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cld and lec23 are disparate mutations that affect maturation of lipoprotein lipase in the endoplasmic reticulum

1999; Elsevier BV; Volume: 40; Issue: 11 Linguagem: Inglês

10.1016/s0022-2275(20)32428-7

1539-7262

Véronique Briquet-Laugier, Osnat Ben-Zeev, Ann L. White, Mark H. Doolittle,

Lipid metabolism and disorders

The mutations cld (combined lipase deficiency) and lec23 disrupt in a similar manner the expression of lipoprotein lipase (LPL). Whereas cld affects an unknown gene, lec23 abolishes the activity of α-glucosidase I, an enzyme essential for proper folding and assembly of nascent glycoproteins. The hypothesis that cld, like lec23, affects the folding/assembly of nascent LPL was confirmed by showing that in cell lines homozygous for these mutations (Cld and Lec23, respectively), the majority of LPL was inactive, displayed heterogeneous aggregation, and had a decreased affinity for heparin. While inactive LPL was retained in the ER, a small amount of LPL that had attained a native conformation was transported through the Golgi and secreted. Thus, Cld and Lec23 cells recognized and retained the majority of LPL as misfolded, maintaining the standard of quality control. Examination of candidate factors affecting protein maturation, such as glucose addition and trimming, proteins involved in lectin chaperone cycling, and other abundant ER chaperones, revealed that calnexin levels were dramatically reduced in livers from cld/cld mice; this finding was also confirmed in Cld cells. We conclude that cld may affect components in the ER, such as calnexin, that play a role in protein maturation. Whether the reduced calnexin levels per se contribute to the LPL deficiency awaits confirmation.—Briquet-Laugier, V., O. Ben-Zeev, A. White, and M. H. Doolittle. cld and lec23 are disparate mutations that affect maturation of lipoprotein lipase in the endoplasmic reticulum. J. Lipid Res. 1999. 40: 2044–2058. The mutations cld (combined lipase deficiency) and lec23 disrupt in a similar manner the expression of lipoprotein lipase (LPL). Whereas cld affects an unknown gene, lec23 abolishes the activity of α-glucosidase I, an enzyme essential for proper folding and assembly of nascent glycoproteins. The hypothesis that cld, like lec23, affects the folding/assembly of nascent LPL was confirmed by showing that in cell lines homozygous for these mutations (Cld and Lec23, respectively), the majority of LPL was inactive, displayed heterogeneous aggregation, and had a decreased affinity for heparin. While inactive LPL was retained in the ER, a small amount of LPL that had attained a native conformation was transported through the Golgi and secreted. Thus, Cld and Lec23 cells recognized and retained the majority of LPL as misfolded, maintaining the standard of quality control. Examination of candidate factors affecting protein maturation, such as glucose addition and trimming, proteins involved in lectin chaperone cycling, and other abundant ER chaperones, revealed that calnexin levels were dramatically reduced in livers from cld/cld mice; this finding was also confirmed in Cld cells. We conclude that cld may affect components in the ER, such as calnexin, that play a role in protein maturation. Whether the reduced calnexin levels per se contribute to the LPL deficiency awaits confirmation.—Briquet-Laugier, V., O. Ben-Zeev, A. White, and M. H. Doolittle. cld and lec23 are disparate mutations that affect maturation of lipoprotein lipase in the endoplasmic reticulum. J. Lipid Res. 1999. 40: 2044–2058. Lipoprotein lipase (LPL), the major enzyme hydrolyzing the triglyceride core of chylomicrons and very low density lipoproteins, is synthesized and secreted by the parenchymal cells of extrahepatic tissues and by neonatal liver cells. In these tissues, LPL is anchored to the capillary endothelium via heparan sulfate proteoglycans, and functions in liberating free fatty acids for storage or oxidation (1Bensadoun A. Lipoprotein lipase.Annu. Rev. Nutr. 1991; 11: 217-237Google Scholar, 2Zechner R. The tissue-specific expression of lipoprotein lipase: implications for energy and lipoprotein metabolism.Curr. Opin. Lipidol. 