Tandem affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in Chinese hamster ovary cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, five function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase were also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57, and glucosidase II, followed by repeated posttranslational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding and may help direct its eventual degradation.
"The present study uncovered an intracellular role of hHL in the production and/or secretion of liver derived apoA-I/HDL. The negative impact of HL expression on the production of these lipoprotein particles may be related to its unusual lengthy maturation time (hours rather than minutes) within the ER/Golgi compartments. Prior to dimerization and gaining catalytic activity, the nascent HL might play an intracellular function in attenuating lumenal lipid droplet formation and apoA-I production. "
[Show abstract][Hide abstract] ABSTRACT: This study was aimed to examine whether expression of human hepatic lipase (hHL) exerted an intracellular effect on hepatic production of apolipoprotein (apo) A-I. The levels of secreted and cell-associated apoA-I were contrasted between primary hepatocytes isolated from Lipc-null and C57BL/6 mice, and between Lipc-null hepa-tocytes transfected with hHL-encoding or control adenovirus. An HSPG-binding deficient hHL protein (hHLmt) was used to determine the impact of cell surface binding on HL action. Accumulation of apoA-I in conditioned media of primary hepatocytes isolated from Lipc-null mice was increased as compared to that from C57BL/6 mice. Metabolic labeling experiments showed that secretion of 35 S-apoA-I from Lipc-null cells was significantly higher than that from C57BL/6 cells. Expression of hHL in Lipc-null hepatocytes, through adenovirus-mediated gene transfer, resulted in decreased synthesis and secretion of 35 S-apoA-I, but not 35 S-apoE, as compared with cells trans-fected with control adenovirus. Expression of HSPG-binding deficient hHLmt in Lipc-null cells also exerted an inhibitory effect on apoA-I production, even though hHLmt displayed impaired exit from the endoplasmic reticu-lum as compared with hHL. Subcellular fractionation revealed that expression of hHL or hHLmt led to increased microsome-association of apoA-I relative to non-transfected control. Expression of hHL negatively impacts hepatic production of apoA-I.
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