Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina


Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.

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  1. 1.

    Wong, W.L., Su, X., Li, X., Cheung, C.M., Klein, R., Cheng, C.Y., Wong, T.Y.: Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob. Health. 2, e106–e116 (2014)

    Article  PubMed  Google Scholar 

  2. 2.

    Barbosa-Sabanero, K., Hoffmann, A., Judge, C., Lightcap, N., Tsonis, P.A., Del Rio-Tsonis, K.: Lens and retina regeneration: new perspectives from model organisms. Biochem. J. 447, 321–334 (2012)

    Article  CAS  PubMed  Google Scholar 

  3. 3.

    Del Rio-Tsonis, K., Tsonis, P.A.: Eye regeneration at the molecular age. Dev. Dyn. 226, 211–224 (2003)

    Article  PubMed  Google Scholar 

  4. 4.

    Haynes, T., Del Rio-Tsonis, K.: Retina repair, stem cells and beyond. Curr. Neurovasc. Res. 1, 231–239 (2004)

    Article  PubMed  Google Scholar 

  5. 5.

    Hayashi, T., Mizuno, N., Ueda, Y., Okamoto, M., Kondoh, H.: FGF2 triggers iris-derived lens regeneration in newt eye. Mech. Dev. 121, 519–526 (2004)

    Article  CAS  PubMed  Google Scholar 

  6. 6.

    Spence, J.R., Aycinena, J.C., Del Rio-Tsonis, K.: Fibroblast growth factor-hedgehog interdependence during retina regeneration. Dev. Dyn. 236, 1161–1174 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Susaki, K., Chiba, C.: MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: is FGF2 an induction factor? Pigment Cell Res. 20, 364–379 (2007)

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Spence, J.R., Madhavan, M., Aycinena, J.C., Del Rio-Tsonis, K.: Retina regeneration in the chick embryo is not induced by spontaneous Mitf downregulation but requires FGF/FGFR/MEK/Erk dependent upregulation of Pax6. Mol. Vis. 13, 57–65 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Schlessinger, J., Plotnikov, A.N., Ibrahimi, O.A., Eliseenkova, A.V., Yeh, B.K., Yayon, A., Linhardt, R.J., Mohammadi, M.: Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell. 6, 743–750 (2000)

    Article  CAS  PubMed  Google Scholar 

  10. 10.

    Sterner, E., Meli, L., Kwon, S.J., Dordick, J.S., Linhardt, R.J.: FGF–FGFR signaling mediated through glycosaminoglycans in microtiter plate and cell-based microarray platforms. Biochemistry. 50, 9009–9019 (2013)

    Article  CAS  Google Scholar 

  11. 11.

    Linhardt, R.J., Toida, T.: Role of glycosaminoglycans in cellular communication. Acc. Chem. Res. 7, 431–438 (2004)

    Article  CAS  Google Scholar 

  12. 12.

    Kim, S.Y., Zhao, J., Liu, X., Fraser, K., Lin, L., Zhang, X., Zhang, F., Dordick, J.S., Linhardt, R.J.: Interaction of Zika virus envelope protein with glycosaminoglycans. Biochemistry. 56, 1151–1162 (2017)

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Kim, S.Y., Zhang, F., Gong, W., Chen, K., Xia, K., Liu, F., Gross, R.A., Wang, J.M., Linhardt, R.J., Cotton, M.L.: Copper regulates the interactions of antimicrobial piscidin peptides from fish mast cells with formyl peptide receptors and heparin. J. Biol. Chem. 293, 15381–15396 (2018)

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Kaprinis, K., Symeonidis, C., Papakonstantinou, E., Tsinopoulos, I., Dimitrakos, S.A.: Decreased hyaluronan concentration during primary rhegmatogenous retinal detachment. Eur. J. Ophthalmol. 26, 633–638 (2016)

    Article  PubMed  Google Scholar 

  15. 15.

    Park, P.J., Shukla, D.: Role of heparan sulfate in ocular diseases. Exp. Eye Res. 110, 1–9 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    McManus, L.M., Mitchell, R.N.: Pathobiology of human disease: a dynamic encyclopedia of disease mechanisms. Elsevier. (2014)

  17. 17.

    Dreyfuss, J.L., Regatieri, C.V., Lima, M.A., Paredes-Gamero, E.J., Brito, A.S., Chavante, S.F., Belfort Jr., R., Farah, M.E., Nader, H.B.: A heparin mimetic isolated from a marine shrimp suppresses neovascularization. J. Thromb. Haemost. 8, 1828–1837 (2010)

    Article  CAS  PubMed  Google Scholar 

  18. 18.

