Proteoglycan Isolation and Analysis

Anne Woods1, John R. Couchman1

1 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.7
DOI:  10.1002/0471143030.cb1007s07
Online Posting Date:  May, 2001
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Abstract

Proteoglycans can be difficult molecules to isolate and analyze due to large mass, charge, and tendency to aggregate or form macromolecular complexes. This unit describes detailed methods for purification of matrix, cell surface, and cytoskeleton‐linked proteoglycans. Methods for analysis of glycoaminoglycan size and type and of core protein species are described.

     
 
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Table of Contents

  • Isolation and Purification of Proteoglycans
  • Basic Protocol 1: Isolation of Proteoglycans from Cultured Cells
  • Alternate Protocol 1: Isolation of Proteoglycan Pools
  • Support Protocol 1: Radiolabeling of Proteoglycans with 35SO4 or [3H]Glucosamine
  • Basic Protocol 2: Anion‐Exchange Chromatography Purification of Proteoglycans with DEAE‐Sephacel
  • Alternate Protocol 2: Isolation of Hydrophobic Proteoglycans
  • Analysis of Proteoglycans
  • Basic Protocol 3: Analysis of Proteoglycans by Size‐Exclusion Chromatography
  • Basic Protocol 4: Analysis of Glycosaminoglycan Size Following Alkaline Elimination
  • Basic Protocol 5: Analysis of Glycosaminoglycan Size Following Papain Digestion
  • Basic Protocol 6: Analysis of Glycosaminoglycan Content and Protein Core by GAG Degradation with Lyases
  • Basic Protocol 7: Treatment with Nitrous Acid to Degrade Heparan Sulfate
  • Basic Protocol 8: Analysis of GAG Type and Core Protein
  • Basic Protocol 9: Analysis of GAG Size
  • Basic Protocol 10: Immunoprecipitation of Proteoglycans
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Isolation of Proteoglycans from Cultured Cells

  Materials
  • Radiolabeled cultured cells in 10‐cm tissue culture dishes (see protocol 3)
  • PBS ( appendix 2A)
  • Urea, ultrapure
  • Solubilization buffer (see recipe) with and without Triton X‐100

Alternate Protocol 1: Isolation of Proteoglycan Pools

  • Serum‐free culture medium
  • Phosphatidylinositol‐specific phospholipase C (PI‐PLC; Boehringer Mannheim, Molecular Probes)
  • Lysis buffer (see recipe) with and without detergent

Support Protocol 1: Radiolabeling of Proteoglycans with 35SO4 or [3H]Glucosamine

  Materials
  • Proteoglycan samples (see protocol 1 or protocol 2)
  • Diethylaminoethyl (DEAE)–Sephacel: 50:50 (v/v) slurry in PBS
  • PBS ( appendix 2A)
  • DEAE‐Sephacel equilibration buffer (see recipe)
  • Conditioned medium from cell line of interest, incubated with cells 24 to 48 hr
  • DEAE low‐pH buffer (see recipe)
  • DEAE elution buffer (see recipe)
  • 2 × 0.5–cm minicolumns

Basic Protocol 2: Anion‐Exchange Chromatography Purification of Proteoglycans with DEAE‐Sephacel

  • DEAE‐Sephacel eluate and column, or column loaded for elution (see protocol 4)
  • DEAE low‐pH buffer (see recipe) without NaCl
  • Hydrophobic chromatography equilibration buffer (see recipe)
  • Hydrophobic chromatography exchange buffer (see recipe)
  • Hydrophobic chromatography washing buffer (see recipe)
  • Hydrophobic chromatography elution buffer (see recipe)
  • Octyl‐Sepharose (Pharmacia): 50:50 (v/v) slurry in PBS containing 0.1% Triton X‐100
  • 0.1% (v/v) Triton X‐100
  • Octyl‐Sepharose equilibration buffer (see recipe) with and without NaCl
  • Octyl‐Sepharose washing buffer (see recipe)
  • Octyl‐Sepharose elution buffer I (see recipe)
  • Octyl‐Sepharose elution buffer II (see recipe)
  • Minicolumn

Alternate Protocol 2: Isolation of Hydrophobic Proteoglycans

  Materials
  • Sepharose 4B (Pharmacia)
  • SEC running buffer (see recipe)
  • Proteoglycan sample (see above)
  • Stock dye solution: 1 mg/ml each blue dextran (Sigma) and N‐2,4‐dinitrophenyl (DNP)–alanine (Sigma)
  • 90 × 1–cm column
  • Column pump

Basic Protocol 3: Analysis of Proteoglycans by Size‐Exclusion Chromatography

  Materials
  • Proteoglycan sample (see protocol 5 or obtain by immunoprecipitation)
  • Ethanol (optional)
  • 1 M NaOH/2 M NaBH 4
  • 10 M acetic acid
  • Dialysis tubing, MWCO 12‐12,000 (optional)

