- Open Access
The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro
BMC Biotechnology volume 21, Article number: 39 (2021)
HSPB5 is an ATP-independent molecular chaperone that is induced by heat shock or other proteotoxic stresses. HSPB5 is cytoprotective against stress both intracellularly and extracellularly. It acts as a potential therapeutic candidate in ischemia-reperfusion and neurodegenerative diseases.
In this paper, we constructed a recombinant plasmid that expresses and extracellularly secrets a HSPB5-Fc fusion protein (sHSPB5-Fc) at 0.42 μg/ml in CHO-K1 cells. This sHSPB5-Fc protein contains a Fc-tag at the C-terminal extension of HSPB5, facilitating protein-affinity purification. Our study shows that sHSPB5-Fc inhibits heat-induced aggregation of citrate synthase in a time and dose dependent manner in vitro. Administration of sHSPB5-Fc protects lens epithelial cells against cisplatin- or UVB-induced cell apoptosis. It also decreases GFP-Httex1-Q74 insolubility, and reduces the size and cytotoxicity of GFP-Httex1-Q74 aggregates in PC-12 cells.
This recombinant sHSPB5-Fc exhibits chaperone activity to protect cells against proteotoxicity.
HSPB5 is a member of a family of small heat shock proteins, a class of ATP-independent molecular chaperones with a conserved alpha-crystallin domain that protects cellular proteostasis by delaying the formation of insoluble protein aggregates through their interactions with destabilized, aggregate-prone proteins [1,2,3,4]. Acting as a chaperone, HSPB5 regulates many cellular processes, such as skeletal protein reorganization , heat shock response , exosome secretion , protein metabolism , anti-apoptosis  and anti-senescence. Dysfunction of HSPB5 is closely associated with proteinopathies (such as Alzheimer’s disease, cataracts, muscle degeneration, heart atrophy, diabetes), tumorigenesis and tumor metastasis [8,9,10,11] and autoimmune diseases . Therefore, HSPB5 is considered a valuable therapeutic target.
HSPB5 protein consists of 175 amino acids, and is constitutively expressed in most tissues at physiological conditions . Its expression is increased by heat shock or other proteotoxic stresses. The amino acids of HSPB5 functionally constitute three regions, a N-terminal region (1–66), a central conserved alpha-crystallin domain (aa 66–149, ACD) and a C-terminal region (149–175) . The C-terminal region contains a conserved IXI motif (aa159–161) and a flexible C-terminal extension . HSPB5 exerts chaperone activity as monomers and dimers rather than as oligomers . HSPB5 inhibits both amorphous and fibrillar protein aggregation through physical interaction , and its chaperone activity is regulated by its intramolecular interaction between the ACD domain and the IXI motif [18, 19], phosphorylation , temperature , pH , and oligomerization with other small heat shock proteins (e.g., HSPB4, HSPB1 and HSPB6) . About 115 proteins are reported to interact with or regulated by HSPB5.
HSPB5 exerts its cytoprotective roles both intracellularly and extracellularly. HSPB5 is secreted extracellularly via a vesicle-mediated unconventional protein secretion system that is regulated by TEMD10 and stress . HSPB5 is secreted by retinal pigment epithelial cells and its secretion is regulated by the phosphorylation of serine 59 in HSPB5 [25, 26]. Administration of recombinant HSPB5 proteins attenuates retinal injury induced by oxidative stress [27, 28], protects heart from ischemic injury  and delays cataract formation in animal models . The recombinant HSPB5 used in research is from heterogenous expression in the engineered E. coli BL21 [31, 32]. Although this bacteria-heteroexpressed HSPB5 exerts chaperone activity, it cannot completely imitate the HSPB5 secreted by mammalian cells due to a lack of posttranslational modification [33, 34]. Earlier, it has been reported that human recombinant lactoferrin, carrying a humanized glycosylation, displays selective antiproliferative effects on cancer cells . This research suggests that natural recombinant proteins possess beneficial biological activities on human health.
In this paper, we generated a recombinant construct that can express and secret a sHSPB5-Fc fusion protein to the supernatant when this construct is transfected into CHO-K1 cells. This sHSPB5-Fc fusion protein, which exists predominantly as dimer and trimer and is regulated by N-linked glycosylation, inhibits Citrate synthase (CS) protein aggregation under heat shock in vitro. Administration of sHSPB5-Fc protein reduces the aggregate size and the cytotoxicity of GFP-Httex1-Q74 in PC-12 cells, and protects lens epithelial cells against cisplatin- and UVB-induced cell apoptosis. These results suggest that this sHSPB5-Fc fusion protein exhibits both chaperone and cytoprotective roles in vitro, and is a possible candidate for studying of its therapeutic potentials.
