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[2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides
N-连接寡糖的[2-3H] 甘露糖标记和分析   

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Abstract

Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-3H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and radioactive detection in a scintillation counter.

Keywords: N-linked oligosaccharide(N-连接寡糖), Mannose-labeling(甘露糖标记), Endoplasmic reticulum associated degradation(内质网相关降解), Glycosylation(糖基化), Mannosidase(甘露糖苷酶)

Background

Following entry of a nascent polypeptide into the ER, it is subjected to several post-translational modifications, which are crucial for folding, maturation and quality control processes. The addition of the core oligosaccharide, Glc3Man9GlcNAc2 to produce N-linked glycoproteins is a very common modification and the first to occur (Benyair et al., 2011). Processing of the precursor N-glycan directs the glycoproteins to maturation and quality control machineries by creating recognition tags while in early secretory compartments (Tannous et al., 2015). At later stages, throughout the secretory pathway the core of the oligosaccharide serves as a platform for expansion of the sugar chains into complex glycans, the structures of which relate to the trafficking and function of the glycoproteins (Kamiya et al., 2012). Because the early N-linked glycan modifications reflect glycoprotein biosynthesis and quality control, the oligosaccharide processing has been the subject of many studies (Avezov et al., 2010; Hosokawa et al., 2010; Ninagawa et al., 2014; Ogen-Shtern et al., 2016). Glycoproteomic methods have greatly improved N-glycan characterization, but they do not allow the study of the dynamics of glycan processing in the early secretory pathway.

Here we describe a simplified pulse-chase method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The method includes radioactive labeling by [2-3H]Man followed by enzymatic removal of oligosaccharides by endo-beta-N-acetylglucosaminidase H (Endo H). Then, N-linked oligosaccharide isolation by molecular filtration and separation by high-performance liquid chromatography (HPLC), which discriminates between high-mannose glycan structures depending on their number of monosaccharide residues. The protocol allows analysis of the dynamics of N-linked glycan modification under different conditions, e.g., after drug treatment or modification of protein levels by overexpression or knockdown. Trimming to shorter species, Man5-6GlcNAc2, is a requirement for glycoprotein targeting to endoplasmic reticulum-associated degradation (ERAD) (Frenkel et al., 2003). Man1A (α1,2 mannosidase 1A ) appears to be involved in endoplasmic reticulum (ER) quality control and required for this trimming, as we present in an example. This is a surprising finding considering that the enzyme was thought to be located in the Golgi complex (Igdoura et al., 1999; Herscovics, 2001); a recent reevaluation locates it in quality control vesicles (Ogen-Shtern et al., 2016).

To conclude, the protocol presented here enables the study of the dynamics of N-linked high-mannose glycan modifications, which has an important role in glycoprotein quality control and trafficking. The advantages of the method are its simplicity, high sensitivity of detection and unique information on the dynamics of N-glycan processing in the early secretory pathway.

Materials and Reagents

  1. 100 mm tissue culture dishes (Corning, catalog number: 430167 )
  2. 1.5 and 2 ml microcentrifuge tubes (Eppendorf)
  3. Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30 (EMD Millipore, catalog number: UFC503024 )
  4. Spherisorb NH2 column, 5 µm, 4.6 x 250 mm (WATERS , catalog number: PSS831115 )
  5. HEK-293 cells (ATCC, catalog number: CRL-1573 )
  6. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 41965039 )
  7. Dulbecco’s modified Eagle’s medium-glucose free (Sigma-Aldrich , catalog number: D5030 )
  8. Fetal bovine serum (FBS) (Biological Industries, catalog number: 04-001-1A-US )
  9. Fetal bovine serum (FBS) , dialyzed (Biological Industries, catalog number: 04-011-1A-US )
  10. Sodium pyruvate solution (100 mM) (Sigma-Aldrich, catalog number: S8636 )
  11. Mannose, D-[2-3H(N)]- (Specific Activity: 15-30 Ci/mmol) (PerkinElmer , catalog number: NET570A )
  12. Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich , catalog number: D1408 )
  13. Protein A-Agarose beads , IPA 300 (REPLIGEN , catalog number: 10-1003 )
  14. Rabbit polyclonal anti-H2 carboxy-terminal produced in our lab (Tolchinsky et al., 1996)
  15. Endo Hf Kit (1,000,000 U/ml) (New England Biolabs, catalog number: P0703S )
  16. Standard oligosaccharide mixture prepared by glycoprotein metabolic labeling with [14C] or [3H] and separation with endo H
  17. Acetonitrile LiChrosolv (gradient grade for liquid chromatography) (Merck, catalog number: 100030 )
  18. Phosphoric acid solution (49-51%, for HPLC) (Sigma-Aldrich , catalog number: 79607 )
  19. Opti-Fluor scintillation fluid ( PerkinElmer, catalog number: 6013199 )
  20. Triton X-100 (BDH, catalog number: 306324N )
  21. Protease inhibitor cocktail (Sigma-Aldrich , catalog number: P2714 )
  22. Sodium deoxycholate (Sigma-Aldrich , catalog number: 30970 )
  23. Sodium dodecyl sulfate (SDS) ( Bio-Rad Laboratories, catalog number: 1610301 )
  24. Sodium phosphate (Na3PO4) (Sigma-Aldrich, catalog number: 342483 )
  25. Buffer A (see Recipes)
  26. Buffer D (see Recipes)
  27. HPLC solvent (see Recipes)

