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PNGase Sensitivity Assay to Study the Folding Status of Proteins
聚糖酶敏感性试验研究蛋白质的折叠状态   

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参见作者原研究论文

本实验方案简略版
The Journal of Cell Biology
Nov 2015

Abstract

This protocol aims to evaluate folding status of proteins, utilizing peptide:N-glycanase (PNGase) sensitivity. In the cytosol, PNGase works as a deglycosylation-enzyme. N-glycans on unfolded/misfolded proteins are more susceptible to PNGase than N-glycans on folded proteins because of the preference of PNGase to non-native proteins. PNGase is endogenously expressed in various cell types, including HCT116 cells, DT40 cells and mouse embryonic fibroblast cells. Partial deglycosylation by PNGase can be detected by faster migration of band in SDS-PAGE. You can compare tightness of the folding among wild-type and mutant proteins of interest. This method can be used with regular molecular and cell biology equipment, but applied only to glycoproteins.

Keywords: PNGase (pngase), Protein folding (蛋白质折叠), Glycoproteins (糖蛋白)

Materials and Reagents

  1. 6 well dish (Corning, Falcon®, catalog number: 353046 ) (Stored at room temperature)
  2. PVDF membrane (GE Healthcare, catalog number: 10600023 ) (Stored at room temperature)
  3. DT40 cell line (DT40 is a B cell line derived from an avian leukosis virus induced bursal lymphoma in a white leghorn chicken) (ATCC, catalog number: CRL-2111 ) (Endogenous expression of cytosolic PNGase)
  4. Homo sapiens colon colorectal carcinoma cell line (HCT116) (ATCC, catalog number: CCL-247 ), HCT116 cells (ATCC, catalog number: CCL-247) (Endogenous expression of cytosolic PNGase)
  5. Opti-mem (Thermo Fisher Scientific, GibcoTM, catalog number: 31985-070 )
  6. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  7. Dulbecco’s modified Eagle’s medium (DMEM) (NACALAI TESQUE, catalog number: 08458-45 ) (Stored at 4 °C)
  8. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  9. 100 U/ml penicillin and 100 μg/ml streptomycin (NACALAI TESQUE) (Stored at -20 °C)
  10. RPMI (NACALAI TESQUE, catalog number: 30263-95 ) (Stored at 4 °C)
  11. Chicken serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16110-082 )
  12. 1 M dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 ) (for reduction of proteins; in water; stored at -20 °C)
  13. 20 mM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) (Promega, catalog number: G7231 ) (for inhibition of PNGase activity; stored at -20 °C)
  14. Nonidet P-40 (NACALAI TESQUE, catalog number: 23640-94 ) (for HCT116; stored at room temperature)
  15. 4% digitonin (Wako Pure Chemical Industries, catalog number: 043-21371 ) (for DT40; in water; stored at -80 °C)
  16. Anti-myc antibody (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 562 ) (Stored at -20 °C)
  17. Anti-β-actin antibody conjugated with HRP (Wako Pure Chemical Industries, catalog number: 017-24573 )
  18. NaCl (Wako Pure Chemical Industries, catalog number: 191-01665 )
  19. Na2HPO4
  20. KCl
  21. KH2PO4
  22. Tris/HCl, pH 8.0 (Sigma-Aldrich, catalog number: T6791 ) (Stored at room temperature)
  23. Glycerol
  24. Bromophenol blue (BPB)
  25. Sodium dodecyl sulfate
  26. Protease inhibitor cocktail (100x) (NACALAI TESQUE, catalog number: 25955-11 ) (for inhibition of various proteases’ activity; in ddH2O; stored at -20 °C)
  27. 10 mM Z-Leu-Leu-Leu-CHO (MG132) (PEPTIDE INSTITUTE, catalog number: 3175-v ) (for inhibition of proteasomal activity; in DMSO; stored at -20 °C)
  28. Phosphate buffered saline (PBS) (see Recipes)
  29. 2x sodium dodecyl sulfate (SDS) sample buffer (pH 6.8) (see Recipes)
  30. Buffer A (Stored at 4 °C) (see Recipes)
  31. Buffer B (see Recipes)

Equipment

  1. High speed refrigerated micro centrifuge (Tomy, model: MX-301 )
  2. mPAGE (ATTO, model: AE-6530 ) (Using hand-made 10% gel)
  3. Transfer equipment (ATTO, model: WSE-4020 )
  4. Heat block (TAITEC, model: DTU-1BN )
  5. Micro porator (Digital Bio, model: MP-100 )