The expression and purification of recombinant HSPB5-Fc proteins
To express a secreted HSPB5 (sHSPB5) in mammalian cells, the constructs of psHSPB5-Fc (Fig. 1A) and empty vector were transiently transfected into CHO-K1 cells individually. The supernatants and cell lysates were immunoblotted with antibodies against Fc-tag or HSPB5 respectively. Anti-Fc and anti-HSPB5 antibodies detected sHSPB5-Fc in both supernatants and cell lysates (Fig. 1B). The protein size of sHSPB5-Fc in the supernatant was smaller than that in the cell lysate, suggesting that the signal peptide of sHSPB5-Fc was cleaved. CHO-K1 is a common cell line used to express recombinant proteins in the medical industry. We subcloned a CHO-K1-sHSPB5-Fc cell line that could produce 0.42 mg/ml of sHSPB5-Fc in the supernatant. The sHSPB5-Fc protein was purified from the supernatants with protein-A affinity column, and the quality of purified sHSPB5-Fc protein was verified with Coomassie blue stain and western blot (Fig. 1C-D), which showed a protein size of 53 K Dalton, consistent with the predicted size. HSPB5 contains a crystalline domain by which HSPB5 forms homo-dimer, trimer and polyoligomers in cells . Fc-tag also forms dimers through the disulfide bond. To characterize the status of sHSPB5-Fc, the sHSP5-Fc protein was heated in protein loading buffer with or without β-Mercaptoethanol (β-ME). The Coomassie blue staining results showed that majority of sHSPB5-Fc protein existed in dimers and trimers, but less sHSP5-Fc forms oligomers in the native loading buffer. The reducing agent β-ME minimized the size of sHSPB5-Fc from oligomers to monomer (Fig. 1E). These results indicated that sHSPB5-Fc protein exists predominantly in dimer and trimer. In addition, we studied that whether sHSPB5-Fc is regulated by glycosylation by treating the sHSPB5-Fc protein with PNGase F, an enzyme catalyzing the cleavage of N-linked polysaccharides from glycoproteins. The results showed that PNGase F increased the mobility of sHSPB5-Fc in a SDS-PAGE gel, suggesting that sHSPB5-Fc undergoes glycosylation (Fig. 1F).
Recombinant sHSPB5-Fc experts chaperone activity in vitro
HSPB5 exerts chaperone activity by interacting with and inhibiting protein aggregates in an ATP-independent manner . Citrate synthase (CS) proteins can form protein aggregates under heat shock condition, and this feature has been used to measure the chaperone activity of small heats shock proteins (e.g., HSPB5 or HSPB1) in vitro . We determined the chaperone activity of sHSPB5-Fc by using heat-induced aggregation of CS in vitro. We incubated CS protein alone or together with sHSPB5-Fc at the indicated ratios (Fig. 2A) at 43 °C in 40 mM HEPES buffer (pH 7.4) for 1 h. The aggregated proteins were measured with spectrometry at OD 320 nm. The results showed that sHSPB5-Fc increased CS protein solubility under heat shock conditions in a dose-dependent manner (Fig. 2A). Using the ratio of CS:sHSPB5-Fc of 1:2.5, we further studied the time course influence on sHSPB5-Fc chaperone activity. The results showed that CS protein alone rapidly formed aggregates at 43 °C within 20 min, after which prolonged heat shock time did not increase CS protein aggregation. This suggested that aggregation plateaued after 20 min. sHSPB5-Fc significantly inhibited aggregation of CS protein at 43 °C. sHSPB5-Fc protein alone did not form aggregates under this heat shock condition (Fig. 2B). Furthermore, the chaperone activity of sHSPB5-Fc was tested in both the soluble and insoluble fractions. The solutions that contain CS protein or sHSPB5-Fc alone, or a mixture of CS and sHSPB5-Fc at a ratio of 1:2.5, were treated at 43 °C for 1 h followed by centrifugation at 15000 g for 15 min to separate the pellets from supernatants. The proteins in the pelleted and soluble fractions were analyzed on SDS-PAGE gel and stained with Coomassie blue (Fig. 2C). The densitometry of protein bands was quantitated using image J software and the ratio of pelleted protein vs soluble proteins was quantified (Fig. 2D). The results showed that most of the CS proteins were in the soluble fraction compared to that in the pelleted fraction at 4 °C, and the ratio of pelleted vs soluble CS protein is 0.27 (Fig. 2C, lanes 1 and 2). However, at 43 °C, the bulk of CS proteins was detected in the pelleted fraction (Fig. 2C, lanes 3 and 4) with a ratio of 4. 2 (Fig. 1D). sHSPB5-Fc reduced the amount of CS proteins in the insoluble fraction (Fig. 2C, lanes 7 and 8), with a ratio of 0.34 (Fig. 2D). sHSPB5-Fc always remained in the soluble fraction under heat shock regardless of the presence of CS protein (Fig. 2C, lanes 5, 6, 7 and 8). Taken together, these results suggest that the recombinant sHSPB5-Fc protein can exhibit chaperone activity in vitro.
The recombinant sHSPB5-Fc protein protects cells from stress-induced apoptosis in vitro
Administration of recombinant HSPB5 was reported to protect RPE cells against stress-induced apoptosis . To test the anti-apoptotic activity of sHSPB5-Fc, the mouse lens epithelial cell line (mLEC) was cultured in media containing 50 or 100 μg/ml of sHSPB5-Fc followed by treatment with sham (DMSO) or 10 μM cisplatin for 24 h. Human IgG was used as control. The cisplatin is a common chemotherapy agent that induces both tumor and normal cells’ apoptosis. Apoptosis was measured by using flow cytometry to identify cells that stained positive for Annexin V/PI and immunoblotting those cells for active caspase 3. The immunoblots showed that sHSPB5-Fc can significantly inhibit cisplatin-induced caspase 3 activation at both the 50 and 100 μg/ml concentrations (Fig. 3A, lanes 3 and 5, and Fig. 3B). Administration of normal immunoglobulin, which is used as a negative control, did not impact cisplatin-induced caspase 3 activation. The flow cytometry results showed that cisplatin itself increased the percentage of cells with double annexin-V/PI positive, but this effect was inhibited by sHSPB5-Fc (Fig. 3C). In addition, we tested the protective role of sHSPB5-Fc protein on UVB-induced mLEC cell apoptosis. The mLEC was treated with 3 J/m3 UVB followed by recovery in media containing sham (IgG) or 50 and 100 μM sHSPB5-Fc for 24 h. The activated caspase 3 was tested with immunoblotted. The results showed that sHSPB5-Fc attenuated UVB-induced caspase 3 activation (Fig. 3D-E). Taken together, our data suggest that the recombinant sHSPB5-Fc can exhibit anti-apoptotic activity.
