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We recently demonstrated that the secretion of two novel endoplasmic reticulum (ER) stress-inducible proteins, cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) and mesencephalic astrocyte-derived neurotrophic factor (MANF), are oppositely regulated by the overexpression of 78 kDa glucose-regulated protein (GRP78). In the present study, we found that the co-transfection of CRELD2 and MANF remarkably enhanced the secretion of CRELD2 without affecting the expression level of GRP78. To identify the structural features of CRELD2 and MANF involved in this process, we generated several CRELD2 and MANF expression constructs. The deletion of the four C-terminal amino acids, either REDL in CRELD2 or RTDL in MANF, abolished the increased secretion of CRELD2 induced by the co-expression of MANF. The deleted mutation of MANF partially abolished the increased secretion of wild type CRELD2 (wtCRELD2) as a positive action of wild type MANF (wtMANF), even when we added the amino acid sequence RTDL at the C-terminus of each mutated MANF construct. Enhanced green fluorescent protein (EGFP), which was tagged with the signal peptide sequence at the N-terminus and four C-terminal amino acids (KEDL, REDL or RTDL), were retained intracellularly, but they did not enhance the secretion of wtCRELD2. Taken together, our data demonstrate that MANF is a factor in regulating the secretion of CRELD2 through four C-terminal amino acids, RTDL and REDL, and the fluctuation of intracellular MANF seems to potentiate the secretion of CRELD2.Key words endoplasmic reticulum; chaperone; 78 kDa glucose-regulated protein (GRP78); cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2); mesencephalic astrocyte-derived neurotrophic factor (MANF)The folding and modification of newly synthesized transmembrane and secretory proteins in the endoplasmic reticulum (ER) is maintained by a variety of mechanisms.1,2) Under some pathophysiological conditions, certain ER functions become disordered and then unfolded and/or misfolded proteins are accumulated in the ER.3,4) Abnormal protein retention results in ER stress and activation of the three canonical ERresident stress sensors; protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), 5) inositol-requiring enzyme 1 (IRE1) 6) and activating transcription factor 6 (ATF6), 7) induces a variety of genes.8-12) Among them, growth arrest and DNA damage-inducible protein 153 (GADD153) is well-known to promote stress-induced cell death by activating caspases. 8,9) ER resident molecular chaperones such as 78 kDa glucoseregulated protein (GRP78) are reported to alleviate the stress by properly folding and degrading unfolded proteins and attenuating ER stress signals. 10,11) We previously identified the cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) gene as a novel ER stress-inducible gene and demonstrate that ATF6 positively regulates the transcription of the CRELD2 gene through a well-conserved ER stress response element (ERSE) in the proximal regio...

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔCMANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔCMANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNFRTDL and CDNFSARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNFKTEL and CDNFHPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANFQTEL and MANFYPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANFKTEL and MANFHPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF.

In this study, we applied a highly sensitive small luciferase, NanoLuc, to establish a knock‐in cell line using the CRISPR/Cas9 system and characterized the endogenous promoter activity of the glucose‐regulated protein 78 (GRP78) gene. The N‐terminal region of the human GRP78 gene was fused to the NanoLuc gene and aligned with the puromycin‐resistant gene through the 2A peptide sequence and used as a knock‐in vector. The selected cells responded to both pharmacological and genetic ER stress and show NanoLuc‐based CRISPR/Cas9 system is a very useful tool to isolate gene‐edited cells and to characterize the endogenous promoter activity for genes of interest.