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

Abstract

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.

Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages

“…The phenotypic differentiation of THP-1 into macrophage-like cells was controlled by flow cytometry analysis of the cell surface adhesion molecule CD11c expression level in untreated and PMA-treated THP-1 cells 21,22,26 59 60 In addition, N-glycans were released from cells glycoproteins using a PVDF-membrane based protocol, as previously described 28 …”

A facile method for cellular N-glycomic profiling by matrix-assisted laser desorption/ionization mass spectrometry

“…More recently, Holst et al have applied an optimized, high-throughput membrane-based enzymatic glycan release for cellular N-glycan analysis with sialic linkage specic carboxyl derivatization method. 20 However, this method has some disadvantages, such as the instability of the generated lactones and byproducts of the derivatives.…”

Broad and thematic remodeling of the surface glycoproteome on isogenic cells transformed with driving proliferative oncogenes

“…There is abundant evidence to suggest that high-mannose type glycans are present at the cell surface, and furthermore, that cancerous cells display increased abundance of highmannose glycans at their cell surface (Holst et al, 2016;Hua et al, 2014). A similar observation was reported in the glycomic comparison of transformed versus human embryonic stem cells (hESC), where high-mannose glycans were observed at significantly higher abundance on plasma membranes of hESCs .…”

“…Thus, over 60% of the N-glycopeptides identified contained less mature high mannose or paucimannose structures originating from processing in the ER or early Golgi. It is not surprising to find these high mannose modifications associated with cell surface proteins as others have seen that high mannose N-glycosylation is abundant at the cell surface and especially associated with oncogene transformation Balog et al, 2012;Holst et al, 2016).…”