HSPB5-Fc can decrease the aggregation and cytotoxicity of GFP-Httex1-polyQ74 in PC-12 cells
Huntington’s disease is a type of neurodegenerative disease caused by the aggregation of huntingtin proteins containing multiple polyQ sequence repeats. Direct inhibition of polyQ aggregation by lefunomide or teriflunomide or by indirectly activating HSF1-mediated heat shock response improves PolyQ-induced neuropathy [36, 37]. It was reported that increased HSPB5 expression in mice improves Huntington disease by reducing poly-Q mediated mHtt aggregation . Thus, we further tested this cytoprotective effect of sHSPB5-Fc in vitro. PC-12 cell line, a neuroblastoma cell line, was transiently transfected with plasmid pEGFP-Httex1-Q74. The cells were then incubated with media containing sHSPB5-Fc at concentrations of 50 and 100 μg/ml or control IgG for 24 h. The results showed that the cells containing pEGFP vector alone expressed GFP protein evenly in both the cytosol and nucleus (Fig. 4A). In contrast, the cells that were transfected with pEGFP-Httex1-Q74 formed GFP-Httex1-Q74 aggregates (Fig. 4A). The administration of sHSPB5-Fc proteins significantly reduced the number of cells with GFP-Httex1-Q74 aggregates (Fig. 4A-B). To determine whether sHSPB5-Fc decreases the insolubility of GFP-Httex1-Q74 in PC-12 cells, a membrane-filtration assay was performed. The cells were treated in the same way as that in Fig. 4A, and then lysed in NP-40 lysis buffer. The pellets were treated with SDS buffer. The SDS-treated NP40-insoluble proteins were pass through a 0.2 μm cellulose acetate membrane using a slot blot apparatus followed by immunoblotting with anti-GFP antibody. The results showed that sHSPB5-Fc protein can significantly reduce the amount of GFP-Httex1-Q74 left on the cellulose acetate membrane (Fig. 4C and D), implying that sHSPB5-Fc can decrease GFP-Httex1-Q74 aggregation.
According to the literature , reducing the size or increasing the solubility of Httex1-Q74 aggregates could reduce the cytotoxicity, Furthermore, we quantitated the diameter of GFP-Httex1-Q74 aggregates in the cells that were treated with normal IgG (sham) or sHSPB5-Fc using the Image J software. Approximately 150 GFP-Httex1-Q74 positive cells were used for analysis (Fig. 5A). The quantitative results showed that recombinant sHSPB5-Fc reduced the diameter of GFP-Httex1-Q74 aggregates compare to control IgG (Fig. 5B). These results suggest that sHSPB5-Fc protein can decrease the aggregate size of GFP-Httex1-Q74 in the PC-12 cell line.
The aggregation of GFP-Httex1-Q74 reportedly cause PC-12 cell cytotoxicity . We have shown that sHSPB5-Fc can reduce the size and number of GFP-Httex1-Q74 aggregates. Therefore, we proposed that sHSPB5-Fc could reduce the cytotoxicity of GFP-Httex1-Q74 in PC-12 cells. To this end, PC-12 cells were transiently transfected with pEGFP- Httex1-Q74 vector and then treated with sHSPB5-Fc protein for 24 h. The activity of caspase 3 and caspase 7 were measured. The results showed that ectopic expression of GFP-Httex1-Q74 induced caspase 3/7 activation, and sHSPB5-F significantly decreased this activity (Fig. 5C). Taken together, these results suggest that sHSPB5-Fc protein can decrease the aggregation and cytotoxicity of Httex1-Q74 in PC-12 cells.
In this paper, we established a CHO-K1 subclone cell line that expresses and secretes human sHSPB5-Fc protein. This sHSPB5-Fc has both chaperone and anti-apoptosis activities, and can reduce the aggregation and cytotoxicity of mHtt proteins in vitro.
Delivery of HSPB5 proteins has shown a cytoprotective role against divergent stresses . Most of the HSPB5 proteins used for in vivo administration are His-tagged HSPB5 generated via hetero-expression in the E. coli BL-21 strain [31, 32]. Although this is an economic way to obtain HSPB5, it is difficult to remove LPS contamination [42, 43], and the product cannot imitate some of the characteristics of mammalian HSPB5. To express sHSPB5 protein in mammalian cells, we utilized the CHO-K1-Fc cell system, a biomedical tool to express and produce recombinant proteins with an Fc-tag. We subcloned HSPB5 cDNA in-frame upstream of the Fc-tag. To improve the secretion of HSPB5-Fc protein, we added a secretion signal peptide sequence to the N-terminus of HSPB5. In this system, approximately 0.42 mg/ml of sHSPB5-Fc protein was obtained from the supernatant. We have shown that this recombinant sHSPB5-Fc protein is soluble, exists in dimer and trimer forms with glycosylation, and exhibits chaperone activity in vitro (Fig. 2). We further tested the cytoprotective role of this sHSPB5-Fc protein and found that it can inhibit cisplatin and UVB induced lens epithelial cell apoptosis. We also tested it neuroprotective roles in vitro, and found that administration of sHSPB5-Fc can reduce the size of the aggregates as well as the cytotoxicity of aggregated GFP-Httex1-Q74 in PC-12 cells (Figs. 3 and 4). These results suggest that sHSPB5-Fc protein possesses both chaperone and cytoprotective roles in vitro.
