The Lysosomal Targeting of Acid Sphingomyelinase Xiaoyan Ni Department of Anatomy and Cell Biology McGiII University Montreal, Canada A thesis submitted to McGiII University in partial fulfillment of the requirements of the degree of Master in Science © Xiaoyan Ni, 2005 Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre référence ISBN: 978-0-494-22756-5 Our file Notre référence ISBN: 978-0-494-22756-5 NOTICE: AVIS: The author has granted a non­ L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell theses le monde, à des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse. While these forms may be included Bien que ces formulaires in the document page cou nt, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada ABSTRACT Acid sphingomyelinase (ASM), a member of the saposin like protein (SAPLIP) family, is a lysosomal hydrolase that converts sphingomyelin to ceramide. The deficient activity of ASM causes a variant form (i.e., type AIB) of the inherited disorder Niemann-Pick disease. The lysosomal targeting mechanism of ASM has not been conclusively identified. Previous studies suggested that ASM could use another membrane-associated receptor as weIl as M6P receptor to target lysosomes. Sortilin, a type 1 transmembrane glycoprotein, belongs to a novel family of receptor proteins. Both the luminal domain and the cytoplasmic domain of sortilin show structural features typical of receptors involved in lysosomal or vacuolar targeting. Using a dominant-negative sortilin construct lacking the cytoplasmic taïl, 1 proved that sortilin was involved in the lysosomal targeting of ASM. Confocal microscopy revealed that truncated sortilin partially inhibited the lysosomal targeting of ASM in COS 7 cells and completely abolished the lysosomal targeting of ASM in I-cells. Pulse-chase experiments also suggested that sortilin was involved in normal sorting of newly synthesized ASM. Over-expression of truncated sortilin accelerated and enhanced the secretion of ASM from COS 7 cells and I-cells. Co-immunoprecipitation assays further confmned the interaction between sortilin and ASM. 1 also observed that the lysosomal transport of ASM was reduced to sorne extent in I-cell disease fibroblasts as compared to normal fibroblasts. In conclusion, both the M6P receptor and sortilin mediate lysosomal targeting of ASM. 1 RÉSUMÉ L'acide sphingomyelinase (ASM), un membre de la famille des protéines saposine (SAPLIP), est un hydrolase lysosomal qui convertit le sphingomyeline en ceramide. L'activité déficiente de l'ASM cause une forme variante (c'est-à-dire le type AIB) de la maladie héréditaire Niemann-Pick. Le transport de l'ASM aux lysosomes n'a pas été déterminé jusqu'à présent. Plusieurs études précédentes ont suggéré que l'ASM pourrait être transporté aux lysosomes par deux mécanismes dont un qui implique le récepteur M6P. Le récepteur sortilin, un glycoprotéine de type I appartient à une nouvelle famille de récepteurs. Le domaine luminal ainsi que le domaine cytoplasmique du récepteur sortilin montre des caractéristiques typiques des récepteurs impliqués dans le transport aux lysosomes ou aux vacuoles. Utilisant un agencement du récepteur sortilin qui ne possédait pas de portion cytoplasmique, nous avons démontré que le sortilin est impliquée dans le transport de l' ASM aux lysosomes. La microscopie confocale a révélé que le sortilin sans portion cytoplasmique a diminué le transport de l' ASM dans des cellules COS-7 et a totalement interrompu ce même transport dans les cellules ICD. Les résultats des expériences pulse-chase suggèrent aussi que le sortilin est impliqué dans le transport normal de l' ASM. La surexpression du sortilin sans portion cytoplasmique a accéléré la sécretion de l' ASM dans des cellules COS-7 et ICD. Des tests de coimmunoprécipitation ont comfirmé l'existence d'interaction entre le sortilin et l'ASM. Nous avons aussi observé que le transport de l'ASM aux lysosomes était réduit dans les fibroblasts de patients atteints de l'ICD comparées à des fibroblasts normaux. Finalement, le récepteur M6P ainsi que le sortilin sont impliqués dans le transport de l' ASM aux lysosomes. ii To my parents and husband who have been a source of inspiration and love. 111 ACKNOWLEDGEMENTS 1 would first like to appreciate my supervisor Dr. Carlos Morales for his expert guidance, continuaI encouragement and support throughout my master studies. And 1 would like to extend gratitude to Dr. Yves Clermont for his constant caring and encouragement during my studies. Jibin Zeng and Jacob Hassan are to be infinitely thanked for their helpful technical assistance on my molecular biology experiments and immunofluorescence staining. Thanks to Archana Srivastava for help trouble shoot with the confocal microscope. 