1997; 8: 77-88Google Scholar). Glycosylation at the conserved N-terminal site in LPL has been shown to be crucial for the formation of active enzyme (3Ben-Zeev O. Stahnke G. Liu G. Davis R.C. Doolittle M.H. Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme.J. Lipid Res. 1994; 35: 1511-1523Google Scholar). Similar to other glycoproteins (4Kornfeld R. Kornfeld S. Assembly of asparagine-linked oligosaccharides.Annu. Rev. Biochem. 1985; 54: 631-664Google Scholar), core oligosaccharide chains, consisting of the 14-saccharide unit Glc3Man9GlcNAc2, are added co-translationally in the endoplasmic reticulum (ER). Early on, these core high mannose chains undergo a series of trimming reactions that involve removal of the terminal glucose by glucosidase I, followed by successive hydrolysis of the inner glucoses by glucosidase II. Inhibition of these trimming reactions by specific glucosidase inhibitors (castanospermine, deoxynojirimycin) severely inhibits catalytic activity of LPL, and most of the inactive enzyme is retained intracellularly (5Ben-Zeev O. Doolittle M.H. Davis R.C. Elovson J. Schotz M.C. Maturation of liporotein lipase: expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.J. Biol. Chem. 1992; 267: 6219-6227Google Scholar, 6Carroll R. Ben-Zeev O. Doolittle M.H. Severson D.L. Activation of lipoprotein lipase in cardiac muscle by glycosylation requires trimming of glucose residues in the endoplasmic reticulum.Biochem. J. 1992; 285: 639-696Google Scholar, 7Masuno H. Blanchette-Mackie E.J. Schultz C.K. Sparth A.E. Scow R.O. Okuda H. Retention of glucose by N-linked oligosaccharide chains impedes expression of lipoprotein lipase activity: effect of castanospermine.J. Lipid Res. 1992; 33: 1343-1349Google Scholar, 8Park J-W. Oh M-S. Yang J-Y. Park B-H. Rho H-W. Lim S-N. Jhee E-C. Kim H-R. Glycosylation, dimerization, and heparin affinity of lipoprotein lipase in 3T3-L1 adipocytes.Biochim. Biophys. Acta. 1995; 1254: 45-50Google Scholar). Addition and removal of the outer glucose residues from core glycans is a prerequisite for entry of nascent glycoproteins into a folding cycle involving lectin chaperones. Specifically, these chaperones (e.g., calnexin, calreticulin) recognize and bind the innermost glucose residue of nascent glycoproteins, after trimming of the two distal glucose residues by glucosidase I and II (9Hammond C. Helenius A. Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment and Golgi apparatus.J. Cell Biol. 1994; 126: 41-52Google Scholar, 10Helenius A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum.Mol. Biol. Cell. 1994; 5: 253-265Google Scholar, 11Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins.J. Biol. Chem. 1995; 270: 4697-4704Google Scholar, 12Spiro R.G. Zhu Q. Bhoyroo V. Söling H-D. Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi.J. Biol. Chem. 1996; 271: 11588-11594Google Scholar). Removal of the final glucose residue by glucosidase II liberates nascent proteins from their lectin anchors. If the released proteins are still improperly folded, binding is resumed after reglucosylation of the protein by UDP-glucose:glycoprotein glucosyltransferase (UGGT) (13Sousa M. Parodi A.J. The molecular basis for the recognition of misfolded proteins by the UDP-glc:glycoprotein glucosyltransferase.EMBO J. 1995; 14: 4196-4203Google Scholar). Thus, cycles of re- and de-glucosylation permit repeated access of nascent glycoproteins to the chaperones (14Hebert D.H. Foellmer B. Helenius A. Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum.Cell. 1995; 81: 425-433Google Scholar). The bound glycoproteins may interact with ERp57, a protein that associates with calnexin/calreticulin (15Zapun A. Darby N.J. Tessier D.C. Michalak M. Bergeron J.J. Thomas D.Y. Enhanced catalysis of ribonuclease B folding by the interaction of calnexin or calreticulin with ERp57.J. Biol. Chem. 1998; 273: 6009-6012Google Scholar, 16Oliver J.D. van der Wal F.J. Bulleid N.J. High S. Interaction of the thiol-dependent reductase ERp57 with nascent glycoproteins.Science. 