Skip to main content

Advertisement

SpringerLink
Log in

Extraction and separation of proteoglycans

  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

Proteoglycans contain a unique carbohydrate component, glycosaminoglycan, which consists of repeating, typically sulfated disaccharides, and is capable of interacting with diverse molecules. Specific, clustered arrangements of sulfate on the glycosaminoglycan backbone form binding sites for many biologically important ligands such as extracellular matrix molecules and growth factors. Core proteins of proteoglycans also show molecular interactions necessary for organizing scaffolds in the extracellular matrix or for anchoring proteoglycans to the plasma membrane. Experimental protocols aiming at extracting maximal amounts of proteoglycans from tissues or cells require disruption of molecular interactions involving proteoglycans by denaturing solvents. Among many of the proteoglycan separation procedures, anion exchange chromatography, which takes advantage of the presence of highly negatively charged glycosaminoglycans in all proteoglycans, serves one of the most convenient general separation techniques.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Notes

  1. Discussions on biochemical and biological characteristics of each glycosaminoglycan can be found in excellent reviews published elsewhere [1–4].

  2. Except hyaluronan, which is synthesized at the plasma membrane, contains many more repeating disaccharides up to ~25,000 per chain, and does not undergo sulfation.

  3. In many studies on biological functions of proteoglycans, cell culture and tissue explant systems in combination with metabolic radiolabeling techniques are employed. In these experiments, monitoring incoporated radioactivity provides excellent measures to follow proteoglycans through extraction and purification steps and to evaluate recoveries at each experimental step. Due to characteristic sulfation in the glycosaminoglycan moiety, [35S]sulfate is an excellent precursor for metabolic labeling of glycosaminoglycans, thus proteoglycans. In many cell culture systems, more than ~90% of radioactive incorporation of 35S from [35S]sulfate is accounted for by the incorporation into glycosaminoglycans. Concomitant use of other radioactive precursors such as those labeled with 3H, which can be differentiated from 35S by spectral analysis, provides further detailed information about molecular structures and metabolic state of the cell. For example, the use of [3H]glucosamine, as shown in the present manuscript, allows labeling hexosamines ([3H]glucosamine is metabolically converted into both [3H]glucosamine and [3H]galactosamine) in all glycoconjugates synthesized by the cell. Further details on metabolic labeling techniques using cell cultures should be consulted elsewhere [5].

  4. Additional consideration should be made when one is attempting to extract cell-associated proteoglycans; i.e., inclusion of sufficient amounts of detergent may be necessary (such as 2% Triton X-100, a non-ionic detergent, or 1% CHAPS, a zwitter-ionic detergent) for the solubilization of proteoglycans [5]. It should be noted that critical micellar concentration of detergents could change drastically in chaotropic solvents such as guanidine HCl or urea [12], and some of the detergents are not soluble in such solvents. Adding the detergent from the beginning of extraction is highly preferred in order to completely disrupt hydrophobic interactions, since irreversible, artificial molecular complexes may be formed between newly exposed hydrophobic sites in proteins during the process of denaturation by 4 M guanidine HCl.

  5. Solvent exchange by Sephadex G-50 chromatography in a disposable pipette—Preswell SephadexG-50, fine (obtained from GE Healthcare Life Science) in hot water off the heater, which achieves sterilization, degassing and shortening of swelling time. An extreme caution should be exercised when adding Sephadex powder to boiling water to avoid flushing. A convenient concentration of gel (50% slurry) can be made by mixing 5 g of Sephadex G-50 with 100 ml water. Bacteriostatic agents (e.g., 0.02% Na azide) should be added for a long-term storage. Pour preswollen Sephadex G-50 into a 10 ml plastic disposable pipette (Falcon), which was cut at the top with a file and plugged with glass wool (no. 3950, Corning) at the bottom, to make 8 ml bed volume. Remove excess water and equilibrate the column with a buffer (8 M urea, 0.20 M NaCl, 0.05 M Na acetate, 0.5% Triton X-100, pH 6.0, a total of 9 ml is sufficient to equilibrate the column). Carefully prepare a flat gel surface with a glass Pasteur pipette and remove excess urea buffer. Apply 2 ml sample and discard the eluent. After the entire sample is in the column, carefully overlay 3 ml of buffer and collect eluent until the entire buffer is in the column (3 ml of V0 fraction collected). This fraction contains proteoglycans and other macromolecules in 8 M urea buffer, while leaving small molecules in the original extract (guanidine HCl, isotope precursors etc.) behind in the column. At this point, column can be safely disposed as a radioactive waste. Dimension of the column may be changed proportionately when sample size varies.