Software

  1. ImageJ

Procedure

  1. Culture adherent HCT116 cells in Dulbecco’s modified Eagle’s medium (glucose 4.5 g/L) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37 °C in a humidified 5% CO2/95% air atmosphere. Culture suspended DT40 cells at a density of 1 x 105-1 x 106 cells per ml in RPMI1640 medium supplemented with 10% fetal bovine serum, 1% chicken serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 39.5 °C in a humidified 5% CO2/95% air atmosphere.
  2. Transfect 8.0 x 105 HCT116 cells (40-60% confluent in 6-well plate) using 1 μg of plasmid DNA, 300 μl of Opti-mem and 10 μl of Lipofectamine 2000 (Invitrogen) for one well according to the manufacturers’ instructions (After the transfection medium is not replaced). To obtain 1.05 x 107 cells transfected cells, electroporate three times 3.5 x 106 DT40 cells with 8 µg plasmid DNA with two pulses at 1,500 V for 15 msec according to the manufacturer’s instructions.
  3. Collect approximately 2.0 x 106 HCT116 cells or 4.0 x 106 DT40 cells for further analysis 24 h (HCT116 cell) or 16 h (DT40 cells) after transfection. For this the cells are washed with 1 ml PBS three times, scraped off, centrifuged at 800 x g for 2 min at 4 °C. After this step, samples from HCT116 cells and DT40 cells are treated in the same manner.
  4. Remove supernatants of samples, suspend cell pellets in 200 μl buffer A and incubate for 20 min on ice to lyse the cells.
    Note: The buffer A must NOT contain Z-VAD-fmk as it would inihibit PNGase activity.
  5. Clarify cell lysates by centrifugation at 17,800 x g for 10 min at 4 °C and transfer the supernatants to new tubes. Pellets are discarded.
  6. To allow PNGase processing of misfolded proteins, incubate the cell lysates further for various periods (a good starting range would be 4 h) on ice. Remove 40-50 µl aliquots at different times, immediately mix with 40-50 μl of buffer B supplemented with 100 mM DTT (for reduction) and 2 µM Z-VAD-fmk (to inhibit the PNGase) and incubate at 100 °C for 5 min. Separate 10 µl of the samples by SDS-PAGE followed by immunoblotting using PVDF membrane and probing with an antibody against the protein of interest. Percentage of SDS-PAGE depends on the molecular weight of the protein of interest. Band intensities can be analyzed and compared to each other by ImageJ or similar programs (Figure 1).


    Figure 1. Severely misfolded mutants were more sensitive to endogenous PNGase. A. Chicken EDEM1/2/3 triple KO cells (gEDEM TKO) were described previously (Ninagawa et al., 2015). The indicated myc-tagged hATF6α(C) variants (indicated on the left side of the blots) were transiently expressed in gEDEM-TKO cells and subjected to PNGase sensitivity assay. After the indicated time points the PNGase reaction was stopped and samples analyzed by a 12% SDS-PAGE followed by immunoblotting with antibodies against the myc-tag. The data shown represents a single representative experiment out of two repeats. Less folded proteins are more sensitive to PNGase, so partially deglycosylated bands were detected at earlier time points in hATF6α(C)-myct mutants, Δ111-119, 182-194 mutant and Δ219-270 mutant. See Ninagawa et al. (2015) for further proteins’ information. B. The graph shows % intensity of upper band/total band of (A). The band intensity was analyzed by imageJ.

Notes

  1. Most cells including HCT116 cells, DT40 cells and mouse embryonic fibroblast cells express PNGase endogenously. Therefore it is not necessary to express PNGase exogenously. Other cell types have to be tested for PNGase expression. PNGase is also expressed in yeast, but we have not checked whether this assay is applicable to yeast cells.
  2. This assay has been used to determine the folding state of several proteins, however, some glycoproteins are not sensitive to PNGase at all.
  3. The use of strong detergents, such as SDS, is not appropriate for this method as PNGase activity can be lost. In our case, we used 1% NP-40 for mammalian cells and 1% digitonin for chicken cells. But you can use 1% NP-40 for chicken cells and 1% digitonin for mammalian cells. Not too strong detergent can be used for PNGase assay.
  4. As control one can include two micromole of the PNGase inhibitor Z-vad-fmk.
  5. You can normalize the sample by the concentration of proteins or cell number. Or you can use anti-β-actin antibody conjugated with HRP for SDS-PAGE to show the equal load of proteins.
  6. We can provide plasmids to express hATF6α(C)-myct 1-302 and Δ280-298 for positive controls (Folded), and Δ111-119, 182-194 and Δ219-270 for negative controls (Less folded).
  7. This assay has been used for exogenously expressed proteins, but should also be applicable to endogenous proteins.
  8. We recommend that when you analyze glycoproteins, you should include Z-VAD-fmk in lysis buffer in order to inhibit PNGase activity after cell lysis.
  9. Recombinant PNGase is also available for PNGase sensitivity assay (Liu et al., 2016).

