Identification of N-glycan oligomannoside isomers in the diatom Phaeodactylum tricornutum

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Characterization of biopharmaceuticals produced from algae

Le virus d'immunodéficience humaine (VIH) affecte plus de 38 millions de personnes dans le monde et est responsable de près de 700 000 décès par an. Parmi les solutions thérapeutiques développées, la recherche d’anticorps monoclonaux est très active depuis plusieurs années. Ces biomédicaments ciblent soit les processus de multiplication du virus, soit la stimulation du système immunitaire afin de détruire les cellules infectées. Dans ce contexte, un anticorps bispécifique anti-VIH a été conçu par la société AlbaJuna Therapeutics SL puis produit par celle-ci dans des cellules HEK et par la société Leaf Expression Systems dans des plantes de tabac. Dans le cadre de mes travaux de thèse, la microalgue Phaeodactylum tricornutum a été transformée génétiquement pour produire cet anticorps anti-VIH. Les glycoprotéines thérapeutiques produites dans des cellules HEK et dans des plantes de tabac ont ensuite été caractérisées par différentes approches analytiques. Ces travaux ont permis de confirmer la séquence protéique dans son ensemble et d’établir que la N-glycosylation est fonction du système d'expression utilisé et du compartiment d’adressage de la protéine thérapeutique. En complément, les structures détaillées des isomères majoritaires de N-glycanes oligomannosidiques présents sur les protéines endogènes de la diatomée Phaeodactylum tricornutum ont été déterminées. Ces oligosaccharides sont identiques à ceux synthétisés par les cellules de mammifères et donc non immunogènes. En conséquence, Phaeodactylum tricornutum peut être considérée comme un système adapté à la production de glycoprotéines dédiées à la thérapie humaine.The human immunodeficiency virus (HIV) affects more that 38 millions of people worldwide and is responsible for about 700 000 deaths per year. Among the therapeutic treatments that are currently investigated, search for monoclonal antibodies is very active since several years. These biopharmaceuticals are developed either to prevent the virus multiplication or to stimulate the immune system in order to kill infected cells. With the aim to develop new anti-HIV specific antibodies, the company AlbaJuna Therapeutics SL has designed a bispecific antibody that was produced by this company in HEK mammalian cells or in tobacco plants by Leaf Expression Systems. In the context of my PhD thesis, the diatom Phaeodactylum tricornutum has been transformed in order to produce this anti-HIV antibody. In parralel, the therapeutic glycoproteins produced in HEK cells and tobacco plants have been characterized. We have confirmed the overall sequence of the protein backbone and demonstrated that the protein N-glycosylation is dependent on the expression system and the compartment where the therapeutic protein was targeted. In addition, we have investigated the structures of oligomannoside isomers N-linked to proteins of Phaeodactylum tricornutum and demonstrated that these oligosaccharides are identical to those synthesized in mammals, and as a consequence are non immunogenic. Thus, Phaeodactylum tricornutum appears to be a suitable expression system for the production of human therapeutic glycoproteins

Analyses structurales d'anticorps multifonctionnels dirigés contre le VIH

International audienc

Production and characterization of biopharmaceuticals in microalgae

International audienc

N- and O-Glycosylation Pathways in the Microalgae Polyphyletic Group

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A Peptidomic Approach to Characterize Peptides Involved in Cerebellar Cortex Development Leads to the Identification of the Neurotrophic Effects of Nociceptin

International audienceThe cerebellum is a brain structure involved in motor and cognitive functions. The development of the cerebellar cortex (the external part of the cerebellum) is under the control of numerous factors. Among these factors, neuropeptides including PACAP or somatostatin modulate the survival, migration and/or differentiation of cerebellar granule cells. Interestingly, such peptides contributing to cerebellar ontogenesis usually exhibit a specific transient expression profile with a low abundance at birth, a high expression level during the developmental processes, which take place within the first two postnatal weeks in rodents, and a gradual decline toward adulthood. Thus, to identify new peptides transiently expressed in the cerebellum during development, rat cerebella were sampled from birth to adulthood, and analyzed by a semi-quantitative peptidomic approach. A total of 33 peptides were found to be expressed in the cerebellum. Among these 33 peptides, 8 had a clear differential expression pattern during development, 4 of them i.e. cerebellin 2, nociceptin, somatostatin and VGF [353-372], exhibiting a high expression level during the first two postnatal weeks followed by a significative decrease at adulthood. A focus by a genomic approach on nociceptin, confirmed that its precursor mRNA is transiently expressed during the first week of life in granule neurons within the internal granule cell layer of the cerebellum, and showed that the nociceptin receptor is also actively expressed between P8 and P16 by the same neurons. Finally, functional studies revealed a new role for nociceptin, acting as a neurotrophic peptide able to promote the survival and differentiation of developing cerebellar granule neurons

Production and characterization of biopharmaceuticals in microalgae Glycobiology

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Towards understanding the regulation of the N-glycosylation pathway in regards to the huge diversity of the N-glycan structures identified in Eukaryotes

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Towards understanding the extensive diversity of protein N‐glycan structures in eukaryotes

International audienceN-glycosylation is an important post-translational modification of proteins that has been highly conserved during evolution and is found in Eukaryota, Bacteria and Archaea. In eukaryotes, N-glycan processing is sequential, involving multiple specific steps within the secretory pathway as proteins travel through the endoplasmic reticulum and the Golgi apparatus. In this review, we first summarize the different steps of the N-glycan processing and further describe recent findings regarding the diversity of N-glycan structures in eukaryotic clades. This comparison allows us to explore the different regulation mechanisms of N-glycan processing among eukaryotic clades. Recent findings regarding the regulation of protein N-glycosylation are highlighted, especially the regulation of the biosynthesis of complex-type N-glycans through manganese and calcium homeostasis and the specific role of transmembrane protein 165 (TMEM165) for which homologous sequences have been identified in several eukaryotic clades. Further research will be required to characterize the function of TMEM165 homologous sequences in different eukaryotic clades

Functional role of ODN, an endogenous peptide released by astrocyte, under oxydatif stress

National audienc