Equipment

  1. Eppendorf centrifuge (Eppendorf, model: 5415 D )
  2. CO2 incubator
  3. -80 °C freezer
  4. LS 6500 Liquid Scintillation Counting systems (Beckman Coulter , model: LS 6500, catalog number: 510720 )
  5. 600E Multisolvent delivery System controller (HPLC) (WATERS , catalog number: WAT062710 )
    Note: This product has been discontinued.
  6. SpeedVac Concentrator 5301, incl. 48 x 1.5/2.0 ml fixed-angle rotor (Eppendorf , model: Concentrator 5301, catalog number: 5301 000.016 )
  7. Frac-100 fraction collector (Amersham Biosciences, model: FRAC-100, catalog number: 18-1000-77 )
  8. Vibra-Cell ultrasonic processors VCX 750 (Sonics and Materials, model: VCX 750 , catalog number: 690-003)

Procedure

  1. For metabolic labeling of N-linked glycans, include a pulse and chase samples (usually 1 to 3) for each condition analyzed, and a negative pulse control sample, to be immunoprecipitated with a preimmune antibody. Here HEK293 cells grown in a 90 mm dish were used for each sample. Transfections of HEK 293 cells (about 2 x 106) were carried out according to the calcium phosphate method, 24 h before the experiment. Other transfection methods can be used.
  2. Remove growth medium from 90 mm dish, scrape the cells with complete DMEM and transfer into a 2 ml Eppendorf tube. Spin the cells (6 sec at 16,000 x g) and rinse with 2 x 1 ml of glucose-free medium. Starve the cells from glucose by incubation in 1 ml of freshly prepared pre-warmed (37 °C) glucose-free medium supplied with 10% dialyzed FBS and 4 mM sodium pyruvate for 30 min in a CO2 incubator at 37 °C (Figure 1). Leave the lid of the tube open during this and subsequent incubations at 37 °C. Labeling in Eppendorf tubes allows the use of small volumes of medium and radioactive precursor in the following steps.


