Glycoconjugate Journal

, Volume 30, Issue 5, pp 497–510 | Cite as

Structural remodeling of proteoglycans upon retinoic acid-induced differentiation of NCCIT cells

  • Leyla Gasimli
  • Hope E. Stansfield
  • Alison V. Nairn
  • Haiying Liu
  • Janet L. Paluh
  • Bo Yang
  • Jonathan S. Dordick
  • Kelley W. Moremen
  • Robert J. Linhardt


Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1,500-fold and 2,800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen.


Glycomics Teratocarcinoma Pluriotent Glycosaminoglycans 




BCA assay

Bicinchoninic acid assay


Ethylene bridged hybrid




Complementary deoxyribonucleic acid




Chondroitin sulfate/dermatan sulfate


4-deoxy-α-L-threo-hex-4-enopryanosyluronic acid






Glial fibrillary acid protein


Glucuronic acid


Iduronic acid




Horseradish peroxidase




Heparin/heparan sulfate




Keratin associated protein 3-2


Liquid chromatography/mass spectrometry




Polyvinyl difluoride


Quantitative reverse transcription-polymerase chain reaction


Retinoic acid


Ribosomal protein S18


Ribonucleic acid






Ultra-performance liquid chromatography


Western immunoblotting









The authors thank Empire State Stem Cell Fund for funding in the form of contract #C024334 and the National Institutes of Health for funding in the form of grant #3R01HL09697203.

Supplementary material

10719_2012_9450_MOESM1_ESM.doc (30 kb)
Supplemental Table 1 RT-PCR primers for NCCIT characterizationa (DOC 29 kb)
10719_2012_9450_MOESM2_ESM.doc (920 kb)
Supplemental Figure 1 HP/HS disaccharide composition analysis of GAGs from NCCIT cells (DOC 919 kb)
10719_2012_9450_MOESM3_ESM.doc (898 kb)
Supplemental Figure 2 HP/HS disaccharide composition analysis of GAGs from NCCIT-RA cells (DOC 898 kb)
10719_2012_9450_MOESM4_ESM.doc (170 kb)
Supplemental Figure 3 CS/DS disaccharide composition analysis of GAGs from NCCIT cells (DOC 170 kb)
10719_2012_9450_MOESM5_ESM.doc (131 kb)
Supplemntal Figure 4 CS/DS disaccharide composition analysis of GAGs from NCCIT-RA cells (DOC 131 kb)


