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Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans

秀丽隐杆线虫糖胺聚糖(GAG)的分离和纯化

Tabea Dierker Tabea Dierker
LK Lena Kjellén

发布: 2017年08月05日第7卷第15期 DOI: 10.21769/BioProtoc.2437 浏览次数: 11628

评审: Peichuan ZhangAnonymous reviewer(s)

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Abstract

The nematode Caenorhabditis elegans is a popular model organism for studies of developmental biology, neurology, ageing and other fields of basic research. Because many developmental processes are regulated by glycosaminoglyans (GAGs) on cell surfaces and in the extracellular matrix, methods to isolate and analyze C. elegans GAGs are needed. Such methods have previously been optimized for other species such as mice and zebrafish. After modifying existing purification protocols, we could recently show that the nematodes also produce chondroitin sulfate, in addition to heparan sulfate, thus challenging the view that only non-sulfated chondroitin was synthesized by C. elegans. We here present our protocol adapted for C. elegans. Since the purification strategy involves separation of non-sulfated and sulfated GAGs, it may also be useful for other applications where this approach could be advantageous.

Keywords: Glycosaminoglycans (糖胺聚糖) search Caenorhabditis elegans (秀丽隐杆线虫) search Proteoglycans (蛋白多糖) search Ion exchange chromatography (离子交换色谱法) search Sulfation (硫酸化) search

Background

Glycosaminoglycans (GAGs) are linear polysaccharide chains of repeating disaccharide units, which are often substituted with sulfate groups. Except for hyaluronan, which is a non-sulfated GAG synthesized at the plasma membrane without being anchored to any protein, all other GAGs are covalently linked to core proteins, thus forming proteoglycans (PGs). The most common GAGs found on PGs are heparan sulfate (HS) and chondroitin sulfate (CS)/dermatan sulfate, containing N-acetyl-glucosamine and N-acetyl-galactosamine, respectively (Zhang, 2010). During their biosynthesis in the Golgi compartment, which is a non-template driven process, they are subject to multiple modifications, including epimerization of glucuronic acid into iduronic acid and sulfation at various positions (Bulow and Hobert, 2006; Zhang, 2010). The sulfation patterns generated are important for the ability of the GAG chains to interact with growth factors and cytokines, which in turn is essential for their ability to influence development and other important physiological processes (Bishop et al., 2007).

In order to analyze their composition, GAGs need to be purified from crude cell or tissue lysates, most commonly achieved by ion exchange chromatography after protease and nuclease digestion (Ledin et al., 2004). Disaccharides, generated by the action of specific GAG lyases, can then be identified using different chromatography methods or mass spectrometry (Shao et al., 2013; Kiselova et al., 2014). We and several other labs have used reversed-phase ion-pairing (RPIP)-HPLC for detection of different types of GAGs in multiple species (Ledin et al., 2004; Lawrence et al., 2008; Yamada et al., 2011; Holmborn et al., 2012).

C. elegans synthesizes HS with modifications similar to those found in mammals and other organisms, but like other ecdysozoa the nematodes do not produce hyaluronan (Yamada et al., 1999; Toyoda et al., 2000; Lawrence et al., 2008; Townley and Bulow, 2011). However, although vast amounts of non-sulfated chondroitin were detected, CS was not previously identified, giving C. elegans a unique position among the ecdysozoa (Yamada et al., 1999; Toyoda et al., 2000).

In our protocol we separated sulfated and non-sulfated GAGs prior to analysis, facilitating detection of CS occurring in much lower quantities than the non-sulfated chondroitin (Dierker et al., 2016). Here, this method is described in detail.