    Figure 1. Flow chart of experimental procedure

  3. Replace the starvation medium with 1 ml of pre-warmed (37 °C) glucose-free medium containing 10% dialyzed FBS, 4 mM sodium pyruvate and 400 μCi of [2-3H]-labeled mannose (stock concentration 1 μCi/1 μl), and incubate the cells in a CO2 incubator at 37 °C for 1 h (pulse).
  4. Remove the labeling medium from each sample, and add 2 ml of PBS at 4 °C to the samples corresponding to pulse and place them on ice, while the samples corresponding to the chase are rinsed 3 times with 1 ml of pre-warmed (37 °C) normal complete culture medium, and then placed in a CO2 incubator at 37 °C with 1 ml of pre-warmed normal complete culture medium for the desired chase periods (usually 30 min to 8 h, depending on the turnover of the studied protein).
  5. Rinse the pulse samples 3 times with 1 ml of ice-cold PBS.
  6. Short-spin the cells at 16, 000 x g (6-10 sec), discard the supernatant and proceed with step 7 or freeze the cell pellet at -80 °C (for up to several months).
  7. Perform steps 5 and 6 also for the chase samples at the end of the chase time.
  8. Remove the cells from the freezer and lyse them by addition of 400 μl of buffer A, vortexing briefly and incubating for 30 min on ice.
  9. Centrifuge the lysates at 16,000 x g for 30 min at 4 °C. Transfer the supernatant to a new 1.5 ml Eppendorf tube and discard the pellet.
  10. For immunoprecipitation of the H2a glycoprotein shown in Data analysis, add 20 μl/sample of Protein A-Agarose beads 1:1 suspension and 5 μl/sample of rabbit polyclonal anti H2a antibody to the supernatant from step 9.
  11. Incubate the samples for 4-16 h at 4 °C, constantly mixing by slow rotation.
  12. Spin down the beads at 16,000 x g for 30 sec, and carefully remove the supernatant.
  13. Wash the beads by adding 500 μl buffer D and vortexing. Spin as in step 12 and remove the supernatant. Repeat the rinse 3 times.
  14. Add 20 μl denaturing buffer (supplied with the NEB endo-H kit) to the bead pellet and boil the samples for 5 min.
  15. Spin down the beads (16,000 x g for 30 sec), and transfer the supernatant into a new Eppendorf tube. Then 0.5 μl of endo-H enzyme is added to each sample along with 2 μl of the reaction buffer (also supplied with the NEB endo-H kit), and the samples are incubated at 37 °C for 3 h.
  16. To separate the released glycans from the protein, the sample is diluted 5 times with double distilled water (DDW) and placed on top of a molecular filter (Microcon Ultracel YM30), with a 30 kDa cut-off, then centrifuged at 14, 000 x g for 3 min (the filter must be washed with 0.5 ml DDW and centrifuged for 3.5 min at 14,000 x g prior loading the sample). Apply 100 μl of DDW to Eppendorf tubes containing the endo-H reaction and transfer to the corresponding molecular filter. Repeat centrifugation of the samples. This washout is repeated 1 more time, keeping the flow through (total throughput is about 300 μl).
  17. Check 10% in a scintillation counter (30 μl). If you get at least 100 cpm, proceed to next step. Less than this amount will give insufficient cpm for further analysis.
  18. Place the tube containing the endo-H reaction flow through (steps 16 and 17) in a SpeedVac concentrator, and dry the samples completely (this can be done up to 45 °C to accelerate this process, usually taking 1-2 h).
  19. To prepare the samples for the HPLC, resuspend the dry pellets in 12 μl of the HPLC solvent (acetonitrile:water, 63:37, 2.5% phosphoric acid).
  20. Adjust the HPLC device (connected to the Spherisorb column) to constant flow of the solvent (1 ml/min) and pressure (a value between 1,000 and 2,000 psi), and place a fraction collector to change a tube every 1 min.
  21. Load the samples into the HPLC device and start the fraction collector simultaneously.
  22. Collect 65 fractions of 1 ml from each sample run and from a run of a standard oligosaccharide mixture. We use a standard mixture prepared by glycoprotein metabolic labeling with [14C] and separation with endo-H (kind gift of Armando Parodi).
  23. Transfer 500 μl from each fraction to a scintillation vial, and mix the contents with 3 ml of water-miscible scintillation fluid (Opti-fluor).
  24. Load the vials and read in a scintillation counter using settings for [3H].
  25. The cpm readout is plotted as a function of fraction number.
  26. The cpm values within a peak represent a specific glycan species, identified according to the parallel run of the standard oligosaccharide mixture.
  27. The sum of cpm values for the fractions within each peak reflects the absolute amount of a specific glycan species. In order to compensate for the fact that species containing more mannose residues acquire more label, the ‘relative molar amount’ of each glycan species is calculated as follows: the absolute amount in cpm of each glycan species is divided by the number of mannose residues that it contains, resulting in a ‘relative molar amount’. This value is then divided by the sum of the relative molar amounts of all glycan species obtained, to obtain the ‘molar percent of total’ for each specific glycan species (Figure 2). An example of the use of this procedure is shown in Figure 3.


    Figure 2. Example of calculation of relative molar amounts

Data analysis

The effect of knockdown of ManIA was tested in cells expressing the ERAD substrate H2a, labeled with [2-3H]Man. Cells were lysed, H2a was immunoprecipitated and treated with endo-H. The N-linked oligosaccharides were separated by HPLC, and fractions were counted in a beta counter. M9 to M5 stand for Man9GlcNAc2 to Man5GlcNAc2. Relative molar amounts of each oligosaccharide species were calculated based on mannose content. We converted the cpm values obtained for each glycan species, the percent of each species relative to the total sum of the relative molar amounts of all species present was then plotted. Only the values for 3 h chase are shown. Three independent biological replicate experiments were performed (Figure 3).


