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. 2019 Jan 8;43(2):167–173. doi: 10.1002/cbin.11082

Quantitative analysis on secretion level of CDNF regulated by two key α‐helices in CDNF protein

Hao Liu 1, Lin Zhong 1, Chunling Zhao 2, Xiaolei Tang 3, Jun Yang 1,✉, Lei Gong 1,✉
PMCID: PMC13397405  PMID: 30548741

Abstract

Cerebral dopamine neurotrophic factor (CDNF) has been considered as potent candidates for the therapy of Parkinson's disease (PD) for which can promote the survival of midbrain dopaminergic neurons. In addition to secret out from cells like other classical neurotrophic factors (NTFs), CDNF can locate in the endoplasmatic reticulum (ER), where they can function as ER stress response protein to regulate ER stress. In our previous studies, we have found two helices, α1 and α7, which can regulate the intracellular trafficking and secretion of CDNF. α1 distruction can significantly retain CDNF protein in the ER, but α7 distruction induce most CDNF protein secreting out the cells. Then α1 and α7 regulate protein trafficking and secretion in opposite side. However, the exact secretion level of CDNF affected by α1 or α7 have not been sensitively quantified. In this study, we used nanoluciferase to quantify the secretion level of CDNF protein so that we could evaluate the impact of α1 and α7 on CDNF secretion or function.

Keywords: CDNF, endoplasmic reticulum stress, luciferase, neurotrophic factor, secretion

Introduction

Cerebral dopamine neurotrophic factor (CDNF) and the related mesencephalic astrocyte‐derived neurotrophic factor (MANF) form an evolutionarily conserved neurotrophic factor (NTF) family which can protect and repair the midbrain dopaminergic neurons (Lindholm et al., 2007; Lindahl et al., 2017). Thereby CDNF/MANF are considered as potent candidates for the therapy of Parkinson's disease (PD) (Lindholm et al., 2016; Tang et al., 2017). Unlike all other classical NTFs that are solely secreted proteins, CDNF/MANF present unique protein structure and function (Lindstrom et al., 2013; Norisada et al., 2016). They can secrete out the cells like other NTFs, but can also locate in the cells (Voutilainen et al., 2015; Garea‐Rodriguez et al., 2016). Thus, Voutilainen et al. (2015) proposed the hypothetical dual mode of action about CDNF/MANF. One mode is they secret out from cells like other NTFs and activate unknown receptors on the plasma membrane and anti‐apoptotic signaling pathways to protect injured neurons from death. The other is they locate in the endoplasmatic reticulum (ER), where they can function as ER stress response protein to regulate ER stress, maintain ER homeostasis and promote the cells survival.

There are eight α‐helices in the crystal structure of CDNF protein. By inserting a proline (P) into respective position to destroy the corresponding α‐helix structure in CDNF, we have found two helices which can regulate its intracellular trafficking and secretion (Sun et al., 2011; Liu et al., 2015b). One is α1 that is located in the N‐terminal saposin‐like domain. Disrupting of α1 can significantly retain CDNF protein in the ER and correspondingly reduce its secretion (Sun et al., 2011). The other is α7 that is located in the C‐terminal SAP‐domain. Disrupting of α7 can induce most CDNF protein secreting out the cells and little retaining in the cell body (Liu et al., 2015b). These findings have been verified by western blot or immunofluorescence staining. In this study, we used nanoluciferase (NanoLuc) which is a 19‐kDa highly active and small luciferase. The specific activity of NanoLuc is 150‐fold greater than that of firefly luciferase (Fluc) and Renilla luciferase (Rluc). So recently, NanoLuc is frequently used to analyze the transcriptional regulation of genes and the intracellular localization or secretion of proteins (Hall et al., 2012). We used pNLF1‐C vector (Promega) to construct CDNF and NanoLuc protein fusion plasmids. After the construction was transfected into cells, we detected the luciferase activity intracellular and extracellular to quantify the secretion level of CDNF protein so that we could evaluate the impact of α1 and α7 on CDNF secretion or function.

