Subcellular distribution of endogenous malectin under rest and stress conditions is regulated by ribophorin I

Subcellular distribution of endogenous malectin under rest and stress conditions is regulated by... Abstract Malectin is a newly discovered endoplasmic reticulum (ER)-resident lectin, which specifically recognizes Glc2Man9GlcNAc2 on newly synthesized glycoproteins. We have previously demonstrated that malectin forms a complex with ribophorin I for selective retention of misfolded glycoproteins inside the cell. Here, we showed that ribophorin I also functions to regulate the subcellular localization of malectin under various conditions. Even though malectin does not contain an ER-retention signal motif, we found that endogenous malectin mainly localizes in the ER, which is disrupted upon suppression of ribophorin I, leading to its movement from ER to Golgi. In contrast, under ER-stress conditions, malectin mainly localizes in the Golgi, which is restored to ER localization by overexpression of ribophorin I. These results indicate that the subcellular localization of malectin is accurately regulated by the expression level of ribophorin I, which will provide further insights into the understanding of the function of malectin. endoplasmic reticulum, lectin, malectin, ribophorin I, subcellular localization Introduction Lectins are carbohydrate-binding proteins that are highly specific for the sugar moieties. They distribute ubiquitously in nature and serve various biological functions in living organisms. Some lectins localize in the endoplasmic reticulum (ER), namely, ER-resident lectins, which are known to play important roles in the quality control of N-glycoproteins (Hebert et al. 2010). In the lumen of the ER, N-glycosylation of proteins is initiated by oligosaccharyltransferase (OST), which catalyzes the transfer of Glc3Man9GlcNAc2 (G3M9) as en bloc from lipid-linked intermediates to asparagine residues within the Asn-X-Ser/Thr motif in newly synthesized peptides (Kelleher and Gilmore 2006). During the folding of nascent glycoproteins, G3M9 is trimmed to remove several glucose and mannose residues to generate G2M9, G1M9, M9, M8 and so on by ER-resident glucosidases and mannosidases (Bernasconi et al. 2008; Gauss et al. 2011; Hosokawa et al. 2008, 2009; Mikami et al. 2010; Sousa et al. 1992; Yamaguchi et al. 2010), which work as signals for cell to recognize the folding status of glycoproteins and take suitable actions. In this process, the ER-resident lectins play important roles by specific recognition of different sugar chains, for example, calnexin/calreticulin recognize G1M9 to facilitate the folding of glycoproteins, VIP36/VIPL bind M9 or M8B on folded glycoproteins and transport them to Golgi (Yamamoto 2014), XTP-3B and OS9 bind M8C, M7B, M6, M5 on misfolded glycoproteins and transport them to cytoplasm for degradation, namely ER-associated degradation (ERAD) (Bernasconi et al. 2008; Hosokawa et al. 2008, 2009; Mikami et al. 2010; Yamaguchi et al. 2010). Malectin is a membrane-anchored ER-resident lectin, which is first identified in Xenopus laevis in 2008 and is highly conserved among animals (Schallus et al. 2008). The fact that malectin specifically recognizes G2M9 has drawn great concern of its functions in glycoprotein quality control system. Many studies have shown that malectin is an ER stress-induced lectin and preferentially associates with folding-defective glycoproteins and reduces their secretion (Chen et al. 2011; Galli et al. 2011; Qin, Hu, et al. 2012; Takeda et al. 2014). Recently, we have demonstrated that malectin forms a complex with ribophorin I for selective retention of misfolded glycoproteins, in which malectin recognizes G2M9 glycan while ribophorin I recognizes the misfolded protein backbone (Qin, Hu, et al. 2012). So far, many studies have shown that malectin localizes in the ER for its function, but it does not have an ER localization signal motif in its amino acid sequence (Nilsson and Warren 1994; Schallus et al. 2008). Thus, we hypothesized that subcellular localization of malectin in the ER may be due to the complex formation with ribophorin I, which is a subunit of ER-resident OST complex (Wilson and High 2007). Considering that malectin is an ER stress-induced lectin, its localization may be changed under the ER-stress conditions. In the present study, we also explored the intercellular localization of malectin under ER stress conditions. Our results showed that malectin localizes in the ER under normal condition, but mainly distributes in the Golgi under ER stress conditions, which is controlled by the expression level of ribophorin I. Results Malectin mainly localizes in the ER under the normal condition Considering all the previous studies on malectin distribution were performed by overexpression of malectin. In this study, we first examined the subcellular localization of endogenous malectin under the normal physiological condition by double label immunofluorescence with the use of malectin antibody with calnexin (ER marker) or protein disulfide isomerase (PDI, ER marker), or GM130 (Golgi marker). The specificity of malectin antibody was confirmed by a full immunoblot experiment using malectin knock out cells (Supplementary Figure S1). As shown in Figure 1A, the distribution pattern of malectin and calnexin is somehow different. In most cells, malectin forms a concentrated dot-like structure, which contrasts with the even distribution of calnexin. Despite these, the yellow spots in the merged image indicate that the majority of malectin is still localized in the ER, possibly in a special subcompartment of the ER rather than the bulk ER. The compact distribution of malectin is reminiscent of Golgi apparatus. To address this possibility, we performed a direction comparison of malectin with a Golgi maker GM130 and we did not see any overlap between these two proteins (Figure 2C). For further confirmation, we also investigated the colocalization of malectin with another commonly used ER maker PDI. As shown in Figure 1B, a relatively high-degree colocalization of malectin and PDI was observed though they were still not completely overlapped. These results confirmed that under the normal condition, malectin mainly localizes in the ER, possibly in a special subcompartment. Fig. 1. View largeDownload slide Malectin localizes in the ER under the normal condition. HeLa cells were fixed, and permeabilized, and double label immunofluorescence was performed. (A) HeLa cells were costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER. The lower panel shows a cell representative of the general population in the upper panel. (B) HeLa cells were costained with anti-PDI antibody and antimalectin antibody for visualization of malectin in the ER. The lower panel shows a cell representative of the general population in the upper panel. (C) HeLa cells were costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi. The lower panel shows a cell representative of the general population in the upper panel. The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 1. View largeDownload slide Malectin localizes in the ER under the normal condition. HeLa cells were fixed, and permeabilized, and double label immunofluorescence was performed. (A) HeLa cells were costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER. The lower panel shows a cell representative of the general population in the upper panel. (B) HeLa cells were costained with anti-PDI antibody and antimalectin antibody for visualization of malectin in the ER. The lower panel shows a cell representative of the general population in the upper panel. (C) HeLa cells were costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi. The lower panel shows a cell representative of the general population in the upper panel. The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 2. View largeDownload slide SiRNA-mediated down-regulation of ribophorin I leads to the localization of malectin in Golgi apparatus. (A) HeLa cells were transfected with negative control siRNA (siNC) or siRNA specific for ribophorin I (siRib I) for 48 h, then the expression of ribophorin I at protein level was analyzed by Western blotting. (B) HeLa cells were transfected with siNC or siRib I for 48 h, then the expression of ribophorin I at mRNA level was analyzed by RT-PCR. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01 (Student’s t test)). (C–F) HeLa cells were transfected with siNC (C and D) or siRib I (E and F) for 48 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (C and E) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (D and F). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 2. View largeDownload slide SiRNA-mediated down-regulation of ribophorin I leads to the localization of malectin in Golgi apparatus. (A) HeLa cells were transfected with negative control siRNA (siNC) or siRNA specific for ribophorin I (siRib I) for 48 h, then the expression of ribophorin I at protein level was analyzed by Western blotting. (B) HeLa cells were transfected with siNC or siRib I for 48 h, then the expression of ribophorin I at mRNA level was analyzed by RT-PCR. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01 (Student’s t test)). (C–F) HeLa cells were transfected with siNC (C and D) or siRib I (E and F) for 48 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (C and E) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (D and F). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. SiRNA-mediated down-regulation of ribophorin I leads to the subcellular localization of malectin in the Golgi apparatus Since malectin does not have an ER localization signal motif in its amino acid sequence, we hypothesized that subcellular localization of malectin in the ER may be due to the complex formation with ribophorin I. To verify this, we examined whether inhibition of ribophorin I expression could affect the distribution of malectin. After transfecting cells with control siRNA (siNC) and ribophorin I-specific siRNA for 48 h, the silencing efficiency of ribophorin I in HeLa cells was investigated by western blotting of total cell lysates using antiribophorin I antibody (Figure 2A) and real-time quantitative PCR (RT-PCR) (Fig. 2B). Compared to that treated with siNC, the expression of ribophorin I was significantly decreased by treatment with ribophorin I siRNA (Figure 2A and B). Along with the siRNA-mediated knockdown of ribophorin I, malectin, which mainly distributes in the ER under normal conditions (Figure 2C and D, Supplementary Figure S2A and B), became mainly localized in the Golgi apparatus with a morphological change to form a more compact shape (Figure 2E and F, Supplementary Figure S2C and D). These results indicated that ribophorin I is important to maintain the subcellular localization of malectin in the ER. Malectin moves from ER to Golgi under ER-stress conditions Our previous studies have shown that the expression of malectin is up-regulated under ER-stress conditions. We wondered whether its localization will be affected under ER-stress conditions. We investigated the localization of malectin after the induction of ER-stress by thapsigargin (TG), which induces the ER-stress by blocking the ability of the cell to pump calcium into ER (Oslowski and Urano 2011). To verify whether ER-stress was really induced by TG, the expression of two ER-stress markers Chop and Bip were investigated after treatment with 3 μM TG for 24 h. As shown in Figure 3A and B, a significantly increased expression of Chop and Bip were observed at protein level and mRNA level, respectively. Under these conditions, malectin, which distributed in the ER under normal condition, became mainly localized to the Golgi apparatus (Figure 3C and D, Supplementary Figure S3A and B). Similar change in the distribution of malectin was observed when cells were treated with 5 μg/mL tunicamycin (TM) for 24 h (Figure 3E and F, Supplementary Figure S3C and D), which also causes ER-stress by causing the accumulation of nonglycosylated proteins in the ER lumen (Oslowski and Urano 2011). Fig. 3. View largeDownload slide Subcellular localization of malectin under ER-stress conditions. HeLa cells were treated with 3 μM thapsigargin (TG) and 5 μg/mL tunicamycin (TM) for 24 h, then the expression of Chop at protein level (A) and Bip at mRNA level (B) was analyzed by Western blotting and RT-PCR, respectively. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01 (Student’s t test)). (C–F) HeLa cells were treated with 3 μM TG (C and D) or 5 μg/mL TM (E and F) for 24 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (C and E) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (D and F). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 3. View largeDownload slide Subcellular localization of malectin under ER-stress conditions. HeLa cells were treated with 3 μM thapsigargin (TG) and 5 μg/mL tunicamycin (TM) for 24 h, then the expression of Chop at protein level (A) and Bip at mRNA level (B) was analyzed by Western blotting and RT-PCR, respectively. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01 (Student’s t test)). (C–F) HeLa cells were treated with 3 μM TG (C and D) or 5 μg/mL TM (E and F) for 24 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (C and E) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (D and F). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Overexpression of ribophorin I restores the localization of malectin to ER under ER-stress conditions Since the subcellular localization of malectin is regulated by ribophorin I, we wondered whether the distribution of malectin in Golgi under ER-stress conditions is due to the disruption of the expression balance between malectin and ribophorin I. We then investigated the expression of malectin and ribophorin I under ER-stress conditions. Consistent with previous report, the expression of malectin was significantly induced upon TG or TM treatment by RT-PCR (Figure 4A). This result was also confirmed at protein level by Western blotting (Figure 4B). In contrast, although the expression of ribophorin I was also induced at mRNA level (Figure 4A), the amount of ribophorin I protein was significantly decreased (Figure 4B). These finding raise a possibility that the distribution of malectin to Golgi is likely to be caused by decreased expression of ribophorin I under ER-stress conditions. To verify this, we next examined the distribution of malectin by overexpression of ribophorin I under the ER-stress conditions. We treated both MOCK and ribophorin I over-expressing cells with 3 μM TG for 24 h. Expression of ribophorin I in HeLa cells was confirmed by western blotting (Figure 4C). Compared to the MOCK cell in which malectin mainly localized in the Golgi upon ER stress (Figure 4D and E, Supplementary Figure S4A and B), overexpression of ribophorin I restored the localization of malectin into the ER even under the ER-stress conditions (Figure 4F and G, Supplementary Figure S4C and D). These results indicated that ribophorin I plays an essential role in control of the subcellular localization of malectin under various conditions. Fig. 4. View largeDownload slide Overexpression of ribophorin I restores the localization of malectin to ER under ER-stress conditions. HeLa cells were treated with 3 μM TG and 5 μg/mL TM for 24 h, then the expression of Malectin (Mal) and ribophorin I (Rib I) at mRNA level (A) and protein level (B) was analyzed by RT-PCR and Western blotting. (C) HeLa cells were transfected with empty vector (Mock) or ribophorin I expression vector (Rib I) for 48 h, then the expression of ribophorin I was analyzed by Western blotting. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, *P < 0.05 (Student’s t test)). (D–G) HeLa cells were transfected with empty plasmid (D and E) or ribophorin I express plasmid (F and G) for 24 h, then treated with 3 μM TG for 24 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (D and F) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (E and G). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 4. View largeDownload slide Overexpression of ribophorin I restores the localization of malectin to ER under ER-stress conditions. HeLa cells were treated with 3 μM TG and 5 μg/mL TM for 24 h, then the expression of Malectin (Mal) and ribophorin I (Rib I) at mRNA level (A) and protein level (B) was analyzed by RT-PCR and Western blotting. (C) HeLa cells were transfected with empty vector (Mock) or ribophorin I expression vector (Rib I) for 48 h, then the expression of ribophorin I was analyzed by Western blotting. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, *P < 0.05 (Student’s t test)). (D–G) HeLa cells were transfected with empty plasmid (D and E) or ribophorin I express plasmid (F and G) for 24 h, then treated with 3 μM TG for 24 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (D and F) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (E and G). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Discussion Malectin is a membrane-anchored ER lectin, which was first identified in Xenopus laevis in 2008 and is highly conserved among animals (Schallus et al. 2008). Because malectin specially recognizes G2M9 which is generated at the very early stage of the processing of N-glycans, its function and cellular localization have drawn much attention. We and other groups have shown that malectin is involved in the quality control of glycoproteins (Chen et al. 2011; Galli et al. 2011; Qin, Hu, et al. 2012; Schallus et al. 2008; Takeda et al. 2014; Yamamoto 2014), however, the localization of endogenous malectin and its regulation mechanism remain elusive. Several studies by overexpression of malectin have revealed that malectin localizes in the ER (Galli et al. 2011; Schallus et al. 2008; Takeda et al. 2014). To our acknowledge, the membrane proteins resident in the ER usually contain the dilysine based ER retention sequence (Nilsson and Warren 1994), however, malectin does not have this motif in its amino acid sequence (Schallus et al. 2008). These facts raised a question on whether the endogenous malectin localizes in the ER and if so, how it is retained in the ER. In our present studies, we investigated the localization of endogenous malectin. Our results showed that malectin forms a concentrated structure inside the cell under normal condition, which is only partially overlapped with calnexin or PDI (Fig. 1), suggesting that maletin is likely to localize in a special subcompartment rather than the bulk ER. Lederkremer et al. proposed that in the ER there is a subcompartment called ER quality control (ERQC), where substrates for ER/proteasomal degradation are concentrated (Kamhi-Nesher et al. 2001). Considering that malectin is involved in retaining misfolded glycoprotein, it is reasonable for malectin to localize in ERQC to execute its function. Since malectin does not have a ER retention signal, researchers have thought that retention of malectin in the ER might be attributed to its interaction with other ER-resident proteins (Schallus et al. 2008). We have previously shown that malectin forms a complex with ribophorin I to play its role in selective recognition of misfolded glycoproteins in the ER (Qin, Hu, et al. 2012). Therefore, in this study, we examined the effects of ribophorin I on the cellular distribution of malectin by down-regulation of the ribophorin I expression. The results showed that ribophorin I really contributes to the localization of malectin in the ER (Figure 2). Considering that the expression of malectin can be induced upon ER-stress, we also investigated the subcellular localization of malectin under ER-stress conditions induced by TG or TM. We observed a significant distribution shift of malectin from ER to the Golgi apparatus (Figure 3), which was likely to be caused by the overexpression of malectin relative to ribophorin I under ER-stress conditions (Figure 4). On the other hand, we and other group have previously shown that overexpressed malectin predominantly localizes in the ER (Galli et al. 2011; Schallus et al. 2008; Takeda et al. 2014). According to our present conclusion, overexpression of malectin should increase the ratio of malectin/ribophorin I, which will lead to the localization of malectin to the Golgi apparatus. Although thus far we have no direct evidence, one possibility for this discrepancy is that the expression level of endogenous ribophorin I is much higher than that of endogenous malectin, which will retain the overexpressed malectin in the ER. We also investigated whether the expression of ribophorin I was induced upon overexpression of malectin, but we did not