1997; 275: 86-88Google Scholar, 17Elliott J.G. Oliver J.D. High S. The thiol-dependent reductase ERp57 interacts specifically with N-glycosylated integral membrane proteins.J. Biol. Chem. 1997; 272: 13849-13855Google Scholar), that may promote folding through its protein–disulfide isomerase activity (18Hirano N. Shibasaki F. Sakai R. Tanaka T. Nishida J. Yazaki Y. Takenawa T. Hirai H. Molecular cloning of the human glucose-regulated protein ERp57/GRP58, a thiol-dependent reductase. Identification of its secretory form and inducible expression by the oncogenic transformation.Eur. J. Biochem. 1995; 234: 336-342Google Scholar) and translgutamination activity (19Chandrashekar R. Tsuji N. Morales T. Ozols V. Mehta K. An ERp60-like protein from the filarial parasite Dirofilaria immitis has both transglutaminase and protein disulfide isomerase activity.Proc. Natl. Acad. Sci. USA. 1998; 95: 531-536Google Scholar). Upon attaining a native conformation, proteins released from the chaperones by glucosidase II are no longer recognized by UGGT, and thus exit from the cycle and continue along the secretory pathway. The components of the folding cycle (e.g., lectin chaperones, ERp57, glucosidase II, and UGGT) comprise a functional complex within the ER referred to as the "calnexin cycle". If any of these components are absent or non-functional, the calnexin cycle is compromised. Thus, in cell lines deficient in glucosidase I (Lec23) or glucosidase II (PhaR2.7) activity, association of nascent glycoproteins with calnexin is virtually nonexistent (20Ora A. Helenius A. Calnexin fails to associate with substrate proteins in glucosidase-deficient cell lines.J. Biol. Chem. 1995; 270: 26060-26062Google Scholar). While initial glycosylation events are crucial in LPL maturation, a naturally occurring mutation in the mouse, cld (combined lipase deficiency), also affects lipase activity posttranslationally, although the mechanism is unknown (reviewed in ref. 21Reue K. Doolittle M.H. Naturally occurring mutations in mice affecting lipid transport and metabolism.J. Lipid Res. 1996; 37: 1387-1405Google Scholar). Located on chromosome 17, this recessive mutation virtually abolishes LPL and hepatic lipase activity, resulting in severe chylomicronemia and death 2–3 days after birth (21Reue K. Doolittle M.H. Naturally occurring mutations in mice affecting lipid transport and metabolism.J. Lipid Res. 1996; 37: 1387-1405Google Scholar, 22Paterniti Jr., J.R. Brown W.V. Ginsberg H.N. Artzt K. Combined lipase deficiency (cld): A lethal mutation on chromosome 17 of the mouse.Science. 1983; 221: 167-169Google Scholar, 23Artzt K. Gene mapping within the T/t complex of the mouse. III: t-Lethal genes are arranged in three clusters on chromosome 17.Cell. 1984; 39: 565-572Google Scholar). Mice homozygous for the mutation synthesize normal levels of lipase protein that is inactive and retained intracellularly (24Masuno H. Blanchette-Mackie E.J. Chernick S.S. Scow R.O. Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipase-deficient cld/cld mice.J. Biol. Chem. 1990; 265: 1628-1638Google Scholar, 25Olivecrona T. Chernick S.S. Bengtsson-Olivecrona G. Paterniti Jr., J.R. Brown W.V. Scow R.O. Combined lipase deficiency (cld/cld) in mice.J. Biol. Chem. 1985; 260: 2552-2557Google Scholar, 26Davis R.C. Ben-Zeev O. Martin D. Doolittle M.H. Combined lipase deficiency in the mouse: lipase transcription, translation and processing.J. Biol. Chem. 1990; 265: 17960-17966Google Scholar). The mutation does not affect the lipase coding sequence, as cld does not co-localize with the lipase structural genes (27Lusis A.J. Taylor B.A. Quon D. Zollman S. LeBoeuf R.C. Genetic factors controlling structure and expression of apolipoproteins B and E in mice.J. Biol. Chem. 1987; 262: 7594-7604Google Scholar, 28Warden C.H. Davis R.C. Yoon M-Y. Hui D-Y. Svenson K. Xia Y-R. Diep A. He K-Y. Lusis A.J. Chromosomal localization of lipolytic enzymes in the mouse: pancreatic lipase, colipase, hormone-sensitive lipase, hepatic lipase, and carboxyl ester lipase.J. Lipid Res. 1993; 34: 1451-1455Google Scholar). Rather, it has been proposed that cld might interfere with early glycan modification events, or with the course of lipase folding carried out in conjuncture with these events (21Reue K. Doolittle M.H. Naturally occurring