Maturation of Hepatic Lipase
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m310051200
ISSN1083-351X
AutoresOsnat Ben-Zeev, Mark H. Doolittle,
Tópico(s)Diet, Metabolism, and Disease
ResumoAmong three lipases in the lipase gene family, hepatic lipase (HL), lipoprotein lipase, and pancreatic lipase, HL exhibits the lowest intracellular specific activity (i.e. minimal amounts of catalytic activity accompanied by massive amounts of inactive lipase mass in the endoplasmic reticulum (ER)). In addition, HL has a distinctive sedimentation profile, where the inactive mass overlaps the region containing active dimeric HL and trails into progressively larger molecular forms. Eventually, at least half of the HL inactive mass in the ER reaches an active, dimeric conformation (t½ = 2 h) and is rapidly secreted. The remaining inactive mass is degraded. HL maturation occurs in the ER and is strongly dependent on binding to calnexin in the early co-/post-translational stages. Later stages of HL maturation occur without calnexin assistance, although inactive HL at all stages appears to be associated in distinct complexes with other ER proteins. Thus, unlike other lipases in the gene family, HL maturation is the rate-limiting step in its secretion as a functional enzyme. Among three lipases in the lipase gene family, hepatic lipase (HL), lipoprotein lipase, and pancreatic lipase, HL exhibits the lowest intracellular specific activity (i.e. minimal amounts of catalytic activity accompanied by massive amounts of inactive lipase mass in the endoplasmic reticulum (ER)). In addition, HL has a distinctive sedimentation profile, where the inactive mass overlaps the region containing active dimeric HL and trails into progressively larger molecular forms. Eventually, at least half of the HL inactive mass in the ER reaches an active, dimeric conformation (t½ = 2 h) and is rapidly secreted. The remaining inactive mass is degraded. HL maturation occurs in the ER and is strongly dependent on binding to calnexin in the early co-/post-translational stages. Later stages of HL maturation occur without calnexin assistance, although inactive HL at all stages appears to be associated in distinct complexes with other ER proteins. Thus, unlike other lipases in the gene family, HL maturation is the rate-limiting step in its secretion as a functional enzyme. Withdrawal: Maturation of hepatic lipase: Formation of functional enzyme in the endoplasmic reticulum is the rate-limiting step in its secretion.Journal of Biological ChemistryVol. 294Issue 33PreviewVOLUME 279 (2004) PAGES 6171–6181 Full-Text PDF Open Access Hepatic lipase (HL) 1The abbreviations used are: HLhepatic lipaseCnxcalnexinCstcastanospermineCxcycloheximideDSPdithiobis[succinimidylpropionate]ELendothelial lipaseendo Hendoglucosidase HERendoplasmic reticulumFFAfree fatty acidsHDLhigh density lipoproteinLPLlipoprotein lipaseNEMN-ethylmaleimidePBSphosphate-buffered salinePLpancreatic lipasePS-PLA1phosphatidylserine phospholipase A1CHOChinese hamster ovary. is a member of the mammalian lipase gene family that includes lipoprotein lipase (LPL), pancreatic lipase (PL), endothelial lipase (EL), phosphatidylserine phospholipase A1 (PS-PLA1), and the most recently discovered lipase H (1Hide W.A. Chan L. Li W.-H. J. Lipid Res. 1992; 33: 167-178Abstract Full Text PDF PubMed Google Scholar, 2Rader D.J. Jaye M. Curr. Opin. Cell Biol. 2000; 11: 141-147Google Scholar, 3Sato T. Aoki J. Nagai Y. Dohmae N. Takio K. Doi T. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 2192-2198Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 4van Groningen J.J. Egmond M.R. Bloemers H.P. Swart G.W. FEBS Lett. 1997; 404: 82-86Crossref PubMed Scopus (23) Google Scholar, 5Jin W. Broedl U.C. Monajemi H. Glick J.M. Rader D.J. Genomics. 2002; 80: 268-273Crossref PubMed Scopus (36) Google Scholar). These enzymes are localized in specific organs, suggesting that they have evolved to play distinct roles in lipid metabolism. Thus, HL is synthesized in the hepatocytes and is secreted to the space of Disse, where it is anchored to the cell surface by heparan sulfate proteoglycans. At this site, HL mediates the conversion of high density lipoprotein (HDL2) to HDL3 particles and transforms intermediate density lipoproteins to low density lipoproteins. HL also participates in the conversion of very low density lipoprotein remnants to low density lipoproteins and enhances the uptake of remnant lipoproteins (6Santamarina-Fojo S. Haudenschild C. Amar M. Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar, 7Shafi S. Brady S.E. Bensadoun A. Havel R.J. J. Lipid Res. 1994; 35: 709-720Abstract Full Text PDF PubMed Google Scholar). Besides the liver, HL is also found on the endothelium of the adrenal and the gonads (8Jansen H. De Greef W.J. Biochem. J. 1981; 196: 739-745Crossref PubMed Scopus (64) Google Scholar) and in macrophages (9Gonzalez-Navarro H. Nong Z. Freeman L. Bensadoun A. Peterson K. Santamarina-Fojo S. J. Lipid Res. 2002; 43: 671-675Abstract Full Text Full Text PDF PubMed Google Scholar), implying that HL may play a direct role in the pathogenesis of atherosclerosis. LPL, on the other hand, is found in heart, skeletal muscle, and adipose tissue, where it hydrolyzes the triglyceride core of chylomicrons and very low density lipoproteins (10Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar). PL hydrolyzes dietary lipids in the intestine (11Winkler F.K. d'Arcy A. Hunziker W. Nature. 1990; 343: 771-774Crossref PubMed Scopus (1037) Google Scholar). EL localizes primarily to the placenta, lung, and macrophages, and PS-PLA1 localizes to platelets, whereas lipase H is found in the intestine (3Sato T. Aoki J. Nagai Y. Dohmae N. Takio K. Doi T. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 2192-2198Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 5Jin W. Broedl U.C. Monajemi H. Glick J.M. Rader D.J. Genomics. 2002; 80: 268-273Crossref PubMed Scopus (36) Google Scholar, 12Hirata K. Dichek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T. J. Biol. Chem. 1999; 274: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The physiological role of the last three lipases is still under investigation. hepatic lipase calnexin castanospermine cycloheximide dithiobis[succinimidylpropionate] endothelial lipase endoglucosidase H endoplasmic reticulum free fatty acids high density lipoprotein lipoprotein lipase N-ethylmaleimide phosphate-buffered saline pancreatic lipase phosphatidylserine phospholipase A1 Chinese hamster ovary. Despite the disparate anatomical location and distinct physiological functions of the lipases in this gene family, sequence homologies and gene structure strongly suggest a common folding pattern. HL, LPL, and EL are particularly closely related, whereas PL, PS-PLA1, and lipase H appear to have branched out earlier from the primordial progenitor (1Hide W.A. Chan L. Li W.-H. J. Lipid Res. 1992; 33: 167-178Abstract Full Text PDF PubMed Google Scholar, 2Rader D.J. Jaye M. Curr. Opin. Cell Biol. 2000; 11: 141-147Google Scholar, 3Sato T. Aoki J. Nagai Y. Dohmae N. Takio K. Doi T. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 2192-2198Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 5Jin W. Broedl U.C. Monajemi H. Glick J.M. Rader D.J. Genomics. 2002; 80: 268-273Crossref PubMed Scopus (36) Google Scholar, 13Kirchgessner T.G. Chuat J.-C. Heinzmann C. Etienne J. Guilhot S. Svenson K. Ameis D. Pilon C. D'Auriol L. Andalibi A. Schotz M.C. Galibert F. Lusis A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9647-9651Crossref PubMed Scopus (185) Google Scholar). In particular, HL, LPL, and EL share homologous heparin-binding domains and also exhibit two conserved N-linked glycosylation sites, located in the N-terminal and in the C-terminal regions, respectively. At least for LPL and HL, it has been shown that the presence of the conserved N-terminal glycan was obligatory for the formation of active lipase (14Ben-Zeev O. Stahnke G. Liu G. Davis R.C. Doolittle M.H. J. Lipid Res. 1994; 35: 1511-1523Abstract Full Text PDF PubMed Google Scholar, 15Wölle J. Jansen H. Smith L.C. Chan L. J. Lipid Res. 1993; 34: 2169-2176Abstract Full Text PDF PubMed Google Scholar). Human HL is distinct from HL in other species, since it contains two additional N-linked glycan chains, whereas EL displays three other potential, but