Efficient glycosylation site utilization by intracellular apolipoprotein B: implications for proteasomal degradation

The balance between the hepatic assembly of apolipoprotein B (apoB) and its presecretory degradation at the level of the endoplasmic reticulum (ER) ma y control the secretion of apoB-containing lipoproteins. in one model, apoB that fails to assemble with lipid undergoes translocation arrest, exposing the protein to the cytosolic proteasome. To examine apoB's translocation b ehavior under various metabolic conditions, glycosylation site utilization studies were performed. A 70-amino acid peptide containing three sites for N-linked glycosylation was appended to the C-terminus of apoB-50 (amino-ter minal 50% of apoB) and expressed in both hepatic and nonhepatic cell lines. When the C-terminal reporter peptide was released by cyanogen bromide clea vage, all of the sites were glycosylated irrespective of cell type, labelin g time, or assembly status. Similar peptide mapping of endogenous apoB-100 expressed in HepG2 cells was performed to monitor glycosylation at Asn resi dues 2752 (apoB-61), 2955 (apoB-65), and 3074 (apoB-68). N-Linked glycosyla tion occurred at a minimum of two of the three sites, a frequency identical to that observed in apoB-100 recovered from cell media. Treatment of cells with proteasome inhibitors produced a 2.5-fold increase in intracellular a poB but failed to cause accumulation of an unglycosylated form. These resul ts indicate that 1) the efficient translocation of apoB into the ER occurs independently of microsomal triglyceride transfer protein and its assembly with lipid and 2) despite its large size and affinity for lipid, delivery o f misassembled apoB to the proteasome requires retrograde translocation fro m the ER lumen to cytosol.