Suppression of β1,3galactosyltransferase β3Gal‐T5 in cancer cells reduces sialyl‐Lewis a and enhances poly N‐acetyllactosamines and sialyl‐Lewis x on O‐glycans

Abstract

We investigated the role of b3Gal-T5, a member of the b1,3galactosyltransferase (b1,3Gal-T) family, in cancerassociated glycosylation, focusing on the expression of sialyl-Lewis a (sLe a , the epitope of CA19.9 antigen), poly N-acetyllactosamines, and sialyl-Lewis x (sLe x ) antigen. A clone permanently expressing an antisense fragment of b3Gal-T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both b1,3Gal-T activity and sLe a expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O-glycans. Endo-b-galactosidase mostly releases NeuAca2-3Galb1-3[Fuca1-4]GlcNAcb1-3Gal and NeuAca2-3Galb1-3GlcNAcb1-3Gal from such O-glycans of BxPC3 membranes, but GlcNAcb1-3Gal and type 2 chain oligosaccharides, including NeuAca2-3Galb1-4[Fuca1-3]GlcNAcb1-3Gal, from those of the antisense clone.Furthermore, BxPC3 cells secrete sLe a in the culture media but not sLe x , while antisense clone secretes mostly sLe x , and accumulation of both antigens is prevented by benzyla-GalNAc. These data indicate that b3Gal-T5 suppression turns synthesis of type 1 chain O-glycans to poly N-acetyllactosamine elongation and termination by sLe x . In other cell lines and clones, b3Gal-T5 transcript, b1,3Gal-T activity, and sLe a antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that b3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors.

Helicobacter pylori chronic infection and mucosal inflammation switches the human gastric glycosylation pathways

“…2A, B). Enhanced expression of β1,3-galactosyltransferase 5 (β3GalT5) has been reported to result in increased biosynthesis of extended type 1 chains on lactosylceramides and glycoproteins in colon carcinoma cells [42,43], while suppression of β3GalT5 in pancreas adenocarcinoma cells reduced the expression of sialyl-Le a [44]. In line with these observations, the induction of β3GalT5, in response to H. pylori infection, can drive the extension of precursor chains towards the biosynthesis of type 1 sequences, therefore contributing for the increased expression of sialyl-Le a (Fig.…”

Selectin Ligands Sialyl-Lewis a and Sialyl-Lewis x in Gastrointestinal Cancers

“…In vivo and in cell models its expression is closely associated with that of Lewis type 1 antigens, including sLe a [36,37]. B3GALT5 acts on N - and O -linked chains of glycoproteins, on glycolipids, and on soluble type 1 chain oligosaccharides [37,38]. In glycolipids, it is specifically responsible for globo-series elongation [39].…”

Comparative Analysis of Retroviral and Native Promoters Driving Expression of β1,3-Galactosyltransferase β3Gal-T5 in Human and Mouse Tissues

“…Human bile duct carcinoma cells HuCC-T1 (Japanese Collection of Research Bioresources 0425) were cultured in RPMI 1640 medium containing the same supplements as above. Human colon adenocarcinoma cells Caco-2 and SW-1116 and human gastric carcinoma cells MKN-45 were cultured as previously described (3,16). Human ileum and colon samples were collected at surgery and kindly donated by Dr. Filippo Mare (Hospital di Circolo, Varese, Italy).…”

“…Mouse total RNA was purchased from Clontech. Total and poly(A) ϩ RNAs were prepared from human tissues or cell lines as previously reported (3,16).…”

“…RT-PCR-Competitive RT-PCR was performed as previously described (3,16). The human and mouse ␤-actin and human ␤3Gal-T5 coding sequence competitors and oligonucleotide primers used were those previously described (3).…”