THE MUTATION GLY(142)-]GLU IN HUMAN LIPOPROTEIN-LIPASE PRODUCES A MISSORTED PROTEIN THAT IS DIVERTED TO LYSOSOMES

Citation
R. Busca et al., THE MUTATION GLY(142)-]GLU IN HUMAN LIPOPROTEIN-LIPASE PRODUCES A MISSORTED PROTEIN THAT IS DIVERTED TO LYSOSOMES, The Journal of biological chemistry, 271(4), 1996, pp. 2139-2146
Citations number
40
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
271
Issue
4
Year of publication
1996
Pages
2139 - 2146
Database
ISI
SICI code
0021-9258(1996)271:4<2139:TMGIHL>2.0.ZU;2-L
Abstract
While the molecular characterization of lipoprotein lipase (LPL) activ ation is progressing, the intracellular processing, transport, and sec retion 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 eluci date the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutag enesis 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 th e mutant G142E hLPL were expressed by transient transfection in COS1 c ells, Using Western blot assays we identified a single band that had t he 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 mutan t LPL was only 25% of the WT protein, indicating defective secretion o f the altered enzyme. Heparin increased LPL secretion in the case of t he WT hLPL but did not have any stimulatory effect when acting on G142 E hLPL-transfected cells. However, heparin-Sepharose chromatography re vealed that both proteins presented the same heparin affinity, Metabol ic 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 leve l of the mutant enzyme, thus indicating its higher intracellular degra dation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeli ng in G142E hLPL-expressing cells. This result was confirmed using imm unoelectron microscopy, which in addition showed gold labeling in Golg i stacks. This finding, together with experiments performed with endog lycosidase H digestion of immunoprecipitated radiolabeled LPL, indicat ed that the mutant enzyme entered the Golgi compartment. The results r eported in this paper show that the G142E hLPL is not efficiently secr eted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal misso rting might be a mechanism of cell quality control of secreted LPL.