Materials and methods

Reagents and antibodies

Dulbecco's modified Eagle's minimum essential medium (DMEM) (Cat. # 11960–044), fetal bovine serum (Cat. # 12484–010), horse serum (Cat. # 26050–070), penicillin‐streptomycin (Cat. # 15140–122), L‐glutamine (Cat. # 21051–024) and G418 (Cat. # 11811–023), Lipofectamine 2000 transfection reagent (Cat. # 11668019) were all obtained from Invitrogen. Mouse anti‐HA (HA.11) antibody (Cat. # H6908) from Covance Research Products. Rabbit antibody against the ER marker calnexin (Cat. # SAB4503258) and rabbit antibody against the Golgi apparatus marker TGN38 (Cat. # T9826) were from Sigma. HRP‐conjugated goat anti‐mouse IgG antibodies (Cat. # 31430), Alexa Fluor 488‐conjugated goat anti‐mouse IgG antibody (Cat. # A32723) and Alexa Fluor 594‐conjugated goat anti‐rabbit IgG antibody (Cat. # A11012) were obtained from Invitrogen. pNLF1 C‐terminal NanoLuc Protein Fusion Vectors (Cat. # N1361) and Nano‐Glo® Luciferase Assay System (Cat. #N1110) were obtained from Promega. All other reagents were from Sigma except as specifically indicated.

Cell culture

PC12 (clone 615) cells were maintained in Dulbecco's modified Eagle's minimum essential medium (DMEM) containing 10% fetal bovine serum, 5% horse serum, supplemented with 100 U/mL penicillin‐streptomycin, and 2 mM l‐glutamine plus 200µg/ml G418.

Construction of plasmids

The plasmids of Wt (wild‐type CDNF), M1 (insert a proline between L44 and N45 to destruct α1) and M7 (insert a proline between L151 and H152 to destruct α7) have been constructed as the full‐length cDNA were subcloned into pcDNA3.1Neo expression vector and the HA epitope tag was added to the 3′ end of the CDNF by PCR method. On this basis, these full‐length cDNA sequence of CDNF and two mutants were amplified by PCR and subcloned into pNLF1‐C vector (Promega) using EcoR V and Xba I (MBI Fermentas) to construct fusion expression plasmids Wt‐NL, M1‐NL, and M7‐NL, respectively. All plasmids were confirmed by DNA sequence.

Western blot

Forty‐eight hours after the plasmids were transfected, cells were washed three times with PBS and incubated in serum‐free DMEM medium for 2 h at 37°C. Then, cell medium and cell lysate were collected and protein concentration was quantified by BCA Protein Assay Kit (Cat. # BCA1‐1KT, Sigma). Cell lysate protein was loaded 15µg and medium protein was loaded 40µg in every lane. Proteins were separated by SDS–PAGE gels, followed by transfered to PVDF membrane (Bio‐Rad, Hercules, CA). Then, membranes were blocked in 10% milk, incubated with mouse HA antibodies in 5% milk (1:1000), following incubated with HRP‐conjugated goat anti‐mouse IgG antibodies in 5% milk (1:2000). The intensity of immunoreactive bands were scanned and quantitated by MetaMorf Image (West Chester, PA) as described before (Sun et al., 2011).

Immunocytofluorescence and quantitative analysis

PC12 cells were subcultured on poly‐D‐lysine coated glass coverslips and transfected with Wt‐NL, M1‐NL and M7‐NL respectively. Forty‐eight hours after transfection, cells were fixed and permeabilized, then blocked by 1% BSA, and incubated with HA antibody (1: 500) followed by Alexa Fluor 488‐conjugated goat anti‐mouse IgG antibody (1: 1000). When performed co‐stainning with ER or Golgi marker proteins, cells were incubated with HA antibody and calnexin antibody or TGN38 antibody followed by Alexa Fluor 488‐conjugated goat anti‐mouse IgG antibody and Alexa Fluor 594‐conjugated goat anti‐rabbit IgG antibody.

