Hepatic lipase maturation: a partial proteome of interacting factors

Abstract

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.-Doolittle, M. H., O. Ben-Zeev, S. Bassilian, J. P. Whitelegge, M. Péterfy, and H. Wong. Hepatic lipase maturation: a partial proteome of interacting factors.

Lipase maturation factor 1 is required for endothelial lipase activity

“…a tobacco etch virus (TEV) protease site and ending in two adjacent IgG-binding domains derived from protein A ( 29 ). Mouse Lmf1 cDNA was subcloned into the pcDNA3.1 expression vector (Invitrogen) containing an N-terminal c-Myc epitope tag ( 25 ).…”

“…For experiments using lipase affi nity purifi cation, a tandem affi nity purifi cation (TAP) tag was synthesized for in-frame integration into an Age I site occurring just after the V5 epitope tag of pcDNA6 ( 29 ). After transfection, the resulting expressed LPL, HL, PL and EL proteins contained a C-terminal V5-TAP tag consisting of the V5 epitope followed by a single calmodulin-binding peptide domain, indicating that Lmf1 transfection restored the activity of these lipases to heterozygous levels.…”

Secretion of triacylglycerol-poor VLDL particles from McA-RH7777 cells expressing human hepatic lipase

“…Intracellular maturation of HL involves conversion of catalytically inactive mass into functional, dimeric conformation prior to secretion ( 20 ), a slow process that occurs in the ER and takes hours to accomplish ( 41 ). The prolonged residence time of HL within the ER/Golgi secretory pathway may exert a negative effect on mobilization of TAG into microsomal lumen for VLDL assembly.…”

“…However, the present mutagenesis study indicates that the catalytic activity of HL is not absolutely required for this effect. A group of HL-interacting proteins have been identifi ed that may play a role in intracellular HL maturation process, none of which, however, appear to be involved in lipid binding or partitioning ( 41 ). Lipid binding regions are present within HL molecules ( 42 ).…”

Lipase Maturation Factor 1 (Lmf1) Is Induced by Endoplasmic Reticulum Stress Through Activating Transcription Factor 6α (Atf6α) Signaling

“…The conversion of nascent lipase polypeptide chain into catalytically active enzyme requires post-translational maturation that involves glycosylation, glycan processing, and protein folding (5). The maturation of lipases is facilitated by both major chaperone systems operating within the ER, the calnexin/calreticulin and 78-kDa glucose-regulated protein (Grp78)/Grp94 systems (6,7). In addition to general chaperones, lipase maturation critically depends on a client-specific chaperone residing in the ER membrane, lipase maturation factor 1 (Lmf1) (8).…”