nonhomologous, glycosylation sites. The requirement for glycosylation at the conserved N-terminal site for optimal HL and LPL enzymatic activity implies that these lipases associate with the lectin chaperones calnexin and/or calreticulin. Nascent proteins bind to these chaperones via the innermost glucose residue of the oligosaccharide chain, following removal of the outer two glucose residues by ER glucosidases I and II (16Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (719) Google Scholar). Indeed, both HL and LPL activity were inhibited by drugs that prevent trimming of the outer glucose molecules (17Verhoeven A.J.M. Jansen H. J. Lipid Res. 1990; 31: 1883-1893Abstract Full Text PDF PubMed Google Scholar, 18Verhoeven A.J. Neve B.P. Jansen H. Biochem. J. 1999; 337: 133-140Crossref PubMed Google Scholar, 19Masuno H. Blanchette-Mackie E.J. Schultz C.K. Sparth A.E. Scow R.O. Okuda H. J. Lipid Res. 1992; 33: 1343-1349Abstract Full Text PDF PubMed Google Scholar, 20Ben-Zeev O. Doolittle M.H. Davis R.C. Elovson J. Schotz M.C. J. Biol. Chem. 1992; 267: 6219-6227Abstract Full Text PDF PubMed Google Scholar), and direct evidence of binding of HL to calnexin was also demonstrated (21Boedeker J.C. Doolittle M. Santamarina-Fojo S. White A.L. J. Lipid Res. 1999; 40: 1627-1635Abstract Full Text Full Text PDF PubMed Google Scholar). Besides association with calnexin, we have recently investigated additional aspects of LPL maturation (i.e. the process of acquisition of enzymatic activity by newly synthesized lipase) (22Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We have demonstrated the concurrent formation of two different LPL pools. One pool became fully active; its maturation occurred in the ER and was rapid, to the extent that no inactive precursor to the active form could be detected at steady state or in pulse-chase experiments. The second LPL pool consisted of inactive, misfolded lipase molecules that associated into large aggregates via interchain disulfide bonds. In accordance with strict quality control, these aggregates were degraded in the ER, whereas the active LPL molecules were readily secreted. The close homology between LPL and HL suggested that the latter might exhibit similar maturation properties, a hypothesis that was investigated in the present study. Our results demonstrate that, like LPL, part of nascent HL remains misfolded and is degraded in the ER, whereas the pool of HL that reaches a functional conformation is secreted. However, in stark contrast to the rapid maturation of LPL, HL achieves an active conformation after prolonged residence in the ER, during which it remains associated with calnexin and with an apparent array of other ER factors in complexes of varied size. Cell Lines and Media—The proline auxotroph derivatives (Pro5) of Chinese hamster ovary (CHO) cells, Lec1 cells, and HepG2 cells were obtained from the American Type Culture Collection. Cell cultures were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, antibiotics (50 units/ml penicillin and 50 μg/ml streptomycin), 0.1 mm nonessential amino acids, and 1.0 mm sodium pyruvate. Expression Constructs—The cDNA of human HL and PL was inserted into the pcDNA6/V5-His expression vector (Invitrogen) as previously described for the human LPL construct (22Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The termination codons (nucleotides 1,498-1,500 of HL and 1,396-1,398 of PL) were replaced with a BamHI site and cloned into the expression vector as a HindIII/BamHI fragment. This placed the 3′-end of human HL and PL in frame with the V5 epitope tag, which was subsequently used for antibody detection of the expressed lipase proteins. For HL, the in-frame cloning replaced the natural termination codon with glycine followed by a 50-amino acid C-terminal adduct: 501STSPVWWNSADIQHSGGRSSLEGPRFEGKPIPNPLLGLDSTRTGHHHHHH550 (the V5 epitope tag is underlined). For PL, the natural termination codon was replaced with leucine followed by the 50-amino acid C-terminal adduct: 467GDPLVQCGGILQISSTVAAARGHPFEGKPIPNPLLGLDSTRTGHHHHHH516. The constructs were driven by a cytomegalovirus promoter and contained a bovine growth hormone polyadenylation