The Mutation Gly142→ Glu in Human Lipoprotein Lipase Produces a Missorted Protein That Is Diverted to Lysosomes

Abstract

While the molecular characterization of lipoprotein lipase (LPL) activation is progressing, the intracellular processing, transport, and secretion signals of LPL are still poorly known. The aim of this paper is to study the involvement of glycine 142 in LPL secretion and to elucidate the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutagenesis in order to produce the G142E hLPL in which the glycine 142 was replaced by a glutamic acid. The wild type human LPL (WT hLPL) and the mutant G142E hLPL were expressed by transient transfection in COS1 cells. Using Western blot assays we identified a single band that had the same molecular weight for both proteins. However, Western blots of culture media did not reveal any specific band for the mutant protein, and ELISA experiments showed that the extracellular mass of the mutant LPL was only 25% of the WT protein, indicating defective secretion of the altered enzyme. Heparin increased LPL secretion in the case of the WT hLPL but did not have any stimulatory effect when acting on G142E hLPL-transfected cells. However, heparin-Sepharose chromatography revealed that both proteins presented the same heparin affinity. Metabolic labeling and radioimmunoprecipitation studies showed that both the WT and the mutant hLPL intracellular levels decreased upon chase time. Furthermore, leupeptin had a greater effect on the intracellular level of the mutant enzyme, thus indicating its higher intracellular degradation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeling in G142E hLPL-expressing cells. This result was confirmed using immunoelectron microscopy, which in addition showed gold labeling in Golgi stacks. This finding, together with experiments performed with endoglycosidase H digestion of immunoprecipitated radiolabeled LPL, indicated that the mutant enzyme entered the Golgi compartment. The results reported in this paper show that the G142E hLPL is not efficiently secreted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal missorting might be a mechanism of cell quality control of secreted LPL.

Maturation of Lipoprotein Lipase in the Endoplasmic Reticulum

“…Nevertheless, the lysosomal pathway was considered, since a mutant form of human LPL lacking activity (G142E) was reported to be degraded in this manner (58). However, in contrast to this mutant form of LPL, lysosomal degradation of the aggregate was ruled out (Fig.…”

Instability of the Amyloidogenic Cystatin C Variant of Hereditary Cerebral Hemorrhage with Amyloidosis, Icelandic Type

“…No internalized cystatin C was observed. Since the majority of the internalized proteins are degraded in lysosomes, the experi- ment was performed in the presence of leupeptin, a lysosomal serine and cysteine protease inhibitor (30). No internalized cystatin C was observed under these conditions (data not shown).…”

The role of O‐linked sugars in determining the very low density lipoprotein receptor stability or release from the cell

“…COS1 cells, used for transfection experiments, were cultured in Dulbecco's modified Eagle's medium (DMEM), with 4.5 g/l glucose and 10% fetal bovine serum. Transient transfection experiments were performed as described [34].…”