Figure 3. Trimming of high mannose residues on an ERAD substrate glycoprotein and the requirement of Man1A. HEK 293 cells cotransfected with an H2a-encoding vector together with pSUPER encoding control anti-lacZ shRNA (A) or anti-Man1A shRNA (B) were pulse-labeled for 1 h with [2-3H]Man in glucose-free medium and chased for 0 or 3 h in complete medium. C. The knockdown of Man1A significantly inhibited extensive mannose trimming, causing the accumulation of Man9GlcNAc2 (M9). One representative experiment of three independent biological replicate experiments is shown. D. Standard [14C] M9 to M5 oligosaccharide mixture.

Notes

Radioactive waste produced in the diverse steps should be collected and stored properly, according to the regulations at your institution.

Recipes

  1. Buffer A
    1% Triton X-100
    0.5% (w/v) sodium deoxycholate
    Protease inhibitor cocktail 2% v/v in PBS
  2. Buffer D
    0.5% Triton X-100
    0.25% (w/v) sodium deoxycholate
    0.5% (w/v) SDS
    Protease inhibitor cocktail 2% (v/v) in PBS

    Note: Stocks (5x) of buffers A and D can be prepared and stored in aliquots at -20 °C. Fresh protease inhibitor cocktail should be added just before use.

  3. HPLC solvent
    63% acetonitrile
    37% DDW
    2.5% phosphoric acid

Acknowledgments

Research related to this work is supported by grants from the Israel Science Foundation (1593/16) and the Recanati Foundation. A related protocol can be found in Avezov et al., 2010.

References

  1. Avezov, E., Ron, E., Izenshtein, Y., Adan, Y. and Lederkremer, G. Z. (2010). Pulse-chase analysis of N-linked sugar chains from glycoproteins in mammalian cells. J Vis Exp (38): 1899.
  2. Benyair, R., Ron, E. and Lederkremer, G. Z. (2011). Protein quality control, retention, and degradation at the endoplasmic reticulum. Int Rev Cell Mol Biol 292: 197-280.
  3. Frenkel, Z., Gregory, W., Kornfeld, S. and Lederkremer, G. Z. (2003). Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2. J Biol Chem 278(36): 34119-34124.
  4. Herscovics, A. (2001). Structure and function of Class I α1,2-mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control. Biochimie 83(8): 757-762.
  5. Hosokawa, N., Tremblay, L. O., Sleno, B., Kamiya, Y., Wada, I., Nagata, K., Kato, K. and Herscovics, A. (2010). EDEM1 accelerates the trimming of α1,2-linked mannose on the C branch of N-glycans. Glycobiology 20(5): 567-575.
  6. Kamiya, Y., Satoh, T. and Kato, K. (2012). Molecular and structural basis for N-glycan-dependent determination of glycoprotein fates in cells. Biochim Biophys Acta 1820(9): 1327-1337.
  7. Igdoura, S. A., Herscovics, A., Lal, A., Moremen, K. W., Morales, C. R. and Hermo, L. (1999). α-mannosidases involved in N-glycan processing show cell specificity and distinct subcompartmentalization within the Golgi apparatus of cells in the testis and epididymis. Eur J Cell Biol 78(7): 441-452.
  8. Ninagawa, S., Okada, T., Sumitomo, Y., Kamiya, Y., Kato, K., Horimoto, S., Ishikawa, T., Takeda, S., Sakuma, T., Yamamoto, T. and Mori, K. (2014). EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step. J Cell Biol 206(3): 347-356.
  9. Ogen-Shtern, N., Avezov, E., Shenkman, M., Benyair, R. and Lederkremer, G. Z. (2016). Mannosidase IA is in quality control vesicles and participates in glycoprotein targeting to ERAD. J Mol Biol 428(16): 3194-205.
  10. Tannous, A., Pisoni, G. B., Hebert, D. N. and Molinari, M. (2015). N-linked sugar-regulated protein folding and quality control in the ER. Semin Cell Dev Biol 41: 79-89.
  11. Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F. and Lederkremer, G. Z. (1996). Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide. J Biol Chem 271(24): 14496-14503.

