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
Article

Abstract

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.

Keywords

Glycomics Teratocarcinoma Pluriotent Glycosaminoglycans 

Abbreviations

Ac

Acetyl

BCA assay

Bicinchoninic acid assay

BEH

Ethylene bridged hybrid

C5Epi

C5Epimerase

cDNA

Complementary deoxyribonucleic acid

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

CS/DS

Chondroitin sulfate/dermatan sulfate

ΔUA

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

GAG

Glycosaminoglycan

GalN

Galactosamine

GFAP

Glial fibrillary acid protein

GlcA

Glucuronic acid

IdoA

Iduronic acid

GlcN

Glucosamine

HRP

Horseradish peroxidase

HFIP

1,1,1,3,3,3-hexafluoro-2-propanol

HP/HS

Heparin/heparan sulfate

HXA

Hexylamine

KRTAP3-2

Keratin associated protein 3-2

LC/MS

Liquid chromatography/mass spectrometry

NDST

N-deacetylase-N-sulfotransferase

PVDF

Polyvinyl difluoride

qRT-PCR

Quantitative reverse transcription-polymerase chain reaction

RA

Retinoic acid

RPS18

Ribosomal protein S18

RNA

Ribonucleic acid

S

Sulfo

TrBA

Tributylamine

UPLC

Ultra-performance liquid chromatography

WB

Western immunoblotting

HS2ST

2-O-sulfotransferase

HS3ST

3-O-sulfotransferase

HS6ST

6-O-sulfotransferase

Supplementary material

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

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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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