The Fc-tag, which is composed of hinge chain-CH2-CH3 domains of human IgG1 heavy chain, is a common tag used to express divergent recombinant proteins in the biomedicine industry [44, 45]. The FDA has approved 11 Fc-fusion proteins to treat different diseases, such as infectious diseases, cancer, and arthrosis . In addition to facilitating protein-A-mediated immunoaffinity purification, Fc-tag also possess immune activity by binding to FcR, and activating the antibody-dependent cell-mediated cytotoxicity (ADCC) and complement systems . A D143A mutation in the Fc-tag impairs its ADCC effect . To get rid of the ADCC effect of sHSPB5-Fc, we mutated D143A in the Fc-tag portion of the sHSPB5-Fc protein. Mutation at this site does not affect HSPB5 chaperone (Fig. 1) or its cytoprotective activity in vitro (Fig. 2). Whether sHSPB5-Fc exhibits cytoprotective roles in vivo is still under investigation in our lab. Because of the Fc-tag, its potential regulation on the immune system should be considered when HSPB5-Fc is administrated in vivo.
The HSPB5 exists as a dimer or a polymer and its function depends on the dimer rather than polyoligomer . To determine the status of sHSPB5-Fc, we treated sHSPB5-Fc in SDS loading buffer with or without β-ME, a reducer to destroy the disulfide bond. We found that the majority of sHSPB5-Fc protein is in dimer and trimer forms, and few sHSPB5-Fc forms polyoligomers (Fig. 1). β-ME destroyed the oligomers of sHSPB5-Fc to monomer (Fig. 1E), suggesting that disulfide bond may be involved in sHSPB5-Fc’ dimerization (Fig. 1E). But this needs more evidence (such as cysteine mutation in Fc) to confirm. Nevertheless, Fc does not impact HSPB5’s chaperone and cytoprotective activities (Figs. 2, 3, 4 and 5).
Glycosylation of proteins is an important posttranslational modification and can contribute to the folding and secretion of proteins . In addition, glycosylation has complex significance on the function of proteins. For example, N-glycosylation can increase protein stability . The N-glycosylation of Asn-297 of murine IgG can enhance immune response. Human recombinant lactoferrin, carrying humanized glycosylation, displays a selective antiproliferative effect on cancer cells . Our data in Fig. 1 suggested that the HSPB5-Fc protein possesses an N-linked glycosylation, which may be beneficial to its biological activity. However, the effect of this glycosylation on HSPB5-Fc is unknown and needs further investigation.
In summary, we established a mammalian expression system by using an industrialized CHO-K1 cell line, which can express and secret sHSPB5-Fc protein in a large scale. This sHSPB5-Fc protein is soluble in dimer or trimer. It can protect cells against cisplatin- or UV-induced cell apoptosis and attenuate the cytotoxicity of GFP-Httex1-Q74 aggregates in vitro.
Antibodies and chemical reagents
The rabbit antibody against HSPB5 was bought from Abcam (Cambridge, USA). The rabbit antibody against cleaved caspase 3 was from Cell Signaling Technology (Danvers, USA). The mouse antibodies against GFP and β-actin were from Proteintech (Wuhan, China). Phalloidin-iFluor 594 was from Abcam. Alexa Fluor 594 goat anti-rabbit IgG was from Thermo Fisher Scientific (Waltham, USA). Citrate synthase, DAPI and 302 serum-free medium were from Sigma (St. Louis, USA). PNGase F was from NEB (Beverly, USA). Caspase-Glo 3/7 assay kit was from Promega (Madison, USA). pEGFP-Httex1-Q74 plasmid was from Addgene (Cambridge, USA). HiTrap Protein A HP column was from GE Healthcare (Little Chalfont, UK).
Chinese hamster ovary (CHO-K1) cells were maintained in our laboratory . The cells were cultured in 302 serum-free medium supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin (Gibco, Grand Island, USA) in an incubator with shaking. Mouse lens epithelial cells (mLEC) were cultured with DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin. The PC-12 cell line was a gift from Xiao Xu, an Associate Professor at Henan University. The PC-12 cells were cultured in RPMI 1640 medium supplemented with 5% FBS, 10% horse serum (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin.
HSPB5-fc protein expression and purification
HSPB5 cDNA was synthesized by using RNA from human lens epithelial cells and subcloned into the pHB-Fc expression vector  at the HindIII (NEB, Beverly, USA) and EcoRI (NEB) restriction sites to generate pHB-HSPB5-Fc. To facilitate HSPB5-Fc secretion, A 57 bp cDNA fragment (ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGCAACTGCAACTGGAGTACATTCA) encoding a signal peptide was inserted into the HindIII restriction site to fused to the 5′ terminal of HSPB5 cDNA (Supplementary sequence), generating the plasmid psHSPB5-Fc. The psHSBP5-Fc construct was transfected into and stably expressed in CHO-K1 cells. The secreted HSPB5-Fc (sHSPB5-Fc) recombinant protein in the supernatant was collected and purified with a HiTrap Protein A HP column in Akta pure 150 (GE Healthcare, Little Chalfont, UK).
