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

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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.

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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

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Leyla Gasimli, Jonathan S. Dordick & Robert J. Linhardt

  2. Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Haiying Liu, Bo Yang & Robert J. Linhardt

  3. Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Jonathan S. Dordick & Robert J. Linhardt

  4. Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY, 12180, USA

    Jonathan S. Dordick & Robert J. Linhardt

  5. Department of Orthopedics, Center for Muscoloskeletal Research, University of Rochester Medical Center, Rochester, NY, 14642, USA

    Hope E. Stansfield

  6. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA

    Alison V. Nairn & Kelley W. Moremen

  7. Nanobioscience Constellation, College of Nanoscale Science and Engineering, University at Albany, SUNY, Albany, NY, 12203, USA

    Janet L. Paluh

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  1. Leyla Gasimli
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Correspondence to Robert J. Linhardt.

Electronic supplementary material

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Supplemental Table 1

RT-PCR primers for NCCIT characterizationa (DOC 29 kb)

Supplemental Figure 1

HP/HS disaccharide composition analysis of GAGs from NCCIT cells (DOC 919 kb)

Supplemental Figure 2

HP/HS disaccharide composition analysis of GAGs from NCCIT-RA cells (DOC 898 kb)

Supplemental Figure 3

CS/DS disaccharide composition analysis of GAGs from NCCIT cells (DOC 170 kb)

Supplemntal Figure 4

CS/DS disaccharide composition analysis of GAGs from NCCIT-RA cells (DOC 131 kb)

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Gasimli, L., Stansfield, H.E., Nairn, A.V. et al. Structural remodeling of proteoglycans upon retinoic acid-induced differentiation of NCCIT cells. Glycoconj J 30, 497–510 (2013). https://doi.org/10.1007/s10719-012-9450-x

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