1 thank my committee meeting advisors: Dr. Louis Hermo, Dr. John Presley and Dr. Michael Greenwood for their professional advice on my research project. Especially, 1 would like to appreciate Dr. Louis Hermo for his constructive criticism on my seminars. 1 also thank Dr. Volkan Seyrantepe for providing me normal fibroblast cell line and thank Dr. C.M .. Peterson for giving me anti-sortilin antibody. Special thanks to Maryssa Canuel and Myriam Block for their excellent French translation of the abstracto Finally, 1 would like to express my most sincere thanks to my husband for his always support and to people in Morales's lab: Jibin Zeng, Maryssa Canuel, Libin Yuan and Ji-Hae Kim, for their enthusiasm, emotional support and friendship. IV TABLE OF CONTENTS Abstract 1 Resume II Dedication III Acknowledgements iv Table of contents v List of figures Vlll Introduction 1 Literature review 4 1 Saposin-like protein (SAPLIP) family 5 1 Members of SAPLIP family 5 2 Structural features of SAPLIPs 6 3 Lipid-binding ability of SAPLIPs 8 4 Role of saposin-like domain in the intracellu1ar targeting 9 ofSAPLIPs 5 Acid sphingomyelinase belongs to the SAPLIP family Il II Targeting of Iysosomai proteins 13 1 Mannose 6-phosphate dependent transport of lysosomal proteins. 13 1.1 Biosynthesis and phosphorylation 13 1.2 Phosphotransferase and I-cell disease 14 1.3 The mannose 6-phosphate (M6P) receptors 16 v 1.5 Final destination of the mannose 6- phosphate receptors 19 2 Mannose 6-phosphate independent transport of lysosomal 19 proteins 2.1 Mannose 6-phosphate independent vacuolar targeting in yeast 19 2.2 Mannose 6-phosphate independent lysosomal targeting in 20 mammalian ceUs III The VpslOp superfamily 22 1 A novel receptor family 22 2 The genes ofhuman Vpsl0p family 22 3 Two common structural features ofVpsl0p receptors 23 4 Vps 1Op family receptors bind unrelated ligands 24 5 Structure and functions of cytoplasmic tails of V ps 1Op receptors 25 6 Sortilin: a multifunction protein 26 IV GGAs are required for the sorting soluble lysosomal proteins 28 1 A novel family proteins interacting with ADP-ribosylation 28 proteins (ARFs) 2 Structure of GGA proteins 29 3 Functions of GGAs in the yeast 29 4 GGAs interacting proteins 30 5 SortiliniGGA-mediated pathways and the sorting of ASM 31 Materials and methods 32 Reagents and antibodies 32 VI DNA constructs 32 Celllines and cell cultures 33 Transfections of COS-7 cells 33 Electroporation 34 Immunofluorescence and confocal microscopy 34 Co-immunoprecipitation assays 35 Metabolic labeling and pulse-chase experiments 35 Results 37 Lysosomal targeting of ASM 37 Sortilin interacts with GGA through its cytoplasmic domain 38 Dominant-negative sortilin altered the lysosomal targeting 39 ofASM ASM targeting is abolished when the M6P and sortilin pathways 40 are blocked Dominant-negative sortilin enhances the secretion of ASM 41 Sortilin binds and interacts with ASM 42 Discussion 44 Conclusion 52 Abbreviation 53 References 57 Appendix - Research Compliance Certificate 66 vu LIST OF FIGURES Figure 1 - Intracellular localization of ASM in I-Cells. Figure 2 - Lysosomal targeting of ASM in nonnal fibroblasts and in I-cells. Figure 3 - Expression of sortilin-EGFP. Figure 4 - Intracellular localization of full-length sortilin and truncated sorti lin. Figure 5 - Effects of truncated GGA on intracellular trafficking of sortilin. Figure 6 - Co-immunoprecipitation assay showing association of sortilin and GGA. Figure 7 - Effects offull-length and truncated sortilins on lysosomal targeting of ASM. Figure 8 - Effect of truncated GGA on lysosomal trafficking of ASM in COS 7 Cells. Figure 9 - Effect oftruncated sortilin on lysosomal targeting of ASM in I-cells. FigurelO- Influence oftruncated sortilin on the sorting of ASM in COS7 cells and I-cells. Figurell- Co-immunoprecipitation showing interaction between sortilin and ASM.