    Jiang, X., Couchman, J.R.: Perlecan and tumor angiogenesis. J. Histochem. Cytochem. 51, 1393–1410 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Regatieri, C.V., Dreyfuss, J.L., Melo, G.B., Lavinsky, D., Hossaka, S.K., Rodrigues, E.B., Farah, M.E., Maia, M., Nader, H.B.: Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression. Braz. J. Med. Biol. Res. 43, 627–633 (2010)

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    Clark, S.J., Bishop, P.N., Day, A.J.: Complement factor H and age-related macular degeneration: the role of glycosaminoglycan recognition in disease pathology. Biochem. Soc. Trans. 38, 1342–1348 (2010)

    Article  CAS  PubMed  Google Scholar 

  21. 21.

    Clark, S.J., Perveen, R., Hakobyan, S., Morgan, B.P., Sim, R.B., Bishop, P.N., Day, A.J.: Impaired binding of the age-related macular degeneration-associated complement factor H 402H allotype to Bruch’s membrane in human retina. J. Biol. Chem. 285, 192–202 (2010)

    Google Scholar 

  22. 22.

    Kelly, U., Yu, L., Kumar, P., Ding, J.D., Jiang, H., Hageman, G.S., Arshavsky, V.Y., Frank, M.M., Hauser, M.A., Rickman, C.B.: Heparan sulfate, including that in Bruch’s membrane, inhibits the complement alternative pathway: implications for age-related macular degeneration. J. Immunol. 185, 5486–5494 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Alibardi, L.: Hyaluronic acid in the tail and limb of amphibians and lizards recreates permissive embryonic conditions for regeneration due to its hygroscopic and immunosuppressive properties. J Exp Zool B Mol Dev Evol. 328, 760–771 (2017)

    Article  CAS  PubMed  Google Scholar 

  24. 24.

    Ouyang, X., Panetta, N.J., Talbott, M.D., Payumo, A.Y., Halluin, C., Longaker, M.T., Chen, J.K.: Hyaluronic acid synthesis is required for zebrafish tail fin regeneration. PLoS One. 12, e0171898 (2017)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Kulyk, W.M., Zalik, S.E., Dimitrov, E.: Hyaluronic acid production and hyaluronidase activity in the newt iris during lens regeneration. Exp. Cell Res. 172, 180–191 (1987)

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    Gardiner, D.M.: Regulation of regeneration by heparan sulfate proteoglycans in the extracellular matrix. Regen Eng Transl Med. 3, 192–198 (2017)

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Phan, A.Q., Lee, J., Oei, M., Flath, C., Hwe, C., Mariano, R., Vu, T., Shu, C., Dinh, A., Simkin, J., Muneoka, K., Bryant, S.V., Gardiner, D.M.: Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum). Regeneration. 2, 182–201 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Ramachandra, R., Namburi, R.B., Dupont, S.T., Ortega-Martinez, O., van Kuppevelt, T.H., Lindahl, U., Spillmann, D.: A potential role for chondroitin sulfate/dermatan sulfate in arm regeneration in Amphiura filiformis. Glycobiology. 27, 438–449 (2017)

    CAS  PubMed  Google Scholar 

  29. 29.

    Becker, C.G, Becker, T.: Repellent guidance of regenerating optic axons by chondroitin sulfate glycosaminoglycans in zebrafish. J. Neurosci. 22, 842–853 (2002)

  30. 30.

    Rauvala, H., Paveliev, M., Kuja-Panula, J., Kulesskaya, N.: Inhibition and enhancement of neural regeneration by chondroitin sulfate proteoglycans. Neural Regen. Res. 12, 687–691 (2017)

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Inatani, M., Tanihara, H.: Proteoglycans in retina. Prog. Retin. Eye Res. 21, 429–447 (2002)

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Joven, A., Simon, A.: Homeostatic and regenerative neurogenesis in salamanders. Prog. Neurobiol. 170, 81–98 (2018)

    Article  CAS  PubMed  Google Scholar 

  33. 33.

    Roy, S., Levesque, M.: Limb regeneration in axolotl: is it superhealing? Sci. World J. 6, 12–25 (2006)

    Article  Google Scholar 

  34. 34.