site. Transfection, Selection, and Harvesting of Cells—CHO cells were stably transfected with the pcDNA6/V5-His vector containing the cDNA of HL, LPL, and PL, using the calcium phosphate kit and instructions provided by Invitrogen. The selection medium contained 10 μg/ml blasticidin. Lipase-expressing colonies were identified by assaying the medium for HL activity (23Briquet-Laugier V. Ben-Zeev O. Doolittle M.H. Methods Mol. Biol. 1999; 109: 81-94PubMed Google Scholar), after incubating the cells for 16 h in the presence of 10 units/ml heparin. Cells were subcultured onto 100-mm plates and kept under blasticidin selection. Prior to experiments, cells were incubated for 6-12 h in medium without blasticidin, containing 10 units/ml heparin. HepG2 cells were transiently transfected with the pcDNA6/V5-HL expression vector using the FuGene 6 transfection agent (Roche Applied Science) with the protocol provided by the manufacturer. Six hours prior to harvesting, fresh medium containing 10 units/ml heparin was added. The cells were harvested 24 h after transfection. For harvesting, cell monolayers were washed twice with phosphate-buffered saline (PBS) and scraped off in 1 ml of PBS. The cell slurry was centrifuged at 1000 × g for 5 min at 4 °C, and the resulting cell pellets were stored at -80 °C. For analysis, pellets from individual plates were suspended in 0.6 ml of lysis buffer (10 mm Tris-HCl, pH 7.5, containing 0.2% sodium deoxycholate and 10 units/ml heparin) and sonicated for 6 s at a force of 2 g. Cellular debris was removed by centrifugation at 1,000 × g for 5 min at 4 °C. Chromatography and Sucrose Gradient Centrifugation—For heparin-Sepharose chromatography, 1 ml of heparin-Sepharose (Amersham Biosciences) was packed into a 1 × 10-cm column and equilibrated with column buffer (10 mm Tris-HCl, pH 7.5, containing 0.1% Triton X-100). Combined cell lysates from six 100-mm plates were applied to the column, and the flow-through was collected. After washing the column with 10 bed volumes of column buffer, HL was eluted in two stages: the addition of 0.5 m NaCl, followed by 1.0 m NaCl in column buffer. Heparin was added to all eluted fractions to a final concentration of 10 units/ml, and samples were maintained at -80 °C until analyzed. β-Ricin chromatography of the active, high affinity HL fraction obtained from heparin-Sepharose chromatography was performed after removing the terminal sialic acid of the glycan chains by neuraminidase digestion (see below). 0.5 ml of Ricinus communis agglutinin (RCA120) linked to agarose (Sigma) were washed with 10 mm Tris-HCl, pH 7.5, containing 0.1% Triton X-100 and 1.0 m NaCl. After application of the sample, unbound HL was collected in the flow-through, and the bound HL was eluted with 0.2 m galactose (Sigma) in the column buffer. Rate zonal centrifugation was carried out using a 12-ml, 5-20% linear sucrose gradient, topped by 0.55 ml of sample. The molecular markers used were ovalbumin (45 kDa), malic dehydrogenase (74 kDa), glucose 6-phosphate dehydrogenase (114 kDa), and catalase (240 kDa). Centrifugation was carried out at 200,000 × g for 22 h at 4 °C. Fractions of 0.48 ml were manually collected from the top, as described (22Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Glycosidase Digestion—For endoglycosidase H (endo H) digestion, samples were denatured by adding SDS to 0.5% and heated at 95 °C for 2 min. After chilling, 10 milliunits of endo H (Roche Applied Science) were added, and the samples were incubated at 37 °C for 16-18 h. For neuraminidase digestion, 50 milliunits of neuraminidase (Calbiochem) were added to 1 ml of HL eluate obtained from heparin-Sepharose chromatography. Incubation was carried out for 90 min on ice prior to β-ricin chromatography. Lipase Assays and Determination of Specific Activity—The activity of HL and LPL was measured using the respective triolein substrates, prepared by sonication (24Doolittle M.H. Ben-Zeev O. Briquet-Laugier V. J. Lipid Res. 1998; 39: 934-942Abstract Full Text Full Text PDF PubMed Google Scholar, 25Nilsson-Ehle P. Schotz M.C. J. Lipid Res. 1976; 17: 536-541Abstract Full Text PDF PubMed Google Scholar). PL was