ABSENCE OF N-GLYCOSYLATION AT ASPARAGINE-43 IN HUMAN LIPOPROTEIN-LIPASE INDUCES ITS ACCUMULATION IN THE ROUGH ENDOPLASMIC-RETICULUM AND ALTERS THIS CELLULAR COMPARTMENT

Lipoprotein lipase (LPL) is the enzyme responsible for the hydrolysis of plasma triglycerides from apolipoprotein C-II-containing lipoprotei ns at the capillary endothelium and it is synthesized in parenchymal c ells of several tissues. Intracellular LPL processing is a major aspec t of LPL regulation. The present study aims to determine the intracell ular accumulation site of the LPL that is not glycosylated at Asn43. H uman LPL (hLPL) cDNA was mutated by site-directed mutagenesis. An Ala residue was substituted for Asn at position 43 of the protein generati ng N43A hLPL. Wild type hLPL and the mutant hLPL were expressed in COS 1 cells. Using immunofluorescence and immunoelectron microscopy we fou nd that wild type hLPL in addition to being secreted into the medium w as present in the rough endoplasmic reticulum (ER), Golgi compartments , and vesicles. Neither LPL activity nor protein was found in medium o f cells expressing the mutant hLPL and all detectable protein was pres ent exclusively in the ER identified with a specific antibody against the protein disulfide isomerase (PDI), an ER marker. In addition, the intracellular distribution of the ER of the cells that expressed the m utant protein was grossly altered. Treatment of COS1 cells with tunica mycin for 24 h had the same effect on wild type hLPL processing and en doplasmic reticulum distribution. Next, we investigated the influence of the accumulation of mutant hLPL on the intracellular transport of t hree other proteins that are N-glycosylated before reaching the plasma membrane: the related B-o,B-+ amino acid transporter (rBAT), the insu lin-regulated glucose transporter (GLUT4), and the placental alkaline phosphatase (FLAP) protein. Coexpression of the mutant hLPL (but not w ild type) caused the accumulation of rBAT and GLUT4 in the ER while FL AP reached the plasma membrane. Our findings demonstrate that glycosyl ation of Asn43 of human lipoprotein lipase in the endoplasmic reticulu m is essential for its efflux from this com compartment and that the r etention of the non-glycosylated LPL induces morphological changes in the ER that could also affect its ability to modify the transport of o ther proteins.