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Characterisation of Semaphorin 3A-Chondroitin Sulphate Interaction in the Central Nervous System Lynda Djerbal

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Characterisation of Semaphorin 3A-Chondroitin Sulphate Interaction in the Central Nervous System Lynda Djerbal Characterisation of semaphorin 3A-chondroitin sulphate interaction in the central nervous system Lynda Djerbal To cite this version: Lynda Djerbal. Characterisation of semaphorin 3A-chondroitin sulphate interaction in the central nervous system. Biochemistry, Molecular Biology. Université Grenoble Alpes, 2018. English. NNT : 2018GREAV041. tel-02144589v2 HAL Id: tel-02144589 https://tel.archives-ouvertes.fr/tel-02144589v2 Submitted on 4 Jun 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE LA COMMUNAUTE UNIVERSITE GRENOBLE ALPES Spécialité : Biologie structurale et nanobiologie Arrêté ministériel : 25 mai 2016 Présentée par « Lynda DJERBAL » Thèse dirigée par Hugues LORTAT-JACOB (DR, UMR5075-IBS- UGA-CNERS-CEA) et Jessica KWOK (DR, université de Leeds) préparée au sein du groupe Structure et Activité des Glycosaminoglycans à l’Institut de Biologie Structurale- UMR5075 dans l'École Doctorale Chimie et Science du Vivant Characterisation of semaphorin 3A-chondroitin sulphate interaction in the central nervous system Thèse soutenue publiquement le «30 Novembre 2018 », devant le jury composé de : Pr Dulce PAPY-GARCIA Professeur d’université, université Paris Est Créteil, Rapporteur Pr Joost VERHAAGEN Professeur, Netherlands institute for neuroscience, Rapporteur Dr Jean-Maurice MALLET Directeur de recherche, ENS Paris, Examinateur Dr Anne IMBERTY Directeur de recherche, CERMAV, Président 1 2 Summary Perineuronal nets (PNNs) are the key regulators of neuronal plasticity and regeneration in the mature central nervous system (CNS). They are a unique and highly organised extracellular matrix (ECM) structure, found around sub-population of neurons, composed mainly of chondroitin sulphate proteoglycan (CSPG). Chondroitin sulphate (CS) is a linear polysaccharide belonging to glycosaminoglycans (GAGs) family. The sulphation pattern defines different types of CS, which interact with different signalling proteins including those regulating axonal outgrowth and guidance such as semaphorin 3A (Sema3A). Sema3A is a secreted chemorepulsive protein found accumulated in the PNN through its interaction with CS. This process is believed to potentiate Sema3A signalling through plexin A1 (PlxnA1) and neuropilin 1 (Nrp1) and regulate plasticity and regeneration. The aim of the thesis project is to characterise the interface of Sema3A-CS interaction. For this purpose, Sema3A is expressed in eukaryote cells and purified. Interestingly, two major forms were obtained: a full length Sema3A (90 kDa) which remains attached to the cell surface GAGs and a truncated form without the C-ter part (65 kDa) which is released to the culture medium. With the use of surface plasmon resonance (SPR), we observed that full length Sema3A binds selectively to CS-E (4,6-disulphated chondroitin) and heparan sulphate with a high affinity (KD in the sub pM range), while the truncated Sema3A does not bind to any GAG. Four putative GAG-binding sequences were identified in the C-ter of Sema3A and mutated using site directed mutagenesis. SPR analysis then revealed that two out of these four sites are required for the binding to CS-E. The importance of these GAG-binding sequences in inhibition of neurites outgrowth of dorsal root ganglion neurons in culture was also reported, indicating thus the importance of GAG-binding in Sema3A signalling. In parallel, the minimal required sequence of Sema3A-binding of CS-E was determined as being a tetrasaccharide. The Sema3A-CS interface was thus characterized. Furthermore, quartz crystal microbalance with dissipation monitoring analysis suggested that Sema3A could crosslink GAG chains. This suggests Sema3A could be involved in stabilising the PNN network and induces mechanical changes on neuronal surface. The detail characterization of Sema3A-CS interaction may enable the design of new strategies aiming at enhancing plasticity and regeneration for neurodegenerative diseases or spinal cord injury. 