see any significant changes in ribophorin I expression level when malectin was overexpressed. Although we still can not rule out other mechanisms involved in localization of malectin, the expression balance between malectin and ribophorin I should play an important role in intracellular localization of malectin. Interestingly, the localization shift of malectin from ER to the Golgi apparatus under ER-stress conditions is quite similar with the ER-resident lectin cargo receptor ERGIC-53 (Appenzeller et al. 1999; Qin, Kawasaki, et al. 2012; Vollenweider et al. 1998), which also raises a possibility that malectin may be also involved in the transportation of glycoproteins between ER and Golgi. Based on the above facts, we proposed a model: under the normal condition, malectin forms a complex with ribophorin I for selective retention of misfolded glycoproteins in the ERQC and transport them to cytoplasm for degradation by ERAD. However, under the ER-stress conditions caused by the accumulation of excessive misfolded proteins, a further retention of misfolded glycoproteins in the ER makes no sense. Overexpression of malectin relative to ribophorin I under these conditions will increase the ratio of free malectin, which will bind G2M9-bearing glycoproteins and transport them to the Golgi to reduce the burden of ER. This is the first study on the subcellular distribution of endogenous malectin and its regulation mechanism. Materials and methods Cells HeLa cells were maintained in DMEM containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified incubator with 5% CO2. Antibodies Monoclonal antimalectin antibody was purchased from sigma. Polyclonal antiribophorin I antibody was purchased from Santa Cruz Biotechnology. Monoclonal anti-β-actin, goat antimouse IgG, and goat antirabbit IgG, antibodies were purchased from Proteintech. Polyclonal anticalnexin antibody was purchased from Abcam. Monoclonal anti-GM130 and polyclonal anti-Chop antibodies were purchased from Cell Signaling Technology. AlexaFluor-488 goat antimouse IgG and AlexaFluor-543 goat antirabbit IgG antibodies were obtained from Invitrogen. Construction of expression plasmid for ribophorin I The coding sequence for human ribophorin I was amplified by PCR using primers: 5′-GAGTTAACGCCTCCTCCGAGGCACCG-3′ and 5′-AAGGAAAAAAGCGGCCGCCTACAGGGCATCCAGGAT-3′ (HpaI and NotI sites are underlined). The amplified DNA was digested with HpaI and NotI and then inserted into the HpaI and NotI sites of a pRcCMV-based vector containing an N-terminal CD8α signal sequence followed by a Myc tag sequence as described previously (Qin, Hu, et al. 2012). Real-time quantitative PCR Total RNA was isolated with Trizol reagent according to the manufacturer’s instrutions. Then RNA samples were reverse transcribed using Primescript™ First-Strand cDNA Synthesis kit following the manufacturer’s protocol (Takara Bio, Dalian, China). Real-time quantitative PCR was performed with SYBR Premex EX Taq (Takara Bio, Dalian, China) using the LightCycler® 480 System (POD; Roche Diagnostics GmbH, Mannheim, Germany). The relative amounts of RNA for the target gene transcripts were normalized against an endogenous gene β-actin. Primer details are listed as following: malectin-F, 5′-GCAAGGACCCTTTGGAAGGC-3′, malectin-R, 5′-GCTGGGACTGTGCAAAGTAG-3′; ribophorin I-F, 5′-TCTTCACCGTTATCATCTATGTTCG-3′, ribophorin I-R, 5′-TGTTCAGTCTCCAGGCTCTTCTT-3′; Bip-F, 5′-CCTGGGTGGCGGAACCTTCGATGTG-3′, Bip-R, 5′-CTGGACGGGCTTCATAGTAGACCGG-3′; β-actin-F, 5′-CTTCCTGGGCATGGAGTCCT-3′, β-actin-R, 5′-GGAGCAATGATCTTGATCTT-3′. The P-values were calculated using a paired Student’s t-test and statistical significance was determined when a P-value was <0.05. Polyacrylamide gel electrophoresis and Western blotting Cellular total protein samples were separated by SDS-polyacrylamide gel electrophoresis according to the method of Laemmli (Laemmli 1970), and transferred to PVDF membrane (Millipore, Bedford, MA) at 15 V for 45 min using semi-dry blotting system (Bio-Rad Laboratories, Hercules, CA). The membrane was incubated with 20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, 0.005% Tween 20 (TBS-T) and 3% BSA for 1 h and then incubated with anti-Chop, antiribophorin I or anti-β-actin antibody for overnight at 4°C followed by washing three times for each 15 min with TBS-T. Next, the membrane was incubated with horseradish peroxidase-conjugated goat antimouse IgG or goat antirabbit IgG for 1 h at room temperature, followed by washing three times for each 15 min with TBS-T. Finally, the membrane was developed in BeyoECL plus (Beyotime Biotechnology Co., Shanghai, China) with a KODAK Gel Logic 1500 Transilluminator Integrated Imaging System. Immunofluorescence microscopy HeLa cells were seeded and cultured into dishes special for confocal and fixed with 4% para-formaldehyde for 20 min at room temperature, followed by permeabilization for 5 min with 0.1% Triton X-100 and blocked for 30 min with 10% FBS at room temperature. Cells were then incubated overnight at 4°C with 5 μg/mL antimalectin antibody and 5 μg/mL anticalnexin antibody for the staining of the ER, or and 5 μg/mL anti-GM130 antibody for the staining of the Golgi apparatus. After washing three times (10 min each) with PBS, cells were incubated for 30 min with secondary antibody conjugated with fluorochromes at 37°C. The specimens were analyzed using a Zeiss LSM 510 Meta laser scanning confocal microscope system. RNA interference One day before transfection, cells were seeded on six-well plates at 30–50% confluency, and then transfected with siRib I or siNC for 48 h by using Lipofectamine 2000 (Invitrogen). siRNA details are listed as following: siRib I: 5′-GGAAGUACGUGAAACCAAAUU-3′; siNC: 5′-UUCUCCGAACGUGUCACGUTT-3′. Supplementary data Supplementary data is available at Glycobiology online. Funding National Natural Science Foundation of China [81602436]; Natural Science Foundation of Guangdong Province [2016A030313102]; China Postdoctoral Science Foundation [2013M540683]; and the Fundamental Research Funds for the Central Universities [21617495]. Conflict of interest statement None declared. Abbreviations ER endoplasmic reticulum ERAD ER-associated degradation ERGIC ER and Golgi intermediate compartment ERQC ER quality control G3M9 Glc3Man9GlcNAc2 OST oligosaccharyltransferase TG thapsigargin TM tunicamycin References Appenzeller C , Andersson H , Kappeler F , Hauri HP . 1999 . 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Google Scholar CrossRef Search ADS PubMed Yamaguchi D , Hu D , Matsumoto N , Yamamoto K . 2010 . Human XTP3-B binds to alpha1-antitrypsin variant null(Hong Kong) via the C-terminal MRH domain in a glycan-dependent manner . Glycobiology . 20 : 348 – 355 . Google Scholar CrossRef Search ADS PubMed Yamamoto K . 2014 . Intracellular lectins are involved in quality control of glycoproteins . Proc Jpn Acad Ser B Phys Biol Sci . 90 : 67 – 82 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycobiology Oxford University Press