assayed with the substrate used for HL, with the following modifications. A mixture of 0.5 μg of colipase (Sigma), 0.8 μmol of taurodeoxycholate (Sigma), and 0.2 μmol of CaCl2 was prepared in a final volume of 50 μl in water. Enzyme source and 10 mm Tris-HCl, pH 7.5, were then added to complete the volume to 100 μl, and the reaction was initiated by the addition of 100 μl of substrate. One milliunit of lipase activity is defined as 1 nmol of free fatty acid (FFA) hydrolyzed per min. Lipase specific activity was calculated by dividing the activity loaded onto electrophoretic gels by the densitometric value of the band obtained by Western blotting. Since densitometry does not provide an absolute concentration, we report the specific activity relative to that of a sample chosen from the blot that was arbitrarily set to a value of 1.0. In instances where HL was fully active, 1 milliunit of active lipase was considered to represent 1 ng of lipase protein. This value was supported by the reported specific activity of purified HL as 55,000 μmol of FFA/mg/h (26Twu J.S. Garfinkel A.S. Schotz M.C. Biochim. Biophys. Acta. 1984; 792: 330-337Crossref PubMed Scopus (42) Google Scholar), corresponding to 0.92 nmol of FFA/ng/min, or nearly 1 milliunit/ng. This correlation of 1 milliunit/1 ng of lipase protein was applied to HL that was secreted or to active intracellular HL with high affinity to heparin (see Figs. 3C and 4A). Specifically, in Fig. 3C, the densitometric value of HL mass secreted after 2 h was converted to the value of its milliunits of enzymatic activity, and the mass of the other samples on the plot was reported relative to that value. Similarly, in Fig. 4A, the densitometric mass value of the peak active fraction eluted at 1.0 m NaCl was converted to the value of milliunits of activity, and the mass of all other samples eluted from the column was reported relative to that value.Fig. 4Isolation of active HL in the ER.A, isolation of intracellular active HL by heparin-Sepharose chromatography. Cell lysates combined from six 100-mm plates were applied onto a heparin-Sepharose column. After collecting the flow-through, HL was sequentially eluted with 0.5 and 1.0 m NaCl. Fractions were analyzed for enzymatic activity (gray triangles), and HL mass was calculated by densitometric scanning of bands obtained by Western blotting (black circles). The mass is reported relative to milliunit activity in the peak fraction eluted at 1.0 m NaCl, since that fraction was fully mature (Fig. 4C), and 1 milliunit represents approximately 1 ng of HL protein (see “Experimental Procedures”). Since the intracellular activity is dramatically low relative to HL mass, the inset shows HL activity alone drawn to scale. For assessment of subcellular location, aliquots from the peak fractions were subjected to endo H analysis (see panels below respective fractions and “Results” for explanation). B, separation of high affinity HL into ER and Golgi forms. Following β-ricin chromatography (see “Experimental Procedures”), the high mannose (ER) HL form was recovered in the flow-through, whereas the complex (Golgi) form was eluted with 0.2 m galactose. Aliquots of the two forms were subjected to endo H digestion and Western blotting. Due to the extremely small amount of the complex form located intracellularly, this form could not be detected in the galactose eluate unless the film was exposed for 1 h. C, specific activity of active HL located in the ER is similar to that of secreted HL. Aliquots of similar enzymatic activity from the β-ricin flow-through, HL secreted from CHO cells, and HL secreted from Lec1 cells were subjected to Western blotting. HL mass was calculated by densitometric scanning, and the specific activity of HL secreted from CHO cells was arbitrarily assigned a value of 1.0. mu, milliunits.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When specific activities of intracellular PL, LPL, and HL were compared (see Fig. 2A), consideration was given to the reported specific activities of the purified lipases (4.3, 0.92, and about 1.0 mmol/min/mg, respectively (26Twu J.S. Garfinkel A.S. Schotz M.C. Biochim. Biophys. Acta. 1984; 792: 330-337Crossref PubMed Scopus (42) Google Scholar, 27Sayari A. Mejdoub H. Gargouri Y. Biochimie (Paris). 