  6. Q-Sepharose chromatography—Q-Sepharose, fast flow (GE Healthcare Life Science) has to be pre-equilibrated with the low salt buffer (8 M urea, 0.20 M NaCl, 0.05 M Na acetate, 0.5% Triton X-100, pH 6.0) used in the NaCl gradient. Two ml of preequilibrated Q-Sepharose (1 ml of Q-Sepharose can bind up to 3–5 mg of proteoglycans) is packed into a small column (10 ml plastic pipette is cut by a file and plugged with glass wool at the bottom). Alternatively, 2 ml of preequilibrated Q-Sepharose is mixed with the sample in 8 M urea buffer (of any volume) and gently shaken for 1 h, then packed into the column; this latter method gives uniform binding of proteoglycans to Q-Sepharose gel resulting in a better flow property, especially when a large quantity of materials (proteoglycan, protein, nucleic acid, etc.) is used. After sample application, the column is washed with 10 ml of the low salt buffer. Then the column is connected to a gradient former (using, for an example, a high salt buffer: 8 M urea, 1.5 M NaCl, 0.05 M Na acetate, 0.5% Triton X-100, pH 6.0) and eluted with approximately a total 40 ml of buffer with a flow rate of 10–15 ml/h. Every 1–2 ml fraction is collected and monitored for NaCl concentration by conductivity measurement, Fig. 2. Eluent fractions are monitored for proteoglycans by radioactivity detection or colorimetric procedures, such as a convenient and safe colorimetric procedure using Safranin O [6, 13] or a classic procedure measuring uronic acid by m-phenylphenol reactants [14], which can be modified to use a microtiter plate for easier handling. Typically, heparan sulfate proteoglycans are eluted in a peak at approximately 0.5 M NaCl and chondroitin sulfate proteoglycans at 0.65 M NaCl. The use of step elution for the purpose of differentially eluting proteoglycan species is not recommended unless salt concentrations in which individual proteoglycans elute are widely separate. Determination of the exact salt concentration which enables clear separation of proteoglycan species may be rather delicate. One of the major technical problems associated with anion exchange chromatography of proteoglycans, especially when purifying molecules are present in small quantities (e.g., isolation of proteoglycans from cell cultures), is poor recovery. This can be, in most cases, overcome by the use of detergents (either non-ionic or zwitter-ionic) in elution buffer. Routinely, the use of 0.5% (w/v) Triton X-100 dramatically improves recovery of proteoglycans (even glycosaminoglycans) from ion exchange columns. Most non-ionic detergents (such as Triton X-100 and NP-40) possess strong absorbance in the UV range, thus making the UV tracing for protein detection difficult. If this causes problems in the analysis, non-UV absorbing, non-ionic detergents such as Genapol X-100® (Calbiochem) can be used with virtually the same chromatographic result. Also, when the removal of detergents in later experimental steps is required, the use of ones with high CMC (such as CHAPS, Calbiochem) in place of Triton X-100 is beneficial.

  7. Superose 6 chromatography—A Superose 6 column (HR 10/30, 1 × 30 cm) is preequilibrated with 4 M guanidine HCl, 50 mM Na acetate, pH 6.0 containing 0.5% Triton X-100 and eluted with the same buffer at a flow rate of 0.4 ml/min and fractions of 0.4 ml are collected for measurement of proteoglycan content (by radioactivity or colorimetric analysis [6, 13, 14]). Inclusion of detergent in the elution buffer is critical to obtain quantitative recovery. Most efficient detergents in this respect have been non-ionic detergent such as Triton X-100.

  8. Annexin affinity chromatography—SH-derivatized Magnetic beads® (obtained from Promega) is conjugated with annexin. Binding of radiolabeled heparan sulfate proteoglycan to the beads is performed in 200 μl of buffer consisting of 10 mM Hepes, 50 mM NaCl, pH 7.0, containing 0.5%Triton X-100 and 1% BSA. Supernatant is removed after incubation at 4°C for 1 h. Washed beads are then eluted with 10 mM Hepes, 500 mM NaCl, 5 mM EDTA, pH 7.0, containing 0.5%Triton X-100, and the eluant is counted for radioactivity.

References

  1. Kimata, K., Habuchi, O., Habuchi, H., Watanabe, H.: Knockout mice and proteoglycans, in comprehensive glycoscience. In: Kamerling J. P., et al. (eds.) Elsevier, Amsterdam (2007)

  2. Shipp, E.L., Hsieh-Wilson, L.C.: Profiling the sulfation specificities of glycosaminoglycan interactions with growth factors and chemotactic proteins using microarrays. Chem. Biol. 14, 195–208 (2007)

    Article  CAS  PubMed  Google Scholar 

  3. Habuchi, H., Habuchi, O., Kimata, K.: Sulfation pattern in glycosaminoglycan: Does it have a code? Glycoconj. J. 21, 47–52 (2004)

    Article  CAS  PubMed  Google Scholar 

  4. In: Conrad, H.E. (ed.) Heparin-Binding Proteins. Academic, San Diego (1998)

  5. Hascall, V.C., Calabro, A., Midura, R.J., Yanagishita, M.: Isolation and characterization of proteoglycans. Methods Enzymol. 230, 390–417 (1994)