Subcellular distribution of endogenous malectin under rest and stress conditions is regulated by ribophorin I

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10.1093/glycob/cwy034
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Abstract

Abstract Malectin is a newly discovered endoplasmic reticulum (ER)-resident lectin, which specifically recognizes Glc2Man9GlcNAc2 on newly synthesized glycoproteins. We have previously demonstrated that malectin forms a complex with ribophorin I for selective retention of misfolded glycoproteins inside the cell. Here, we showed that ribophorin I also functions to regulate the subcellular localization of malectin under various conditions. Even though malectin does not contain an ER-retention signal motif, we found that endogenous malectin mainly localizes in the ER, which is disrupted upon suppression of ribophorin I, leading to its movement from ER to Golgi. In contrast, under ER-stress conditions, malectin mainly localizes in the Golgi, which is restored to ER localization by overexpression of ribophorin I. These results indicate that the subcellular localization of malectin is accurately regulated by the expression level of ribophorin I, which will provide further insights into the understanding of the function of malectin. endoplasmic reticulum, lectin, malectin, ribophorin I, subcellular localization Introduction Lectins are carbohydrate-binding proteins that are highly specific for the sugar moieties. They distribute ubiquitously in nature and serve various biological functions in living organisms. Some lectins localize in the endoplasmic reticulum (ER), namely, ER-resident lectins, which are known to play important roles in the quality control of N-glycoproteins (Hebert et al. 2010). In the lumen of the ER, N-glycosylation of proteins is initiated by oligosaccharyltransferase (OST), which catalyzes the transfer of Glc3Man9GlcNAc2 (G3M9) as en bloc from lipid-linked intermediates to asparagine residues within the Asn-X-Ser/Thr motif in newly synthesized peptides (Kelleher and Gilmore 2006). During the folding of nascent glycoproteins, G3M9 is trimmed to remove several glucose and mannose residues to generate G2M9, G1M9, M9, M8 and so on by ER-resident glucosidases and mannosidases (Bernasconi et al. 2008; Gauss et al. 2011; Hosokawa et al. 2008, 2009; Mikami et al. 2010; Sousa et al. 1992; Yamaguchi et al. 2010), which work as signals for cell to recognize the folding status of glycoproteins and take suitable actions. In this process, the ER-resident lectins play important roles by specific recognition of different sugar chains, for example, calnexin/calreticulin recognize G1M9 to facilitate the folding of glycoproteins, VIP36/VIPL bind M9 or M8B on folded glycoproteins and transport them to Golgi (Yamamoto 2014), XTP-3B and OS9 bind M8C, M7B, M6, M5 on misfolded glycoproteins and transport them to cytoplasm for degradation, namely ER-associated degradation (ERAD) (Bernasconi et al. 2008; Hosokawa et al. 2008, 2009; Mikami et al. 2010; Yamaguchi et al. 2010). Malectin is a membrane-anchored ER-resident lectin, which is first identified in Xenopus laevis in 2008 and is highly conserved among animals (Schallus et al. 2008). The fact that malectin specifically recognizes G2M9 has drawn great concern of its functions in glycoprotein quality control system. Many studies have shown that malectin is an ER stress-induced lectin and preferentially associates with folding-defective glycoproteins and reduces their secretion (Chen et al. 2011; Galli et al. 2011; Qin, Hu, et al. 2012; Takeda et al. 2014). Recently, we have demonstrated that malectin forms a complex with ribophorin I for selective retention of misfolded glycoproteins, in which malectin recognizes G2M9 glycan while ribophorin I recognizes the misfolded protein backbone (Qin, Hu, et al. 2012). So far, many studies have shown that malectin localizes in the ER for its function, but it does not have an ER localization signal motif in its amino acid sequence (Nilsson and Warren 1994; Schallus et al. 2008). Thus, we hypothesized that subcellular localization of malectin in the ER may be due to the complex formation with ribophorin I, which is a subunit of ER-resident OST complex (Wilson and High 2007). Considering that malectin is an ER stress-induced lectin, its localization may be changed under the ER-stress conditions. In the present study, we also explored the intercellular localization of malectin under ER stress conditions. Our results showed that malectin localizes in the ER under normal condition, but mainly distributes in the Golgi under ER stress conditions, which is controlled by the expression level of ribophorin I. Results Malectin mainly localizes in the ER under the normal condition Considering all the previous studies on malectin distribution were performed by overexpression of malectin. In this study, we first examined the subcellular localization of endogenous malectin under the normal physiological condition by double label immunofluorescence with the use of malectin antibody with calnexin (ER marker) or protein disulfide isomerase (PDI, ER marker), or GM130 (Golgi marker). The specificity of malectin antibody was confirmed by a full immunoblot experiment using malectin knock out cells (Supplementary Figure S1). As shown in Figure 1A, the distribution pattern of malectin and calnexin is somehow different. In most cells, malectin forms a concentrated dot-like structure, which contrasts with the even distribution of calnexin. Despite these, the yellow spots in the merged image indicate that the majority of malectin is still localized in the ER, possibly in a special subcompartment of the ER rather than the bulk ER. The compact distribution of malectin is reminiscent of Golgi apparatus. To address this possibility, we performed a direction comparison of malectin with a Golgi maker GM130 and we did not see any overlap between these two proteins (Figure 2C). For further confirmation, we also investigated the colocalization of malectin with another commonly used ER maker PDI. As shown in Figure 1B, a relatively high-degree colocalization of malectin and PDI was observed though they were still not completely overlapped. These results confirmed that under the normal condition, malectin mainly localizes in the ER, possibly in a special subcompartment. Fig. 1. View largeDownload slide Malectin localizes in the ER under the normal condition. HeLa cells were fixed, and permeabilized, and double label immunofluorescence was performed. (A) HeLa cells were costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER. The lower panel shows a cell representative of the general population in the upper panel. (B) HeLa cells were costained with anti-PDI antibody and antimalectin antibody for visualization of malectin