Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Resource
  • Published:

The GAGOme: a cell-based library of displayed glycosaminoglycans

Abstract

Glycosaminoglycans (GAGs) are essential polysaccharides in normal physiology and disease. However, understanding of the contribution of specific GAG structures to specific biological functions is limited, largely because of the great structural heterogeneity among GAGs themselves, as well as technical limitations in the structural characterization and chemical synthesis of GAGs. Here we describe a cell-based method to produce and display distinct GAGs with a broad repertoire of modifications, a library we refer to as the GAGOme. By using precise gene editing, we engineered a large panel of Chinese hamster ovary cells with knockout or knock-in of the genes encoding most of the enzymes involved in GAG biosynthesis, to generate a library of isogenic cell lines that differentially display distinct GAG features. We show that this library can be used for cell-based binding assays, recombinant expression of proteoglycans with distinct GAG structures, and production of distinct GAG chains on metabolic primers that may be used for the assembly of GAG glycan microarrays.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Graphic depiction of the GAGOme approach.
Fig. 2: Overview of the genetic regulation of GAG biosynthesis.
Fig. 3: Summary overview of effects of KO/KI engineering on HPLC disaccharide profiles of total cell lysates.
Fig. 4: Exploring the binding specificities of GAG-binding proteins by using flow cytometry with GAGOme sublibraries.

Similar content being viewed by others

References

  1. Esko, J. D. & Selleck, S. B. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435–471 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Mizumoto, S., Yamada, S. & Sugahara, K. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins. Curr. Opin. Struct. Biol. 34, 35–42 (2015).

    Article  CAS  PubMed  Google Scholar 

  3. Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu. Rev. Biochem. 83, 129–157 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Mizumoto, S., Yamada, S. & Sugahara, K. Human genetic disorders and knockout mice deficient in glycosaminoglycan. Biomed. Res. Int. 2014, 495764 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Gama, C. I. et al. Sulfation patterns of glycosaminoglycans encode molecular recognition and activity. Nat. Chem. Biol. 2, 467–473 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Fu, L., Suflita, M. & Linhardt, R. J. Bioengineered heparins and heparan sulfates. Adv. Drug Deliv. Rev. 97, 237–249 (2016).

    Article  CAS  PubMed  Google Scholar 

  7. Esko, J. D., Stewart, T. E. & Taylor, W. H. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. USA 82, 3197–3201 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang, L., Lawrence, R., Frazier, B. A. & Esko, J. D. CHO glycosylation mutants: proteoglycans. Methods Enzymol. 416, 205–221 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Okayama, M., Kimata, K. & Suzuki, S. The influence of p-nitrophenyl β-d-xyloside on the synthesis of proteochondroitin sulfate by slices of embryonic chick cartilage. J. Biochem. 74, 1069–1073 (1973).

    CAS  PubMed  Google Scholar 

  10. Li, G. et al. Glycosaminoglycanomics of cultured cells using a rapid and sensitive LC-MS/MS approach. ACS Chem. Biol. 10, 1303–1310 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Yang, Z. et al. Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat. Biotechnol. 33, 842–844 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Koike, T., Izumikawa, T., Tamura, J. & Kitagawa, H. FAM20B is a kinase that phosphorylates xylose in the glycosaminoglycan-protein linkage region. Biochem. J. 421, 157–162 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Koike, T., Izumikawa, T., Sato, B. & Kitagawa, H. Identification of phosphatase that dephosphorylates xylose in the glycosaminoglycan-protein linkage region of proteoglycans. J. Biol. Chem. 289, 6695–6708 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sugahara, K., Yamashina, I., De Waard, P., Van Halbeek, H. & Vliegenthart, J. F. Structural studies on sulfated glycopeptides from the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans of swarm rat chondrosarcoma. Demonstration of the structure Gal(4-O-sulfate)β-1-3Gal-β-1-4XYL-β-1-O-Ser. J. Biol. Chem. 263, 10168–10174 (1988).

    Article  CAS  PubMed  Google Scholar 

  15. Ueno, M., Yamada, S., Zako, M., Bernfield, M. & Sugahara, K. Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence. J. Biol. Chem. 276, 29134–29140 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. de Waard, P., Vliegenthart, J. F., Harada, T. & de Sugahara, K. Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues. J. Biol. Chem. 267, 6036–6043 (1992).