Recipes

  1. Phosphate buffered saline (PBS)
    137 mM NaCl
    8.1 mM Na2HPO4
    2.68 mM KCl
    1.47 mM KH2PO4
  2. 2x sodium dodecyl sulfate (SDS) sample buffer (pH 6.8)
    100 mM Tris/HCl (pH 6.8)
    20% glycerol
    0.2% bromophenol blue (BPB)
    4% sodium dodecyl sulfate
  3. Buffer A
    50 mM Tris/HCl (pH 8.0)
    1% NP-40 or 1% digitonin
    150 mM NaCl
    Protease inhibitor cocktail (Add before use)
    20 μM MG132 (Add before use)
    Stored at 4 °C
    Note: 1% NP-40 is for mammalian cells such as HCT116 cells and 1% digitonin is for chicken cells such as DT40 cells.
  4. Buffer B
    1x SDS sample buffer (Stored at room temperature)
    100 mM dithiothreitol (Add before use)
    2 μM Z-VAD-fmk (Add before use)

Acknowledgments

This protocol was adapted from and used in Ninagawa et al. (2015).

References

  1. Liu, Y. C, Fujimori, D. G. and Weissman, J. S. (2016). Htm1p-Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation. Proc Natl Acad Sci U S A. 113(28): E4015-4024.
  2. Ninagawa, S., Okada, T., Sumitomo, Y., Horimoto, S., Sugimoto, T., Ishikawa, T., Takeda, S., Yamamoto, T., Suzuki, T., Kamiya, Y., Kato, K. and Mori, K. (2015). Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway. J Cell Biol 211(4): 775-784.

简介

该协议旨在评估蛋白质的折叠状态,利用肽:N-聚糖酶(PNGase)灵敏度。 在细胞质中,PNGase作为去糖基化酶。 由于PNG酶对非天然蛋白的偏好,解折叠/错折叠蛋白上的N-聚糖比折叠蛋白上的N-聚糖更易受PNG酶的影响。 PNGase在多种细胞类型中内源表达,包括HCT116细胞,DT40细胞和小鼠胚胎成纤维细胞。 通过PNGase的部分去糖基化可以通过在SDS-PAGE中更快的条带迁移来检测。 您可以比较感兴趣的野生型和突变蛋白之间折叠的紧密度。 该方法可以与常规的分子和细胞生物学设备一起使用,但仅应用于糖蛋白。