简介

其生物合成后不久,糖蛋白的N-连接寡糖的修饰与糖蛋白折叠状态相关。 可以通过脉冲追踪分析[2- 3 H]甘露糖标记的糖蛋白,通过酶切除标记的N-聚糖来检测这些变化,根据大小通过HPLC分离和放射性 在闪烁计数器中检测。
【背景】在将新生多肽进入ER后,进行若干翻译后修饰,这对于折叠,成熟和质量控制过程至关重要。加入核糖寡糖Glc 3 N 3 GlcNAc 2以产生N-连接的糖蛋白是非常常见的修饰,首先发生(Benyair等人,2011)。前体N-聚糖的加工通过在早期分泌室(Tannous等人,2015)中创建识别标签来引导糖蛋白成熟和质量控制机制。在后期阶段,在整个分泌途径中,寡糖的核心作为将糖链扩展成复合聚糖的平台,其结构涉及糖蛋白的运输和功能(Kamiya等人, ,2012)。由于早期N-连接的聚糖修饰反映糖蛋白生物合成和质量控制,寡糖加工已成为许多研究的主题(Avezov等人,2010; Hosokawa等人。,2010; Ninagawa等人,2014; Ogen-Shtern等人,2016)。糖蛋白组学方法大大改善了N-聚糖的表征,但它们不允许研究早期分泌途径中聚糖加工的动力学。
 这里我们描述一种用于分离和分析代谢标记的N-连接寡糖的简化脉冲追踪方法。该方法包括通过[2- H] Man的放射性标记,然后通过内切-β-N-乙酰氨基葡糖苷酶H(Endo H)酶促除去寡糖。然后,通过高效液相色谱(HPLC)的分子过滤和分离进行N-连接的寡糖分离,其根据其单糖残基的数量区分高甘露糖聚糖结构。该方案允许分析不同条件下的N-连接聚糖修饰的动力学,例如在药物治疗之后或通过过表达或敲低修饰蛋白质水平。修剪为较短的物种,人5-6 GlcNAc 糖蛋白靶向内质网相关降解(ERAD)的要求(Frenkel et al。 ,2003)。 Man1A(α1,2甘露糖苷酶1A)似乎参与内质网(ER)质量控制,并且需要这种修剪,如我们在一个例子中所示。考虑到该酶被认为位于高尔基复合体(Igdoura et al。,1999; Herscovics,2001)),这是一个令人惊奇的发现。最近的重新评估将其定位于质量控制囊泡(Ogen-Shtern et al。,2016)。
 总而言之,本文提出的方案能够研究N-连接的高甘露糖聚糖修饰的动力学,其在糖蛋白质量控制和贩运中具有重要作用。该方法的优点是其简便性高,检测灵敏度高,对早期分泌途径中N-聚糖加工动力学的独特信息。

关键字:N-连接寡糖, 甘露糖标记, 内质网相关降解, 糖基化, 甘露糖苷酶

材料和试剂

  1. 100毫米组织培养皿(康宁,目录号:430167)
  2. 1.5和2ml微量离心管(Eppendorf)
  3. Microcon Amicon Ultra 0.5 ml 30K或Centricon ultracel YM-30(EMD Millipore,目录号:UFC503024)
  4. Spherisorb NH2柱,5μm,4.6×250mm(WATERS,目录号:PSS831115)
  5. HEK-293细胞(ATCC,目录号:CRL-1573)
  6. Dulbecco修改的Eagle's培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:41965039)
  7. Dulbecco修改的Eagle's中等葡萄糖(Sigma-Aldrich,目录号:D5030)
  8. 胎牛血清(FBS)(Biological Industries,目录号:04-001-1A-US)
  9. 胎牛血清(FBS),透析(Biological Industries,目录号:04-011-1A-US)
  10. 丙酮酸钠溶液(100mM)(Sigma-Aldrich,目录号:S8636)
  11. 甘露糖,D- [2- 3 H(N)] - (比活度:15-30Ci / mmol)(PerkinElmer,目录号:NET570A)
  12. Dulbecco的磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D1408)
  13. 蛋白A琼脂糖珠,IPA 300(REPLIGEN,目录号:10-1003)
  14. 我们实验室生产的兔多克隆抗H2羧基末端(Tolchinsky等,1996)
  15. Endo H 试剂盒(1,000,000 U / ml)(New England Biolabs,目录号:P0703S)
  16. 通过糖蛋白代谢标记用[14 C]或[3 H]制备的标准寡糖混合物,并用内容物H分离
  17. 乙腈LiChrosolv(梯度级液相色谱)(Merck,目录号:100030)
  18. 磷酸溶液(HPLC为49-51%)(Sigma-Aldrich,目录号:79607)
  19. Opti-Fluor闪烁液(PerkinElmer,目录号:6013199)
  20. Triton X-100(BDH,目录号:306324N)
  21. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P2714)
  22. 脱氧胆酸钠(Sigma-Aldrich,目录号:30970)
  23. 十二烷基硫酸钠(SDS)(Bio-Rad Laboratories,目录号:1610301)
  24. 磷酸钠(Na 3 PO 4)(Sigma-Aldrich,目录号:342483)
  25. 缓冲液A(参见食谱)
  26. 缓冲区D(见配方)
  27. HPLC溶剂(参见食谱)