    Alunni, A., Bally-Cuif, L.: A comparative view of regenerative neurogenesis in vertebrates. Development. 143, 741–753 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Sun, Y.B., Xiong, Z.J., Xiang, X.Y., Liu, S.P., Zhou, W.W., Tu, X.L., Zhong, L., Wang, L., Wu, D.D., Zhang, B.L., Zhu, C.L.: Whole-genome sequence of the Tibetan frog Nanorana parkeri and the comparative evolution of tetrapod genomes. Proc. Natl. Acad. Sci. 112, 1257–1262 (2015)

    Article  CAS  Google Scholar 

  36. 36.

    Nowoshilow, S., Schloissnig, S., Fei, J.F., Dahl, A., Pang, A.W., Pippel, M., Winkler, S., Hastie, A.R., Young, G., Roscito, J.G., Falcon, F.: The axolotl genome and the evolution of key tissue formation regulators. Nature. 554, 50–55 (2018)

    Article  CAS  PubMed  Google Scholar 

  37. 37.

    Svistunov, S.A., Mitashov, V.I.: Proliferative activity of the pigment epithelium and regenerating retinal cells in Ambystoma mexicanum. Ontogenez. 14, 597–606 (1983)

    CAS  PubMed  Google Scholar 

  38. 38.

    Voss, S.R., Epperlein, H.H., Tanaka, E.M.: Ambystoma mexicanum, the axolotl: a versatile amphibian model for regeneration, development, and evolution studies. Cold Spring Harb Protoc. 8, pdb.emo128, (2009)

  39. 39.

    Linhardt, R.J., Turnbull, J.E., Wang, H.M., Loganathan, D., Gallagher, J.T.: Examination of the substrate specificity of heparin and heparan sulfate lyases. Biochemistry. 29, 2611–2617 (1990)

    Article  CAS  PubMed  Google Scholar 

  40. 40.

    Wang, H., He, W., Jiang, P., Yu, Y., Lin, L., Sun, X., Koffas, M., Zhang, F., Linhardt, R.J.: Construction and functional characterization of truncated versions of recombinant keratanase II from Bacillus circulans. Glycoconj. J. 34, 643–649 (2017)

    Article  CAS  PubMed  Google Scholar 

  41. 41.

    Kundu, J., Michaelson, A., Talbot, K., Baranov, P., Young, M.J., Carrier, R.L.: Decellularized retinal matrix: natural platforms for human retinal progenitor cell culture. Acta Biomater. 31, 61–70 (2016)

    Article  CAS  PubMed  Google Scholar 

  42. 42.

    Wright, A.F., Chakarova, C.F., El-Aziz, M.M., Bhattacharya, S.S.: Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait. Nat Rev Genet. 11, 273–284 (2010)

    Article  CAS  PubMed  Google Scholar 

  43. 43.

    Grzybowski, A.: General structure and function of the retina. Acta Ophthalmol. 94, (2016)

  44. 44.

    Sanchez, I., Martin, R., Ussa, F., Fernandez-Bueno, I.: The parameters of the porcine eyeball. Graefes Arch. Clin. Exp. Ophthalmol. 249, 475–482 (2011)

    Article  PubMed  Google Scholar 

  45. 45.

    Porrello, K., Lavail, M.M.: Immunocytochemical localization of chondroitin sulfates in the interphotoreceptor matrix of the normal and dystrophic rat retina. Curr. Eye Res. 5, 981–993 (1986)

    Article  CAS  PubMed  Google Scholar 

  46. 46.

    Singhal, S., Lawrence, J.M., Bhatia, B., Ellis, J.S., Kwan, A.S., MacNeil, A., Luthert, P.J., Fawcett, J.W., Perez, M.T., Khaw, P.T., Limb, G.A.: Chondroitin sulfate proteoglycans and microglia prevent migration and integration of grafted Müller stem cells into degenerating retina. Stem Cells. 26, 1074–1082 (2008)

    Article  PubMed  Google Scholar 

  47. 47.

    Suzuki, T., Akimoto, M., Imai, H., Ueda, Y., Mandai, M., Yoshimura, N., Swaroop, A., Takahashi, M.: Chondroitinase ABC treatment enhances synaptogenesis between transplant and host neurons in model of retinal degeneration. Cell Transplant. 16, 493–503 (2007)

    Article  PubMed  Google Scholar 

  48. 48.

    Ichijo, H., Kawabata, I.: Roles of the telencephalic cells and their chondroitin sulfate proteoglycans in delimiting an anterior border of the retinal pathway. J. Neurosci. 21, 9304–9314 (2001)

    Article  CAS  PubMed  Google Scholar 

  49. 49.

