Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase

Lipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 h of recovery period after the depletion of LPL with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.     

Glycosylation of lipoprotein lipase in human subcutaneous and omental adipose tissues.

Human adipose tissues from the abdomen (subcutaneous), thigh (subcutaneous) and omentum were incubated for 2 h with [35S]methionine. Then glycosylation of lipoprotein lipase (LPL) was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endoglycosidase H (endo H)-digested subunits of the 35S-labeled lipase. Adipose tissues from the abdomen, thigh, and omentum all synthesized LPL subunits with Mr = 57,000 composed of two types of subunits. One type was partially endo H-sensitive yielding a product with Mr = 55,000, indicating that it had one endo H-resistant and one endo H-sensitive oligosaccharide chain. The other type of subunit was totally endo H-sensitive yielding a product with Mr = 52,000. Subcutaneous adipose tissues contained nearly equal amounts of partially and totally endo H-sensitive subunits of LPL, whereas omental adipose tissues contained mainly partially endo H-sensitive subunits of LPL.     

Retention of glucose by N-linked oligosaccharide chains impedes expression of lipoprotein lipase activity: effect of castanospermine.

The effect of castanospermine (CSTP), an inhibitor of glucosidase I, on processing, activity, and secretion of lipoprotein lipase was studied in 3T3-L1 adipocytes. Processing was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endoglycosidase H (endo H)-digested subunits of lipoprotein lipase from cells incubated 1-2 h with [35S]methionine. Lipoprotein lipase in untreated cells consisted of two groups of subunits, M(r) = 55,000-58,000 and M(r) = 53,000-55,000. The heavier subunits were endo H-resistant, whereas the others were either totally or partially endo H-sensitive. The lipase secreted by untreated cells contained primarily endo H-resistant subunits. Immunofluorescent studies showed that lipoprotein lipase accumulated in Golgi in untreated cells. CSTP, 100 micrograms/ml for 18 h, decreased intracellular lipase activity by 80% and decreased secretion of lipase activity by 91%. Most of the lipase subunits in CSTP-treated cells were totally endo H-sensitive with M(r) = 57,000, some were partially endo H-sensitive, and a trace was endo-H resistant. Totally endo H-sensitive subunits in CSTP-treated cells had a M(r) 2,000-4,000 larger than that in untreated cells, indicating impaired trimming of sugar residues from oligosaccharide chains of the lipase in CSTP-treated cells. The small amount of lipase secreted by CSTP-treated cells consisted primarily of partially endo H-sensitive subunits, with one sensitive and one resistant chain per subunit. Immunofluorescent studies showed that lipoprotein lipase was excluded from Golgi in CSTP-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of nordihydroguaiaretic acid on the secretion of lipoprotein lipase

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, inhibits the secretion of proteins and causes the redistribution of resident Golgi proteins into the endoplasmic reticulum (ER). In this study, the effect of NDGA on lipoprotein lipase (LPL) secretion was investigated in 3T3-L1 adipocytes, and compared with those of brefeldin A (BFA), a well-known fungal metabolite that exhibits similar ER-Golgi redistribution. Both BFA and NDGA blocked secretions of LPL. In the presence of BFA, the active and dimeric LPL was accumulated in adipocytes. After endoglycosidase H (endo H) digestion, the proportion of LPL subunits with partially endo H-sensitive oligosaccharide was significantly increased with BFA. However, in the presence of NDGA, the cellular LPL became inactive, and only the endo H-sensitive fraction of the LPL subunit was observed. An increase of the aggregated forms was observed in the fractions of the sucrose-density gradient ultracentrifugation. These properties of LPL in the NDGA-treated cells were similar to those of LPL that is retained in ER, and the effects of NDGA could not be reversed by BFA. These results indicate that the inhibitory mechanism of NDGA on the LPL secretion is functionally different from the ER-Golgi redistribution that is induced by BFA.     

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.     

Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipase-deficient cld/cld mice

Combined lipase deficiency (cld) is a recessive mutation which causes a severe deficiency of lipoprotein lipase and hepatic lipase activities and lethal hypertriacylglycerolemia within 3 days in newborn mice. The effect of this genetic defect on lipoprotein lipase was studied in primary cultures of brown adipocytes derived from tissue of newborn mice. Cells cultured from cld/cld mice replicated, accumulated triacylglycerol, and differentiated into adipocytes at normal rates. Lipoprotein lipase activity in unaffected cells was detectable on Day 0 of confluence and increased to 1.3 units/mg DNA by Day 6, while that in cld/cld cells was less than 4% of that in unaffected cells on Days 4-6. Unaffected cells released 1.2% of their lipase activity in 30 min in the absence of heparin, and 11% in 10 min in the presence of heparin, whereas cld/cld cells released no lipase activity. cld/cld cells contained 2-3 times as much lipoprotein lipase protein as unaffected cells, and released no lipase protein to the medium. Immunofluorescent lipoprotein lipase was not detectable in unaffected adipocytes unless lipase secretion was blocked with monesin, causing retention of the lipase in Golgi. cld/cld adipocytes, in contrast, contained immunofluorescent lipoprotein lipase distributed in a diffuse reticular pattern, indicating retention of lipase in endoplasmic reticulum. Lipoprotein lipase immunoprecipitated from cells incubated 1-3 h with [35S]methionine was digested with or without endoglycosidase H (endo H) or F, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipoprotein lipase in unaffected cells (Mr = 56,000-58,000) consisted of three glycosylated forms, of which the most prevalent was endo H-resistant, the next was totally endo H-sensitive, and the least was partially endo H-sensitive. In contrast, lipoprotein lipase in cld/cld cells (Mr = 56,000) consisted of a single, totally endo H-sensitive form. Lipoprotein lipase in both groups of cells contained two oligosaccharide chains. Chromatography studies with heparin-Sepharose indicated that at least some of the lipoprotein lipase in cld/cld cells was dimerized. The findings demonstrate that brown adipocytes cultured from cld/cld mice synthesize lipoprotein lipase with two high mannose oligosaccharide chains, but it is inactive and retained in endoplasmic reticulum. Whether the cld mutation affects primarily processing of oligosaccharide chains of lipoprotein lipase in endoplasmic reticulum, transport of the lipase from the reticulum, or some other process, is to be resolved.     

Intracellular transport, sorting, and turnover of acetylcholinesterase. Evidence for an endoglycosidase H-sensitive form in Golgi apparatus, sarcoplasmic reticulum, and clathrin-coated vesicles and its rapid degradation by a non-lysosomal mechanism

Tissue-cultured muscle cells synthesize several oligomeric forms of acetylcholinesterase (AChE) destined for the cell surface or secretion. Previous studies on the biogenesis of AChE polypeptide chains have shown that only a small fraction become assembled into catalytically active oligomers which transit the Golgi apparatus and acquire endoglycosidase H (endo H) resistance. Most of the AChE polypeptides remain endo H-sensitive and are rapidly degraded intracellularly. We now show that all newly synthesized AChE polypeptides are transported from the rough endoplasmic reticulum to the Golgi apparatus where they acquire N-acetylglucosamine. However, approximately 80% of these AChE polypeptides remain endo H-sensitive and are degraded intracellularly with a half-life of about 1.5 h by a mechanism which is insensitive to lysosomotropic agents. These endo H-sensitive AChE molecules can be chased into clathrin-coated vesicles and/or the sarcoplasmic reticulum prior to degradation. Pulse-chase studies of isotopically labeled or catalytically active AChE molecules suggest that there are at least two discreet populations of clathrin-coated vesicles which leave the Golgi, one whose origin is cis/medial and one whose origin is trans. These studies indicate the existence of a post-rough endoplasmic reticulum, non-lysosomal degradative pathway for intra-luminal proteins and suggest that post-translational events at the levels of protein sorting and degradation may play a role in regulating the abundance of exportable proteins.     

The non-classical N-glycan processing pathway of bovine brain ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is brain specific and not due to mannose-6-phosphorylation

Ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is a glycosylphosphatidylinositol (GPI)-anchored alkaline lysophospholipase C which is predominantly expressed in brain myelin and kidney. Due to shedding of the GPI-anchor eNPP6 occurs also as a soluble isoform (s-eNPP6). eNPP 6 consists of two identical monomers of 55 kDa joined by a disulfide bridge, and possesses four N-glycans in each monomer. In brain s-eNPP6 the N-glycans are mainly hybrid and high mannose type structures, reminiscent of processed mannose-6-phosphorylated glycans. Here we completed characterization of the site-specific glycan structures of bovine brain s-eNPP6, and determined the endo H-sensitivity glycan profiles of s-eNPP6 from bovine liver and kidney. Whereas in brain s-eNPP6 all of the N-glycans were endo H-sensitive, in liver and kidney only one of the glycosylation sites was occupied by an endo H-sensitive glycan, likely N406, which is located within the cleft formed by the dimer interface. Thus, the non-classical glycan processing pathway of brain eNPP 6 is not due to mannose-6-phosphorylation, suggesting that there is an alternative Golgi glycan-processing pathway of eNPP6 in brain. The resulting brain-specific expression of accessible hybrid and oligomannosidic glycans may be physiologically important within the cell–cell communication system of the brain.     

