Biosynthesis of the cancer-related sialyl-α2,6-lactosaminyl epitope in colon cancer cell lines expressing β-galactoside α2,6-sialyltransferase under a constitutive promoter

An elevation of b-galactoside a2,6-sialyltransferase(ST6Gal.I) enzyme activity and an increased a2,6-sialyla-tion of cell membranes are among the most prominentglycosylation changes associated with colon cancer; bothmodifications correlate with a worse prognosis. In ourprevious studies, we have frequently observed a discrepancybetween the ST6Gal.I level within a colon cancer sample orcell line and the respective level of reactivity with thea2,6-sialyl-specific lectin from Sambucus nigra (SNA). Inthis study, we have investigated quantitatively thebiosynthesis of the sialyl-a2,6-lactosaminyl epitope in twocolon cancer cell types expressing the ST6Gal.I cDNAunder the control of a constitutive promoter. By measuringthe amount of ST6Gal.I mRNA using competitive RT-PCR,the expression of a2,6-sialylated lactosaminic structureswith SNA and anti-CDw75 Ig, and the presence ofunsubstituted lactosaminic termini by Erythrina cristagallilectin, we reached the following conclusions: (a) a highproportion of the cell surface lactosaminic termini remainsunsubstituted, even in the presence of a very high ST6Gal.Iactivity. This proportion is strongly dependent on the celltype; (b) ST6Gal.I-transfected colon cancer cells do notexpress the CDw75 epitope; (c) the level of ST6Gal.Ienzyme activity only partially correlates with the mRNAlevel; (d) despite the control by a constitutive promoter, theST6Gal.I mRNA is not constantly expressed over time; and(e) a very large portion of the enzyme molecules is secretedin the extracellular milieu. These results indicate that post-transcriptional and post-translational mechanisms play apivotal role in the control of a2,6-sialylation in colon cancercells.Keywords: colon cancer; glycosyltransferases; glycosyla-tion; a2,6-sialyltransferase; CDw75.The biosynthesis of the oligosaccharide moieties ofglycoconjugates is a complex process and the fine regulationof this process is only partially understood. Although it isgenerally thought that the biosynthesis of a given oligo-saccharide structure is regulated mainly by the expression ofthe cognate glycosyltransferase, the enzyme–productrelationship might in some cases correlate very poorly[1,2], suggesting that other factors play a relevant role.b-Galactoside a2,6-sialyltransferase (ST6Gal.I) is theonly enzyme able to catalyze the a2,6-sialylation oflactosamine (Galb1,4GlcNAc) [3], a structure commonlyfound in the antennae of N-linked chains but present also inO-linked chains and in glycolipids (reviewed in [4]). One ofthe most characteristic glycosylation changes in coloncancer is represented by an elevation of ST6Gal.I enzymeactivityincancertissues,incomparisonwiththesurrounding normal mucosa [5]. Clinical studies indicatethat ST6Gal.I enzyme activity is further increased inmetastatic tissues [6] and that a high level of ST6Gal.I inprimary cancer is associated with a poor prognosis [7]. Also,the level of a2,6-sialylation of membrane glycoconjugates,as determined by the binding of NeuAca2,6Gal-specificlectins from Sambucus nigra (SNA) and Tricosanthesjaponica, is increased in colon cancer [8–10] and a strongreactivity is associated with a worse prognosis [11]. Thus, itis important to clarify the relationship between these twoparameters for potential clinical use. In recent studies, wehave observed that the level of ST6Gal.I enzyme activity ina given colon cancer sample only partially correlates withthe level of SNA reactivity, suggesting the existence of othermechanisms of regulation [12,13]. Moreover, while theincrease of ST6Gal.I enzyme activity in colon cancer is ageneral phenomenon (the exceptions constituting ,10% ofall cases), some studies reported an increase of ST6Gal.ImRNA only in a subset of cases [1,14]. Together, these datasuggest that multiple factors control the expression of theoligosaccharide product. Many of these factors might be cellspecific, for example, the stability of the mRNA, theefficiency of the translational machinery, the stability andthe post-translational processing of enzyme proteins, theexpression of other glycosyltransferases involved in thebiosynthesis of backbone oligosaccharide structures.We have undertaken this study to investigate quantitat-ively the sequence of biochemical events, from mRNAexpression to the final oligosaccharide product, in a modelsystem designed to minimize the contribution of these