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N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression*

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Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.

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Author contributions: S.H., A.M.D., Y.R., and M.W. the designed research; S.H., A.J.D., G.W.v., S.J.v., and J.J.G. performed the research; C.A.K., W.E.M., and R.A.T. contributed new reagents or analytic tools; S.H., J.J.G., and C.A.K. analyzed the data; S.H., G.W.v., Y.R., and M.W. wrote the paper; and A.J.D. performed method optimization.

*

This work was supported by the European Union (Seventh Framework Programme HighGlycan project, grant number: 278535).

This article contains supplemental material Supplemental Tables S1-S8 and Supplemental Figs. S1-S7.

1

The abbreviations used are:

    CRC

    colorectal cancer

    DHB

    2,5-dihydroxybenzoic acid

    dHex

    deoxyhexose

    DMEM

    Dulbecco's Modified Eagle

    F

    Fuc, fucose

    FCS

    fetal calf serum

    GalNAc

    N-acetylgalactosamine

    GlcNAc

    N-acetylglucosamine

    GuHCl

    guanidine hydrochloride

    H

    Hex, hexose

    N

    HexNAc, N-acetylhexosamine

    HILIC

    hydrophilic interaction liquid chromatography

    LC-ESI-MS

    liquid chromatography-electrospray ionization-mass spectrometry

    MALDI-TOF-MS

    matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

    MS/MS

    tandem mass spectrometry

    NeuAc

    N-acetylneuraminic acid

    PCA

    principal component analysis

    PNGase F

    peptide N-glycosidase F

    RPMI

    Roswell Park Memorial Institute

    SPE

    solid phase extraction.