Changes in enzyme activity and the expression levels of a(1,6)fucosyltransferase [a(1,6)FT] have been reported in certain types of malignant transformations. To develop a better understanding of the role of a(1,6)FT in human colorectal carcinoma (CRC), we analysed the enzyme activity in healthy and tumour tissues. a(1,6)FT activity was considerably higher in tumour tissue than in healthy tissue and was related to gender, lymph node metastasis, type of growth and tumour stage. We also observed a significant increase in the a(1,6)FT expression in tumour tissues as compared to healthy and transitional tissues, inflammatory lesions and adenomas. The immunohistochemical expression in tumour tissues was correlated with the degree of infiltration through the intestinal wall. Finally, a statistical correlation was found between enzyme activity and expression obtained by Western blot in colorectal tumours when compared in the same patient. All these findings demonstrate an alteration of a(1,6)FT activity and expression in CRC. ' 2008 Wiley-Liss, Inc.Key words: a(1,6)fucosyltransferase; FUT8; colorectal cancer It is well documented that N-linked oligosaccharides on glycoproteins are structurally altered during malignant transformation.

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SummaryBackgroundA 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.MethodsIn 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.ResultsThe 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.ConclusionAll 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.

Abstract. lewis b and lewis y (le) antigens are known to be elevated in colorectal tumours. Alterations in the catalytic behaviour of gDP-l-fucose:β-D-galactoside α(1,2) fucosyltransferase [α(1,2)Ft, ec: 2.4.1.69], the key enzyme in their synthesis, have been suggested as being responsible for these changes. In particular, an aberrant tumour-specific α(1,2)Ft activity that converts le a and le x to le b and le y determinants, respectively, has been reported in colorectal cancer tissues. to clarify the catalytic function of this enzyme during colorectal tumorigenesis, we analyzed α(1,2)Ft activity levels in healthy and tumour colon specimens using different acceptor substrates and determined the kinetic properties of the enzyme. to complete the study, the aberrant le a /le x α(1,2) fucosylation was determined in healthy and tumour colorectal tissues. A correlation analysis between the activity levels and various standard clinicopathological features, such as tumour stage, was also carried out to elucidate the role of these activities in tumour progression. The results obtained confirm the enhanced α(1,2)fucosylation in colorectal neoplastic tissues and the importance of the aberrant le a /le x α(1,2)Ft activity in this increase. However, taking into account the high levels of le a /le x fucosylation observed in healthy control tissues, we must rule out the idea of a colorectal tumour-specific α(1,2) FT. On the other hand, no significant association was observed between α(1,2)Ft activity levels and the clinicopathological characteristics. overall, our results suggest that α(1,2)Ft activity plays a critical role in the accumulation of le b and le y antigens in human colorectal carcinoma. IntroductionAlterations in glycosylation patterns are one of the characteristics associated with differentiation and malignant transformation (1). the oligosaccharide portions of cellular glycoconjugates, glycolipids and glycoproteins, convey specific immunodeterminants, such as the blood group and related antigens (2). these antigens are known to play an important role in cell recognition, interaction, adhesion and motility. Accordingly, changes in these antigens during carcinogenesis may be crucial in tumour progression and may be involved in extravasation and metastatic phenomena (3).ABo and related determinants are the result of the sequential addition of monosaccharide units to one of the five disaccharide precursors described. type 1 [galβ (1,3)glcnac β1-r] and 2 [gal β(1,4)glcnac β1-r] precursors produce H 1 and H 2 structures, respectively, after the addition of a fucose in α(1,2) linkage to the terminal β-galactosyl residue. H 1 and H 2 determinants can subsequently be converted into le b and le y difucosylated antigens by fucosylation in α(1,4) or α(1,3) linkage, respectively. their positional isomers, the le a and le x antigens, show a similar structure, but the antigens are not fucosylated in α(1,2) (4). Specific alterations of Lewis antigens have been reported in multiple carcinomas and have been correlated with the pr...

The α(1,6)fucose residue attached to the N-glycoprotein core is suspected to play an essential role in the progression of several types of cancer. Lectins remain the first choice for probing glycan modifications, although they may lack specificity. Thus, efforts have been made to identify new lectins with a narrower core fucose detection profile. Here, we present a comparison of the classical Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA) and Aspergillus oryzae lectin (AOL) with the newer Pholiota squarrosa lectin (PhoSL), which has been described as being specific for core fucosylated N-glycans. To this end, we studied the binding profiles of the four lectins using mammalian glycan arrays from the Consortium of Functional Glycomics. To validate their glycan specificity, we probed AOL, LCA and PhoSL in western-blot assays using protein extracts from eight common colorectal cancer lines and colorectal biopsies from a small cohort of patients with colorectal cancer. The results showed that i) LCA and PhoSL were the most specific lectins for detecting the presence of core fucose in a concentration-dependent manner; ii) PhoSL exhibited the highest N-glycan sequence restriction, with preferential binding to core fucosylated paucimannosidic-type N-glycans, iii) the recognition ability of PhoSL was highly influenced by the presence of terminal N-acetyl-lactosamine; iv) LCA bound to paucimannosidic, bi-antennary and tri-antennary core fucosylated N-glycans; and v) AOL and AAL exhibited broader specificity towards fucosylation. Together, our results support the choice of LCA as the most appropriate lectin for core fucose detection, as validated in protein extracts from colorectal cancer cell lines and tissue specimens from patients with colorectal cancer.