For oligomerization of sHSPB5-Fc, the sHSPB5-Fc protein was heated in SDS-loading buffer (10% SDS, 0.5% bromophenol blue and 50% glycerol in 250 mM Tris-HCl, pH 6.8) with or without 5% β-Mercaptoethanol (β-ME) for 10 min. The samples were separated in SDS-PAGE gels followed by Coomassie blue staining. For glycosylation analysis, the sHSPB5-Fc protein was incubated with buffer containing PNGase F for 1 h at 37 °C. The proteins were separated in SDS-PAGE gel followed by Coomassie blue staining.
Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma). Thirty to fifty microgram of proteins were separated by electrophoresis on SDS-PAGE gels, and then transferred to PVDF membranes. The membranes were blocked in 5% BSA/TBST for 1 h followed by incubating with the appropriate primary antibodies at 4 °C overnight. After washing three times in TBST buffer, the membranes were incubated in blocking buffer containing the appropriate HRP-conjugated secondary antibody. The membrane was washed in TBST buffer for 3–4 times and developed in ECL buffer. The signals were exposed to X-ray films.
In vitro chaperone activity analysis
Citrate synthase (CS, 1 mg/ml) alone or combined with HSPB5-Fc was incubated at 43 °C in 40 mM HEPES buffer (pH 7.4) for the indicated times. The protein aggregates were measured at an absorbance wavelength of 320 nm in Varioskan Flash (Thermo electron corporation, Waltham, USA).
Filter retardation assay
Cells expressing GFP alone or GFP-Httex1-polyQ74 were lysed with NP40 lysis buffer containing protease inhibitor cocktail. The cell lysates were centrifuged at 12,000 rpm/min for 5 min, and the supernatant was collected for western blot. The pellets were washed twice with NP40 lysis buffer followed by incubation with 10 U DNase I (NEB, Beverly, USA) in buffer containing 20 mM Tris-HCl, 15 mM MgCl2, pH 8.0 at 37 °C for 1 h to remove genomic DNA. After this, the pellets were combined with 2% SDS, 20 mM EDTA and 50 mM DTT and boiled at 98 °C for 5 min . Fifty microgram of protein was filtered through a cellulose acetate (CA) membrane (0.2 μm) (Toyo Roshi Kaisha Ltd., Tokyo, Japan) using a slot blot unit (Midwestz, Beijing, China). The membranes were washed with 1% SDS and blocked in 5% non-fat milk/TBST. After this, the CA membrane was incubated with mouse anti-GFP antibody followed by HRP-conjugated goat anti-mouse secondary antibody. The membranes were developed in ECL buffer, and the signals were exposed to X-ray films.
Immunofluorescent staining assay
PC-12 cells were transiently transfected with construct expressing GFP-Httex1-polyQ74 or pEGFP vector. The cells were fixed in 4% PFA for 20 min in room temperature followed by washing in PBS twice. The cells were then incubated with permeable buffer for 2 min and stained with buffer containing phalloidin-iFluor 594 (1:1000 dilution in PBS; Abcam) . Nuclei were stained with DAPI (blue). The signals were visualized and captured using the confocal microscope (R1, Nikon, Japan).
Annexin V/PI apoptosis assay by flow cytometry
Apoptosis was measured using an Annexin V-FITC/PI detection kit (KeyGEN BioTECH, Nanjing, China) following kit’s protocol. Briefly, cells were digested with non-EDTA trypsin. After washing with PBS, the cells were resuspended with binding buffer mixed with FITC-conjugated Annexin V and PI for 15 min at room temperature. After washing, the signal was measured by flow cytometry (BD Biosciences; San Jose, CA, USA) with excitation and emission wavelengths at 488 nm and 530 nm.
Caspase 3/7 activity detection
The caspase 3/7 activity was measured by using the Caspase-Glo 3/7 kit (Promega, Madison, USA) following kit’s protocol. Briefly, the caspase 3/7 reagents were prepared by equally mixing the caspase 3/7 substrate and buffer freshly before use. One hundred microliter of mixed buffer was added to cells cultured in 96 well plates. After gently mixing, the plates were left RT for 1 h, and the luminescent signals, which reflect caspase-3/7 activities, were measured with Varioskan Flash (Thermo electron corporation, Waltham, USA).
Image J was used to quantify the densitometry of immunoblot bands. SPSS 17.0 and GraphPad Prism 5 were used for data analysis. A Student’s t-test was used for statistical analysis. p < 0.05 was considered to be statistically significant.
Availability of data and materials
All authors declare that the data supporting the findings of this study are available within the article and supplementary file.
Rajagopal P, Tse E, Borst AJ, Delbecq SP, Shi L, Southworth DR, et al. A conserved histidine modulates HSPB5 structure to trigger chaperone activity in response to stress-related acidosis. eLife. 2015;4:e07304. https://0-doi-org.brum.beds.ac.uk/10.7554/eLife.07304.
Dimauro I, Antonioni A, Mercatelli N, Caporossi D. The role of alphaB-crystallin in skeletal and cardiac muscle tissues. Cell Stress Chaperones. 2018;23(4):491–505. https://0-doi-org.brum.beds.ac.uk/10.1007/s12192-017-0866-x.