Different oligosaccharide processing of the membrane-integrated and the secretory form of gp 80 in rat liver.

Rat liver synthesizes a glycoprotein with Mr of 80.000 (gp 80) which is partly inserted into the plasma membrane and partly secreted into the serum. The membrane-integrated and the secretory form of this glycoprotein have an identical peptide pattern, but different N-linked glycans. Whereas gp 80 from the serum is glycosylated with complex-type oligosaccharides, gp 80 from the plasma membrane has high mannose glycans. Phase separation with Triton X-114 showed that membrane-integrated gp 80 contains hydrophobic portions, whereas secretory gp 80 has hydrophilic properties. Intracellular transport and oligosaccharide processing of gp 80 were studied in vivo in the endoplasmic reticulum, the Golgi apparatus and plasma membranes of rat liver and in serum using pulse-chase labeling with L-[35S]methionine and immunoprecipitation. Peak labeling of gp 80 was reached in the endoplasmic reticulum 10 min after the pulse, in the Golgi apparatus 20 min later, and in the plasma membrane after 2 h; in the serum the specific radioactivity was steadily increasing during the experiment. Gp 80 of the endoplasmic reticulum was completely sensitive to endo-beta-N-glucosaminidase H (endo H), but simultaneously occurred in the Golgi apparatus in an endo H-sensitive and endo H-resistant form. The endo H-sensitive form was transported to the plasma membrane, the endo H-resistant species secreted into the serum. Conversion from the endo H-sensitive to the endo H-resistant form was completed within 10 min after transfer of gp 80 to the Golgi apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein lipase (LPL) strongly links native and oxidized low density lipoprotein particles to decorin-coated collagen. Roles for both dimeric and monomeric forms of LPL

Low density lipoprotein (LDL) and oxidized LDL are associated with collagen in the arterial intima, where the collagen is coated by the small proteoglycan decorin. When incubated in physiological ionic conditions, decorin-coated collagen bound only small amounts of native and oxidized LDL, the interaction being weak. When decorin-coated collagen was first allowed to bind lipoprotein lipase (LPL), binding of native and oxidized LDL increased dramatically (23- and 7-fold, respectively). This increase depended on strong interactions between LPL that was bound to the glycosaminoglycan chains of the collagen-bound decorin and native and oxidized LDL (kDa 12 and 5.9 nM, respectively). To distinguish between binding to monomeric (inactive) and dimeric (catalytically active) forms of LPL, affinity chromatography on heparin columns was conducted, which showed that native LDL bound to the monomeric LPL, whereas oxidized LDL, irrespective of the type of modification (Cu(2+), 2, 2'-azobis(2-amidinopropane)hydrochloride, hypochlorite, or soybean 15-lipoxygenase), bound preferably to dimeric LPL. However, catalytic activity of LPL was not required for binding to oxidized LDL. Finally, immunohistochemistry of atherosclerotic lesions of human coronary arteries revealed specific areas in which LDL, LPL, decorin, and collagen type I were present. The results suggest that LPL can retain LDL in atherosclerotic lesions along decorin-coated collagen fibers.     

Membrane traffic in animal cells: cellular glycoproteins return to the site of Golgi mannosidase I