Materials and Reagents

  1. Pipette tips (SARSTEDT, catalog numbers: 70.1114.100 , 70.760.502 , 70.762.200 )
  2. Tissue paper
  3. 15 ml tubes (SARSTEDT, catalog number: 62.554.502 )
  4. 50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
  5. 1.5 ml Screwlock tubes (SARSTEDT, catalog number: 72.692.100 )
  6. Syringes 1 ml (Terumo Medical, catalog number: SS+01T1 )
  7. Syringes 2 ml (BD, catalog number: 300185 )
  8. MicrolanceTM 3 18 G needles (VWR, catalog number: 613-3945 )
  9. MicrolanceTM 3 23 G needles (VWR, catalog number: 613-3923 )
  10. MicrolanceTM 3 27 G needles (VWR, catalog number: 613-3834 )
  11. 10 ml round-bottom tubes
  12. Parafilm
  13. 0.45 µm filters (for example EMD Millipore, catalog number: HAWP04700 )
  14. Petri dishes 10 cm (SARSTEDT, catalog number: 82.1473.001 )
  15. 10 ml Poly-Prep® Chromatography columns (Bio-Rad Laboratories, catalog number: 7311550 )
  16. DEAE Sephacel (GE Healthcare, catalog number: 17050001 )
  17. 1.5 ml reaction tubes (SARSTEDT, catalog number: 72.690.001 )
  18. 2 ml reaction tubes (SARSTEDT, catalog number: 72.695.500 )
  19. NAP-10 columns (GE Healthcare, catalog number: 17-0854-02 )
  20. HPLC tubes (VWR, catalog number: 548-0440 )
  21. Locks (Fisher Scientific, catalog number: 11521434 )
    Note: This product has been discontinued.
  22. C. elegans and bacterial strains
    Note: All C. elegans strains as well as E. coli strain HB101 were obtained from the Caenorhabditis Genetics Center (CGC) https://cbs.umn.edu/cgc/home.
  23. Sodium hypochlorite (NaClO 6-14% active Cl) (Sigma-Aldrich, catalog number: 13440 )
  24. Protease XIV (Sigma-Aldrich, catalog number: P5147-5G )
  25. Benzonase (Merck, catalog number: 70746-3 )
  26. Heparin lyase I, II, III (IBEX Pharmaceuticals, catalog numbers: 50-010 , 50-011 , 50-012 )
  27. Quant-iTTM Broad Range Assay Kit (purchased from Molecular Probes)
  28. Calcium chloride (CaCl2•2H2O) (Merck, catalog number: 1.02382.0500 )
  29. Potassium hypochlorite (KOH) (Sigma-Aldrich, catalog number: P1767 )
  30. Potassium hydroxide (KOH) (Merck, catalog number: 1.05021.0250 )
  31. Magnesium sulfate heptahydrate (MgSO4•7H2O) (Merck, catalog number: 1.05886 )
  32. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  33. Ethanol (SOLVECO, catalog number: 1015 )
  34. Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 1.04873.1000 )
  35. di-Potassium hydrogen phosphate trihydrate (K2HPO4•3H2O) (Merck, catalog number: A257899 )
    Note: This product has been discontinued.
  36. 1 M K2HPO4
  37. Sodium chloride (NaCl) (Riedel-de Haën, catalog number: 31439 )
  38. Agarose (Sigma-Aldrich, catalog number: A9539 )
  39. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  40. Bacteriological agar (VWR, catalog number: 84609.0500 )
  41. LB broth 1.1 G per tablet (Sigma-Aldrich, catalog number: L7275-500TAB )
  42. Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
  43. di-Sodium hydrogen phosphate dodecahydrate (Na2HPO4•12H2O) (Merck, catalog number: 1.06579.100 )
  44. Magnesium chloride hexahydrate (MgCl2•6H2O) (Sigma-Aldrich, catalog number: M2670-500G )
  45. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  46. Tris ultrapure (AppliChem, catalog number: A1086.1000 )
  47. 6 N HCl
  48. Sodium acetate (NaOAc) (Merck, catalog number: 1.06268.1000 )
  49. Silver nitrate (AgNO3) (Sigma-Aldrich, catalog number: S6506-5G )
  50. Acetic acid (Merck, catalog number: 1.00063.2500 )
  51. HEPES (Sigma-Aldrich, catalog number: H4034 )
  52. Chondroitinase ABC (Amsbio, catalog number: AMS.E1028-10 ; Sigma-Aldrich, catalog number: C3667 )
  53. Media for C. elegans growth and handling (see Recipes)
    1. 1 M CaCl2
    2. 5 N KOH
    3. 1 M MgSO4
    4. Cholesterol solution
    5. 1 M potassium phosphate buffer pH 6.0 (KPO4)
    6. Rich Nematode Growth medium (RNGM)
    7. LB agar
    8. 2x YT medium
    9. M9 buffer
  54. Buffers for GAG purification (see Recipes)
    1. 20% ethanol
    2. 1 M MgCl2
    3. Stock solutions
      i.10% Triton X-100
      ii.5 M NaCl
      iii.1 M CaCl2
      iv.1 M Tris-HCl
    4. Protease buffer
    5. DEAE elution buffer
    6. DEAE wash buffers
    7. DEAE wash buffer, pH 8
    8. DEAE wash buffer, pH 4
    9. 0.1% (w/v) AgNO3
    10. 5x chondroitinase ABC buffer
    11. 2x heparinase lyase buffer

Equipment

  1. Pipettes (for example Eppendorf, model: Research® plus )
  2. 125 ml Erlenmeyer flask (autoclaved)
  3. Rubber bulb
  4. 10 ml glass pipettes (autoclaved, plugged)
  5. Centrifuge
  6. Incubator for C. elegans (Lovibond Thermostatic Cabinet)
  7. Stereo microscope (Nikon Instruments, model: SMZ745 , eyelens: C-W10xB/22, magnification 2.5x to 50x)
  8. Hybridization oven (Hybaid Shake ‘n’ Stack)
  9. 37 °C incubator (Heraeus Instruments)
  10. SpeedVac (Labconco Freezer Dry System connected to Savant SpeedVac concentrator or Savant Instruments, model: SC11OA SpeedVac® Plus )
  11. HPLC system
    1. Merck Hitachi FL Detector L-7485 (Hitachi, model: Model L-7485 )
    2. Interface D-7000 (Hitachi, model: Model D-7000 )
    3. Autosampler L-7200 (Hitachi, model: Model L-7200 )
    4. Pump L-7100 (Hitachi, model: Model L-7100 )
    5. Column Oven L-7350 (Hitachi, model: Model L-7350 )
    6. Reagent pumps: Shimadzu LC-10AD (Shimadzu, model: LC-10AD ); Column: Phenomenex Luna 5u C18(2) 100A (Shimadzu)

Software

  1. D-7000 HPLC System Manager (HSM) software version 4.1
  2. GraphPad Prism version 5.0b for Mac OS X (GraphPad Software, San Diego California USA, www.graphpad.com

Procedure

English
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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Dierker, T. and Kjellén, L. (2017). Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans. Bio-protocol 7(15): e2437. DOI: 10.21769/BioProtoc.2437.

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生物化学 > 糖类 > 多糖

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Hi, are these methods suitable to be used for separation and...
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