Studies on the role(s) of post-translational modification(s) of human apolipoprotein B during very low density lipoprotein assembly and secretion.

The role of two post-translational modifications, N-linked glycosylation and palmitoylation, of human apolipoprotein B (apoB) in the biosynthesis of hepatic apoB-containing very low density lipoproteins (VLDL) was investigated. Working with tunicamycin-treated rat hepatoma McA-RH7777 cells stably expressing human apoB variants, we found that inhibition of N-linked glycosylation decreased the secretion of apoB variants, without affecting translation. Detailed biochemical analysis was performed following site-specific mutagenesis at consensus N-linked glycosylation sites (using asparagine-to-glutamine substitution) within recombinant human apoB variants (i.e. apoB17, -B37, -B48, and -B50). Four notable features associated with the requirement of N-linked oligosaccharides during apoB biosynthesis were observed: (a) N-linked oligosaccharides were required for efficient secretion of the apoB polypeptide. (b) N-linked oligosaccharides were required for the assembly and secretion of the apoB-containing VLDL. (c) Removal of N-linked oligosaccharides was associated with accumulation of total intracellular apoB without specific apoB retention in the ER, and (d) Expression of mutant apoB lacking N-glycans was associated with changes in mass or activity of microsomal triglyceride transfer protein (MTP). Similar biochemical analysis was performed following site-specific mutagenesis at potential palmitoylation sites (using cysteine-to-serine substitution) within human apoB48. The cysteine-to-serine substitution had no effect on the secretion of apoB48-containing lipoproteins or apoB intracellular distribution. Thus, while N-linked glycosylation at the amino terminus of apoB represents an important requisite for proper biogenesis of apoB-containing VLDL, palmitoylation is not required for apoB-containing VLDL assembly and secretion