Fluorescence images were acquired by a HQ2 cool CCD camera

mounted on a Nikon Eclipse TE2000‐U microscope equipped with a motorized Z drive, and assigned to a pseudo color (green, red). All of the images were collected using a 60× objective lens and processed by MetaMorph software. Two regions were delineated within each cell to denote cell body and processes. Staining intensities in these two regions were integrated using MetaMorph software. To quantitate the proportion of co‐localization between CDNF and the ER marker‐calnexin and the Golgi marker‐TGN38, multiple optical confocal images were obtained across the z‐axis using a Zeiss LSM510 microscope (Carl Zeiss, Thornwood, NY, USA), and the slice containing the maximal fluorescent intensity was chosen. The proportion of co‐localization was quantitated using the co‐localization module in the MetaMorph software. Approximately 25 cells were examined at random. Three independent experiments were repeated.

Luciferase assay

Twenty‐four hours after transfection, cells were incubated in the serum‐free medium for 4 h at 37°C. Then the culture medium were collected and cells were lysis with passive lysis buffer (PLB). After centrifugation, the extracellular and intracellular luciferase activities was measured using a NanoLuc assay system (Promega) according to the manufacturer's instructions. The total luciferase activity in both medium and cell lysate were calculated and the NanoLuc activity ratio of extracellular to intracellular reflected the secret level of every plasmid. Three independent experiments were repeated.

Statistical analysis

Statistical analyses were performed using SPSS 19.0. Data are presented as mean ± standard deviation (SD). Significant difference between groups was analyzed by unpaired Student's t‐test. Differences were considered statistically different if P < 0.05.

Results

NanoLuc‐fused CDNF protein can express normally in PC12 cells

In our previous studies, we have found that the secretion of M1 protein reduced significantly compared with Wt but the secretion of M7 protein showed higher than Wt (Sun et al., 2011; Liu et al., 2015b). In this study, we constructed fusion plasmids of Wt, M1, and M7 with NanoLuc separately as shown in Figure 1A. To detect the expression of NanoLuc‐fused CDNF proteins, three constructions were electrotransfected in PC12 cells. The results of western blot showed that M1‐NL showed significantly reduced secretion level when compared with Wt‐NL, but the secretion level of M7‐NL showed higher than that of Wt‐NL (*P < 0.05, **P < 0.01) (Figures 1B and 1C). So that the fused proteins of CDNF with NanoLuc could express normally in PC12 cells and the addition of NanoLuc did not induce substantial variation in the secretion level of CDNF mutants.

Figure 1.

Figure 1

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Expression of NanoLuc‐fused CDNF proteins. (A) Numbering corresponds to CDNF amino acid residues. The eight α‐helices were indicated with blue. The eight conserved cysteines were indicated with white bars. The M1 and M7 mutants had been constructed by inserting proline into α1 and α7 respectively. (B) After transfection, cell lysate and medium were prepared and analyzed by immunoblotting with anti‐HA antibody. (C) The value of medium versus cell lysate from Wt‐NL was set to 100%. The value of secretion of two mutants was normalized to Wt‐NL. Plotted data were expressed as mean ± SEM from three separate experiments (*P < 0.05, **P < 0.01, Student's t‐test).

α1 and α7 alter intracellular distribution of NanoLuc‐fused CDNF protein

Furthermore, we performed immunocytofluorescence to detect the intracellular distribution of NanoLuc‐fused CDNF proteins (Wt‐NL, M1‐NL, M7‐NL) and CDNF proteins without NanoLuc tag (Wt, M1, M7). Our results showed that both Wt and Wt‐NL proteins exhibited a punctate staining pattern distributed in the cell body and extended to the distal processes in PC12 cells (Figures 2A Wt and 2B Wt‐NL). And compared with Wt or Wt‐NL separately, M1 or M1‐NL proteins showed a significantly greater distribution in the cell body and decreased distribution in the distal regions of processes (**P < 0.01) (Figures 2A M1, 2B M1‐NL and 2C), but most M7 or M7‐NL proteins distributed in the distal parts of processes and little located in the cell body(**p< 0.05) (Figures 2A M7, 2B M7‐NL and 2C). And there were not significant difference between NanoLuc‐fused proteins and without NanoLuc tag proteins. Therefore, NanoLuc‐fused proteins showed similar intracellular distribution to CDNF proteins without NanoLuc tag.

