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Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor

SPPL2a抑制剂处理细胞后的CD74N端片段聚集的检测

Rubén Martínez-Barricarte Rubén Martínez-Barricarte
XK Xiao-Fei Kong
JC Jean-Laurent Casanova

发布: 2019年06月05日第9卷第11期 DOI: 10.21769/BioProtoc.3254 浏览次数: 6187

评审: Gal HaimovichVasiliki KoliarakiAnonymous reviewer(s)

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Abstract

The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different subsets of PBMCs and immortalized B cells, in which SPPL2a is chemically inhibited and CD74-NTF levels are then assessed by flow cytometry or western blotting. The chemical inhibition of SPPL2a has been described elsewhere, but this is the first time that this inhibition has been reported as a protocol.

Keywords: SPPL2a (SPPL2a) search CD74-NTF (CD74-NTF) search Inhibition (抑制剂) search Chemical (化学的) search L-685,458 (L-685,458) search

Background

Signal peptide peptidase-like 2A (SPPL2a) is a transmembrane intracellular protease from the GxGD family (Voss et al., 2013). In both humans and mice, the absence of this enzyme results in the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF) in MHC class II-positive cells (Beisner et al., 2013; Bergmann et al., 2013; Schneppenheim et al., 2013; Kong et al., 2018). This accumulated CD74-NTF is selectively toxic to B cells in mice (Beisner et al., 2013; Bergmann et al., 2013; Schneppenheim et al., 2013) and conventional dendritic cells type 2 (cDC2) in both humans (CD1c+ DCs) and mice (CD11c+CD11c+ DCs). We identified the cells most sensitive to CD74-NTF accumulation and gained insight into the mechanism of CD74-NTF-mediated cytotoxicity, using a specific chemical inhibitor of SPPL2a (L-685,458) (Huttl et al., 2015) in peripheral blood mononuclear cells (PBMCs) and Epstein Barr virus-immortalized B cells (EBV-B) from healthy controls. We analyzed CD74-NTF accumulation by fluorescence-activated cell sorting (FACS) or western blotting (WB).

