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
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.