Figure 2.

Figure 2

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Intracellular distribution of NanoLuc‐fused CDNF proteins. (A) Intracellular distribution of CDNF proteins without NanoLuc tag in PC12 cells. Scale bar, 10 µm. (B) Intracellular distribution of NanoLuc‐fused CDNF proteins in PC12 cells. Scale bar, 10 µm. (C) Quantitative analysis was performed as described under “Materials and methods.” The proportion of total fluorescence localized to the cell body or processes is presented as a mean ± SEM determined from analysis of three independent experiments (*P < 0.05, **P < 0.01, Student's t‐test).

α1 and α7 affect sub‐localization of NanoLuc‐fused CDNF protein in PC12 cells

To further assess protein sub‐localization in the cell body, we detected the localization of NanoLuc‐fused proteins in the ER and Golgi apparatus by co‐immunostainning with calnexin (a marker of ER) (Figures 3A and 3B) and TGN38 (a marker of trans‐Golgi network) (Figures 3C and 3D) separately. The results showed that M1‐NL co‐localized greater with calnexin but less with TGN significantly than that of Wt‐NL, but for M7‐NL, which co‐localization with calnexin and with TGN both showed less percentage significantly than that of Wt‐NL. The results suggest that M1‐NL may be blocked in the ER and can not transport to Golgi and also can not secrete out the cells, but M7‐NL can transport from ER to Golgi and secrete out the cells successfully. Therefore, NanoLuc addition can not affect the intracellular trafficking of CDNF proteins.

Figure 3.

Figure 3

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Sub‐localization of NanoLuc‐fused CDNF protein in PC12 cells. (A) Co‐localization of the ER marker, calnexin, was visualized by co‐staining with anti‐HA and anti‐calnexin antibodies. Scale bar, 10 µm. (C) Co‐localization of CDNF and the Golgi apparatus marker, TGN38, was visualized by co‐staining with anti‐HA and anti‐TGN38 antibodies. Scale bar, 10 µm. (B and D) Quantitative analysis of results was conducted by scoring 25 cells for each CDNF construct. The proportion of colocalization between CDNF and ER or Golgi is presented as a mean ± SEM determined from analysis of three independent experiments (*P < 0.05, **P < 0.01, Student's t‐test).

α1 and α7 regulate the NanoLuc‐fused CDNF protein secretion

NanoLuc is a small and highly stable enzyme which can generate a bright intense glow‐type blue luminescence (Zhang et al., 2018). Therefore, detecting the NanoLuc activity of NanoLuc‐fused protein can evaluate the protein transport and secretion efficiently. In this study, we quantified the secretion of CDNF and its mutants by detecting the luciferase activity of NanoLuc‐fused CDNF proteins. The results of in the medium showed that the luciferase activity of M1‐NL reduced significantly but activity of M7‐NL increased than that of Wt‐NL (Figure 4A). And in the cell lysate, we found that the luciferase activity of M1‐NL is higher and activity of M7‐NL is lower than that of Wt‐NL (Figure 4B). We calculated the ratio of the activity in the medium and that in the cell lysate to get the secretion level of CDNF mutants. As shown in Figure 4C, M1‐NL secretion was decreased by 75% approximately and M7‐NL secretion was increase twofolds approximately than Wt‐NL secretion. Therefore, the addition of NanoLuc greatly facilitates the quantification of secretion level of CDNF proteins. These results will be helpful for further study on the regulation of α1 and α7 on CDNF secretion and action mode.