in the ER. The lower panel shows a cell representative of the general population in the upper panel. (C) HeLa cells were costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi. The lower panel shows a cell representative of the general population in the upper panel. The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 1. View largeDownload slide Malectin localizes in the ER under the normal condition. HeLa cells were fixed, and permeabilized, and double label immunofluorescence was performed. (A) HeLa cells were costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER. The lower panel shows a cell representative of the general population in the upper panel. (B) HeLa cells were costained with anti-PDI antibody and antimalectin antibody for visualization of malectin in the ER. The lower panel shows a cell representative of the general population in the upper panel. (C) HeLa cells were costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi. The lower panel shows a cell representative of the general population in the upper panel. The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 2. View largeDownload slide SiRNA-mediated down-regulation of ribophorin I leads to the localization of malectin in Golgi apparatus. (A) HeLa cells were transfected with negative control siRNA (siNC) or siRNA specific for ribophorin I (siRib I) for 48 h, then the expression of ribophorin I at protein level was analyzed by Western blotting. (B) HeLa cells were transfected with siNC or siRib I for 48 h, then the expression of ribophorin I at mRNA level was analyzed by RT-PCR. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01 (Student’s t test)). (C–F) HeLa cells were transfected with siNC (C and D) or siRib I (E and F) for 48 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (C and E) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (D and F). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 2. View largeDownload slide SiRNA-mediated down-regulation of ribophorin I leads to the localization of malectin in Golgi apparatus. (A) HeLa cells were transfected with negative control siRNA (siNC) or siRNA specific for ribophorin I (siRib I) for 48 h, then the expression of ribophorin I at protein level was analyzed by Western blotting. (B) HeLa cells were transfected with siNC or siRib I for 48 h, then the expression of ribophorin I at mRNA level was analyzed by RT-PCR. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01 (Student’s t test)). (C–F) HeLa cells were transfected with siNC (C and D) or siRib I (E and F) for 48 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (C and E) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (D and F). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. SiRNA-mediated down-regulation of ribophorin I leads to the subcellular localization of malectin in the Golgi apparatus Since malectin does not have an ER localization signal motif in its amino acid sequence, we hypothesized that subcellular localization of malectin in the ER may be due to the complex formation with ribophorin I. To verify this, we examined whether inhibition of ribophorin I expression could affect the distribution of malectin. After transfecting cells with control siRNA (siNC) and ribophorin I-specific siRNA for 48 h, the silencing efficiency of ribophorin I in HeLa cells was investigated by western blotting of total cell lysates using antiribophorin I antibody (Figure 2A) and real-time quantitative PCR (RT-PCR) (Fig. 2B). Compared to that treated with siNC, the expression of ribophorin I was significantly decreased by treatment with ribophorin I siRNA (Figure 2A and B). Along with the siRNA-mediated knockdown of ribophorin I, malectin, which mainly distributes in the ER under normal conditions (Figure 2C and D, Supplementary Figure S2A and B), became mainly localized in the Golgi apparatus with a morphological change to form a more compact shape (Figure 2E and F, Supplementary Figure S2C and D). These results indicated that ribophorin I is important to maintain the subcellular localization of malectin in the ER. Malectin moves from ER to Golgi under ER-stress conditions Our previous studies have shown that the expression of malectin is up-regulated under ER-stress conditions. We wondered whether its localization will be affected under ER-stress conditions. We investigated the localization of malectin after the induction of ER-stress by thapsigargin (TG), which induces the ER-stress by blocking the ability of the cell to pump calcium into ER (Oslowski and Urano 2011). To verify whether ER-stress was really induced by TG, the expression of two ER-stress markers Chop and Bip were investigated after treatment with 3 μM TG for 24 h. As shown in Figure 3A and B, a significantly increased expression of Chop and Bip were observed at protein level and mRNA level, respectively. Under these conditions, malectin, which distributed in the ER under normal condition, became mainly localized to the Golgi apparatus (Figure 3C and D, Supplementary Figure S3A and B). Similar change in the distribution of malectin was observed when cells were treated with 5 μg/mL tunicamycin (TM) for 24 h (Figure 3E and F, Supplementary Figure S3C and D), which also causes ER-stress by causing the accumulation of nonglycosylated proteins in the ER lumen (Oslowski and Urano 2011). Fig. 3. View largeDownload slide Subcellular localization of malectin under ER-stress conditions. HeLa cells were treated with 3 μM thapsigargin (TG) and 5 μg/mL tunicamycin (TM) for 24 h, then the expression of Chop at protein level (A) and Bip at mRNA level (B) was analyzed by Western blotting and RT-PCR, respectively. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01 (Student’s t test)). (C–F) HeLa cells were treated with 3 μM TG (C and D) or 5 μg/mL TM (E and F) for 24 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (C and E) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (D and F). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 3. View largeDownload slide Subcellular localization of malectin under ER-stress conditions. HeLa cells were treated with 3 μM thapsigargin (TG) and 5 μg/mL tunicamycin (TM) for 24 h, then the expression of Chop at protein level (A) and Bip at mRNA level (B) was analyzed by Western blotting and RT-PCR, respectively. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01 (Student’s t test)). (C–F) HeLa cells were treated with 3 μM TG (C and D) or 5 μg/mL TM (E and F) for 24 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (C and E) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (D and F). