The scientific community shows an increasing interest in qualitative and quantitative alterations that different glycoproteins undergo during the malignant transformation process. This interest is fundamentally focused on the detection of new tumour biomarkers for diagnosis, prognosis and follow-up of the disease, as well as on the development of new therapeutic targets. Thus, a large number of biomolecules currently used as tumour markers are glycoproteins [24, 25].
In particular, the study of the α(1,6)fucosylated proteins expressed in tumour cells has achieved high importance. Several researchers have reported the specific alteration of α(1,6)FT activity and expression in malignant processes, such as HCC [26, 27], thyroid papillary carcinoma  and ovarian adenocarcinoma . Recently, we have also reported the alteration of the enzyme in CRC  and its implication in the disease progression (submitted data). In this sense, it is important to mention the critical role of α(1,6)FT activity in the modulation of different growth factor receptors such as EGFR, TGFβR or VEGFR. These molecules, strongly implicated in the carcinogenesis process and considered gold therapeutic targets for the treatment of solid tumours, need to be core-fucosylated to activate their intracellular signaling pathways [6–8]. Likewise, recent reports have shown that α(1,6)fucosylation of different adhesion molecules, such as adhesins or integrins, modify their functional activity, and could probably act as relevant factors for the acquisition of the migratory phenotype by the epithelial tumour cells [6, 10, 11]. Therefore, the identification and characterization of core-fucosylated proteins differentially expressed in tumour cells is considered of great usefulness to discover new tumour biomarkers and therapeutic agents.
In the present study we combined a LCA-affinity chromatography with SDS-PAGE and mass spectrometry in order to identify α(1,6)fucosylated proteins differentially expressed in the tumour tissue of 5 CRC patients. We identified a group of proteins candidates to be α(1,6)fucosylated and specifically regulated in colorectal tumours. Validating our approach, all the identified proteins have been described as glycoproteins, and most of them as α(1,6)fucosylated proteins. We selected three of them, the GRP94, the pIgR and the IgGFcBP in order to validate their altered expression during colorectal carcinogesis.
The GRP94 or endoplasmin is the most abundant glycoprotein in the endoplasmic reticulum. It belongs to the family of the heat-shock proteins, and together with GRP78 assists the folding and assembly of a wide range of proteins. The GRP94 also shows ATPase activity and plays an essential role in the cellular protection against different stress situations. Under pathological conditions, such as tumour growth, GRP94 is dramatically up-regulated as a survival mechanism , being its overexpression associated with a more aggressive tumour phenotype and a poor evolution of the disease [31, 32]. In CRC, gene and protein GRP94 expression are strongly increased in both animal models and human tumours [33, 34], and therefore GRP94 has been proposed as a useful diagnostic and prognostic marker for the disease. Concordantly, after an immunoblot analysis of the GRP94 expression in paired healthy and tumour colorectal specimens, we observed a significant increase in tumour tissue. In this sense, it is conceivable that up-regulation of GRP94 allows the correct folding of several oncogenic products promoting the colorectal carcinogenesis, although this increase has also been related with the acquisition of survival mechanisms by tumour cells to stand up lethal conditions, such as glucose starvation and hypoxia, and with the ability to form new distant solid tumours . It also important to remark that although this protein is known to be glycosylated our study suggest for the first time its α(1,6)fucosylated status, however further studies should be developed to demonstrate this it.
In the same band where GRP94 was detected, MS analysis also identified the potential presence of pIgR. This glycosylated receptor is located in the basolateral membrane of the oral and gastrointestinal epithelial cells. Its main function is the transport of IgA and IgM polymeric forms across the epithelial cell membranes . Recently, the α(1,6)fucosylation of this protein has been demonstrated in both, healthy and tumour hepatic specimens , confirming the results obtained in our study for colonic tissue. Interestingly, the importance of pIgR is not restricted to immunology, since changes in its expression, either increase or decrease, have been described in different types of tumours . However, after our comparative immunoblot analysis between paired specimens of healthy and tumour mucosa, no significant differences were found in spite of the previous studies indicating an early decrease of pIgR expression during colorectal carcinogenesis . It seems that the absence of pIgR in the tumour cells could induce a depletion of the immune response that promotes their malignant potential; nevertheless, its role in cancer development remains unknown.
Finally, we analyzed by Western blot and immunohistochemistry the expression of IgGFcBP, a protein secreted by the mucosa epithelial cells and present in the body fluids . It specifically recognizes the constant fraction of IgG, and plays a relevant role in the structural maintenance of the mucosa through the binding to the MUC2 mucin . Despite the theoretical molecular mass of IgGFcBP is ~500 kDa , SDS-PAGE reports Mr ~100-80 kDa depending on the tissue analyzed. In our study two bands of 108 and 52 kDa were detected, suggesting a tissue specific proteolytic process. Interestingly, the two bands showed a clear decrease in tumour specimens, statistically significant for the 108 KDa band. These results were confirmed by immunohistochemistry, since a positive IgGFcBP expression was observed in all the healthy specimens analyzed, while only 3 early staged tumours were found positive (with the expression mainly confined to the non-infiltrating epithelia). Concordantly with the specific expression of IgGFcBP in the goblet cells of the mucosa, the 3 positive tumours present higher percentage of mucinous component than the other specimens. It should be of great interest check the status of the IgGFcBP expression in mucilaginous tumours to demonstrate the independence between the tumour histology and the IgGFcBP downregulation. Furthermore, the analysis of 6 polyps showed expression of IgGFcBP in 4 of the specimens. Supporting our results, the lost of expression of this α(1,6)fucosylated protein in tumour specimens has also been reported in hepatic tissues  and the same tumour decrease of IgGFcBP has been described in animal models of CRC and human polyps [40, 41]. The biological significance of the down-regulation of IgGFcBP in tumour cells is uncertain. In this sense, the binding between this protein and IgG could protect it from the action of bacterial proteases , promoting the immunological response of the mucosa. Thus, the IgGFcBP overexpression has been detected in ulcerous colitis and Cröhn patients . Consequently, it is thought that low secreted levels of this protein in tumour cells could facilitate the immune evasion. However, the absence of IgGFcBP in the colon mucosa could also lead to the structural disorganization of intestinal mucus, promoting the exposure of mucosa to carcinogenic agents and, therefore, the appearance of premalignant lesions in the epithelium. Taking into account that increased levels of serum IgGFcBP have been observed in inflammatory processes, a possible decrease in CRC could be of great value as a biomarker for this neoplasia.