mutations in mice affecting lipid transport and metabolism.J. Lipid Res. 1996; 37: 1387-1405Google Scholar). In this study, we attempt to elucidate the underlying basis of the lipase deficiency caused by the cld mutation. To accomplish this aim, a cell line derived from cld/cld mice (Cld) was established and compared with the Lec23 cell line deficient in glucosidase I activity (29Ray M.K. Yang J. Sundaram S. Stanley P. A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in α-glucosidase I.J. Biol. Chem. 1991; 34: 22818-22825Google Scholar). This allowed a side-by-side comparison of the effect of the two mutations on lipase activity, as well as on lipase structural characteristics such as assembly state and heparin affinity. Based on these criteria, the majority of LPL failed to attain a native conformation, supporting the hypothesis of improper folding/assembly as the underlying defect of both mutations. We then tested possible candidate processes in the ER that could explain lipase misfolding, including ER-to-Golgi transport, glucose addition and removal, and components of the calnexin cycle and other ER chaperones. Of these, only calnexin levels were found to be substantially decreased in microsomal-enriched fractions from either cld/ cld livers or the Cld fibroblast cell line. Whether reduced calnexin levels in Cld cells contribute to the lipase deficiency awaits further experimentation. However, as cld reduces calnexin levels, and lec23 affects binding of glycoproteins to calnexin, it is tempting to speculate that both mutations may impair maturation of LPL by limiting its access to the calnexin cycle. Ionomycin and brefeldin A were purchased from Calbiochem and Epicentre Technologies, respectively. Castanospermine (Cs), deoxynojirimycin, and endoglycosidase H (endo H) were from Genzyme. Endoglycosidase D (endo D), collagenase A, and insulin were from Boehringer Mannheim Biochemicals. Jack bean α-mannosidase was from Oxford GlycoSystems. Heparin-Sepharose was from Pharmacia. Biotinylated rabbit anti-chicken IgG, PVDF blotting membranes, and SuperSignal® chemiluminescent substrate were from Pierce. Hyperfilm-ECL photographic film was from Amersham. Antibodies against calnexin, calreticulin, BiP, GRP94 and PDI were purchased from Affinity Bioreagents, Inc. or from Stressgen Biotechnologies Corp. The antibody against ERp57 was a kind gift from M. Kito, Kyoto University. Lec23, the mutant Chinese hamster ovary (CHO) cell line deficient in α-glucosidase I activity (29Ray M.K. Yang J. Sundaram S. Stanley P. A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in α-glucosidase I.J. Biol. Chem. 1991; 34: 22818-22825Google Scholar), was a generous gift from Pamela Stanley. Pro5, used as a control cell line for Lec23, was obtained from the American Type Culture Collection (ATCC). Fibroblast cell lines were derived from skin explants removed from cld/ cld and unaffected (+/?) neonatal mice. After washing in sterile PBS, 10–20 of these skin explants (2–3 mm in size) were placed with the dermal side down onto a collagen-coated 60-mm dish containing 1 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS), penicillin–streptomycin, sodium pyruvate, glutamine, and non-essential amino acids. After 3 days, the volume of growth medium was increased to 3 ml, and non-adherent explants were removed. The medium was changed weekly until a substantial outgrowth of cells was observed. At this time adherent skin explants were removed, and when the outgrowing cells reached 50% confluency the cells were passaged. The cultures were maintained in the same growth medium except for lowering serum supplementation to 10% FBS. After about 20 passages, the primary cultures were overgrown by foci of spontaneously immortalized fibroblasts. At this time, FBS was replaced by 10% calf serum and the cell lines were passaged at split ratios of 1:8. Cells maintained for over 200 passages retained normal growth characteristics and morphology. Immortalized fibroblasts derived from either unaffected or cld/ cld mice did not express detectable lipase activity. To estimate chromosome number per cell, Het and Cld cells were arrested in metaphase using 0.1 mm