2000; 82: 153-159Crossref PubMed Scopus (37) Google Scholar, 28Smith L.C. Pownall H.J. Borgström B. Brockman H.L. Lipases. Elsevier, Amsterdam1984: 263-305Google Scholar)). Based on these reported specific activities, if fully active PL was assigned a value of 1.0, fully active LPL and HL would have a specific activity of 0.23. Instead, we found relative values of 1.0, 0.13, and 0.004 for PL, LPL, and HL, respectively, indicating that intracellular LPL was 55% active and intracellular HL was only 2% active. Co-immunoprecipitation—For detection of HL-calnexin associates, lipase samples were incubated overnight with 40 μg of anti-calnexin IgG isolated by protein A chromatography from anti-calnexin antiserum (Stressgen). Purified staphylococcus A (24Doolittle M.H. Ben-Zeev O. Briquet-Laugier V. J. Lipid Res. 1998; 39: 934-942Abstract Full Text Full Text PDF PubMed Google Scholar) was then added, and the samples were incubated for 30 min at 4 °C on a rotary shaker. Following centrifugation, the staphylococcus A pellets were washed twice with lysis buffer (10 mm Tris-HCl, pH 7.5, 0.2% sodium deoxycholate, 10 units/ml heparin) and once with deionized water. Bound proteins were released by suspending the precipitates in 10 mm Tris-HCl, pH 7.5, containing 1% SDS, and heating at 95 °C for 2 min. PAGE and Western Blotting—Unless otherwise stated, cell lysates, medium, or proteins released from co-immunoprecipitates were SDS- and heat-denatured (95 °C for 2 min) in the presence of 0.4 m β-mercaptoethanol (Sigma) and subjected to PAGE using 7% Tris-glycine gels. For cross-linked lysates containing multicomponent complexes, electrophoresis was carried out without reducer in 3-8% gradient Tris acetate gels (Novex). Western blotting was performed as previously described (24Doolittle M.H. Ben-Zeev O. Briquet-Laugier V. J. Lipid Res. 1998; 39: 934-942Abstract Full Text Full Text PDF PubMed Google Scholar), using the horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen; 1:2,500). Quantitation of the lipase bands was carried out by densitometric scanning of the Western blots using the NIH Image analysis program for the Macintosh. Cross-linking of HL—Cells in 100-mm plates were washed twice with PBS at room temperature, followed by incubation for 20 min on ice with cold PBS containing 20 mmN-ethylmaleimide (NEM; Calbiochem). After an additional wash with cold PBS without NEM, cells were harvested, resuspended in PBS (0.45 ml/cell pellet obtained from one plate), and kept on ice. 50 μl of a freshly prepared solution containing 20 mm dithiobis(succinimidylpropionate) (DSP; Pierce) in Me2SO were added to the cells (2 mm final concentration), and the slurry was incubated on ice for 1 h with intermittent shaking. In control plates, an equivalent amount of Me2SO without DSP was added, and the cells were incubated in a similar manner. The cross-linking was quenched by the addition of 1 m glycine (50 mm final concentration) and 1 m NEM (40 mm final concentration) and an additional incubation on ice for 15 min. The cells were pelleted by centrifugation at 12,000 × g for 3 min. HL expressed in CHO cells exhibited only small amounts of intracellular lipase activity; after 5 h of incubation in the presence of heparin, less than 15% of the total HL activity resided in the cells, whereas the remainder was secreted. Based on the intracellular quality control mechanism that ensures secretion of only functional proteins (29Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (587) Google Scholar), we considered secreted HL as the fully mature form of the protein. Accordingly, this form underwent all necessary co- and post-translational modifications, attained a correct tertiary structure, assembled into dimers (30Hill J.S. Davis R.C. Yang D. Wen J. Philo J.S. Poon P.H. Phillips M.L. Kempner E.S. Wong H. J. Biol. Chem. 1996; 271: 22931-22936Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and exhibited full specific activity (26Twu J.S. Garfinkel A.S. Schotz M.C. Biochim. Biophys. Acta. 1984; 792: 330-337Crossref PubMed Scopus (42) Google