    Article  CAS  PubMed  Google Scholar 

  6. Hascall, V.C., Kimura, J.H.: Proteoglycans: Isolation and characterization. Methods Enzymol. 82, 769–800 (1982)

    Article  CAS  PubMed  Google Scholar 

  7. Yanagishita, M., Midura, R.J., Hascall, V.C.: Proteoglycans: Isolation and purification from tissue cultures. Methods Enzymol 138, 279–289 (1987)

    Article  CAS  PubMed  Google Scholar 

  8. In: Iozzo, R.V. (ed.) Proteoglycan Protocols. Humana, New Jersey (2001)

  9. Yanagishita, M., Hascall, V.C.: Proteoglycans synthesized by rat ovarian granulosa cells in culture; isolation, fractionation, and characterization of proteoglycans associated with the cell layer. J. Biol. Chem. 259, 10260–10269 (1984)

    CAS  PubMed  Google Scholar 

  10. Oike, Y., Kimata, K., Shinomura, T., Nakazawa, K., Suzuki, S.: Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-beta-d-galactosidase (keratanase). Biochem. J. 191, 193–207 (1980)

    CAS  PubMed  Google Scholar 

  11. Fisher, L.W., Termine, J.D., Dejter Jr., S.W., Whitson, S.W., Yanagishita, M., Kimura, J.H., Hascall, V.C., Kleinman, H.K., Hassell, J.R., Nilsson, B.: Proteoglycans of developing bone. J. Biol. Chem. 258, 6588–6594 (1983)

    CAS  PubMed  Google Scholar 

  12. Midura, R.J., Yanagishita, M.: Chaotropic solvents increase the critical micellar concentrations of detergents. Anal. Biochem. 228, 318–322 (1995)

    Article  CAS  PubMed  Google Scholar 

  13. Lammi, M., Tammi, M.: Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O. Anal. Biochem. 168, 352–357 (1988)

    Article  CAS  PubMed  Google Scholar 

  14. Blumenkarantz, N., Asboe-Hansen, G.: New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489 (1973)

    Article  Google Scholar 

  15. Midura, R.J., Hascall, V.C.: Analysis of proteoglycans synthesized by corneal explants from embryonic chicken II. Structural characterization of keratan sulfate and dermatan sulfate proteoglycans from corneal stroma. J. Biol. Chem. 264, 1423–1430 (1989)

    CAS  PubMed  Google Scholar 

  16. Ishitsuka, R., Kojima, K., Utsumi, H., Ogawa, H., Matsumoto, I.: Glycosaminoglycan binding properties of Annexin IV, V, and VI. J. Biol. Chem. 273, 9935–9941 (1998)

    Article  CAS  PubMed  Google Scholar 

  17. Heinegård, D., Wieslander, J., Sheehan, J., Paulsson, M., Sommarin, Y.: Separation and characterization of two populations of aggregating proteoglycans from cartilage. Biochem. J. 225, 95–106 (1985)

    PubMed  Google Scholar 

  18. Couchman, J.R., Tapanadechopone, P.: Detection of proteoglycan core proteins with glycosaminoglycan lyases and antibodies. In: Iozzo, R.V. (ed.) Proteoglycan Protocols, pp. 329–333. Humana, New Jersey (2001)

    Chapter  Google Scholar 

  19. Militsopoulou, M., Lamari, F., Karamanos, N.K.: Capillary electrophoresis: A tool for studying interactions of glycans/proteoglycans with growth factors. J. Pharm. Biomed. Anal. 32, 823–828 (2003)

    Article  CAS  PubMed  Google Scholar 

  20. Karousou, E.G., Viola, M., Genasetti, A., Vigetti, D., De Luca, G., Karamanos, N.K., Passi, A.: Application of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides for analysis of hyaluronan and chondroitin sulfate in human and animal tissues and cell cultures. Biomed. Chromatogr. 19, 761–765 (2005)

    Article  CAS  PubMed  Google Scholar 

  21. Calabro, A., Benavides, M., Tammi, M., Hascall, V.C., Midura, R.J.: Microananlysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE). Glycobiology 10, 273–281 (2000)

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biochemistry, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, 1–5–45 Yushima, Bunkyo-ku, Tokyo, 113–8549, Japan

    Masaki Yanagishita, Katarzyna Anna Podyma-Inoue & Miki Yokoyama

Authors
  1. Masaki Yanagishita
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katarzyna Anna Podyma-Inoue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miki Yokoyama
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masaki Yanagishita.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yanagishita, M., Podyma-Inoue, K.A. & Yokoyama, M. Extraction and separation of proteoglycans. Glycoconj J 26, 953–959 (2009). https://doi.org/10.1007/s10719-008-9138-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-008-9138-4

Keywords

Search

Navigation