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Overexpression of ribophorin I restores the localization of malectin to ER under ER-stress conditions Since the subcellular localization of malectin is regulated by ribophorin I, we wondered whether the distribution of malectin in Golgi under ER-stress conditions is due to the disruption of the expression balance between malectin and ribophorin I. We then investigated the expression of malectin and ribophorin I under ER-stress conditions. Consistent with previous report, the expression of malectin was significantly induced upon TG or TM treatment by RT-PCR (Figure 4A). This result was also confirmed at protein level by Western blotting (Figure 4B). In contrast, although the expression of ribophorin I was also induced at mRNA level (Figure 4A), the amount of ribophorin I protein was significantly decreased (Figure 4B). These finding raise a possibility that the distribution of malectin to Golgi is likely to be caused by decreased expression of ribophorin I under ER-stress conditions. To verify this, we next examined the distribution of malectin by overexpression of ribophorin I under the ER-stress conditions. We treated both MOCK and ribophorin I over-expressing cells with 3 μM TG for 24 h. Expression of ribophorin I in HeLa cells was confirmed by western blotting (Figure 4C). Compared to the MOCK cell in which malectin mainly localized in the Golgi upon ER stress (Figure 4D and E, Supplementary Figure S4A and B), overexpression of ribophorin I restored the localization of malectin into the ER even under the ER-stress conditions (Figure 4F and G, Supplementary Figure S4C and D). These results indicated that ribophorin I plays an essential role in control of the subcellular localization of malectin under various conditions. Fig. 4. View largeDownload slide Overexpression of ribophorin I restores the localization of malectin to ER under ER-stress conditions. HeLa cells were treated with 3 μM TG and 5 μg/mL TM for 24 h, then the expression of Malectin (Mal) and ribophorin I (Rib I) at mRNA level (A) and protein level (B) was analyzed by RT-PCR and Western blotting. (C) HeLa cells were transfected with empty vector (Mock) or ribophorin I expression vector (Rib I) for 48 h, then the expression of ribophorin I was analyzed by Western blotting. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, *P < 0.05 (Student’s t test)). (D–G) HeLa cells were transfected with empty plasmid (D and E) or ribophorin I express plasmid (F and G) for 24 h, then treated with 3 μM TG for 24 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (D and F) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (E and G). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Fig. 4. View largeDownload slide Overexpression of ribophorin I restores the localization of malectin to ER under ER-stress conditions. HeLa cells were treated with 3 μM TG and 5 μg/mL TM for 24 h, then the expression of Malectin (Mal) and ribophorin I (Rib I) at mRNA level (A) and protein level (B) was analyzed by RT-PCR and Western blotting. (C) HeLa cells were transfected with empty vector (Mock) or ribophorin I expression vector (Rib I) for 48 h, then the expression of ribophorin I was analyzed by Western blotting. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, *P < 0.05 (Student’s t test)). (D–G) HeLa cells were transfected with empty plasmid (D and E) or ribophorin I express plasmid (F and G) for 24 h, then treated with 3 μM TG for 24 h, then they were fixed and costained with anticalnexin antibody and antimalectin antibody for visualization of malectin in the ER (D and F) or costained with anti-GM130 antibody and antimalectin antibody for visualization of malectin in the Golgi (E and G). The nucleus were stained with hochest33342 and a merged image is shown in the right panel. Scale bars, 10 μm. Discussion Malectin is a membrane-anchored ER lectin, which was first identified in Xenopus laevis in 2008 and is highly conserved among animals (Schallus et al. 2008). Because malectin specially recognizes G2M9 which is generated at the very early stage of the processing of N-glycans, its function and cellular localization have drawn much attention. We and other groups have shown that malectin is involved in the quality control of glycoproteins (Chen et al. 2011; Galli et al. 2011; Qin, Hu, et al. 2012; Schallus et al. 2008; Takeda et al. 2014; Yamamoto 2014), however, the localization of endogenous malectin and its regulation mechanism remain elusive. Several studies by overexpression of malectin have revealed that malectin localizes in the ER (Galli et al. 2011; Schallus et al. 2008; Takeda et al. 2014). To our acknowledge, the membrane proteins resident in the ER usually contain the dilysine based ER retention sequence (Nilsson and Warren 1994), however, malectin does not have this motif in its amino acid sequence (Schallus et al. 2008). These facts raised a question on whether the endogenous malectin localizes in the ER and if so, how it is retained in the ER. In our present studies, we investigated the localization of endogenous malectin. Our results showed that malectin forms a concentrated structure inside the cell under normal condition, which is only partially overlapped with calnexin or PDI (Fig. 1), suggesting that maletin is likely to localize in a special subcompartment rather than the bulk ER. Lederkremer et al. proposed that in the ER there is a subcompartment called ER quality control (ERQC), where substrates for ER/proteasomal degradation are concentrated (Kamhi-Nesher et al. 2001). Considering that malectin is involved in retaining misfolded glycoprotein, it is reasonable for malectin to localize in ERQC to execute its function. Since malectin does not have a ER retention signal, researchers have thought that retention of malectin in the ER might be attributed to its interaction with other ER-resident proteins (Schallus et al. 2008). We have previously shown that malectin forms a complex with ribophorin I to play its role in selective recognition of misfolded glycoproteins in the ER (Qin, Hu, et al. 2012). Therefore, in this study, we examined the effects of ribophorin I on the cellular distribution of malectin by down-regulation of the ribophorin I expression. The results showed that ribophorin I really contributes to the localization of malectin in the ER (Figure 2). Considering that the expression of malectin can be induced upon ER-stress, we also investigated the subcellular localization of malectin under ER-stress conditions induced by TG or TM. We observed a significant distribution shift of malectin from ER to the Golgi apparatus (Figure 3), which was likely to be caused by the overexpression of malectin relative to ribophorin I under ER-stress conditions (Figure 4). On the other hand, we and other group have previously shown that overexpressed malectin predominantly localizes in the ER (Galli et al. 2011; Schallus et al. 2008; Takeda et al. 2014). According to our present conclusion, overexpression of malectin should increase the ratio of malectin/ribophorin I, which will lead to the localization of malectin to the Golgi apparatus. Although thus far we have no direct