    Article  PubMed  Google Scholar 

  17. Kitagawa, H. et al. Sulfation of the galactose residues in the glycosaminoglycan-protein linkage region by recombinant human chondroitin 6-O-sulfotransferase 1. J. Biol. Chem. 283, 27438–27443 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Wen, J. et al. Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis. Proc. Natl. Acad. Sci. USA 111, 15723–15728 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tone, Y. et al. 2-O-phosphorylation of xylose and 6-O-sulfation of galactose in the protein linkage region of glycosaminoglycans influence the glucuronyltransferase-I activity involved in the linkage region synthesis. J. Biol. Chem. 283, 16801–16807 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Uyama, T., Kitagawa, H., Tamura Ji, J. & Sugahara, K. Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate. J. Biol. Chem. 277, 8841–8846 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Uyama, T. et al. Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/dermatan sulfate. J. Biol. Chem. 278, 3072–3078 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. & Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Izumikawa, T., Uyama, T., Okuura, Y., Sugahara, K. & Kitagawa, H. Involvement of chondroitin sulfate synthase-3 (chondroitin synthase-2) in chondroitin polymerization through its interaction with chondroitin synthase-1 or chondroitin-polymerizing factor. Biochem. J. 403, 545–552 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Izumikawa, T. et al. Identification of chondroitin sulfate glucuronyltransferase as chondroitin synthase-3 involved in chondroitin polymerization: chondroitin polymerization is achieved by multiple enzyme complexes consisting of chondroitin synthase family members. J. Biol. Chem. 283, 11396–11406 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Kitagawa, H., Shimakawa, H. & Sugahara, K. The tumor suppressor EXT-like gene EXTL2 encodes an α1,4-N-acetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme for the chain initiation of heparan sulfate. J. Biol. Chem. 274, 13933–13937 (1999).

    Article  CAS  PubMed  Google Scholar 

  26. Kim, B. T. et al. Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4- N-acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis. Proc. Natl. Acad. Sci. USA 98, 7176–7181 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lind, T., Tufaro, F., McCormick, C., Lindahl, U. & Lidholt, K. The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate. J. Biol. Chem. 273, 26265–26268 (1998).

    Article  CAS  PubMed  Google Scholar 

  28. McCormick, C., Duncan, G., Goutsos, K. T. & Tufaro, F. The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate. Proc. Natl. Acad. Sci. USA 97, 668–673 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pacheco, B., Malmström, A. & Maccarana, M. Two dermatan sulfate epimerases form iduronic acid domains in dermatan sulfate. J. Biol. Chem. 284, 9788–9795 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Maccarana, M. et al. Biosynthesis of dermatan sulfate: chondroitin-glucuronate C5-epimerase is identical to SART2. J. Biol. Chem. 281, 11560–11568 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Pinhal, M. A. et al. Enzyme interactions in heparan sulfate biosynthesis: uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo. Proc. Natl. Acad. Sci. USA 98, 12984–12989 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Salanti, A. et al. Targeting human cancer by a glycosaminoglycan binding malaria protein. Cancer Cell 28, 500–514 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Turnbull, J. E., Fernig, D. G., Ke, Y., Wilkinson, M. C. & Gallagher, J. T. Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate. J. Biol. Chem. 267, 10337–10341 (1992).

    Article  CAS  PubMed  Google Scholar 

  34. Pye, D. A., Vives, R. R., Turnbull, J. E., Hyde, P. & Gallagher, J. T. Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity. J. Biol. Chem. 273, 22936–22942 (1998).

    Article  CAS  PubMed  Google Scholar 

  35. Avnur, Z. & Geiger, B. Immunocytochemical localization of native chondroitin sulfate in tissues and cultured cells using specific monoclonal antibody. Cell 38, 811–822 (1984).

    Article  CAS  PubMed  Google Scholar 

  36. Ito, Y. et al. Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library. Glycobiology 15, 593–603 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Persson, A., Ellervik, U. & Mani, K. Fine-tuning the structure of glycosaminoglycans in living cells using xylosides. Glycobiology 28, 499–511 (2018).

    Article  CAS  PubMed  Google Scholar 

  38. Esko, J. D. et al. Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I. J. Biol. Chem. 262, 12189–12195 (1987).

    Article  CAS  PubMed  Google Scholar 

  39. Kolset, S. O. & Tveit, H. Serglycin—structure and biology. Cell. Mol. Life Sci. 65, 1073–1085 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Datta, P. et al. Bioengineered Chinese hamster ovary cells with Golgi-targeted 3-O-sulfotransferase-1 biosynthesize heparan sulfate with an antithrombin-binding site. J. Biol. Chem. 288, 37308–37318 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Blixt, O. et al. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. USA 101, 17033–17038 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Stopschinski, B. E. et al. Specific glycosaminoglycan chain length and sulfation patterns are required for cell uptake of tau versus α-synuclein and β-amyloid aggregates. J. Biol. Chem. 293, 10826–10840 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Baik, J. Y. et al. Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin. Metab. Eng. 14, 81–90 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Paulson, J. C., Blixt, O. & Collins, B. E. Sweet spots in functional glycomics. Nat. Chem. Biol. 2, 238–248 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Song, X., Heimburg-Molinaro, J., Cummings, R. D. & Smith, D. F. Chemistry of natural glycan microarrays. Curr. Opin. Chem. Biol. 18, 70–77 (2014).

