Identification of α(1,6)fucosylated proteins differentially expressed in human colorectal cancer

Abstract

Background: A universal hallmark of cancer cells is the change in their glycosylation phenotype. One of the most frequent alterations in the normal glycosylation pattern observed during carcinogenesis is the enhancement of α(1,6)linked fucose residues of glycoproteins, due to the up-regulation of the α(1,6)fucosyltransferase activity. Our previous results demonstrated the specific alteration of this enzyme activity and expression in colorectal cancer, suggesting its implication in tumour development and progression.

Methods: In the current work we combined a LCA-affinity chromatography with SDS-PAGE and mass spectrometry in order to identify α(1,6)fucosylated proteins differentially expressed in colorectal cancer. This strategy allowed the identification of a group of α(1,6)fucosylated proteins candidates to be involved in CRC malignancy.

Results: The majority of the identified proteins take part in cell signaling and interaction processes as well as in modulation of the immunological response. Likewise, we confirmed the increased expression of GRP94 in colorectal cancer tissue and the significant down-regulation of the IgGFcBP expression in tumour cells.

Conclusion: All these results validate the importance of core-fucosylated proteins profile analysis to understand the mechanisms which promote cancer onset and progression and to discover new tumour markers or therapeutic targets.

Figure 1

Chromatographic profile. Representative chromatographic profile of α(1,6)fucosylated proteins enrichment from healthy (A) and tumour (B) colorectal specimens.

Figure 2

Different expression of α(1,6)fucosylated proteins in healthy and tumour colorectal tissues. (A) Representative Coomassie brilliant blue stained 1D-SDS-PAGE gel of the total protein fraction (TF) and the following chromatographic fractions obtained; FI (non-retained proteins), FII (unspecific retained proteins) and FIII [α(1,6)fucosylated proteins] and the corresponding lectin blot in a pair of healthy and tumour tissue. (B) Patern of bands obtained in the FIII fraction of a pair of healthy and tumour samples. Bands 5 and 8 were excised and submitted to mass spectrometry (MS) analysis.

Figure 3

GRP94 expression in healthy and tumour colorectal tissues. (A) Validation of the 1D-SDS-PAGE results for GRP94 by immunoblotting in 20 paired samples of healthy (H) and tumour (T) tissue. (B) Statistically significant increase of the GRP94 expression found in tumour tissue. *p < 0.05, according to Wilcoxon's test.

Figure 4

pIgR expression in healthy and tumour colorectal tissues. (A) Validation of the 1D-SDS-PAGE results for pIgR by immunoblotting in 12 paired samples of healthy (H) and tumour (T) tissue. (B) The statistical analysis showed no significant differences between healthy and tumour tissues according to Wilcoxon's test.

Figure 5

IgGFcBP expression in healthy and tumour colorectal tissues. (A) Validation of the 1D-SDS-PAGE results for IgGFcBP by immunoblotting in 20-paired samples of healthy (H) and tumour (T) tissue. The Western blot allowed the visualization of two underexpressed isoforms of ~108 and ~52 kDa. (B) For the ~108 kDa band, this decrease was statistically significant. *p < 0.05, according to Wilcoxon's test.

Figure 6

Immunohistochemistry analysis of IgGFcBP expression. (A) Healthy colon tissue with a specific IgGFcBP staining localized in the intracellular mucus of globelet cells. (B) CRC tissue with a positive IgGFcBP staining present in the non-invasive zone. (C) Mixed hyperplastic/adenomatous polyp with the IgGFcBP positive glands in the non dysplastic areas.