title: Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity.
abstract: Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF's intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: K
authors.initials: K
authors.lastname: Mätlik
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: Li-ying
authors.initials: LY
authors.lastname: Yu
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: A
authors.initials: A
authors.lastname: Eesmaa
authors.affiliation: Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, PO Box 65, Helsinki 00014, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Hellman
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: P
authors.initials: P
authors.lastname: Lindholm
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: J
authors.initials: J
authors.lastname: Peränen
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: E
authors.initials: E
authors.lastname: Galli
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: J
authors.initials: J
authors.lastname: Anttila
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Saarma
authors.affiliation: Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, PO Box 65, Helsinki 00014, Finland.|Department of Biological and Environmental Sciences, Nanoscience center, University of Jyvaskyla, PO Box 35, FI-40014, Jyvaskyla, Finland.|Department of Chemistry, Nanoscience center, University of Jyvaskyla, PO Box 35, FI-40014, Jyvaskyla, Finland.
authors.forename: P
authors.initials: P
authors.lastname: Permi
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Airavaara
authors.affiliation: Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.|Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia.
authors.forename: U
authors.initials: U
authors.lastname: Arumäe
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002470:Cell Survival; D003545:Cysteine / Q000235:genetics; D004195:Disease Models, Animal; D005047:Etoposide / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D009154:Mutation / Q000235:genetics; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D020521:Stroke / Q000473:pathology; D013329:Structure-Activity Relationship; D017783:Superior Cervical Ganglion / Q000166:cytology
chemical_list: C099695:MANF protein, human; D009414:Nerve Growth Factors; D018696:Neuroprotective Agents; D011994:Recombinant Proteins; D005047:Etoposide; D003545:Cysteine