INTRACELLULAR EARLY AND LATE MODIFICATIONS OF HUMAN APOLIPOPROTEIN-A-II - EFFECT OF GLUTAMINE-+1 TO LEUCINE SUBSTITUTION

It has been shown previously that apoA-II undergoes several intracellu lar modifications in HepG2 cells (Hussain & Zannis, 1990). In the pres ent study, we have generated permanent cell lines in mouse C127 cells which express the normal apoA-II gene and a mutated form in which Gln1 was substituted with Leu (Leu+1). This modification was designed to prevent cyclization of the N-terminal glutamine of apoA-II and thus id entify the isoproteins which are precursors and products of the N-term inal cyclization reaction. The C127-expression cells were also utilize d to study the cellular compartments where the apoA-II modifications o ccur as well as the importance of the modifications for apoA-II traffi cking and secretion. We have found that apoA-II (Gln+1) synthesized by C127 and HepG2 cells had similar isoproteins. In both cell types, unm odified pro-apoA-II, designated isoprotein 3, had a similar isoelectri c point as the cell-free translation product of apoA-II mRNA, suggesti ng that isoprotein 3 results from cleavage of the signal peptide. Isop rotein 3 represents an unmodified apoA-II isoprotein and undergoes an early modification into a more acidic isoprotein 1, which differs from isoprotein 3 by two negative charges. Brefeldin A treatment of the ce lls did not prevent the formation of isoprotein 1, suggesting that thi s modification occurs in a pre-Golgi compartment. Neuraminidase treatm ent of secreted apoA-II isoproteins did not affect isoprotein 1, indic ating that it is not sialylated isoprotein. Isoprotein 1 undergoes fur ther modifications which are consistent with cleavage of the propeptid e, N-terminal cyclization and sialylation most likely resulting from O -glycosylation. The Gln+1 to Leu substitution prevented N-terminal cyc lization as evidenced by the lack of conversion of isoprotein 1a to 0 in the intracellular and secreted mutant (Leu+1) forms of apoA-II. Fur thermore, the Gln+1 to Leu substitution also impaired cleavage of the propeptide. ApoA-II undergoes extensive intracellular degradation in C 127 cells and to a lesser extent in HepG2 cells. The kinetics of deple tion of intracellular apoA-II were similar in HepG2 cells as well as i n C127 cells expressing the wild-type and the mutant apoA-II isoprotei ns.