Basic Protocol 4: Analysis of Glycosaminoglycan Size Following Alkaline Elimination

  Materials
  • Proteoglycan sample (see protocol 5 or obtain by immunoprecipitation)
  • Papain digestion buffer (see recipe)
  • 20 mg/ml papain (Sigma)
  • 65°C water bath
  • Additional reagents and equipment for dialysis or ethanol precipitation (see protocol 7)

Basic Protocol 5: Analysis of Glycosaminoglycan Size Following Papain Digestion

  Materials
  • Proteoglycan sample (see protocol 5 or obtain by immunoprecipitation)
  • Chondroitinase buffer (see recipe)
  • Heparinase III buffer (see recipe)
  • Chondroitin sulfate type C or B (chondroitin 6‐sulfate or dermatan sulfate; Sigma)
  • Chondroitinase ABC (EC 4.2.2.4), ACII (EC 4.2.2.5), or B (no EC number) (Seikagaku America)
  • Heparinase III (a.k.a. heparitinase or heparitinase I; EC 4.2.2.8; Seikagaku America)
  • Additional reagents and equipment for dialysis or ethanol precipitation (see protocol 7)

Basic Protocol 6: Analysis of Glycosaminoglycan Content and Protein Core by GAG Degradation with Lyases

  Materials
  • 1 N H 2SO 4
  • 0.114 g/ml Ba(NO 2) 2
  • Proteoglycan sample (see protocol 5 or obtain by immunoprecipitation)
  • 10 N NaOH

Basic Protocol 7: Treatment with Nitrous Acid to Degrade Heparan Sulfate

  Materials
  • HPLC running buffer (see recipe)
  • Proteoglycan sample, enzymatically or chemically treated (see protocol 7Basic Protocols 4 and protocol 85)
  • DEAE‐Sephacel column (see protocol 4)
  • PD‐10 gel‐filtration column (Amersham Pharmacia Biotech)
  • High‐performance liquid chromatograph (HPLC) with TSK 4000 column (TosoHaas)
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Figures

Videos

Literature Cited

Literature Cited
   Bernfield, M., Gotte, M., Park, P.W., Reizes, O., Fitzgerald, M.L., Lincecum, J., and Zako, M. 1999. Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68:729‐778.
   Oh, E.S., Woods, A., and Couchman, J.R. 1997. Syndecan‐4 proteoglycan regulates the distribution and activity of protein kinase C. J. Biol. Chem. 272:8133‐8136.
   Schmidtchen, A., Sundler, R., and Fransson, L.A. 1990. A fibroblast heparan sulphate proteoglycan with a 70 kDa core protein is linked to membrane phosphatidylinositol. Glycoconjugate J. 7:563‐572.
   Serru, V., LeNaour, F., Billard, M., Azorsa, D.O., Lanza, F., Boucheix, C., and Rubinstein, E. 1999. Selective tetraspan‐integrin complexes (CD81/α4β1, CD151/α3β1, CD151/α6β1) under conditions disrupting tetraspan interactions. Biochem. J. 340:103‐111.
   Vives, R.R., Pye, D.A., Salmavirta, M., Hopwood, J.J., Lindahl, U., and Gallagher, J.T. 1999. Sequence analysis of heparan sulphate and heparin oligosaccharides. Biochem. J. 339:767‐773.
   Woods, A. and Couchman, J.R. 1998. Syndecans: Synergistic activators of cell adhesion. Trends Cell Biol. 8:189‐192.
   Woods, A., Couchman, J.R., and Höök, M. 1985. Heparan sulfate proteoglycans of rat embryo fibroblasts. A hydrophobic form may link cytoskeleton and matrix components. J. Biol. Chem. 260:10872‐10879.
Key References
   Bernfield et al., 1999. See above.
  An excellent review of cell surface glypican and syndecan proteoglycans.
   Gallagher, 1997. Structure‐activity relationships of heparan sulfate. Biochem. Soc. Trans. 25:1206‐1209.
  An expert review of heparan sulfate glycosaminoglycan structure and function.
   Iozzo, R.V. 1998. Matrix proteoglycans: From molecular design to cellular function. Annu. Rev. Biochem. 67:609 ‐652.
  An in‐depth, informative review of extracellular matrix proteoglycans.
   Lindahl, U., Kusche‐Gullberg, M., and Kjellén, L. 1998. Regulated diversity of heparan sulfate. J. Biol. Chem. 273:24979‐24982.
  A concise review of heparan sulfate glycosaminoglycan complexity and biosynthesis.
Internet Resources
   http://www.glycoforum.gr.jp
  A Web site supported by Seikagaku Corp. that contains articles on glycoscience, including proteoglycans and links to other sites.
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