关键字:pngase, 蛋白质折叠, 糖蛋白

材料和试剂

  1. 6孔皿(Corning,Falcon ,目录号:353046)(在室温下贮存)
  2. PVDF膜(GE Healthcare,目录号:10600023)(在室温下保存)
  3. DT40细胞系(DT40是来源于白喉毒鸡中的禽白血病病毒诱导的淋巴管淋巴瘤的B细胞系)(ATCC,目录号:CRL-2111)(胞质PNGase的内源性表达)
  4. 智人结肠结肠直肠癌细胞系(HCT116)(ATCC,目录号:CCL-247),HCT116细胞(ATCC,目录号:CCL-247)(胞质PNGase的内源性表达)
  5. Opti-mem(Thermo Fisher Scientific,Gibco TM ,目录号:31985-070)
  6. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen TM ,目录号:11668019)
  7. Dulbecco改良Eagle培养基(DMEM)(NACALAI TESQUE,目录号:08458-45)(在4℃保存)
  8. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10270-106)
  9. 100 U/ml青霉素和100μg/ml链霉素(NACALAI TESQUE)(-20℃保存)
  10. RPMI(NACALAI TESQUE,目录号:30263-95)(在4℃贮存)
  11. 鸡血清(Thermo Fisher Scientific,Gibco TM ,目录号:16110-082)
  12. 将1M二硫苏糖醇(DTT)(Wako Pure Chemical Industries,目录号:041-08976)(用于还原蛋白质;在水中;储存在-20℃下)
  13. 20mM苄氧羰基 - 缬氨酰 - 丙氨酰 - 天冬氨酰基 - [O-甲基] - 氟甲基酮(Z-VAD-fmk)(Promega,目录号:G7231)(用于抑制PNGase活性;贮存在-20℃)
  14. Nonidet P-40(NACALAI TESQUE,目录号:23640-94)(对于HCT116;在室温下储存)
  15. 4%洋地黄皂苷(Wako Pure Chemical Industries,目录号:043-21371)(对于DT40;在水中;储存在-80℃)
  16. 抗myc抗体(医疗和生物实验室,目录号:562)(-20℃保存)
  17. 与HRP偶联的抗β-肌动蛋白抗体(Wako Pure Chemical Industries,目录号:017-24573)
  18. NaCl(Wako Pure Chemical Industries,目录号:191-01665)
  19. Na HPO 4
  20. KCl
  21. KH 2 PO 4
  22. Tris/HCl,pH8.0(Sigma-Aldrich,目录号:T6791)(在室温下贮存)
  23. 甘油
  24. 溴酚蓝(BPB)
  25. 十二烷基硫酸钠
  26. 蛋白酶抑制剂混合物(100x)(NACALAI TESQUE,目录号:25955-11)(用于抑制各种蛋白酶活性;在ddH 2 O中;储存在-20℃)
  27. 10mM Z-Leu-Leu-Leu-CHO(MG132)(PEPTIDE INSTITUTE,目录号:3175-v)(用于抑制蛋白酶体活性;在DMSO中;储存在-20℃)
  28. 磷酸盐缓冲盐水(PBS)(见Recipes)
  29. 2x十二烷基硫酸钠(SDS)样品缓冲液(pH 6.8)(参见配方)
  30. 缓冲液A(储存在4°C)(参见配方)
  31. 缓冲液B(参见配方)

设备

  1. 高速冷藏微量离心机(Tomy,型号:MX-301)
  2. mPAGE(ATTO,型号:AE-6530)(使用手工制备的10%凝胶)
  3. 传输设备(ATTO,型号:WSE-4020)
  4. 加热块(TAITEC,型号:DTU-1BN)
  5. 微孔器(Digital Bio,型号:MP-100)

软件

  1. ImageJ

程序

  1. 在37℃,潮湿的5%CO 2中,将补充有10%胎牛血清和抗生素(100U/ml青霉素和100μg/ml链霉素)的Dulbecco改良的Eagle培养基(葡萄糖4.5g/L)中的贴壁HCT116细胞培养> 2/95%空气气氛。培养物在补充有10%胎牛血清,1%鸡血清的RPMI1640培养基中以1×10 5 -1×10 6个细胞/ml的密度悬浮DT40细胞,抗生素(100U/ml青霉素和100μg/ml链霉素)在39.5℃下在潮湿的5%CO 2/95%空气气氛中培养。
  2. 使用1μg质粒DNA,300μlOpti-mem和10μlLipofectamine 2000(Invitrogen),转染8.0×10 5个HCT116细胞(在6孔板中40-60%汇合)根据厂家说明(转染后培养基不更换)。为了获得1.05×10 7个细胞转染的细胞,用8μg质粒DNA电穿孔3次3.5×10 6个DT40细胞,两次脉冲1500V,15msec,根据制造商的说明
  3. 在转染后收集约2.0×10 6个HCT116细胞或4.0×10 6个DT40细胞用于进一步分析24小时(HCT116细胞)或16小时(DT40细胞)。为此,用1ml PBS洗涤细胞三次,刮去,在4℃下以800×g离心2分钟。在该步骤之后,以相同的方式处理来自HCT116细胞和DT40细胞的样品
  4. 取出样品的上清液,将细胞沉淀悬浮在200μl缓冲液A中,并在冰上孵育20分钟以裂解细胞。
    注意:缓冲区A不能包含Z-VAD-fmk,因为它会抑制PNGase活动。
  5. 通过在4℃下以17,800×g离心10分钟澄清细胞裂解物,并将上清液转移到新管中。废弃颗粒。
  6. 为了允许PNGase加工错误折叠的蛋白质,将细胞裂解物在冰上进一步孵育不同时间(良好的起始范围为4小时)。在不同时间取出40-50微升等分试样,立即与40-50微升缓冲液B混合,补充100 mM DTT(用于还原)和2μMZ-VAD-fmk(抑制PNG酶),并在100°C孵育5分钟。通过SDS-PAGE分离10μl样品,然后使用PVDF膜进行免疫印迹,并用针对目标蛋白的抗体进行探测。 SDS-PAGE的百分比取决于目标蛋白质的分子量。可以通过ImageJ或类似程序(图1)分析和比较谱带强度