colcemid, swollen in hypotonic solution (0.04 m KCl, 0.025 m sodium citrate), and fixed in ice-cold acetic methanol (30Freshney R.I. Culture of Animal Cells. A Manual of Basic Technique. Alan R. Liss, Inc., New York1987Google Scholar). Chromosome number was determined in about 50 metaphase-arrested cells, by counting chromosomes under a phase-contrast microscope. As expected for immortalized cells, both cell lines exhibited heteroploidy, predominately in the subtetraploid range; in some cells aneuploidy was also evident. Because of the abnormal chromosomal counts and the possibility of genetic deletions, both cell lines were genotyped for the microsatellite markers D17Au126, D17Au57, and D17Au100 that are linked to cld on chromosome 17 (31Ebersole T. Lai F. Artzt K. New molecular markers for the distal end of the t-complex and their relationships to mutations affecting mouse development.Genetics. 1992; 131: 175-182Google Scholar). All three markers were present in both unaffected and affected cell lines, indicating that chromosome 17 in the region of cld was intact. In addition, the polymorphic nature of these markers was used to genotype the cells lines: the cell lines derived from normal and affected mice were found to be heterozygous and homozygous, respectively, for the region of chromosome 17 containing the cld mutation. Based on these genotypes, the cell lines are referred to as Het and Cld. Human cDNA for LPL (32Wion K.L. Kirchgessner T.G. Lusis A.J. Schotz M.C. Lawn R.M. Human lipoprotein lipase complementary DNA sequence.Science. 1987; 235: 1638-1641Google Scholar) and PL (33Lowe M.E. Rosenblum J.L. Strauss A.W. Cloning and characterization of human pancreatic lipase cDNA.J. Biol. Chem. 1989; 264: 20042-20048Google Scholar) were subcloned into pRc/RSV and pcDNA1/Neo expression vectors driven by enhancer/promoter sequences from the Rous sarcoma virus long terminal repeat and the human cytomegalovirus, respectively. Both vectors contain a neomycin resistance gene used for selection of G418-resistant stable cell lines. The LPL tandem repeat (LPLTR) expression construct (a generous gift from Howard Wong) contains in succession the sequence coding for the LPL signal peptide, residues 1–448 (the complete LPL sequence), an 8 amino acid linker (GSIEGRLE), LPL residues 1–448, and 20 bases of the LPL 3′ untranslated region cloned into the pcDNA3 expression vector (34Wong H. Yang D. Hill J.S. Davis R.S. Nikazy J. Schotz M.C. A molecular biology-based approach to resolve the subunit orientation of lipoprotein lipase.Proc. Natl. Acad. Sci. USA. 1997; 94: 5594-5598Google Scholar). Transient transfection was carried out by electroporation (35Durstenfeld A. Ben-Zeev O. Reue K. Stahnke G. Doolittle M.H. Molecular characterization of human hepatic lipase deficiency: in vitro expression of two naturally occurring mutations.Arterioscler. Thromb. 1994; 14: 381-385Google Scholar). Briefly, 2–5 × 107 cells were exposed to a single voltage pulse (0.33 V, 960 μF) in the presence of 40 μg plasmid DNA, comprised of 30 μg of lipase DNA mixed with 10 μg of a β-galactosidase reporter construct (pCH110). After electroporation, the cell suspensions were distributed into 3 × 60 mm tissue culture plates. The medium was changed after 4 h, and the cells were harvested after reaching confluency, within 24–48 h. To obtain stably transfected cell lines, cells were transfected using co-precipitates of CaPO4 and plasmid DNA (pRc/RSV constructs for Het/Cld cell lines; pcDNA1/Neo constructs for Pro5/Lec23 cell line) according to manufacturer's instruction (Invitrogen). Cells expressing resistance to antibiotics were selected at a concentration of 400–800 μg/ml G418 (Gibco). Individual antibiotic-resistant colonies were then screened for lipase activity, and the highest expressing clones were retained and expanded for subsequent studies. For most experiments, cells were subcultured into 60-mm plates containing 3 ml medium. Treatment of the cells with heparin (10 U/ml), with cytoskeleton-perturbing agents (3 μm ionomycin or 5 μg/ml BFA), or with glucosidase inhibitors (1 mm Cs or 2 mm deoxynojirimycin) was carried out by removing the culture medium, replacing with 2 ml fresh medium containing the pertinent reagent, and incubating the plates for an additional 4.5–5 h. After the experiment, media were recovered and the cell layers were washed twice with phosphate-buffered saline (PBS). The cells were scraped from the plate in 1 ml PBS, centrifuged for 5 min at 4°C, and the pellets were stored at –80°C until use. A detailed description of the sucrose gradient centrifugation protocol has been reported elsewhere (36Ben-Zeev O. Doolitlle M.H. Determining lipase subunit structure by sucrose gradient centrifugation.in: Doolittle M. Reue K. Lipase and Phospholipase Protocols. 