设备

  1. Eppendorf离心机(Eppendorf,型号:5415 D)
  2. CO 2 孵化器
  3. -80°C冰箱
  4. LS 6500液体闪烁计数系统(Beckman Coulter,型号:LS 6500,目录号:510720)
  5. 600E多溶剂输送系统控制器(HPLC)(WATERS,目录号:WAT062710)
    注意:本产品已停产。
  6. SpeedVac集中器5301,包括48×1.5 / 2.0ml固定角转子(Eppendorf,型号:Concentrator 5301,目录号:5301 000.016)
  7. Frac-100馏分收集器(Amersham Biosciences,型号:FRAC-100,目录号:18-1000-77)
  8. Vibra-Cell超声波处理器VCX 750(Sonics and Materials,型号:VCX 750,目录号:690-003)

程序

  1. 200的X- 200 X- 200 200 200 200 200 200 CE 200 200 200 200 200 -40 200 200 -40 200 200 200 200 200 200 200:1992 200 200 Chanolololol。这里使用在90mm培养皿中生长的HEK293细胞。在实验前24小时,根据磷酸钙方法进行HEK 293细胞(约2×10 6)的转染。可以使用其他转染方法。
  2. 从90毫米的培养皿中取出生长培养基,用完全的DMEM刮洗细胞并转移到2ml的Eppendorf管中。旋转细胞(6,000秒16,000 x g),并用2 x 1毫升无葡萄糖培养基冲洗。通过在1升新鲜制备的预热(37℃)无葡萄糖的培养基中,将10%透析的FBS和4mM丙酮酸钠在CO 2中温育30分钟,将细胞从葡萄糖中分离出来,培养箱在37°C(图1)。在37℃下孵育过程中,将管盖打开。在Eppendorf管中的标记允许在以下步骤中使用小体积的中等和放射性前体。

    X-454545454545 X-45454545 X- 20045 X-45454545新新新新新新新新新旗新新200新新新新旗新新旗新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新旗新新新新新新新旗新新旗新新旗新新旗新新旗新新旗新新新新新新新旗新新旗新新旗新新旗旗号 图1.实验程序流程图