    Brittis, P.A., Canning, D.R., Silver, J.: Chondroitin sulfate as a regulator of neuronal patterning in the retina. Science. 255, 733–736 (1992)

    Article  CAS  PubMed  Google Scholar 

  50. 50.

    Bakalash, S., Rolls, A., Lider, O., Schwartz, M.: Chondroitin sulfate-derived disaccharide protects retinal cells from elevated intraocular pressure in aged and immunocompromised rats. Invest. Ophthalmol. Vis. Sci. 48, 1181–1190 (2007)

    Article  PubMed  Google Scholar 

  51. 51.

    Tate, D.J., Oliver, P.D., Miceli, M.V., Stern, R., Shuster, S., Newsome, D.A.: Age-dependent change in the hyaluronic acid content of the human chorioretinal complex. Arch. Ophthalmol. 111, 963–967 (1993)

    Article  CAS  PubMed  Google Scholar 

  52. 52.

    Hollyfield, J.G., Rayborn, M.E., Tammi, M., Tammi, R.: Hyaluronan in the interphotoreceptor matrix of the eye: species differences in content, distribution, ligand binding and degradation. Exp. Eye Res. 66, 241–248 (1998)

    Article  CAS  PubMed  Google Scholar 

  53. 53.

    Inoue, Y., Yoneda, M., Miyaishi, O., Iwaki, M., Zako, M.: Hyaluronan dynamics during retinal development. Brain Res. 1256, 55–60 (2009)

    Article  CAS  PubMed  Google Scholar 

  54. 54.

    Solursh, M., Vaerewyck, S.A., Reiter, R.S.: Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes. Dev. Biol. 41, 233–244 (1974)

    Article  CAS  PubMed  Google Scholar 

  55. 55.

    Munaim, S.I., Klagsbrun, M., Toole, B.P.: Hyaluronan-dependent pericellular coats of chick embryo limb mesodermal cells: induction by basic fibroblast growth factor. Dev. Biol. 143, 297–302 (1991)

    Article  CAS  PubMed  Google Scholar 

  56. 56.

    Vatne, H.O., Syrdalen, P.: The use of sodium hyaluronate (Healon) in the treatment of complicated cases of retinal detachment. Acta Ophthalmol. 64(169–172), (1986)

  57. 57.

    Lipton, S.A., Wagner, J.A., Madison, R.D., D’Amore, P.A.: Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture. Proc. Natl. Acad. Sci. U. S. A. 85, 2388–2392 (1988)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Nagy, T., Reh, T.A.: Inhibition of retinal regeneration in larval Rana by an antibody directed against a laminin–heparan sulfate proteoglycan. Brain Res. Dev. 81, 131–134 (1994)

    Article  CAS  Google Scholar 

  59. 59.

    Schubert, D., LaCorbiere, M.: Isolation of a cell-surface receptor for chick neural retina adherons. J. Cell Biol. 100, 56–63 (1985)

    Article  CAS  PubMed  Google Scholar 

  60. 60.

    Carri, N.G., Perris, R., Johansson, S., Ebendal, T.: Differential outgrowth of retinal neurites on purified extracellular matrix molecules. J. Neurosci. Res. 19, 428–439 (1988)

    Article  CAS  PubMed  Google Scholar 

  61. 61.

    Chernousov, M.A., Carey, D.J.: N-syndecan (Syndecan 3) from neonatal rat brain binds basic fibroblast growth factor. J. Biol. Chem. 268, 16810–16814 (1993)

    CAS  PubMed  Google Scholar 

  62. 62.

    Ornitz, D.M., Itoh, N.: The fibroblast growth factor signaling pathway. Wiley Interdiscip. Rev. Dev. Biol. 4, 215–266 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Keenan, T.D., Pickford, C.E., Holley, R.J., Clark, S.J., Lin, W., Dowsey, A.W., Merry, C.L., Day, A.J., Bishop, P.N.: Age-dependent changes in heparan sulfate in human Bruch's membrane: implications for age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 55, 5370–5379 (2014)

    Article  CAS  PubMed  Google Scholar 

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This research was funded by the NIH in the form of grants DK111958, CA231074, HL125371 (to RJL) and by grant NSF-CBET #1606128 (to RLC).

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Correspondence to Rebecca L. Carrier or Robert J. Linhardt.

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Kim, S.Y., Kundu, J., Williams, A. et al. Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina. Glycoconj J 36, 165–174 (2019).

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  • Amphibian
  • Axolotl
  • Glycosaminoglycans
  • Regeneration
  • Retina