INHIBITION OF N-LINKED GLYCOSYLATION RESULTS IN RETENTION OF INTRACELLULAR APO[A] IN HEPATOMA-CELLS, ALTHOUGH NONGLYCOSYLATED AND IMMATURE FORMS OF APOLIPOPROTEIN[A] ARE COMPETENT TO ASSOCIATE WITH APOLIPOPROTEIN B-100 IN-VITRO

Apolipoprotein[a] (apo[a]) is a highly polymorphic glycoprotein that f orms a covalent complex with apolipoprotein B-100 (apoB-100), producin g a lipoprotein species referred to as lipoprotein[a] (Lp[a]). We have studied the effects of alterations in glycosylation of apo[a] on its intracellular processing and secretion as well as its ability to assoc iate with low density lipoprotein (LDL) apoB-100, HepG2 cells transfec ted with a 6 kringle IV (16 K-IV) apo[a] minigene were treated with tu nicamycin, an inhibitor of N-linked glycosylation, which eliminated ap o[a]-B-100 complexes from the media. Tunicamycin treatment also reduce d secretion of the 6 K-IV apo[a] protein from transfected McA-RH7777 c ells by similar to 50%, but completely eliminated secretion of apo[a] species containing 9 and 17 K-IV repeats, Mixing experiments, performe d with radiolabeled media (+/-tunicamycin) from transfected McA-RH7777 cells, demonstrated no alteration in the extent of association of apo [a] with human LDL, Similar mixing experiments using culture media fro m glycosylation-defective mutant chinese hamster ovary (CHO) cells tra nsfected with the same apo[a] minigene showed identical results, Apo[a ] secretion was demonstrated in all mutant cell lines in the absence o f either N- or O-linked (or both) glycosylation, The mechanisms underl ying the reduced secretion of apo[a] from transfected hepatoma cells w ere examined by pulse-chase radiolabeling and apo[a] immunoprecipitati on. Tunicamycin treatment altered the efficiency of precursor apo[a] p rocessing from the ER by increasing its ER retention time, The increas ed accumulation of precursor apo[a] in the ER was associated with alte rations in the kinetics of association with two resident endoplasmic r eticulum (ER) chaperone proteins, calnexin and BiP. These findings sug gest that the glycosylation state and size of apo[a] appear to play a role in regulating its efficient exit from the endoplasmic reticulum, However, neither N- nor O-linked glycosylation of apo[a] exerts a majo r regulatory role in its covalent association with apoB-100.