evidence, one possibility for this discrepancy is that the expression level of endogenous ribophorin I is much higher than that of endogenous malectin, which will retain the overexpressed malectin in the ER. We also investigated whether the expression of ribophorin I was induced upon overexpression of malectin, but we did not see any significant changes in ribophorin I expression level when malectin was overexpressed. Although we still can not rule out other mechanisms involved in localization of malectin, the expression balance between malectin and ribophorin I should play an important role in intracellular localization of malectin. Interestingly, the localization shift of malectin from ER to the Golgi apparatus under ER-stress conditions is quite similar with the ER-resident lectin cargo receptor ERGIC-53 (Appenzeller et al. 1999; Qin, Kawasaki, et al. 2012; Vollenweider et al. 1998), which also raises a possibility that malectin may be also involved in the transportation of glycoproteins between ER and Golgi. Based on the above facts, we proposed a model: under the normal condition, malectin forms a complex with ribophorin I for selective retention of misfolded glycoproteins in the ERQC and transport them to cytoplasm for degradation by ERAD. However, under the ER-stress conditions caused by the accumulation of excessive misfolded proteins, a further retention of misfolded glycoproteins in the ER makes no sense. Overexpression of malectin relative to ribophorin I under these conditions will increase the ratio of free malectin, which will bind G2M9-bearing glycoproteins and transport them to the Golgi to reduce the burden of ER. This is the first study on the subcellular distribution of endogenous malectin and its regulation mechanism. Materials and methods Cells HeLa cells were maintained in DMEM containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified incubator with 5% CO2. Antibodies Monoclonal antimalectin antibody was purchased from sigma. Polyclonal antiribophorin I antibody was purchased from Santa Cruz Biotechnology. Monoclonal anti-β-actin, goat antimouse IgG, and goat antirabbit IgG, antibodies were purchased from Proteintech. Polyclonal anticalnexin antibody was purchased from Abcam. Monoclonal anti-GM130 and polyclonal anti-Chop antibodies were purchased from Cell Signaling Technology. AlexaFluor-488 goat antimouse IgG and AlexaFluor-543 goat antirabbit IgG antibodies were obtained from Invitrogen. Construction of expression plasmid for ribophorin I The coding sequence for human ribophorin I was amplified by PCR using primers: 5′-GAGTTAACGCCTCCTCCGAGGCACCG-3′ and 5′-AAGGAAAAAAGCGGCCGCCTACAGGGCATCCAGGAT-3′ (HpaI and NotI sites are underlined). The amplified DNA was digested with HpaI and NotI and then inserted into the HpaI and NotI sites of a pRcCMV-based vector containing an N-terminal CD8α signal sequence followed by a Myc tag sequence as described previously (Qin, Hu, et al. 2012). Real-time quantitative PCR Total RNA was isolated with Trizol reagent according to the manufacturer’s instrutions. Then RNA samples were reverse transcribed using Primescript™ First-Strand cDNA Synthesis kit following the manufacturer’s protocol (Takara Bio, Dalian, China). Real-time quantitative PCR was performed with SYBR Premex EX Taq (Takara Bio, Dalian, China) using the LightCycler® 480 System (POD; Roche Diagnostics GmbH, Mannheim, Germany). The relative amounts of RNA for the target gene transcripts were normalized against an endogenous gene β-actin. Primer details are listed as following: malectin-F, 5′-GCAAGGACCCTTTGGAAGGC-3′, malectin-R, 5′-GCTGGGACTGTGCAAAGTAG-3′; ribophorin I-F, 5′-TCTTCACCGTTATCATCTATGTTCG-3′, ribophorin I-R, 5′-TGTTCAGTCTCCAGGCTCTTCTT-3′; Bip-F, 5′-CCTGGGTGGCGGAACCTTCGATGTG-3′, Bip-R, 5′-CTGGACGGGCTTCATAGTAGACCGG-3′; β-actin-F, 5′-CTTCCTGGGCATGGAGTCCT-3′, β-actin-R, 5′-GGAGCAATGATCTTGATCTT-3′. The P-values were calculated using a paired Student’s t-test and statistical significance was determined when a P-value was <0.05. Polyacrylamide gel electrophoresis and Western blotting Cellular total protein samples were separated by SDS-polyacrylamide gel electrophoresis according to the method of Laemmli (Laemmli 1970), and transferred to PVDF membrane (Millipore, Bedford, MA) at 15 V for 45 min using semi-dry blotting system (Bio-Rad Laboratories, Hercules, CA). The membrane was incubated with 20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, 0.005% Tween 20 (TBS-T) and 3% BSA for 1 h and then incubated with anti-Chop, antiribophorin I or anti-β-actin antibody for overnight at 4°C followed by washing three times for each 15 min with TBS-T. Next, the membrane was incubated with horseradish peroxidase-conjugated goat antimouse IgG or goat antirabbit IgG for 1 h at room temperature, followed by washing three times for each 15 min with TBS-T. Finally, the membrane was developed in BeyoECL plus (Beyotime Biotechnology Co., Shanghai, China) with a KODAK Gel Logic 1500 Transilluminator Integrated Imaging System. Immunofluorescence microscopy HeLa cells were seeded and cultured into dishes special for confocal and fixed with 4% para-formaldehyde for 20 min at room temperature, followed by permeabilization for 5 min with 0.1% Triton X-100 and blocked for 30 min with 10% FBS at room temperature. Cells were then incubated overnight at 4°C with 5 μg/mL antimalectin antibody and 5 μg/mL anticalnexin antibody for the staining of the ER, or and 5 μg/mL anti-GM130 antibody for the staining of the Golgi apparatus. After washing three times (10 min each) with PBS, cells were incubated for 30 min with secondary antibody conjugated with fluorochromes at 37°C. The specimens were analyzed using a Zeiss LSM 510 Meta laser scanning confocal microscope system. RNA interference One day before transfection, cells were seeded on six-well plates at 30–50% confluency, and then transfected with siRib I or siNC for 48 h by using Lipofectamine 2000 (Invitrogen). siRNA details are listed as following: siRib I: 5′-GGAAGUACGUGAAACCAAAUU-3′; siNC: 5′-UUCUCCGAACGUGUCACGUTT-3′. Supplementary data Supplementary data is available at Glycobiology online. Funding National Natural Science Foundation of China [81602436]; Natural Science Foundation of Guangdong Province [2016A030313102]; China Postdoctoral Science Foundation [2013M540683]; and the Fundamental Research Funds for the Central Universities [21617495]. Conflict of interest statement None declared. Abbreviations ER endoplasmic reticulum ERAD ER-associated degradation ERGIC ER and Golgi intermediate compartment ERQC ER quality control G3M9 Glc3Man9GlcNAc2 OST oligosaccharyltransferase TG thapsigargin TM tunicamycin References Appenzeller C , Andersson H , Kappeler F , Hauri HP . 1999 . 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GlycobiologyOxford University Press

Published: Apr 2, 2018

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