  3. 用含有10%透析的FBS,4mM丙酮酸钠和400μCi的[2- 3> H]标记的甘露糖的1ml预热(37℃)无葡萄糖培养基代替饥饿培养基X- 200 200 200 200 200 200 -40 200 200 -40 200 200 X- 200 200 200 X- 200 200 200 -40 200 200 200 200 200 200 200:下目录200:200454545“
  4. X- 20045 X- 200 X- 200 200 X- 200 X- 200 X- 200 X- 200 X- 200 X- 200 X- 200 X- 200 X- 200 X- 200 X- 200 X- 200 X- 200 200 200 200 200 200: ℃)正常的完全培养基,然后在37℃下加入1ml预热的正常完全培养基的CO 2培养箱中达到所需的追踪时间(通常为30分钟至8小时) ,取决于所研究的蛋白质的周转量)
  5. 用1ml冰冷的PBS冲洗脉冲样品3次。
  6. 新新新新旗新新新旗新新新旗新新新旗新新旗旗新新新旗新新旗旗新200 200 200 200 200 200:200旗新旗旗新新旗旗新200 200 200 200 200 200 200 200:刑200新新新新旗新新旗新新旗旗新200 200 200 200 200 200:
  7. 在追逐时间结束时也执行步骤5和6
  8. 从冷冻箱中取出细胞,加入400μl缓冲液A进行裂解,短暂涡旋,冰上孵育30分钟。
  9. 在4℃下以16,000×g离心裂解物30分钟。将上清液转移到新的1.5 ml Eppendorf管中,弃去沉淀。
  10. 对于数据分析中所示的H2a糖蛋白的免疫沉淀,向步骤9的上清液中加入20μl/μl蛋白A琼脂糖珠1:1的悬浮液和5μl/兔多克隆抗H2a抗体样品。
  11. 在4℃下孵育样品4-16小时,通过缓慢旋转持续混合。
  12. 以16,000 x g 旋转珠子30秒,小心取出上清液。
  13. 通过加入500μl缓冲液D和涡旋洗涤珠子。如步骤12旋转并除去上清液。重复冲洗3次。
  14. 将20μl变性缓冲液(NEB endo-H试剂盒提供)加入珠粒,煮沸样品5分钟。
  15. 将珠子(16,000 x g 旋转30秒),并将上清液转移到新的Eppendorf管中。然后向每个样品中加入0.5μl内切酶酶,并加入2μl反应缓冲液(也随NEB endo-H试剂盒提供),样品在37℃下孵育3小时。
  16. 为了将释放的聚糖与蛋白质分离,将样品用双蒸水(DDW)稀释5倍,并置于分子过滤器(Microcon Ultracel YM30)的顶部,以30kDa的截止值离心,然后以14,000 xg 3分钟(过滤器必须用0.5ml DDW洗涤,并在加载样品之前以14,000 xg离心3.5分钟)。将100μl的DDW加入到含有H反应的Eppendorf管中并转移到相应的分子过滤器中。重复样品离心。这次冲洗再次重复1次,保持流量(总生产量约为300μl)
  17. 在闪烁计数器(30μl)中检查10%。如果达到至少100厘泊,请继续下一步。少于此数量将不足以进行进一步分析。
  18. 将含有内-H反应流的管(步骤16和17)置于SpeedVac浓缩器中,并将样品完全干燥(这可以在45℃下完成,以加速该过程,通常需要1-2小时)。
  19. 为了制备HPLC样品,将干燥的沉淀重悬于12μl的HPLC溶剂(乙腈:水,63:37,2.5%的磷酸)中。
  20. 调整HPLC装置(连接到Spherisorb色谱柱)以保持溶剂的恒定流量(1 ml / min)和压力(1,000至2,000 psi的值),并放置一个级分收集器,每1分钟更换一次。 />
  21. 将样品装入HPLC装置并同时启动级分收集器。
  22. 从每个样品运行和一批标准寡糖混合物中收集65份1ml的级分。我们使用通过糖蛋白代谢标记制备的标准混合物,并用内切-H(Armando Parodi的礼物)分离。
  23. 从每个部分转移500μl到闪烁瓶,并将内容物与3 ml水混溶性闪烁液(Opti-fluor)混合。
  24. 加载小瓶,并使用[3 H]的设置读入闪烁计数器。
  25. cpm读数作为分数的函数作图。
  26. 峰值内的cpm值表示根据标准寡糖混合物的平行运行鉴定的特定聚糖物种。
  27. 每个峰内部分的cpm值的总和反映了特定聚糖物种的绝对量。为了补偿含有更多甘露糖残基的物质获得更多标签的事实,每个聚糖物种的“相对摩尔数”如下计算:每个聚糖物质的cpm中绝对量除以甘露糖残基数它含有“相对摩尔量”。然后将该值除以所获得的所有聚糖物质的相对摩尔量的总和,以获得每个特定聚糖物质的“总摩尔百分比”(图2)。使用此过程的示例如图3所示。


    图2.计算相对摩尔量的示例

数据分析

在表达ERAD底物H2a的细胞中测试了ManIA的击倒作用,用[2- 3 H] Man标记。将细胞裂解,将H2a免疫沉淀并用内切-H处理。通过HPLC分离N-连接的寡糖,并在β计数器中计数级分。 M9至M5代表Man <5> GlcNAc 2 的Man 9 GlcNAc 2 。基于甘露糖含量计算每种寡糖物质的相对摩尔量。我们转化了每个聚糖物种获得的cpm值,然后绘制每个物种的百分比相对于存在的所有物质的相对摩尔量的总和的总和。只显示3 h追逐的值。进行了三次独立的生物复制实验(图3)

图3.修剪ERAD底物糖蛋白上的高甘露糖残基和Man1A的要求。 用H2S编码载体与pSUPER编码对照抗lacZ shRNA(A)或抗Man1A shRNA(B)共转染的HEK 293细胞用[2- 3' / sup> H]在无葡萄糖培养基中的人,并在完全培养基中追踪0或3小时。 C. Man1A的敲低显着抑制了广泛的甘露糖修剪,导致Man 9 / GlcNAc 2(M9)的积累。显示了三个独立生物重复实验的一个代表性实验。 D.标准品[M]〜M5寡糖混合物。