    Article  CAS  PubMed  Google Scholar 

  46. Rogers, C. J. et al. Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches. Proc. Natl. Acad. Sci. USA 108, 9747–9752 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 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 

  48. Geissner, A. & Seeberger, P. H. Glycan arrays: from basic biochemical research to bioanalytical and biomedical applications. Annu. Rev. Anal. Chem. 9, 223–247 (2016).

    Article  CAS  Google Scholar 

  49. Kudelka, M. R. et al. Cellular O-glycome reporter/amplification to explore O-glycans of living cells. Nat. Methods 13, 81–86 (2016).

    Article  CAS  PubMed  Google Scholar 

  50. Varki, A. et al. Symbol nomenclature for graphical representations of glycans. Glycobiology 25, 1323–1324 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Narimatsu, Y. et al. A validated gRNA library for CRISPR–Cas9 targeting of the human glycosyltransferase genome. Glycobiology 28, 295–305 (2018).

    Article  CAS  PubMed  Google Scholar 

  52. Lonowski, L. A. et al. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis. Nat. Protoc. 12, 581–603 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Yang, Z. et al. Fast and sensitive detection of indels induced by precise gene targeting. Nucleic Acids Res. 43, e59 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Bahr, S., Cortner, L., Ladley, S. & Borgschulte, T. Evaluating the effect of chromosomal context on zinc finger nuclease efficiency. BMC Proc. 7, 3 (2013).

    Article  Google Scholar 

  55. Maresca, M., Lin, V. G., Guo, N. & Yang, Y. Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res. 23, 539–546 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Volpi, N., Galeotti, F., Yang, B. & Linhardt, R. J. Analysis of glycosaminoglycan-derived, precolumn, 2-aminoacridone-labeled disaccharides with LC-fluorescence and LC-MS detection. Nat. Protoc. 9, 541–558 (2014).

    Article  CAS  PubMed  Google Scholar 

  57. Lawrence, R., Lu, H., Rosenberg, R. D., Esko, J. D. & Zhang, L. Disaccharide structure code for the easy representation of constituent oligosaccharides from glycosaminoglycans. Nat. Methods 5, 291–292 (2008).

    Article  CAS  PubMed  Google Scholar 

  58. Czajkowsky, D. M., Hu, J., Shao, Z. & Pleass, R. J. Fc-fusion proteins: new developments and future perspectives. EMBO Mol. Med. 4, 1015–1028 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Lundbeck Foundation, Læge Sofus Carl Emil Friis og hustru Olga Doris Friis’ Legat, the Novo Nordisk Foundation, Lund University, Kirsten og Freddy Johansen Fonden, the European Commission (GlycoImaging H2020-MSCA-ITN-721297; BioCapture H2020-MSCA-ITN-722171), the UCPH Excellence Programme for Interdisciplinary Research (CDO2016), and the Danish National Research Foundation (DNRF107) (all to H.C.). C.G was supported through FCT, POPH (Programa Operacional Potencial Humano) SFRH/BPD/96510/2013. A.S., T.M.C., and T.G. were supported through the ERC MalOnco Program, the Danish Cancer Society, and the Lundbeck Foundation.

Author information

Authors and Affiliations

Authors

Contributions

Y.-H.C., Y.M., Y.N., T.M.C., H.C., and Z.Y. conceived and designed the study; Y.-H.C., Y.M., Y.N., C.G., R.K., C.S., A.M., E.P.B., and Z.Y. contributed with experimental data and data interpretation; T.M.C., C.B.S., T.G., and A.S. contributed to the VAR2CSA studies; A.P., D.W., and U.E. contributed to the xyloside-priming studies; Y.-H.C., Y.M., H.C., and Z.Y. wrote the manuscript; and all authors edited and approved the final version of the manuscript.

Corresponding authors

Correspondence to Yang Mao, Henrik Clausen or Zhang Yang.

Ethics declarations

Competing interests

The University of Copenhagen has filed patent application EP/2017/061385 on the basis of this work. Y.N., C.S., E.P.B., H.C., and Y.Z. are named inventors on the PCT application.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–9 and Supplementary Note 1

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, YH., Narimatsu, Y., Clausen, T.M. et al. The GAGOme: a cell-based library of displayed glycosaminoglycans. Nat Methods 15, 881–888 (2018). https://doi.org/10.1038/s41592-018-0086-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-018-0086-z

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research