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. 2016 Jan;15(1):124-40.
doi: 10.1074/mcp.M115.051235. Epub 2015 Nov 4.

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

Stephanie Holst et al. Mol Cell Proteomics. 2016 Jan.

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.

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Figures

Fig. 1.
Fig. 1.
MALDI-TOF-MS N-glycome spectra of two exemplary colorectal cancer (CRC) cell lines: (A) DLD-1, and (B) T84. N-glycans were detected as sodium adduct ions [M+Na]+ over a mass range of m/z 1000–4500. Spectra show relative intensities with zoom on the mass range m/z 2500–4500. Major glycan peaks are annotated and represent compositions. The presence of structural isomers cannot be excluded. Linkage positions of sialic acid residues are indicated by differing angles.
Fig. 2.
Fig. 2.
Relative quantification of derived glycan traits. Summed relative intensities from MALDI-TOF-MS analysis according to glycan classes: (A) N-glycan types; (B) high-mannose N-glycan content on the cell surface of CRC cell lines versus the content in the residual pellets; (C) hexose (Hex)/N-acetylhexosamines (HexNAc) ratios: Equal amounts of Hex and HexNAc of complex- and hybrid-type N-glycans (Hex = HexNAc) indicates presence of either bisection or terminal HexNAc, whereas HexNAc>Hex ratio points additionally to the presence of terminal HexNAc in form of e.g. LacdiNAc structures; (D) antennary of complex- and hybrid-type N-glycans; (E) fucosylation distinguishing mono-fucosylated (MonoFuc, one fucose) and multi-fucosylated (MultiFuc, 2–5 fucoses) on complex- and hybrid-type N-glycans; (F) purely α2,6- and α2,3-sialylated complex- and hybrid-type N-glycans as well as glycans with mixed sialylation. Error bars display the standard deviation between 2–3 biological replicates. Relative intensities were rescaled to 100% for calculation of traits corresponding to complex- and hybrid-type N-glycans (A, C-F). *DNA profiling studies (57, 58) have shown that DLD-1, HCT-15, HCT-8 and HRT-18G share a single profile; ** variants/sister cell lines; *** same patient;**** derivatives.
Fig. 3.
Fig. 3.
MS/MS analysis of N-glycans. (A) MALDI-TOF/TOF-MS/MS spectrum of m/z 1743.58 [M+Na]+ from CRC cell line RKO confirming the high-mannose composition Hex8HexNAc2; (B) LC-ESI-MS/MS spectrum of m/z 1141.94 [M+2H]2+ from cell line CaCo2 confirms the composition Hex3HexNAc6dHex3 comprising two LacdiNAc (GalNAcβ1–4GlcNAcβ1-) antennae as indicated by the fragment ion m/z 407.16 [HexNAc2]+ and its fucosylated variant with m/z 553.18 [HexNAc2dHex1]+. The fragment ion m/z 699.29 [HexNAc2dHex2]+ may be the result of a fucose re-arrangement (59); (C) LC-ESI-MS/MS/MS spectrum of 991.04 [M+2H]3+ confirming the composition Hex6HexNAc7NeuAc1dHex1 in CaCo2 cells showing a specific fragment ion at m/z 860.64 [M+H]+ (Hex1HexNAc2NeuAc1). MS3 (zoom) of the fragment ion m/z 860.64 [M+H]+ from cell line CaCo2 confirms the presence of Sda-antigen (NeuAcα2–3[GalNAcβ1–4]Galβ1–4GlcNAc-R); (D) MALDI-TOF/TOF-MS/MS spectrum of m/z 2669.97 [M+Na]+ from CRC cell line HCT8 confirming the tri-fucosylated N-glycan with the composition Hex6HexNAc6dHex3 and an additional agalactosylated antenna or bisected GlcNAc; (E) MALDI-TOF/TOF-MS/MS spectrum of m/z 1982.71 [M+Na]+ from CRC cell line HCT116 confirming the composition of the complex type N-glycan Hex5HexNAc4α2,6NeuAc1 with fragment ion m/z 706.70 [M+Na]+ (Hex1HexNAc1α2,6NeuAc1). The presence of structural isomers cannot be excluded. Annotation was performed using GlycoWorkbench 2.1 stable build 146 (http://www.eurocarbdb.org/).
Fig. 4.
Fig. 4.
Principal component analysis (PCA). Averages of biological replicates per glycan and cell line were unit-variance scaled and used for multivariate data analysis in SIMCA V13 (Umetrics, Sweden). The PCA model resulted in 13 principal components (PCs) explaining 84.4% variation (R2X(cum)) within the data and a good prediction power Q2(cum) of 65.4%. Cross validation was performed on technical replicates distributed into six groups. (A) PCA score plot of PC1 (19.8%) against PC2 (13.5%) colored according to cell lines; (B and C) loading plot of PCA model displaying PC1 (19.8%) against PC2 (13.5%) colored according specific glycan features: (B) ratio between the number of hexoses (Hex) and the number of N-acetylhexosamines (HexNAc) with Hex>HexNAc representing fully galactosylated antennae, while HexNAc≥Hex indicates terminal HexNAc with indications for bisection (HexNAc = Hex) and LacdiNAc epitopes (GalNAcβ1, 4GlcNAcβ1-; HexNAc>Hex) and (C) fucosylation distinguishing mono-fucosylated (Mono Fuc) versus multi-fucosylated (Multi Fuc) versus non-fucosylated (No ID) N-glycans. *DNA profiling studies (57, 58) have shown that DLD-1, HCT-15, HCT-8 and HRT-18G share a single profile; ** variants/sister cell lines; *** same patient;**** derivatives.
Fig. 5.
Fig. 5.
Glycan trait correlations with CDX1/villin mRNA. (A) PCA score plot of an unsupervised PCA model displaying principal components (PC) 1 and 2 (see Fig. 4A) colored according to stage of the original tumor. (B) Score plot of PC1 versus PC2 colored according to CDX1/villin-mRNA expression: CDX1/villin-positive (+) versus CDX1/villin-negative (-). Expression of mRNA of CDX1/villin was retrieved from (–35). Potential glycan traits associated with CDX1/villin-mRNA expression based on loadings of the PCA model (See Figs 4B and 4C) and were calculated by summing relative intensities from MALDI-TOF-MS analysis for averages of 2–3 biological replicates. A Mann–Whitney nonparametric test with significance level α = 0.05 was performed. CDX1/villin-negative (CDX1-) and CDX1/villin-positive (CDX1+) were significantly different with increased levels in CDX1+ cells of C) N-glycans with higher amount of N-acetylhexosamine (HexNAc) than hexoses (Hex), p value .0471, and D) multi-fucosylation, p value .0026.

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