    图1.严重错折叠突变体对内源PNGase更敏感。A.先前描述了鸡EDEM1/2/3三KO细胞(gEDEM TKO)(Ninagawa等人)。 ,2015)。将指示的myc标记的hATF6α(C)变体(在印迹的左侧指示)在gEDEM-TKO细胞中瞬时表达,并进行PNGase敏感性测定。在指定的时间点后,停止PNGase反应并通过12%SDS-PAGE分析样品,然后用针对myc标签的抗体进行免疫印迹分析。所示数据表示两个重复中的单个代表性实验。较少折叠的蛋白质对PNG酶更敏感,因此在hATF6α(C)-myct突变体,Δ111-119,182-194突变体和Δ219-270突变体中的较早时间点检测到部分去糖基化条带。见Ninagawa 。 (2015)进一步的蛋白质信息。 B.该图显示(A)的上带/总带的%强度。通过imageJ分析条带强度。

笔记

  1. 包括HCT116细胞,DT40细胞和小鼠胚胎成纤维细胞在内的大多数细胞内源表达PNGase。因此,不需要外源表达PNGase。其他细胞类型必须测试PNGase表达。 PNGase也在酵母中表达,但我们尚未检查该测定是否适用于酵母细胞
  2. 该测定已经用于测定几种蛋白质的折叠状态,然而,一些糖蛋白对PNG酶完全不敏感。
  3. 使用强洗涤剂例如SDS不适合于该方法,因为PNGase活性可能丧失。在我们的例子中,我们对哺乳动物细胞使用1%NP-40,对于鸡细胞使用1%洋地黄皂苷。但你可以使用1%NP-40鸡细胞和1%毛地黄皂苷的哺乳动物细胞。不太强的洗涤剂可用于PNGase测定。
  4. 作为对照,可以包括两微摩尔的PNG酶抑制剂Z-vad-fmk。
  5. 您可以通过蛋白质浓度或细胞数量使样品标准化。或者您可以使用与HRP结合的抗β-肌动蛋白抗体进行SDS-PAGE以显示蛋白质的等量。
  6. 我们可以提供质粒来表达用于阳性对照(折叠)的hATF6α(C)-myct 1-302和Δ280-298,以及用于阴性对照(折叠较少)的Δ111-119,182-194和Δ219-270。
  7. 该测定法已用于外源表达的蛋白质,但也应适用于内源性蛋白质
  8. 我们建议,当你分析糖蛋白,你应该包括Z-VAD-fmk在裂解缓冲液中,以抑制PNGase活性细胞裂解后。
  9. 重组PNG酶也可用于PNGase敏感性测定(Liu等人,2016)。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    137 mM NaCl 8.1mM Na 2 HPO 4
    2.68mM KCl
    1.47mM KH 2 PO 4 sub/
  2. 2x十二烷基硫酸钠(SDS)样品缓冲液(pH 6.8) 100mM Tris/HCl(pH6.8)
    20%甘油 0.2%溴酚蓝(BPB)
    4%十二烷基硫酸钠
  3. 缓冲区A
    50mM Tris/HCl(pH8.0) 1%NP-40或1%洋地黄皂苷
    150mM NaCl 蛋白酶抑制剂混合物(使用前加入)
    20μMMG132(使用前添加)
    储存在4°C
    注意:1%NP-40用于哺乳动物细胞如HCT116细胞,1%洋地黄皂苷用于鸡细胞如DT40细胞。
  4. 缓冲区B
    1x SDS样品缓冲液(室温保存)
    100 mM二硫苏糖醇(使用前加入)
    2μMZ-VAD-fmk(使用前添加)

致谢

该协议改编自Ninagawa等人使用。 (2015)。

参考文献

  1. Liu,Y. C,Fujimori,DG和Weissman,JS(2016)。  Htm1p-Pdi1p是折叠敏感性甘露糖苷酶,其标记用于ER相关蛋白降解的N-糖蛋白。美国国家科学院院刊。 113(28):E4015-4024。
  2. Ninagawa,S.,Okada,T.,Sumitomo,Y.,Horimoto,S.,Sugimoto,T.,Ishikawa,T.,Takeda,S.,Yamamoto,T.,Suzuki,T.,Kamiya, Kato,K. and Mori,K.(2015)。 
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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。