Scholar). Thus, secreted HL was used as the standard in evaluating the maturity of the intracellular HL protein. Cells Exhibit Massive Amounts of Inactive HL Protein—Fully mature HL is a homodimer, with a predicted molecular mass of 120 kDa. To compare the assembly state of intracellular HL with that of the mature, secreted form, cell lysates and medium containing similar amounts of HL activity were subjected to density gradient centrifugation. As expected, HL from both sources displayed a superimposable distribution of enzymatic activity, sedimenting at a region corresponding to a dimer (103 kDa; see Fig. 1, graph). However, the similarity between cells and medium did not extend to the HL mass. As shown in Fig. 1 (bottom panels), while the medium exhibited barely detectable amounts of HL protein that overlapped with the activity (fractions 10-12), comparable amounts of activity in the corresponding fractions of the cell lysate exhibited massive amounts of HL protein. Thus, the intracellular HL specific activity (activity/units of HL mass) was very low. In addition, rather than sedimenting exclusively as a dimer, intracellular HL protein was spread almost continuously across the gradient, including large aggregates at the bottom (fraction 26). Clearly, the vast majority of HL mass in the cells was not functional and exhibited a diverse range of molecular weights. Intracellular HL Differs in Sedimentation Pattern from Other Members in the Lipase Gene Family—The low levels of intracellular HL activity accompanied by the broad distribution of mass across the gradient suggested that correct HL folding and assembly in these cells was either impaired or considerably prolonged. To determine whether this abnormal or delayed maturation was HL-specific, we compared the intracellular activity of HL with that of two related lipases, PL and LPL. Thus, cell lysates containing PL, LPL, and HL with known enzymatic activity were subjected to Western blotting (Fig. 2A). The progressive decrease in the migration of LPL and HL relative to PL was due to the difference in carbohydrate content, as human PL contains one glycan chain, LPL has two chains and human HL has four chains. When the carbohydrate was removed by endo H digestion, the migration of the three lipases was similar (data not shown). The amount of protein in each lane was measured by scanning densitometry, and specific activities were calculated. Considering the relative specific activity of PL as 100% (see “Experimental Procedures” for details), intracellular LPL was 55% active, and intracellular HL was only 2% active (Fig. 2A, histogram). In contrast to HL, the relatively high specific activity of PL and LPL suggested that the intracellular lipase protein contained in large part mature, fully functional molecules. This distinction was evident in the sedimentation pattern of the three lipases. As shown in Fig. 2B, a perfect correlation existed in the sedimentation profile of PL enzymatic activity and protein, corresponding to that of an expected monomer (11Winkler F.K. d'Arcy A. Hunziker W. Nature. 1990; 343: 771-774Crossref PubMed Scopus (1037) Google Scholar). The molecular size of the peak fraction was ∼59 kDa, in close agreement with the predicted PL molecular mass of 53 kDa. LPL intracellular protein, as was previously described (22Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), was separated into the fully mature dimer form that overlapped the enzymatic activity and an inactive aggregate at the bottom of the gradient. In contrast, the sedimentation pattern of HL was not so clearly defined. Although the majority of HL protein migrated to a region that could loosely be interpreted as a homodimer, it was associated with very little enzymatic activity. Therefore, protein accumulation at this sedimentation range could at best represent a mixture of some homodimeric, active HL, and inactive HL that is either dimeric or monomeric in association with proteins of similar molecular weight. In addition, as seen here and in Fig. 1, HL protein trailed into larger molecular forms. Clearly, these cells contained very littl
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