Carra S, Rusmini P, Crippa V, Giorgetti E, Boncoraglio A, Cristofani R, et al. Different anti-aggregation and pro-degradative functions of the members of the mammalian sHSP family in neurological disorders. Philos Trans R Soc London B Biol Sci. 2013;368(1617):20110409.
Sprague-Piercy MA, Wong E, Roskamp KW, Fakhoury JN, Freites JA, Tobias DJ, et al. Human alpha B-crystallin discriminates between aggregation-prone and function-preserving variants of a client protein. Bba-Gen Subjects. 2020;1864(3):129502.
Yin B, Tang S, Xu J, Sun JR, Zhang XH, Li YB, et al. CRYAB protects cardiomyocytes against heat stress by preventing caspase-mediated apoptosis and reducing F-actin aggregation. Cell Stress Chaperones. 2019;24(1):59–68. https://0-doi-org.brum.beds.ac.uk/10.1007/s12192-018-0941-y.
Gangalum RK, Bhat AM, Kohan SA, Bhat SP. Inhibition of the expression of the small heat shock protein alpha B-Crystallin inhibits exosome secretion in human retinal pigment epithelial cells in culture. J Biol Chem. 2016;291(25):12930–42. https://0-doi-org.brum.beds.ac.uk/10.1074/jbc.M115.698530.
D'Agostino M, Lemma V, Chesi G, Stornaiuolo M, Serio MC, D'Ambrosio C, et al. The cytosolic chaperone alpha-crystallin B rescues folding and compartmentalization of misfolded multispan transmembrane proteins. J Cell Sci. 2013;126(18):4160–72.
Zhu ZH, Reiser G. The small heat shock proteins, especially HspB4 and HspB5 are promising protectants in neurodegenerative diseases. Neurochem Int. 2018;115:69–79. https://0-doi-org.brum.beds.ac.uk/10.1016/j.neuint.2018.02.006.
Molnar KS, Dunyak BM, Su B, Izrayelit Y, McGlasson-Naumann B, Hamilton PD, et al. Mechanism of action of VP1-001 in cryAB(R120G)-associated and age-related cataracts. Invest Ophthalmol Vis Sci. 2019;60(10):3320–31. https://0-doi-org.brum.beds.ac.uk/10.1167/iovs.18-25647.
Toft DJ, Fuller M, Schipma M, Chen F, Cryns VL, Layden BT. alpha B-crystallin and HspB2 deficiency is protective from diet-induced glucose intolerance. Genom Data. 2016;9:10–7. https://0-doi-org.brum.beds.ac.uk/10.1016/j.gdata.2016.03.010.
Caporossi D, Parisi A, Fantini C, Grazioli E, Cerulli C, Dimauro I. AlphaB-crystallin and breast cancer: role and possible therapeutic strategies. Cell Stress Chaperones. 2020;26(1):19–28. https://0-doi-org.brum.beds.ac.uk/10.1007/s12192-020-01175-0.
Kuipers HF, Yoon J, van Horssen J, Han MH, Bollyky PL, Palmer TD, et al. Phosphorylation of alpha B-crystallin supports reactive astrogliosis in demyelination. P Natl Acad Sci USA. 2017;114(9):E1745–54. https://0-doi-org.brum.beds.ac.uk/10.1073/pnas.1621314114.
Boelens WC. Cell biological roles of alpha B-crystallin. Prog Biophys Mol Biol. 2014;115(1):3–10. https://0-doi-org.brum.beds.ac.uk/10.1016/j.pbiomolbio.2014.02.005.
Bagneris C, Bateman OA, Naylor CE, Cronin N, Boelens WC, Keep NH, et al. Crystal structures of alpha-crystallin domain dimers of alphaB-crystallin and Hsp20. J Mol Biol. 2009;392(5):1242–52. https://0-doi-org.brum.beds.ac.uk/10.1016/j.jmb.2009.07.069.
Delbecq SP, Jehle S, Klevit R. Binding determinants of the small heat shock protein, alphaB-crystallin: recognition of the 'IxI' motif. EMBO J. 2012;31(24):4587–94. https://0-doi-org.brum.beds.ac.uk/10.1038/emboj.2012.318.
Hayashi J, Carver JA. The multifaceted nature of alphaB-crystallin. Cell Stress Chaperones. 2020;25(4):639–54. https://0-doi-org.brum.beds.ac.uk/10.1007/s12192-020-01098-w.
Hochberg GKA, Ecroyd H, Liu C, Cox D, Cascio D, Sawaya MR, et al. The structured core domain of alpha B-crystallin can prevent amyloid fibrillation and associated toxicity. P Natl Acad Sci USA. 2014;111(16):E1562–70. https://0-doi-org.brum.beds.ac.uk/10.1073/pnas.1322673111.
Pasta SY, Raman B, Ramakrishna T, Rao CM. The IXI/V motif in the C-terminal extension of alpha-crystallins: alternative interactions and oligomeric assemblies. Mol Vis. 2004;10(78):655–62.
Jehle S, Vollmar BS, Bardiaux B, Dove KK, Rajagopal P, Gonen T, et al. N-terminal domain of alpha B-crystallin provides a conformational switch for multimerization and structural heterogeneity. P Natl Acad Sci USA. 2011;108(16):6409–14. https://0-doi-org.brum.beds.ac.uk/10.1073/pnas.1014656108.
