title: Intracellular trafficking and secretion of mouse mesencephalic astrocyte-derived neurotrophic factor.
abstract: Recently, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent cell death under some pathophysiological conditions. MANF, also referred to as arginine rich, mutated in early stage of tumors (Armet), was identified as an endoplasmic reticulum (ER) stress-inducible factor. Using RT-PCR, we found two variants of MANF mRNA: wild type, which contains exon 1 (wt-MANF), and one lacking exon 1, which is presumably not secreted (ΔΝ-MANF) in Neuro2a cells. The latter has a putative translational start site upstream of the second exon in the mouse MANF gene. Comparing the expression of wt-MANF with that of ΔΝ-MANF, we found that the amount of intracellular ΔΝ-MANF was much lower than that of wt-MANF. Furthermore, ΔΝ-MANF was not detected in the culture medium after its transient transfection into Neuro2a cells. Deletion of several α-helices of mouse MANF decreased its intracellular stability and secretion. Secretion of wt-MANF was almost completely inhibited by either treatment with brefeldin A (BFA), which disrupts the Golgi apparatus structure, or overexpression of a dominant negative Sar1 (Sar1[H79G]), which is reported to impair COPII-mediated transport from the ER to the Golgi apparatus. In addition, the enforced expression of glucose-regulated protein 78 kDa (GRP78) attenuated the secretion of wt-MANF and led to its intracellular accumulation. MANF lacking the four C-terminal amino acids (ΔC-MANF) accumulated at low levels in the cells, but its intracellular level was increased by GRP78 overexpression. The amount of ΔC-MANF in the culture medium was partially down-regulated after co-transfection of GRP78. Substitution of the amino acids RTDL at the C-terminus of mouse MANF with KDEL, the canonical ER localization signal in GRP78, markedly decreased MANF secretion and its secretion was further attenuated by GRP78 overexpression. Taken together, our data show that the secretion of MANF is regulated via COPII-mediated transport and that its C-terminus could be responsible for its retention in the ER through GRP78. The alternate isotype, ΔΝ-MANF, may be less stable in cells than wt-MANF and may not be secreted extracellularly.
authors: Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. oohashi@gifu-u.ac.jp
authors: Kentaro
authors: K
authors: Oh-Hashi
authors: Kensuke
authors: K
authors: Tanaka
authors: Hisashi
authors: H
authors: Koga
authors: Yoko
authors: Y
authors: Hirata
authors: Kazutoshi
authors: K
authors: Kiuchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D022181:COP-Coated Vesicles / Q000378:metabolism; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005091:Exons; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; C534671:MANF protein, mouse; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D020126:Brefeldin A; D019284:Thapsigargin; D020559:Monomeric GTP-Binding Proteins