The recycling of cellular glycoproteins to the site of Golgi mannosidase I, an enzyme of asparagine-linked oligosaccharide synthesis, was studied in K562 human erythroleukemia cells. Cells were metabolically labeled in the presence of deoxymannojirimycin, a reversible inhibitor of Golgi mannosidase I. This generates glycoproteins with immature oligosaccharides in their normal locations. Transport to the mannosidase I compartment was then assessed by testing for the conversion of oligosaccharides into mature forms during reculture without deoxymannojirimycin. Transferrin receptor (TfR) was acted on by mannosidase I during reculture, suggesting that it returned to the region of the Golgi complex where this enzyme resides. The slow rate of this transport (t1/2 greater than 6 h) implies that it is probably different than TfR movement during transferrin internalization (t1/2 = 10-20 min) and TfR transport to the sialyltransferase compartment in the Golgi complex (t1/2 = 2-3 h) (Snider, M. D., and O. C. Rogers, 1985, J. Cell Biol., 100:826-834). The total cell glycoprotein pool was also transported to the mannosidase I compartment with a half-time of 4 h. Because this transport is 5-10 times faster than the rate of de novo glycoprotein synthesis in these cells, it is likely that most of the glycoprotein traffic through the Golgi complex is composed of recycling molecules.     

The order of processing events in mouse mammary tumor virus envelope protein maturation: implications for the location of the glucocorticoid-regulated step.

Treatment of the W7MG1 mouse T lymphoma cell line with glucocorticoid stimulates directly or indirectly two observable steps in the processing of mouse mammary tumor virus (MMTV) envelope glycoprotein precursor Pr74: cleavage of Pr74 to yield the mature glycoprotein products gp52 and gp33, and processing of the N-linked oligosaccharides to endoglycosidase H (endo H)-resistant forms found on the mature products but not on the precursor. Therefore, the primary hormone-regulated event in this pathway must occur at or before the point where MMTV envelope proteins become endo H resistant. Pulse-chase analyses identified a novel endo H-resistant 80-kDa species (designated gp80) as a processing intermediate. Therefore, in contrast to conclusions drawn for the envelope proteins of several other retroviruses, proteolytic cleavage of MMTV envelope proteins occurs after acquisition of endo H resistance. Also, proteolytic cleavage cannot be the primary hormone-regulated step. Second, inhibition of mannosidase II by the drug swainsonine did not prevent Pr74 from being proteolytically processed, thus demonstrating that conversion of oligosaccharide chains from endo H-sensitive to -resistant forms was not a prerequisite for proteolytic cleavage. Therefore, the requisite hormone-regulated event in MMTV glycoprotein processing must precede both acquisition of endo H resistance and proteolytic cleavage. This places the regulated event in the endoplasmic reticulum or early Golgi.     

In vitro translation and processing of human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase

Human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase (gamma-GT), a 120 ka single-chain glycoprotein, is much larger than the expected precursor of the dimeric enzyme in other human tissues. However, the Hep G2 gamma-GT mRNA encodes a 63 kDa peptide, similar to that of rat gamma-GT mRNA product and to the predicted, unglycosylated precursor of the enzyme in human tissues. Translation in presence of dog pancreas microsomes results in processing of the 63 kDa to an 80 kDa core-glycosylated species which is subsequently cleaved to 58 and 22 kDa subunits resembling those in other human tissues. The unusually large Mr of gamma-GT in Hep G2 would thus seem to be due to further glycosylation and processing in the Golgi. A deficiency of the processing protease is the most likely reason for the persistence of the single-chain form of gamma-GT in Hep G2 cells.     

Maturation of lipoprotein lipase in the endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates.

The maturation of lipoprotein lipase (LPL) into a catalytically active enzyme was believed to occur only after its transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To test this hypothesis, LPL located in these two subcellular compartments was separated and compared. Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. The latter forms were further separated by beta-ricin chromatography and were found to have comparable activities per unit of LPL mass. Thus, LPL must reach a functional conformation in the ER. Active LPL, regardless of its cellular location, exhibited the expected dimer conformation. However, inactive LPL, found only in the ER, was highly aggregated. Kinetic analysis indicated a concurrent formation of LPL dimer and aggregate and indicated that the two forms have dissimilar fates. Whereas the dimer remained stable even when confined to the ER, the aggregate was degraded. Degradation rates were not affected by proteasomal or lysosomal inhibitors but were markedly reduced by ATP depletion. Lowering the redox potential in the ER by dithiothreitol caused the dimer to associate with calnexin, BiP, and protein-disulfide isomerase to form large, inactive complexes; dithiothreitol removal induced complex dissociation with restoration of the functional LPL dimer. In contrast, the LPL aggregate was only poorly associated with ER chaperones, appearing to be trapped in an irreversible, inactive conformation destined for ER degradation.     