Ciano M, Allocca S, Ciardulli MC, della Volpe L, Bonatti S, D’Agostino M. Differential phosphorylation-based regulation of alpha B-crystallin chaperone activity for multipass transmembrane proteins. Biochem Bioph Res Co. 2016;479(2):325–30. https://0-doi-org.brum.beds.ac.uk/10.1016/j.bbrc.2016.09.071.
Raman B, Ramakrishna T, Rao CM. Temperature dependent chaperone-like activity of alpha-crystallin. FEBS Lett. 1995;365(2–3):133–6. https://0-doi-org.brum.beds.ac.uk/10.1016/0014-5793(95)00440-K.
Baboolall KD, Kaudeer YB, Gershenson A, O'Hara PB. pH Dependence of Oligomerization and Functional Activity of Alpha B Crystallin. Biophys J. 2020;118(3):510a.
Baranova EV, Weeks SD, Beelen S, Bukach OV, Gusev NB, Strelkov SV. Three-dimensional structure of alpha-Crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6. J Mol Biol. 2011;411(1):110–22. https://0-doi-org.brum.beds.ac.uk/10.1016/j.jmb.2011.05.024.
Zhang M, Liu L, Lin X, Wang Y, Li Y, Guo Q, et al. A translocation pathway for vesicle-mediated unconventional protein secretion. Cell. 2020;181(3):637–52 e615. https://0-doi-org.brum.beds.ac.uk/10.1016/j.cell.2020.03.031.
Sreekumar PG, Kannan R, Kitamura M, Spee C, Barron E, Ryan SJ, et al. alpha B Crystallin is apically secreted within exosomes by polarized human retinal pigment epithelium and provides neuroprotection to adjacent cells. PLoS One. 2010;5(10):e12578. https://0-doi-org.brum.beds.ac.uk/10.1371/journal.pone.0012578.
Gangalum RK, Atanasov IC, Zhou ZH, Bhat SP. alpha B-Crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells. J Biol Chem. 2011;286(5):3261–9. https://0-doi-org.brum.beds.ac.uk/10.1074/jbc.M110.160135.
Zhou P, Kannan R, Spee C, Sreekumar PG, Dou GR, Hinton DR. Protection of retina by alpha B Crystallin in sodium iodate induced retinal degeneration. PLoS One. 2014;9(5):e98275. https://0-doi-org.brum.beds.ac.uk/10.1371/journal.pone.0098275.
Kannan R, Sreekumar PG, Hinton DR. Alpha crystallins in the retinal pigment epithelium and implications for the pathogenesis and treatment of age-related macular degeneration. Bba-Gen Subjects. 2016;1860(1):258–68. https://0-doi-org.brum.beds.ac.uk/10.1016/j.bbagen.2015.05.016.
Velotta JB, Kimura N, Chang SH, Chung J, Itoh S, Rothbard J, et al. alpha B-Crystallin improves murine cardiac function and attenuates apoptosis in human endothelial cells exposed to ischemia-reperfusion. Ann Thorac Surg. 2011;91(6):1907–13. https://0-doi-org.brum.beds.ac.uk/10.1016/j.athoracsur.2011.02.072.
Christopher KL, Pedler MG, Shieh B, Ammar DA, Petrash JM, Mueller NH. Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death. Bba-Mol Cell Res. 2014;1843(2):309–15. https://0-doi-org.brum.beds.ac.uk/10.1016/j.bbamcr.2013.11.010.
Zhu ZH, Li RY, Stricker R, Reiser G. Extracellular alpha-crystallin protects astrocytes from cell death through activation of MAPK, PI3K/Akt signaling pathway and blockade of ROS release from mitochondria. Brain Res. 2015;1620:17–28.
Klopstein A, Santos-Nogueira E, Francos-Quijorna I, Redensek A, David S, Navarro X, et al. Beneficial effects of alpha B-Crystallin in spinal cord contusion injury. J Neurosci. 2012;32(42):14478–88. https://0-doi-org.brum.beds.ac.uk/10.1523/JNEUROSCI.0923-12.2012.
Colby CB, Inoue M, Thompson M, Tan YH. Immunologic differentiation between E. coli and CHO cell-derived recombinant and natural human beta-interferons. J Immunol. 1984;133(6):3091–5.
Lee J, Lee EN, Yoon J, Chung SM, Prasad H, Susin C, et al. Comparative study of Chinese hamster ovary cell versus Escherichia coli-derived bone morphogenetic Protein-2 using the critical-size Supraalveolar Peri-implant defect model. J Periodontol. 2013;84(3):415–22. https://0-doi-org.brum.beds.ac.uk/10.1902/jop.2012.110369.
Nakamura-Bencomo S, Gutierrez DA, Robles-Escajeda E, Iglesias-Figueroa B, Siqueiros-Cendón TS, Espinoza-Sánchez EA, et al. Recombinant human lactoferrin carrying humanized glycosylation exhibits antileukemia selective cytotoxicity, microfilament disruption, cell cycle arrest, and apoptosis activities. Investig New Drugs. 2021;39(2):400–15. https://0-doi-org.brum.beds.ac.uk/10.1007/s10637-020-01020-2.
Fuentealba RA, Marasa J, Diamond MI, Piwnica-Worms D, Weihl CC. An aggregation sensing reporter identifies leflunomide and teriflunomide as polyglutamine aggregate inhibitors. Hum Mol Genet. 2012;21(3):664–80. https://0-doi-org.brum.beds.ac.uk/10.1093/hmg/ddr500.
