Biological roles of the bisecting GlcNAc of N-glycans in hepatocarcinogenesis and liver regeneration

Abstract

In this thesis, the role of the bisecting GlcNAc of complex N-glycans was investigated during hepatocarcinogenesis and liver regeneration after partial hepatectomy (PH). The bisecting GlcNAc is transferred to N-glycans by N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. Previously, this laboratory showed that mice homozygous for a targeted Mgat3 gene mutation (Mgat3T37/ T37) that produces truncated, inactive GlcNAc-TIII display markedly retarded progression of liver tumors induced by diethylnitrosamine (DEN) and phenobarbital (PB) treatment. In order to determine whether this phenotype was due to the loss of GlcNAc-TIII activity, I repeated this experiment in mice with a targeted mutation (Mgat3Delta /Delta) that deleted the Mgat3 gene coding exon. Mgat3Delta /Delta mice also exhibited retarded tumor progression, but to a lesser degree than Mgat3T37 /T37 mice. This difference was more significant when PB was omitted and the DEN concentration was increased five-fold. Mgat3Delta/Delta mice also exhibited impaired liver regeneration after 70% PH, based on BrdU incorporation and regenerated liver mass. In contrast to the situation in rats, expression of the Mgat3 gene was not induced in tumor tissues of wild type mice, nor in wild type mouse liver following PH.;A transgenic mouse line overexpressing G1cNAc-TIII in liver driven by the major urinary protein (MUP) promoter, was generated and characterized. Despite high expression of GlcNAc-TIII, there was no significant difference in hepatocarcinogenesis, nor in liver regeneration between wild type and transgenic mice. It is proposed that the impaired liver regeneration and retarded tumor progression in mice lacking GlcNAc-TIII are due to the absence of the bisecting GlcNAc residues on N-glycans of circulating glycoprotein(s) from a tissue other than liver. Determination of apoptotic cells using the TUNEL assay did not show any differences between wild type and Mgat3 Delta/Delta mice during hepatocarcinogenesis, and thus the glycoprotein factor(s) is proposed to be growth promoting rather than apoptosis-inducing. In order to produce candidate glycoproteins with and without a bisecting GlcNAc, LEC10 CHO mutants expressing GlcNAc-TIII were characterized for use in glycosylation engineering.