109. Humana Press, Totowa, NJ1998: 257-266Google Scholar). Briefly, gradients of 5–20% sucrose (11.7 ml) were prepared in LPL lysis buffer (50 mm NH4OH·HCl buffer, pH 8.1, containing 10 U/ml heparin and 0.2% deoxycholate). Gradients were overlaid with a sample of cell lysate (approx. 2 mg cellular protein) containing two internal molecular size markers: 35 units glucose-6-phosphate dehydrogenase (G-6-PDH, 114 kDa) and 15 units malic dehydrogenase (MDH, 74 kDa). Each sedimentation experiment included a separate gradient containing additional molecular size markers: 200 μg cytochrome C (12 kDa), 200 μg ovalbumin (45 kDa), and 400 μg catalase (240 kDa) in LPL lysis buffer. Gradients were centrifuged at 4°C in a Beckman SW 41 Ti rotor for 22 h at 200,000 g, and fractionated into 26 aliquots of 480 μl, starting from the top; fractions were stored at –80°C until assayed. One ml heparin-Sepharose (Pharmacia Biotech.), suspended and washed in distilled water, was packed into 1 × 10 cm columns and equilibrated with column buffer (10 mm Tris-HCl, pH 7.3, containing 0.1% Triton X-100). Samples subjected to chromatography were cell lysates obtained from five or six 100-mm plates or 8 ml of medium from Cld cells incubated overnight with 10 U/ml heparin; Triton X-100 was added to the medium prior to chromatography (final concentration, 0.1%). The column was washed with 10 bed-volumes of column buffer, and LPL elution was carried out in two stages: addition of 0.75 m NaCl, followed by 2 m NaCl, in the column buffer. The eluate was collected in 0.6-ml fractions. To preserve LPL activity, heparin (10 U/ml) was added to all eluted fractions and samples were immediately frozen at –80°C until assayed. The detailed protocol of LPL immunodetection by Western blotting has been described (37Doolittle M.H. Ben-Zeev O. Briquet-Laugier V. Enhanced detection of lipoprotein lipase by combining immunoprecipitation with Western blot analysis.J. Lipid Res. 1998; 39: 934-942Google Scholar). Briefly, LPL immunoprecipitates, generally representing 10 mu activity, were dissociated from Staph A pellets using 30 μl 0.5% SDS in 50 mm sodium phosphate buffer, pH 5.75. For samples subjected to digestion by endoglycosidase (endo) H (Boehringer), 2.5 mu of endo H were added and the sample was incubated overnight at 37°C. When the amount of NaCl in the samples varied (e.g., in heparin-Sepharose eluates), the salt concentration of all samples was adjusted to equivalence. After PAGE and transfer of the proteins onto polyvinylidene difluoride (PVDF) membranes, blots were submerged for 1 h in blocking buffer (2% casein hydrolysate in 50 mm Tris-HCl, pH 7.5, 0.1% Tween-20) and incubated overnight with chicken anti-bovine milk LPL (0.3–0.4 μg/ml in 10 mm sodium phosphate, pH 7.2, containing 0.15 m NaCl and 0.1% Triton X-100 (PBS-T)). After washing, the membrane was incubated for 30 min with biotinylated rabbit anti-chicken IgG (1:10,000 in PBS-T), washed again, and incubated for 10 min with horseradish peroxidase (HRP)–streptavidin (1:5,000) in PBS-T. After a last series of washes, the blots were then developed by chemiluminescence and exposed to photographic film. Densitometric scanning of lipase bands obtained by chemiluminescence was carried out with the aid of an AMBIS Radioanalytic Imaging System. Integration of the scanned bands was performed using the QuantProbe™ Software, version 4.31. LPL isolated by immunoprecipitation was incubated overnight at 37°C with jack bean mannosidase (50 mU) in a 60 μl volume containing 100 mm sodium acetate, pH 5.0, 2% Triton X-100, and 0.25% SDS. At the end of incubation, 40 μl of a glycerol/SDS stock (20% glycerol, 5% SDS, 0.01% bromophenol blue) was added prior to Western blotting. Liver microsomes from unaffected and cld/cld neonatal mice were isolated as previously described (38Blobel G. Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.J. Cell Biol. 1975; 67: 835-851Google Scholar). For microsome-enriched fractions from Cld and Het cells, two 100-mm plates of each cell line were harvested (0.8 ml/plate) in 20 mm HEPES buffer, pH 7.5, containing 0.25 m sucrose, 1 mm EDTA, and protease inhibitors (leupeptin and pepstatin, 10 μm). Cell suspensions were homogenized in a 2-ml Dounce homogenizer with 10 strokes using a loose-fitting pestle followed by 12 strokes using a tight-fitting pestle. The homogenates were subjected to a 10-min centrifugation at 2,500 g, followed by a 20 min centrifugation of the supernatant at 12,000 g. The microsomal-enriched fractions were stored at –80°C; prior to use, they were resuspended in by sonication for 6 sec at 1 gram-force in 20 mm HEPES buffer, pH 7.5, containing 0.2% deoxycholate (sodium salt) and protease inhibitors (leupeptin and pepstatin, 1 μg/ml). Glucosidase II was assayed as described (39Trombetta S.E. Simons J.F. Helenius A. Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound noncatalytic HDEL-containing subunit.J. Biol. Chem. 1996; 271: 27509-27516Google Scholar). The incubation mixture contained, in a total volume of 50 μl, 20 mm HEPES buffer, pH 7.5, 10 mm p nitrophenyl α-d-glucopyranoside, and sample. As a control for the specificity of the reaction, 2 mm deoxynojirimycin or castanospermine was included in similar assay mixtures. After incubating for 60 min at 37°C, the reaction was stopped by addition of 50 μl of 2 m Tris base, and the absorbance was monitored at 405 nm. UGGT was assayed based on Trombetta, Bosch, and Parodi (40Trombetta S.E. Bosch M. Parodi A.J. Glucosylation of glycoproteins by mammalian, plant, fungal, and trypanosomatid protozoa microsomal membranes.Biochemistry. 1989; 28: 8108-8116Google Scholar), using as substrate either native or denatured thyroglobulin; the latter was obtained by dialyzing the protein (20 mg/ml) for 8 h in 10 mm Tris-HCl, pH 8.0, containing 8 m urea, followed by dialysis in the same buffer without urea for 24 h with a change of buffer after 4 h. For the UGGT assay, the incubation mixture contained in a final volume of 100 μl: 20 mm Tris-HCl, pH 8.0, 300 μg native or denatured thyroglobulin, 10 mm CaCl2, 0.6% Triton X-100, 5 μm UDP-[14C]Glc, 40 μm deoxynojirimycin, and 50-100 μg microsomal protein. Reactions were carried out at 37°C for 40 min, and stopped by addition of 1 ml of 10% trichloroacetic acid (TCA). The mixtures were placed in a 60°C water bath for 10 min, allowed to cool, and poured onto glass filters (2.4 cm). The filters were washed with 10% TCA, dried by rinsing with 95% ethanol and acetone, and placed into scintillation vials containing 1 ml Solvable:H2O 1:1. After overnight incubation at room temperature, scintillation fluid was added and radioactivity was measured. For all lipase assays, cell lysates were prepared by sonication in 50 mm NH4OH-HCl buffer, pH 8.1, containing 10 U/ml heparin and either 0.2% deoxycholate or 0.1% Triton X-100. LPL was assayed using a triolein substrate prepared by polytron emulsification (41Nilsson-Ehle P. Schotz M.C. A stable, radioactive substrate emulsion for assay of lipoprotein lipase.J. Lipid Res. 1976; 17: 536-541Google Scholar). PL was assayed using an emulsion of triolein prepared by sonication (42Briquet-Laugier V. Ben-Zeev O. Doolittle M.H. Determining lipoprotein lipase and hepatic lipase activity using radiolabeled substrates.in: Doolittle M. Reue K. Lipase and Phospholipase Protocols. 109. Humana Press, Totowa, NJ1998: 81-94Google Scholar) with the following modifications: a mixture of 0.5 μg colipase (Boehringer), 0.8 μmol tauro-deoxycholic acid (Sigma), 0.2 μmol CaCl2 was prepared in a final volume of 50 μl water; enzyme source and ammonium buffer (50 mm ammonium-HCl, pH 8.0) were then added to complete the volume to 100 μl, and the reaction was initiated by addition of subs