Gomez-Pastor R, Burchfiel ET, Neef DW, Jaeger AM, Cabiscol E, McKinstry SU, et al. Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington’s disease. Nat Commun. 2017;8(1):14405. https://0-doi-org.brum.beds.ac.uk/10.1038/ncomms14405.
Oliveira AO, Osmand A, Outeiro TF, Muchowski PJ, Finkbeiner S. alpha B-Crystallin overexpression in astrocytes modulates the phenotype of the BACHD mouse model of Huntington's disease. Hum Mol Genet. 2016;25(9):1677–89. https://0-doi-org.brum.beds.ac.uk/10.1093/hmg/ddw028.
Ripaud L, Chumakova V, Antonin M, Hastie AR, Pinkert S, Korner R, et al. Overexpression of Q-rich prion-like proteins suppresses polyQ cytotoxicity and alters the polyQ interactome. Proc Natl Acad Sci U S A. 2014;111(51):18219–24. https://0-doi-org.brum.beds.ac.uk/10.1073/pnas.1421313111.
Takahashi T, Katada S, Onodera O. Polyglutamine diseases: where does toxicity come from? What is toxicity? Where are we going? J Mol Cell Biol. 2010;2(4):180–91. https://0-doi-org.brum.beds.ac.uk/10.1093/jmcb/mjq005.
Budnar P, Singh NP, Rao CM. HSPB5 (alpha B-crystallin) confers protection against paraquat-induced oxidative stress at the organismal level in a tissue-dependent manner. Cell Stress Chaperones. 2021;26(1):229–39. https://0-doi-org.brum.beds.ac.uk/10.1007/s12192-020-01171-4.
Cardoso LS, Araujo MI, Goes AM, Pacifico LG, Oliveira RR, Oliveira SC. Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis. Microb Cell Factories. 2007;6(1):1. https://0-doi-org.brum.beds.ac.uk/10.1186/1475-2859-6-1.
Maldonado RF, Sa-Correia I, Valvano MA. Lipopolysaccharide modification in gram-negative bacteria during chronic infection. FEMS Microbiol Rev. 2016;40(4):480–93. https://0-doi-org.brum.beds.ac.uk/10.1093/femsre/fuw007.
Lee GW, Fecko JK, Yen A, Donaldson D, Wood C, Tobler S, et al. Improving the expression of a soluble receptor: fc fusion protein in CHO cells by coexpression with the receptor ligand. Cell Tech Cell Prod. 2007:29–39. https://0-doi-org.brum.beds.ac.uk/10.1007/978-1-4020-5476-1_4.
Jafari R, Zolbanin NM, Rafatpanah H, Majidi J, Kazemi T. Fc-fusion proteins in therapy: an updated view. Curr Med Chem. 2017;24(12):1228–37. https://0-doi-org.brum.beds.ac.uk/10.2174/0929867324666170113112759.
Levin D, Golding B, Strome SE, Sauna ZE. Fc fusion as a platform technology: potential for modulating immunogenicity. Trends Biotechnol. 2015;33(1):27–34. https://0-doi-org.brum.beds.ac.uk/10.1016/j.tibtech.2014.11.001.
Wang YH, Zhang HL, Wang ZZ, Wei YX, Wang ML, Liu MC, et al. Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats. Sci Transl Med. 2020;12(540):eaaw3172.
Xu C, Ng DT. Glycosylation-directed quality control of protein folding. Nat Rev Mol Cell Biol. 2015;16(12):742–52. https://0-doi-org.brum.beds.ac.uk/10.1038/nrm4073.
Menon RP, Nethisinghe S, Faggiano S, Vannocci T, Rezaei H, Pemble S, et al. The role of interruptions in polyQ in the pathology of SCA1. PLoS Genet. 2013;9(7):e1003648. https://0-doi-org.brum.beds.ac.uk/10.1371/journal.pgen.1003648.
Iglesias-Figueroa BF, Siqueiros-Cendon TS, Gutierrez DA, Aguilera RJ, Espinoza-Sanchez EA, Arevalo-Gallegos S, et al. Recombinant human lactoferrin induces apoptosis, disruption of F-actin structure and cell cycle arrest with selective cytotoxicity on human triple negative breast cancer cells. Apoptosis. 2019;24(7–8):562–77. https://0-doi-org.brum.beds.ac.uk/10.1007/s10495-019-01539-7.
We thank Xiao Xu from the Department of Physiology at Henan University for providing the PC-12 cell line.
This work is supported by grants from the National Nature Science Foundation of China (No. 81900843, 81570825, U1604171, 81702721), Medical Science and Technology Development Project of Henan Health Commission (No. SBGJ202003055), Science and Technology Development Project of Henan Province (No. 202102310398, 192102310321, 202102310377), Hunan Province Natural Science Funds for Young scholars (2018JJ3816) and Key Project of Science and Technology of Henan Education Committee (No. 18A310011).
Ethics approval and consent to participate
This research does not involve any ethical issues.
Consent for publication
The authors declare no conflict of interest with respect to the research, authorship, and publication of this article.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Li, J., Yu, J., Xue, W. et al. The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro. BMC Biotechnol 21, 39 (2021). https://0-doi-org.brum.beds.ac.uk/10.1186/s12896-021-00700-y
- Affinity purification
- Chaperone activity