INCREASED ALPHA-2,6 SIALYLATION OF N-GLYCANS IN A TRANSGENIC MOUSE MODEL OF HEPATOCELLULAR-CARCINOMA

Liver cancer is one of the most frequent and lethal malignancies world wide, Early detection is hampered by the absence of reliable markers, Mice transgenic for the SV40 large T antigen under the control of a li ver-specific promoter spontaneously develop well-differentiated hepato cellular carcinomas between 8 to 10 weeks of age, They are excellent m odels to investigate the alterations of protein expression in the earl y stages of tumor development and to follow these changes during tumor progression, In the present study, we analyzed the glycosylation chan ges occurring during tumor development in transgenic mice expressing t he SV40 T antigen under the control of the antithrombin III promoter, The analysis of serum and liver glycoproteins by an ELISA type assay, using the lectin from Sambucus nigra (SNA) as a probe, revealed the pr esence of increased levels of Neu5Ac alpha 2,6Gal beta 1,4GlcNAc on N- glycans in the tumor-bearing transgenic mice as compared to controls, On serum glycoproteins the increase in alpha 2,6 sialylation followed tumor progression, reaching up to 10 times control levels, However, si gnificantly higher SNA binding (2-fold) could already be observed on s erum glycoproteins from mice exhibiting only microscopically small neo plastic foci, On liver membrane glycoproteins, the increase in alpha 2 ,6 sialylation was less pronounced, reaching two to three times contro l values in 6-month-old mice, Western blotting of serum and liver prot eins with radiolabeled SNA showed that all glycoproteins that bind the lectin in controls exhibit larger amounts of Neu5Ac alpha 2,6Gal beta 1,4GlcNAc on N-glycans in the tumor-bearing mice. This general increa se in alpha 2,6 sialylation on all glycoproteins is due to the increas ed activity of the galactoside:alpha 2,6 sialyltransferase (ST6Gal I), which specifically transfers Neu5Ac residues in alpha 2,6 linkage to Gal beta 1,4GlcNAc units on N-glycans, As for the structures synthesiz ed by the enzyme, the increase of ST6Gal I activity in the serum as we ll as in liver microsomes of the transgenic mice followed tumor progre ssion, Interestingly, the activity of the galactoside:alpha 2,3 sialyl transferase (ST3Gal III), which uses the same acceptor substrate (Gal beta 1,4GlcNAc), was unchanged in the earlier stages of tumor developm ent hut decreased in the serum and in liver microsomes from later stag es. Using a rat ST6Gal I cDNA as a probe, Northern blots of total RNA extracted from the livers of control and transgenic mice revealed an i ncreased (4-fold) expression of the ST6Gal I gene. The single transcri pts detected in both normal and cancerous liver showed identical size.