笔记

应根据您所在机构的规定,收集和储存在不同步骤中生产的放射性废物。

食谱

  1. 缓冲区A
    1%Triton X-100
    0.5%(w / v)脱氧胆酸钠
    蛋白酶抑制剂混合物2%v / v in PBS
  2. 缓冲区D
    0.5%Triton X-100
    0.25%(w / v)脱氧胆酸钠
    0.5%(w / v)SDS
    蛋白酶抑制剂混合物2%(v / v)在PBS中

    注意:可以制备储存(5x)缓冲液A和D,并以-20℃的等分试样储存。新鲜的蛋白酶抑制剂鸡尾酒应该在使用前加入。

  3. HPLC溶剂
    63%乙腈
    37%DDW
    2.5%磷酸

致谢

与这项工作有关的研究得到以色列科学基金会(1593/16)和Recanati基金会的资助。相关协议可以在Avezov等人,2010中找到。

参考

  1. Avezov,E.,Ron,E.,Izenshtein,Y.,Adan,Y。和Lederkremer,GZ(2010)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm .nih.gov / pubmed / 20424595“target =”_ blank“>哺乳动物细胞中糖链蛋白的N-连接糖链的脉冲追踪分析。 Vis Vis (38):1899 。
  2. Benyair,R.,Ron,E.和Lederkremer,GZ(2011)。&nbsp; 蛋白质质控制,保留和在内质网中的降解。 Rev Cell Mol Biol 292:197-280。 >
  3. Frenkel,Z.,Gregory,W.,Kornfeld,S.and Lederkremer,GZ(2003)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/ pubmed / 12829701“target =”_ blank“>哺乳动物糖蛋白的内质网相关性降解涉及到Man 6-5&gt; GlcNAc 2 的糖链。 > J Biol Ch em 278(36):34119-34124。
  4. Herscovics,A.(2001)。&nbsp; 结构和功能I型α1,2-甘露糖苷酶参与糖蛋白合成和内质网质量控制。生物化学 83(8):757-762。
  5. Hosokawa,N.,Tremblay,LO,Sleno,B.,Kamiya,Y.,Wada,I.,Nagata,K.,Kato,K。和Herscovics,A。(2010)。&lt; a class = -insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/20065073“target =”_ blank“> EDEM1加速了N-聚糖的C分支上α1,2连接的甘露糖的修剪。糖生物学 20(5):567-575。
  6. Kamiya,Y.,Satoh,T。和Kato,K。(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/22240168” target =“_ blank”>细胞中糖蛋白命运的N-聚糖依赖性测定的分子和结构基础。 Biochim Biophys Acta 1820(9):1327-1337。
  7. Igdoura,SA,Herscovics,A.,Lal,A.,Moremen,KW,Morales,CR和Hermo,L。(1999)。&nbsp; 参与N-聚糖加工的α-甘露糖苷酶显示细胞特异性和睾丸和附睾细胞高尔基体内不同的亚区域。 Eur J Cell Biol 78(7):441-452。
  8. Ninagawa,S.,Okada,T.,Sumitomo,Y.,Kamiya,Y.,Kato,K.,Horimoto,S.,Ishikawa,T.,Takeda,S.,Sakuma,T.,Yamamoto, Mori,K.(2014)。 EDEM2启动哺乳动物糖蛋白ERAD通过催化第一个甘露糖修剪步骤。细胞生物 206(3):347-356。
  9. Ogen-Shtern,N.,Avezov,E.,Shenkman,M.,Benyair,R.and Lederkremer,GZ(2016)。&lt; a class =“ke-insertfile”href =“http://www.sciencedirect Manoosidase IA处于质量控制囊泡中,并参与靶向ERAD的糖蛋白。 J Mol Biol 428(16) :3194-205。
  10. Tannous,A.,Pisoni,GB,Hebert,DN和Molinari,M。(2015)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed / 25534658“target =”_ blank“> ER中的N连接的糖蛋白折叠和质量控制。 Semin Cell Dev Biol 41:79-89。
  11. Tolchinsky,S.,Yuk,MH,Ayalon,M.,Lodish,HF and Lederkremer,GZ(1996)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih人类脱唾液酸糖蛋白受体亚基的膜结合与分泌形式。近年来的五肽的作用.J Biol Chem。271(24):14496-14503。
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引用:Shenkman, M., Ogen-Shtern, N. and Lederkremer, G. Z. (2017). [2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides. Bio-protocol 7(14): e2393. DOI: 10.21769/BioProtoc.2393.
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