Materials and Reagents

  1. 1.5 ml microcentrifuge tube (Fisher Scientific, catalog number: 21-402-905)
  2. 24-well plate (Stemcell, catalog number: 38021)
  3. Plate with 96 V-bottomed wells (Denville Scientific, catalog number: B1201-S)
  4. EBV-B cells were produced in-house, as previously described (Hui-Yuen et al., 2011) 
  5. PBMCs were isolated from fresh blood, as previously described (Panda et al., 2012; Panda and Ravindran, 2013)
  6. Tris (MP Biomedicals, catalog number: 103133)
  7. Tween 20 (Fisher BioReagents, catalog number: BP337-500)
  8. WB stripping buffer (Thermo Fisher Scientific, catalog number: PI-21063)
  9. 20% SDS (VWR Life Sciences, catalog number: 0837-200ML) 
  10. 4-12% CriterionTM, XT Bis-Tris gel (Bio-Rad, catalog number: 345-0124)
  11. 5% non-fat milk powder (Bio-Rad, catalog number: 170-6404)
  12. Anti-CD11c-PerCP-Cy5.5 antibody (BioLegend, BU15, catalog number: 307624)
  13. Anti-CD123-PE-Cy7 antibody (BioLegend, 6H6, catalog number: 30601)
  14. Anti-CD141-PE antibody (BioLegend, M80, catalog number: 344104)
  15. Anti-CD1c-APC-Cy7 antibody (BioLegend, L161, catalog number: 331520)
  16. Anti-CD20-PE antibody (BD Biosciences, 2H7, catalog number: 555623)
  17. Anti-CD56-Alexa700 antibody (BD Biosciences, B159, catalog number: 557919)
  18. Anti-CD74 antibody (Abcam, catalog number: ab97479)
  19. Anti-GAPDH HRP-conjugated antibody (Santa Cruz, catalog number: sc47724 HRP)
  20. Anti-HLA-DR-V450 antibody (BD Biosciences, L243, catalog number: 642285)
  21. Anti-mouse-Alexa 488 secondary antibody (Life Technologies, catalog number: A-21131)
  22. Anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Life Sciences, catalog number: NA934V)
  23. BD Cytofix/Cytoperm (BD, catalog number: 554722)
  24. BioRad protein assay dye reagent concentrate (BioRad, catalog number: 5000006)
  25. Bovine serum albumin (BSA) (Calbiochem or Millipore)
  26. Bromphenol blue (Sigma-Aldrich, catalog number: B75808-5G)
  27. CD14 microbeads (Miltenyi Biotec, catalog number: 130-050-201)
  28. CD19 microbeads (Miltenyi Biotec, catalog number: 130-050-301)
  29. CD3 microbeads (Miltenyi Biotec, catalog number: 130-050-101)
  30. CD56 microbeads (Miltenyi Biotec, catalog number: 130-050-401)
  31. cOmplete protease inhibitor cocktail (Roche, catalog number: 11873580001)
  32. DAPT gamma secretase inhibitor (R&D Systems, 2634/10)
  33. DMSO (MP Biomedicals, catalog number: 196055)
  34. 0.5 M EDTA (Fisher Scientific, catalog number: BP2482-1)
  35. Ethanol 200 Proof (Decon Laboratories, catalog number 2716)
  36. Full-Range Rainbow Molecular Weight Marker (GE Healthcare Life Sciences, catalog number: RPN88E)
  37. Glycerol (Fisher Chemical, catalog number: G33-500)
  38. Glycine (Fisher BioReagents, catalog number: BP381-1)
  39. Heat-inactivated fetal calf serum (FCS) (Thermo Fisher Scientific, catalog number: A3382001)
  40. L-685,458 (EMD Millipore, catalog number: 565771)
  41. Live/DEAD Fixable Aqua (Life Technologies, catalog number: L34957)
  42. NaCl (Fisher Scientific, catalog number: S271-3)
  43. NP40 (Sigma-Aldrich, catalog number: I8896-100ML)
  44. NuPAGETM MES SDS Running Buffer (20x) (Thermo Fisher Scientific, catalog number: NP0002)
  45. PBS (Mediatech, catalog number: 21-0310 CVR)
  46. Pierce ECL substrate (Thermo Fisher Scientific, catalog number: 32106)
  47. PVDF membrane (Millipore, catalog number: IPVH00010)
  48. RPMI medium (Life Technologies, catalog number: 61870-127)
  49. Saponin (Sigma-Aldrich, catalog number: 47036-50G-F)
  50. Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750-100G)
  51. Tris-HCl
  52. Culture media (see Recipes)
  53. RIPA buffer (see Recipes)
  54. 5x Laemmli loading buffer (see Recipes)
  55. TBS-T (see Recipes)
  56. Blocking buffer (see Recipes)
  57. FACS buffer (see Recipes)
  58. Intracellular staining buffer (see Recipes)
  59. Transfer buffer (see Recipes)

Equipment

  1. Amersham Imager 600 (GE Healthcare, catalog number: 29083461 )
  2. BD LSR-II (BD Biosciences) with BDFACSDIVA software (BD Biosciences) and FlowJo software for data analysis.
  3. Centrifuge 5418 R (Eppendorf, catalog number: 022620304)
  4. Criterion Vertical Electrophoresis cells (Bio-Rad, catalog number: 1656001)
  5. Eppendorf centrifuge 5810R (Eppendorf, catalog number: 022625004)
  6. Fisher Scientific Vortex Mixer (Fisher Scientific, catalog number: 02215365) 
  7. FormaTM Series 3 Water Jacketed CO2 Incubator (Thermo Fisher, catalog number: 4110)
  8. Labnet Rocking Platform Shaker, Single, 230 VAC (Labnet, catalog number UX-51401-07)
  9. Pipette 4-pack (2.5,10, 100 and 1,000 μl volumes) and pipette tips (Eppendorf, catalog number: 05-403-151)
  10. PowerPacTM HC High-Current Power Supply (BioRad, catalog number: 1645052)
  11. Trans-Blot® SD Semi-Dry Transfer Cell (BioRad, catalog number: 1703940)

Procedure

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文章信息

版权信息

© 2019 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Martinez-Barricarte, R., Kong, X. and Casanova, J. (2019). Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor. Bio-protocol 9(11): e3254. DOI: 10.21769/BioProtoc.3254.

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分类

免疫学 > 免疫细胞功能 > 淋巴细胞

免疫学 > 免疫细胞功能 > 树突细胞

生物化学 > 蛋白质 > 免疫检测

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