  1. 1.
    Davies, J.A., Fisher, C.E., et al.: Glycosaminoglycans in the study of mammalian organ development. Biochem. Soc. Trans. 29, 166–171 (2001)PubMedCrossRefGoogle Scholar
  2. 2.
    Linhardt, R.J., Toida, T.: Role of glycosaminoglycans in cellular communication. Accts. Chem. Res. 37, 431–438 (2004)CrossRefGoogle Scholar
  3. 3.
    Lanctot, P.M., Gage, F.H., et al.: The glycans of stem cells. Curr. Opin. Chem. Biol. 11, 373–380 (2007)PubMedCrossRefGoogle Scholar
  4. 4.
    Andrews, P.W., Martin, M.M., et al.: Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin. Biochem. Soc. Trans. 33, 1526–1530 (2005)PubMedCrossRefGoogle Scholar
  5. 5.
    Damjanov, I.: Teratocarcinoma stem cells. Cancer Surv. 9, 303–319 (1990)PubMedGoogle Scholar
  6. 6.
    Pierce, G.B.: Carcinoma is to embryology as mutation is to genetics. Amer. Zool. 25, 707–712 (1985)Google Scholar
  7. 7.
    Teshima, S., Shimosato, Y., et al.: Four new human germ cell lines. Lab. Investig. 59, 328–336 (1988)PubMedGoogle Scholar
  8. 8.
    Damjanov, I., Horvat, B., et al.: Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, NCCIT. Lab. Investig. 68, 220–232 (1993)PubMedGoogle Scholar
  9. 9.
    Esko, J.D.: Genetic analysis of proteoglycan structure, function and metabolism. Curr. Opin. Cell Biol. 3, 805–816 (1991)PubMedCrossRefGoogle Scholar
  10. 10.
    Linhardt, R.J., Claude, S.: Hudson Award address in carbohydrate chemistry. Heparin: structure and activity. J. Medic. Chem. 46, 2551–2564 (2003)CrossRefGoogle Scholar
  11. 11.
    Skidmore, M.A., Guimond, S.E., et al.: The activities of heparan sulfate and its analogue heparin are dictated by biosyntehsis, sequence and conformation. Connect. Tissue Res. 49, 140–144 (2008)PubMedCrossRefGoogle Scholar
  12. 12.
    Sugahara, K., Kitagawa, H.: Heparin and heparan sulfate biosynthesis. JUBMB Life. 54, 163–175 (2002)CrossRefGoogle Scholar
  13. 13.
    Packer, A.I., Mailutha, K.G., et al.: Regulation of the Hoxa4 and Hoxa5 genes in the embryonic mouse lung by retinoic acid and TGFβ1: implications for lung development and patterning. Dev. Dynam. 217, 62–74 (2000)CrossRefGoogle Scholar
  14. 14.
    Lee, D.C.W., Chan, K.W., et al.: RET receptor typosine kinase isoforms in kidney function and disease. Oncogene 21, 5582–5592 (2002)PubMedCrossRefGoogle Scholar
  15. 15.
    Kumar, S., Duester, G.: Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2. Dev. Biol. 340, 67–74 (2010)PubMedCrossRefGoogle Scholar
  16. 16.
    Dahlstrand, J., Zimmerman, L.B., et al.: Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments. J. Cell Sci. 103, 589–597 (1992)PubMedGoogle Scholar
  17. 17.
    Couchman, J.R.: Transmembrane signaling proteoglycans. Annu. Rev. Cell. Dev. Biol. 26, 89–114 (2010)PubMedCrossRefGoogle Scholar
  18. 18.
    Esko, J.D., Kimata, K., et al.: Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2009)Google Scholar
  19. 19.
    Hanemann, C.O., Kuhn, G., et al.: Expression of decorin mRNA in the nervous system of rat. J. Histochem. Cytochem. 41, 1383–1391 (1993)PubMedCrossRefGoogle Scholar
  20. 20.
    Minor, K., Tang, X., et al.: Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons. Neurobiol. Dis. 32, 88–95 (2008)PubMedCrossRefGoogle Scholar
  21. 21.
    Troup, S., Njue, C., et al.: Reduced expression of the small leucine-rich proteoglycans, lumican, and decorin is associated with poor outcome in node-negative invasive breast cancer. Clin. Cancer Res. 9, 207–214 (2003)PubMedGoogle Scholar
  22. 22.
    Lemons, M.L., Barua, S., et al.: Adaptation of sensory neurons to hyalectin and decorin proteoglycans. J. Neurosci. 25, 4964–4973 (2005)PubMedCrossRefGoogle Scholar
  23. 23.
    Weber, C.K., Sommer, G., et al.: Biglycan is overexpressed in pancreatic cancer and induces G1-arrest in pancreatic cancer cell lines. Gastroenterology 121, 657–667 (2001)PubMedCrossRefGoogle Scholar
  24. 24.
    Saunders, S., et al.: Expression of the cell surface proteoglycan glypican-5 is developmentally regulated in kidney, limb, and brain. Dev. Biol. 190, 78–93 (1997)PubMedCrossRefGoogle Scholar
  25. 25.
    Williamson, D., Selfe, S., et al.: Cancer Res. 67, 57 (2007)PubMedCrossRefGoogle Scholar
  26. 26.
    Capurro, M.I., Shi, W., et al.: Processing by convertases is not required for glypican-3-induced stimulation of hepatocellular carcinoma growth. J. Biol. Chem. 280, 41201–41206 (2005)PubMedCrossRefGoogle Scholar
  27. 27.
    Filmus, J.: Glypicans in growth control and cancer. Glycobiology 11, 19R–23R (2001)PubMedCrossRefGoogle Scholar
  28. 28.
    Sung, Y.K., Hwang, S.Y., et al.: Glypican-3 is overexpressed in human hepatocellular carcinoma. Cancer Sci. 94, 259–262 (2005)CrossRefGoogle Scholar
  29. 29.
    Lai, J.P., Sandhu, D.S., et al.: Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma. Hepatology 47, 1211–1222 (2008)PubMedCrossRefGoogle Scholar
