3A- and STT3B-specific acceptors is decreased in fibroblasts carrying the corresponding mutated gene and expression of the STT3A (p.Val626Ala) allele in STT3A-deficient HeLa cells does not rescue glycosylation. No additional cases were found in our collection or in reviewing various databases. The STT3A mutation significantly impairs glycosylation of the biomarker transferrin, but the STT3B mutation only slightly affects its glycosylation. Additional cases of STT3B-CDG may be missed by transferrin analysis and will require exome or genome sequencing

Glycan–protein interactions determine kinetics of N-glycan remodeling

A hallmark of N-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemical in vitro analysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of the N-glycans for their processing enzymes derived from the MD simulations, N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure of the glycoprotein PDI affected its N-glycan remodeling in a site-specific way. We propose that the observed differential N-glycan reactivities depend on the surrounding protein tertiary structure and lead to different glycan structures in the same protein through kinetically controlled processing pathways