Figure 4.

Figure 4

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Luciferase assay. Cells were transfected with Wt‐NL, M1‐NL or M7‐NL. Twenty‐four hours after transfection, the culture medium was replaced and cells were incubated in the serum‐free medium for an additional 4 h. The culture medium (A) and cell lysate (B) were collected, and the luciferase activity in each sample was measured. The secreted ratio of CDNF in each case was calculated as described before (C). The values represent the mean ± SD from three independent cultures (*P < 0.05, **P < 0.01, Student's t‐test).

Discussion

Although CDNF/MANF proteins can be secreted like other classical NTFs (Oh‐Hashi et al., 2012; Henderson et al., 2013; Garea‐Rodriguez et al., 2016), they also can function in the ER as ER stress response protein (Cheng et al., 2013; Liu et al., 2015a; Kim et al., 2017). The protective effect of MANF in ER stress has been demonstrated (Apostolou et al., 2008; Glembotski et al., 2012; Zhao et al., 2013). But for CDNF, there has not found any ER stress response element in the CDNF promoter and little significant found has been reported about the effect of CDNF in ER stress. Our recently study has found that CDNF expression can be upregulated by tunicamycin in H9c2 cells and CDNF overexpression can protect H9c2 cells from apoptosis induced by tunicamycin (in press) (Liu et al., 2017). But whether this protective role of CDNF on H9c2 is related with ER stress still need more studies.

There are two domains, N‐terminal saposin‐like domain and C‐terminal SAP‐domain, in the 3D structure CDNF/MANF proteins (Lindstrom et al., 2013). In our previous studies, we found α1 and α7 which locate in the N‐terminal and C‐terminal respectively can regulate the intracellular trafficking and secretion of CDNF in PC12 cells. (Sun et al., 2011; Liu et al., 2015b). α1 mutation led to most CDNF protein retained in the ER and could not traffic to Golgi apparatus, resulting in the reduced secretion. But α7 mutation caused most CDNF protein secreted out the cells and little retained in the cells body. Then α1 and α7 regulate protein trafficking and secretion in opposite side. But the function of the two mutants still unknown. For α1 mutant, most protein retained in the ER, then whether it can protect cells from apoptosis as ER stress response protein more efficiently? And for α7 mutant, most protein secreted out the cells, so whether it can promote the cell survival as secreted NTFs better? Otherwise, maybe neither α1 mutant nor α7 mutant can function normally. All these problems need to be addressed further.

In this study, we quantified the secretion level of CDNF protein by NanoLuc luciferase. We found that α1 mutation reduced the protein secretion seriously but α7 mutation induced the protein secreting double to wild‐type CDNF. By this NanoLuc luciferase, we analysed the extracellular and intracellular level of CDNF protein more accurately. Furthermore, these NanoLuc‐based approach will help us to explore the role of two helix on the dual action mode of CDNF, which may reveal the mechanism of CDNF on cell survival or apoptosis.

Conclusions

In conclusion, our data indicate that we can quantify the secretion level of CDNF protein by NanoLuc luciferase, so that we can evaluate the impact of α1 and α7 on CDNF secretion or function.

Conflict of interest

The authors have no conflict of interest to disclose.

Acknowledgments and funding

This work was supported by grants from National Natural Science Foundation of China (No. 81701246), Key Research and Development Program of Shandong Province (No. 2017GSF218107 and 2017GSF218115), Natural Science Foundation of Shandong Province (No. ZR2016HM72 and ZR2015HM028), University Science and technology Program of Shandong Province (No. J17KB109), Medical and Health Development Program of Shandong Province (No. 2016WS0711 and No. 2015WS0017).

Hao Liu and Lin Zhong authors contributed equally to this study.

Contributor Information

Jun Yang, Email: yangjun196401@163.com.

Lei Gong, Email: leigong27@163.com.

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