  30. 30.
    Bishop, J.R., Schuksz, M., et al.: Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030–1037 (2007)PubMedCrossRefGoogle Scholar
  31. 31.
    Aikawa, J., Esko, J.D.: Molecular cloning and expression of a third member of the heparan sulfate/heparin GlcNAcN-Deacetylase/ N-Sulfotransferase family. J. Biol. Chem. 274, 2690–2695 (1999)PubMedCrossRefGoogle Scholar
  32. 32.
    Aikawa, J., Grobe, K., et al.: Multiple isozymes of heparan sulfate/heparin GlcNAc N-deacetylase/GlcN N-sulfotransferase. Structure and activity of the fourth member, NDST4. J. Biol. Chem. 276, 5876–5882 (2001)PubMedCrossRefGoogle Scholar
  33. 33.
    Grobe, K., Ledin, J., et al.: Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes. Biochim. Biophys. Acta 1573, 209–215 (2002)PubMedCrossRefGoogle Scholar
  34. 34.
    Kusche-Gullberg, M., Kjellén, L.: Sulfotransferases in glycosaminoglycan biosynthesis. Curr. Opin. Struc. Biol. 13, 605–611 (2003)CrossRefGoogle Scholar
  35. 35.
    Filmus, J., Capurro, M.I., et al.: Glypicans. Genome Biol. 8, 224 (2008)CrossRefGoogle Scholar
  36. 36.
    Kraushaar, D.C., Yamaguchi, Y., et al.: Heparan sulfate is required for embryonic stem cells to exit from self-renewal. J. Biol. Chem. 285, 5907–5916 (2010)PubMedCrossRefGoogle Scholar
  37. 37.
    Funderburgh, J.L.: Keratan sulfate biosynthesis. IUBMB Life. 54, 187–194 (2002)PubMedCrossRefGoogle Scholar
  38. 38.
    Funderburgh, J.L., Caterson, B., et al.: Keratan sulfate proteoglycan during embryonic development of the chicken cornea. Dev. Biol. 116, 267–277 (1986)PubMedCrossRefGoogle Scholar
  39. 39.
    Brown, J.J., Papaioannou, V.E.: Ontogeny of hyaluronan secretion during early mouse development. Development 117, 483–492 (1993)PubMedGoogle Scholar
  40. 40.
    Toole, B.P.: Hyaluronan in morphogenesis. Sem. Cell. Dev. Biol. 12, 79–87 (2001)CrossRefGoogle Scholar
  41. 41.
    Paluh, J.L., Dai, G., et al.: In search of the Holy Grail: engineering the stem cell niche. Eur. Pharm. Rev. 16, 28–33 (2011)Google Scholar
  42. 42.
    Li, B., Liu, H., et al.: Analysis of glycosaminoglycans in stem cell glycomics. Methods Mol. Biol. Humana Press 690, 285–300 (2011)CrossRefGoogle Scholar
  43. 43.
    Nairn, A.V., Kinoshita-Toyoda, A., et al.: Glycomics of proteoglycan biosynthesis in murine embryonic stem cell differentiation. J. Proteome Res. 6, 4374–4387 (2007)PubMedCrossRefGoogle Scholar
  44. 44.
    Nairn, A.V., York, W.S., et al.: Regulation of glycan structures in animal tissues: transcript profiling of glycan-related genes. J. Biol. Chem. 25, 17298–17313 (2008)CrossRefGoogle Scholar
  45. 45.
    Pfaffl, M.W.: A new mathematical model for relative quantification in real-time PCR. Nucleic Acids Res. 29, 2002–2007 (2001)CrossRefGoogle Scholar
  46. 46.
    Zhang, F., Sun, P., et al.: Microscale isolation and analysis of heparin from plasma using an anion-exchange spin column. Anal. Biochem. 353, 284–286 (2006)PubMedCrossRefGoogle Scholar
  47. 47.
    Shaya, D., Tocili, A., et al.: Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product. J. Biol. Chem. 281, 15525–15535 (2006)PubMedCrossRefGoogle Scholar
  48. 48.
    Yoshida, E., Arakawa, S., et al.: Cloning, sequencing, and expression of the gene from bacillus circulans that codes for a heparinase that degrades both heparin and heparan sulfate. Biosci. Biotech. Biochem. 66, 1873–1879 (2002)CrossRefGoogle Scholar
  49. 49.
    Solakyildirim, K., Zhang, F., et al.: Ultraperformance liquid chromatography with electrospray ionization ion trap mass spectrometry for chondroitin disaccharide analysis. Anal. Biochem. 397, 24–28 (2010)PubMedCrossRefGoogle Scholar
  50. 50.
    Yang, B., Weyers, A., et al.: Ultra-performace ion pairing liquid chromatography with on-line electrospray ion trap mass spectrometry for heparin disaccharide analysis. Anal. Biochem. 415, 59–66 (2011)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Leyla Gasimli
    • 1
  • Hope E. Stansfield
    • 5
  • Alison V. Nairn
    • 6
  • Haiying Liu
    • 2
  • Janet L. Paluh
    • 7
  • Bo Yang
    • 2
  • Jonathan S. Dordick
    • 1
    • 3
    • 4
  • Kelley W. Moremen
    • 6
  • Robert J. Linhardt
    • 1
    • 2
    • 3
    • 4
  1. 1.Department of Biology, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  2. 2.Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  3. 3.Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  4. 4.Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  5. 5.Department of Orthopedics, Center for Muscoloskeletal ResearchUniversity of Rochester Medical CenterRochesterUSA
  6. 6.Complex Carbohydrate Research CenterUniversity of GeorgiaAthensUSA
  7. 7.Nanobioscience Constellation, College of Nanoscale Science and EngineeringUniversity at Albany, SUNYAlbanyUSA

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