Post-translational processing and activation of insulin and EGF proreceptors

We have investigated the role of glycosylation on the post-translational processing of the insulin, and EGF proreceptor polypeptides. Following translation of the insulin proreceptor, by 3T3-L1 adipocytes, about 1.5 h are required for its conversion into active receptor; an additional 1.5 h are needed for the active receptor to reach the plasma membrane. During this 3-hour period the proreceptor undergoes a complex series of processing events, glycosylation being an essential processing step. Thus, treatment of 3T3-L1 adipocytes with tunicamycin caused the loss of cellular insulin binding activity and the accumulation of an inactive aglyco-proreceptor. Similarly, it was demonstrated in human A431 epidermoid carcinoma cells that the initial EGF-proreceptor (160 kDa) translation product undergoes a slow (t 1/2 = 30 min) processing step by which ligand (EGF) binding activity was acquired. It was shown that N-linked core oligosaccharide addition is essential for this critical processing step and the acquisition of EGF binding activity. This was found not to require the conversion of high mannose chains to complex chains which have been capped with fucose and sialic acid. Possible explanations for this activation in terms of translocation of intermediates and/or formation of disulfide bonds are discussed. To investigate post-translational processing of normal insulin proreceptor and the role of glycosylation in active receptor formation, metabolic labeling experiments were conducted. The first 35S-methionine-labeled intermediate detected is a 190 kDa polypeptide (proreceptor) which is rapidly (t 1/2 = 15 min) processed into a 210 kDa species. Both polypeptides contain N-linked core oligosaccharide chains, but in the latter case these chains appear to contain terminal N-acetylglucosamine. The 210 kDa precursor is converted slowly (t 1/2 = 2 h) by proteolytic processing into a 125 kDa (alpha') and 83 kDa (beta') species. Immediately prior to insertion into the plasma membrane, 3 h after its synthesis, the alpha' and beta' precursors are converted to mature receptor comprised of alpha-(135 kDa) and beta-(95 kDa) subunits. The 125 kDa alpha'- and 83 kDa beta'-subunit precursors are endoglycosidase H-sensitive and their oligosaccharide chains do not contain terminal sialic acid. Just prior to insertion into the plasma membrane the alpha' and beta' precursors are sialylated, apparently in the Golgi apparatus, giving rise to the 135 kDa alpha and 95 kDa beta receptor subunits and become Endo H-resistant and neuraminidase-sensitive. A proposed sequence of post-translational processing events for the insulin proreceptor is shown in Figure 10.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain.

Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using insight ii based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations - a closed, inactive form and an open, active form - differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl-terminal domain, allow substrate access to the active site. To dissect the structure-function relationships of the LPL carboxyl-terminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a 'head-to-tail' orientation, two inactive LPL mutants - a catalytic site mutant (S132T) and a substrate-recognition mutant (W390A/W393A/W394A) - were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection, 50% of the wild-type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substrate-recognition site on the other.     

Endoplasmic reticulum-associated degradation-induced dissociation of class II invariant chain complexes containing a glycosylation-deficient form of p41

The quality control system in the secretory pathway can identify and eliminate misfolded proteins through endoplasmic reticulum-associated degradation (ERAD). ERAD is thought to occur by retrotranslocation through the Sec61 complex into the cytosol and degradation by the proteasome. However, the extent of disassembly of oligomeric proteins and unfolding of polypeptide chains that is required for retrotranslocation is not fully understood. In this report we used a glycosylation mutant of the p41 isoform of invariant chain (Ii) to evaluate the ability of ERAD to discriminate between correctly folded and misfolded subunits in an oligomeric complex. We show that loss of glycosylation at position 239 of p41 does not detectably affect Ii trimerization or association with class II but does result in a defect in endoplasmic reticulum export of Ii that ultimately leads to its degradation via the ERAD pathway. Although class II associated with the mutated form of p41 is initially retained in the endoplasmic reticulum, it is subsequently released and traffics through the Golgi to the plasma membrane. ERAD-mediated degradation of the mutant p41 is dependent on mannose trimming and inhibition of mannosidase I stabilizes Ii. Interestingly, inhibition of mannosidase I also results in prolonged association between the mutant Ii and class II, indicating that complex disassembly and release of class II is linked to mannosidase-dependent ERAD targeting of the misfolded Ii. These results suggest that the ERAD machinery can induce subunit disassembly, specifically targeting misfolded subunits to degradation and sparing properly folded subunits for reassembly and/or export.     

Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport

The biosynthesis and turnover of lipoprotein lipase (LPL) have been investigated in adipose 3T3-F442A cells labeled with [35S]methionine. Pulse-chase experiments, endo-beta-N-acetylglucosaminidase H treatment, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have indicated that LPL is synthesized in the endoplasmic reticulum as a glycoprotein of Mr = 55,500 bearing two N-oligosaccharide side chains of the high mannose-type. This precursor form of LPL is transported within 10 min to the Golgi apparatus, and this event is accompanied by the formation of a mature species of Mr = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a Mr = 51,000 protein similar to that observed after treatment of the Mr = 55,500 precursor form or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells since, after a labeling period of 2 h, only the Mr = 58,000 species is detected. It is shown that only 20% of the newly synthesized molecules of Mr = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL takes place whereas the intracellular degradation is arrested. Heparin is able to mobilize intracellular LPL without changing the rate of LPL export from the endoplasmic reticulum to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae shows that the Mr = 55,500 precursor form is present as a monomer (s = 4.1 S), whereas the Mr = 58,000 mature form is present as a homodimer (s = 6.8 S) to which LPL activity is associated. The results are interpreted as LPL being transiently stored under a dimeric form before its degradation. A sorting process of LPL in the Golgi apparatus, followed by its entry either mainly in a regulated pathway or in a constitutive pathway, is proposed.     

Effects of brefeldin A on the processing of viral envelope glycoproteins in murine erythroleukemia cells.

This paper documents the effects of brefeldin A (BFA) on the processing and transport of viral envelope glycoproteins in a retrovirus-transformed murine erythroleukemia (MEL) cell line. BFA is a fungal metabolite that disrupts intracellular membrane traffic at the endoplasmic reticulum (ER)-Golgi complex junction. In MEL cells, BFA inhibited the processing of the newly synthesized precursor, gPr90env, of the murine leukemia virus envelope protein, gp70, and curtailed the budding of virions into the culture medium by blocking the transport of this protein out of the ER. The block resulted in the intracellular accumulation of gPr90env and two putative products of its processing (78 and 66 kDa). The results of endoglycosidase (endo) H and D digestion of the viral glycoproteins in the presence and absence of BFA indicated that (i) there was no glycoprotein processing during the first approximately 2 h of the BFA block; (ii) active Golgi enzymes relocated to the ER in approximately 2 h during BFA treatment, resulting in the production of partially endo H-resistant forms of the spleen focus-forming virus glycoprotein, gp55 (in controls, this glycoprotein was generally retained in the ER as an endo H-sensitive entity); and (iii) proteolytic processing of gPr90env to gp70 occurred prior to the acquisition of endo H resistance and at approximately the same time as endo D sensitivity (i.e. in a cis Golgi compartment). In control cells, the spleen focus-forming virus glycoprotein, gp55, underwent turnover with a half-life of approximately 5 h. In contrast, its turnover was considerably slower during BFA treatment (t 1/2 = approximately 20 h), suggesting that transport of gp55 out of the ER was required for its degradation or that BFA afforded it protection from proteolysis within the ER.     

Structural basis for activation of fibroblast growth factor signaling by sucrose octasulfate

Sucrose octasulfate (SOS) is believed to stimulate fibroblast growth factor (FGF) signaling by binding and stabilizing FGFs. In this report, we show that SOS induces FGF-dependent dimerization of FGF receptors (FGFRs). The crystal structure of the dimeric FGF2-FGFR1-SOS complex at 2.6-A resolution reveals a symmetric assemblage of two 1:1:1 FGF2-FGFR1-SOS ternary complexes. Within each ternary complex SOS binds to FGF and FGFR and thereby increases FGF-FGFR affinity. SOS also interacts with the adjoining FGFR and thereby promotes protein-protein interactions that stabilize dimerization. This structural finding is supported by the inability of selectively desulfated SOS molecules to promote receptor dimerization. Thus, we propose that SOS potentiates FGF signaling by imitating the dual role of heparin in increasing FGF-FGFR affinity and promoting receptor dimerization. Hence, the dimeric FGF-FGFR-SOS structure substantiates the recently proposed "two-end" model, by which heparin induces FGF-FGFR dimerization. Moreover, the FGF-FGFR-SOS structure provides an attractive template for the development of easily synthesized SOS-related heparin agonists and antagonists that may hold therapeutic potential.