3 4 Acknowledgements Je remercie les membres du Jury : Joost VERHAAGEN, Dulce PAPY-GARCIA, Jean-Maurice MALLET et Anne IMBERTY d’avoir accepté d’évaluer mes travaux de thèse. Je ne pourrais jamais remercier assez mon directeur de thèse Hugues. Quand je suis venue pour l’entretien et je t’ai vu pour la première fois, je me suis dit « il a l’air très bien ce directeur de thèse» et après trois ans je le pense toujours. Merci de m’avoir fait confiance dès le début et de m’avoir donné l’opportunité de faire une thèse dans des conditions « optimales ». Merci pour tout ce que j’ai appris durant ces trois années de thèse. Merci pour tes encouragements et tes conseils pour les manips, rédaction, présentations... Merci de t’être toujours rendu disponible et cela malgré ton emploi du temps chargé. J’ai vraiment apprécié le travail avec toi sur le plan scientifique et humain. Je suis très contente que mon post-doc sera toujours en collaboration avec toi. Infiniment Merci Hugues ! Un très grand merci à mon autre directeur de thèse Jessica. Je t’ai aimé dès notre premier skype pour l’entretien. Je me suis aussi dit « qu’est-ce qu’elle est gentille ! » Et cela ne s’est fait que confirmer au cours de ces trois années. Et je n’oublierai pas ce que tu m’as dit au début de ma thèse « fais toutes les erreurs que t’as à faire pendant la thèse, car c’est pendant la thèse qu’on apprend » et cela m’a mis à l’aise. J’apprécie ton dynamisme et ton esprit scientifique. Merci pour tout ce que tu m’as appris. Merci pour ta confiance, ton soutien, tes encouragements et ta gentillesse. Merci pour l’opportunité que tu m’as offerte de poursuivre sur la même thématique que ma thèse et surtout sur ma protéine préférée «Sema3A ». Je suis très contente de poursuivre en post-doc avec toi. Je remercie les membres de ma deuxième famille « SAGAG ». Rabia, merci beaucoup pour ton amour, ta gentillesse et tes délicieux gâteaux. Je me rappellerai toujours du premier jour quand je t’ai vu à l’entretien, t’étais adorable comme toujours et je me suis dit j’aimerais bien être prise dans cette équipe. Et merci de m’avoir appris à faire les WB et dot blot. Romain, t’es le chercheur le plus cool que je connaisse et cela je l’ai su, avant même de te rencontrer, dans le premier mail que tu m’as envoyé. J’apprécie énormément ton enthousiasme, ton raisonnement scientifique et ton sens de l’humour et merci. Merci de m’avoir appris « heparin-beads approach » et l’analyse disaccharidique. Evelyne, je suis chanceuse que t’as intégré le SAGAG avant que je parte, je suis très contente de t’avoir connue. Merci pour ta gentillesse, ta bonté et ton aide. Rabia et Evelyne, je ne dirai pas vous êtes les mamans du SAGAG mais plutôt les grandes sœurs. Yoan, quand je t’es vu la première fois je me suis dit « pas très souriant, ce post-doc» mais en fait c’est juste une première impression, t’es sympa et très serviable. Merci d’avoir toujours pris le temps de discuter les difficultés que j’ai rencontrées surtout pendant la purification de la Sema. Merci de m’avoir montré tous les logiciels et tous les sites requis pour mon projet. Rana, ma petite Rana, j’ai su qu’on allait devenir amies avant même de te rencontrer. Tout aurait été moins drôle si tu n’étais pas là. Je suis très contente de t’avoir rencontrée. Merci pour tous les moments agréables passés au labo surtout après 19h, au bureau, au coin café en dehors du travail. Merci pour ta complicité dans tout et surtout ton amitié. Merci à tous les membres du SAGAG d’avoir contribué à ma formation scientifique. On dit « il faut tout un village pour élever un enfant » et moi je dis «il faut tout un groupe de recherche pour 5 former un étudiant ». Vous allez beaucoup me manquer mais je reviendrai vous voir. Je m’arrête là pour le SAGAG sinon je vais écrire une deuxième thèse de remerciements … Je remercie les HBB de la « pause 17:00 » : Rana (tu vois c’est comme les groupes de FB et WhatsApp, t’es partout), Rida, je suis très contente d’avoir fait ta connaissance. T’es la libanaise la plus drôle que je connaisse après Rana biensûr. T’es une fille formidable. Merci pour tous les moments géniaux qu’on a passé ensemble à l’IBS ou en dehors de l’IBS et merci pour le GreenMango du mois de juillet. Kevin, infatigable Kevin, j’étais ravie de rédiger ma thèse au même temps que toi. Merci pour les amandes et ton sens de l’humour. Guillaume, merci pour ta gentillesse, et les friandises du distributeur et ton sens de détection de fautes d’orthographe. Rime ma compatriote, merci pour ton soutien, ta gentillesse et ta bonté. Ilham, mon autre compatriote, contente de t’avoir rencontrée. Merci Elodie pour ta gentillesse. Merci aux voisins du SAGAG, IRPAS, pour les pauses café du matin, leur gentillesse et de partager le matériel de leur labo. Merci à tous les amis que j’ai rencontrés à l’IBS : Muge, Tomas, Laura, Catarina, Amal, Stefaniia, Aldo, Simon, Silvia, Quentin, Marko, Justine… Je tiens à remercier également tous les gens qui m’ont aidé à la réalisation des manips. Merci à Ralf RICHTER de m’avoir accueillie dans son laboratoire à Leeds.
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