Increased CMP‐NeuAc:Galβ1,4GlcNAc‐R α2,6 sialyltransferase activity in human colorectal cancer tissues

Abstract

The sialyltransferase activities of 10 human colorectal specimens were compared with those of the corresponding adjacent normal mucosa. Using asialofetuin as an acceptor we found, in tumor tissues of 9 out of 10 patients, an increased sialyltransferase activity towards the N-linked chains as determined upon peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase) treatment. On the contrary, the activity towards the O-linked chains was not significantly changed. When the specificity of the sialyltransferase acting on N-linked chains was investigated by using N-acetyl-lactosamine (Gal beta 1,4GlcNAc) as an acceptor, we found that the alpha 2,6 sialyltransferase activity expressed by both normal and tumor colorectal tissues was far higher than the alpha 2,3-activity and that alpha 2,6 was the only sialyltransferase activity increased in tumor tissues. Kinetic analysis revealed that normal and tumor alpha 2,6 sialyltransferases have the same apparent Km for the acceptor substrate (469 and 465 microns), but normal enzyme has a higher Km for CMP-NeuAc (303 microns) than the tumor enzyme (50 microns). The higher affinity of tumor enzyme for the nucleotide-sugar might partially explain its increased activity in tumor tissues. In addition, tumor tissues contain a lower amount of sialic acid despite the increase in alpha 2,6 sialyltransferase activity.

Alterations of CMP-NeuAc:Asialofetuin Sialyltransferase Activities in Human Colorectal Adenocarcinoma

“…This could be simply explained because the apparent K m for CMP-NeuAc in tumour tissue was 2.5-fold smaller than in control tissue (table 1). This finding is in keeping with that obtained by Dall’Olio et al [19], who described a 6-fold decrease in the K m of α(2,6)-sialyltransferase activity in colon adenocarcinoma with respect to healthy tissue. However, it is very important to distinguish the acceptor sequence employed to measure a concrete glycosyltransferase activity because the results could vary considerably, depending on the actual structure in question.…”

“…2), we found that the incorporation of NeuAc into N-linked chains was higher in tumour than in healthy tissue, whereas onto O-linked chains activity remained unaltered (table 3). Accordingly, it must be the incorporation into N-linked chains what determines the increase in sialyltransferase activity detected in tumour tissue, in agreement with previous reports [19]. After the examination of the type of isomers formed onto ASF, our results demonstrate (as is shown in table 4) that most of the NeuAc is incorporated into α(2,6)-linkages as well in healthy as in tumour tissue.…”

“…However, it is very important to distinguish the acceptor sequence employed to measure a concrete glycosyltransferase activity because the results could vary considerably, depending on the actual structure in question. It should therefore be considered that those authors reported that decrease employing the oligosaccharide structure Galβ(1,4)GlcNAc- as an acceptor sequence [19]. In our case, this structure belongs to the final part of the complex N-chains of ASF and it has been proposed that plays a role in colon cancer progression [1, 26].…”

“…Previous reports have demonstrated that CMP-NeuAc:asialofetuin sialyltransferase is affected in human colon tumour processes [7, 9, 19], but currently the type and the origin of these alterations remain obscure. In the present work, in order to evaluate CMP-NeuAc:asialofetuin sialyltransferase activity we first established the optimal assay conditions.…”

“…The reaction was stopped by the addition of 1 ml of 1% (w/v) FTA in 0.5 M HCl. N-linked chains were obtained enzimatically as described by Dall’Olio et al [19]. Briefly, samples were centrifuged at 2,000 g for 10 min and the resulting pellet was washed once with 1 ml of 1% (w/v) FTA in 0.5 M HCl, and three times with methanol (1 ml).…”