title: Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model.
abstract: Mammalian MANF and CDNF proteins are evolutionarily conserved neurotrophic factors that can protect and repair mammalian dopaminergic neurons in vivo. In Drosophila, the sole MANF protein (DmManf) is needed for the maintenance of dopaminergic neurites and dopamine levels. Although both secreted and intracellular roles for MANF and CDNF have been demonstrated, very little is known about the molecular mechanism of their action. Here, by using a transgenic rescue approach in the DmManf mutant background we show that only full-length MANF containing both the amino-terminal saposin-like and carboxy-terminal SAP-domains can rescue the larval lethality of the DmManf mutant. Independent N- or C-terminal domains of MANF, even when co-expressed together, fail to rescue. Deleting the signal peptide or mutating the CXXC motif in the C-terminal domain destroys the activity of full-length DmManf. Positively charged surface amino acids and the C-terminal endoplasmic reticulum retention signal are necessary for rescue of DmManf mutant lethality when DmManf is expressed in a restricted pattern. Furthermore, rescue experiments with non-ubiquitous expression reveals functional differences between the C-terminal domain of human MANF and CDNF. Finally, DmManf and its C-terminal domain rescue mammalian sympathetic neurons from toxin-induced apoptosis in vitro demonstrating functional similarity of the mammalian and fly proteins. Our study offers further insights into the functional conservation between invertebrate and mammalian MANF/CDNF proteins and reveals the importance of the C-terminal domain for MANF activity in vivo.
authors.affiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Riitta
authors.initials: R
authors.lastname: Lindström
authors.forename: Päivi
authors.initials: P
authors.lastname: Lindholm
authors.forename: Jukka
authors.initials: J
authors.lastname: Kallijärvi
authors.forename: Li-Ying
authors.initials: LY
authors.lastname: Yu
authors.forename: T Petteri
authors.initials: TP
authors.lastname: Piepponen
authors.forename: Urmas
authors.initials: U
authors.lastname: Arumäe
authors.forename: Mart
authors.initials: M
authors.lastname: Saarma
authors.forename: Tapio I
authors.initials: TI
authors.lastname: Heino
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis / Q000502:physiology; D004298:Dopamine / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004331:Drosophila melanogaster / Q000254:growth & development / Q000502:physiology*; D005804:Genes, Lethal; D006801:Humans; D007814:Larva / Q000378:metabolism; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: C588382:CDNF protein, Drosophila; D029721:Drosophila Proteins; C539344:MANF protein, Drosophila; D009414:Nerve Growth Factors; D004298:Dopamine
references.citation: Neuron. 1999 Mar;22(3):451-61
references.pmid: 10197526
references.citation: Mol Cell Neurosci. 2008 Nov;39(3):356-71
references.pmid: 18718866
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D706-14
references.pmid: 22127867
references.citation: BMC Genomics. 2012 Apr 11;13:134
references.pmid: 22494833
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2429-34
references.pmid: 19164766
references.citation: Nat Cell Biol. 2003 Apr;5(4):352-7
references.pmid: 12652309
references.citation: Cell. 1995 Jul 14;82(1):67-76
references.pmid: 7606787
references.citation: Mol Cell Biochem. 2012 Apr;363(1-2):35-41
references.pmid: 22120531
references.citation: J Biol Chem. 2011 Jan 28;286(4):2675-80
references.pmid: 21047780
references.citation: Biochem J. 2005 Jul 15;389(Pt 2):249-57
references.pmid: 15992358
references.citation: FEBS Lett. 2010 Apr 16;584(8):1536-42
references.pmid: 20214902
references.citation: Curr Opin Cell Biol. 2011 Apr;23(2):239-52
references.pmid: 21288706
references.citation: Cell Cycle. 2007 Apr 1;6(7):830-5
references.pmid: 17387279
references.citation: J Biol Chem. 2012 Jul 27;287(31):25893-904
references.pmid: 22637475
references.citation: Dev Neurobiol. 2010 Apr;70(5):360-71
references.pmid: 20186704
references.citation: EMBO Rep. 2005 Jan;6(1):28-32
references.pmid: 15643448
references.citation: J Biol Chem. 1994 Oct 21;269(42):26559-67
references.pmid: 7929381
references.citation: J Neurosci. 2009 Jul 29;29(30):9651-9
references.pmid: 19641128
references.citation: J Mol Neurosci. 2003 Apr;20(2):173-88
references.pmid: 12794311
references.citation: Protein Eng Des Sel. 2009 Apr;22(4):233-41
references.pmid: 19258449
references.citation: Circ Res. 2008 Nov 21;103(11):1249-58
references.pmid: 18927462
references.citation: Cell Transplant. 2012;21(6):1213-23
references.pmid: 21943517
references.citation: Exp Cell Res. 2008 Aug 1;314(13):2454-67
references.pmid: 18561914
references.citation: Exp Neurol. 2010 Sep;225(1):104-13
references.pmid: 20685313
references.citation: Development. 1993 Jun;118(2):401-15
references.pmid: 8223268
references.citation: Synapse. 2006 May;59(6):321-9
references.pmid: 16437537
references.citation: Cell Struct Funct. 2007;32(1):41-50
references.pmid: 17507765
references.citation: Cell. 1994 Jan 14;76(1):77-87
references.pmid: 8287481
references.citation: Nature. 2007 Jul 5;448(7149):73-7
references.pmid: 17611540
references.citation: Biotechniques. 2004 May;36(5):784-8, 790
references.pmid: 15152597