Retention of mutant low density lipoprotein receptor in endoplasmic reticulum (ER) leads to ER stress

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). More than 50% of these mutations lead to receptor proteins that are completely or partly retained in the endoplasmic reticulum (ER). The mechanisms involved in the intracellular processing and retention of mutant LDLR are poorly understood. In the present study we show that the G544V mutant LDLR associates with the chaperones Grp78, Grp94, ERp72, and calnexin in the ER of transfected Chinese hamster ovary cells. Retention of the mutant LDLR was shown to cause ER stress and activation of the unfolded protein response. We observed a marked increase in the activity of two ER stress sensors, IRE1 and PERK. These results show that retention of mutant LDLR in ER induces cellular responses, which might be important for the clinical outcome of familial hypercholesterolemia.     

The conversion of apoB100 low density lipoprotein/high density lipoprotein particles to apoB100 very low density lipoproteins in response to oleic acid occurs in the endoplasmic reticulum and not in the Golgi in McA RH7777 cells

The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [35S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.     

Transmembrane lipid transfer is crucial for providing neutral lipids during very low density lipoprotein assembly in endoplasmic reticulum

Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [14C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [14C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.     

Apolipoprotein B production and very low density lipoprotein secretion by calf liver slices.

Secretion of triglycerides by the liver in ruminants as components of very low density lipoproteins particles is low as compared with that in primates or rodents. The rate-limiting steps for the hepatic export of very low density lipoproteins have been studied in liver slices to determine the origin of the low lipotropic capacity of calf liver compared to that of rat liver. The rates of production of apolipoprotein B (apo B) and albumin as well as the rate of secretion of VLDL-apolipoproteins were measured during 12-h incubation of liver slices in organo-culture using [35S]methionine-cysteine labeling. Hepatic apo B production was similar in the two animal species but the VLDL-apolipoprotein secretion rate for calf liver slices amounted to only 20% of that observed for rat liver slices. Although calf and rat liver slices synthesized similar amounts of total protein, the hepatic production of albumin, measured in cells and media, was much higher in calf than rat liver slices (around 2.7-fold), whereas the rate secretion of albumin was similar in the two species. Our results showed that the slow rate of secretion of VLDL by calf liver cells was not consecutive to a low rate of synthesis of apo B but rather to a defect in VLDL assembly and/or secretion.     

Apolipoprotein AII is a regulator of very low density lipoprotein metabolism and insulin resistance

Apolipoprotein AII (apoAII) transgenic (apoAIItg) mice exhibit several traits associated with the insulin resistance (IR) syndrome, including IR, obesity, and a marked hypertriglyceridemia. Because treatment of the apoAIItg mice with rosiglitazone ameliorated the IR and hypertriglyceridemia, we hypothesized that the hypertriglyceridemia was due largely to overproduction of very low density lipoprotein (VLDL) by the liver, a normal response to chronically elevated insulin and glucose. We now report in vivo and in vitro studies that indicate that hepatic fatty acid oxidation was reduced and lipogenesis increased, resulting in a 25% increase in triglyceride secretion in the apoAIItg mice. In addition, we observed that hydrolysis of triglycerides from both chylomicrons and VLDL was significantly reduced in the apoAIItg mice, further contributing to the hypertriglyceridemia. This is a direct, acute effect, because when mouse apoAII was injected into mice, plasma triglyceride concentrations were significantly increased within 4 h. VLDL from both control and apoAIItg mice contained significant amounts of apoAII, with approximately 4 times more apoAII on apoAIItg VLDL. ApoAII was shown to transfer spontaneously from high density lipoprotein (HDL) to VLDL in vitro, resulting in VLDL that was a poorer substrate for hydrolysis by lipoprotein lipase. These results indicate that one function of apoAII is to regulate the metabolism of triglyceride-rich lipoproteins, with HDL serving as a plasma reservoir of apoAII that is transferred to the triglyceride-rich lipoproteins in much the same way as VLDL and chylomicrons acquire most of their apoCs from HDL.     

Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very low density lipoprotein uptake

We investigated the roles of lipoprotein lipase and apolipoprotein E (apoE) secreted from human monocyte-derived macrophages in the uptake of very low density lipoproteins (VLDL). ApoCII-deficient VLDL were isolated from a patient with apoCII deficiency. The lipolytic conversion to higher density and the degradation of the apoCII-deficient VLDL by macrophages were very slight, whereas the addition of apoCII enhanced both their conversion and degradation. This suggests that the lipolysis and subsequent conversion of VLDL to lipoproteins of higher density are essential for the VLDL uptake by macrophages. VLDL incubated with macrophages obtained from subjects with E3/3 phenotype (E3/3-macrophages) showed a 17-fold greater affinity in inhibiting the binding of 2 micrograms/ml 125I-low density lipoprotein (LDL) to fibroblasts than native VLDL, whereas the incubation of VLDL with macrophages obtained from a subject with E2/2 phenotype (E2/2-macrophages) did not cause any increase in their affinity. Furthermore, 3 micrograms/ml 125I-VLDL obtained from a subject with E3/3 phenotype were degraded by E3/3-macrophages to a greater extent than by E2/2-macrophages (2-fold), indicating that VLDL uptake is influenced by the phenotype of apoE secreted by macrophages. From these results, we conclude that both lipolysis by lipoprotein lipase and incorporation of apoE secreted from macrophages alter the affinity of VLDL for the LDL receptors on the cells, resulting in facilitation of their receptor-mediated endocytosis.     

Regulation of ApoB secretion by the low density lipoprotein receptor requires exit from the endoplasmic reticulum and interaction with ApoE or ApoB

Apolipoprotein B (apoB) is required for the hepatic assembly and secretion of very low density lipoprotein (VLDL). The LDL receptor (LDLR) promotes post-translational degradation of apoB and thereby reduces VLDL particle secretion. We investigated the trafficking pathways and ligand requirements for the LDLR to promote degradation of apoB. We first tested whether the LDLR drives apoB degradation in an endoplasmic reticulum (ER)-associated pathway. Primary mouse hepatocytes harboring an ethyl-nitrosourea-induced, ER-retained mutant LDLR secreted comparable levels of apoB with LDLR-null hepatocytes, despite reduced secretion from cells expressing the wild-type LDLR. Additionally, treatment of cells with brefeldin A inhibited LDLR-dependent degradation. However, this rescue was reversible, and degradation of apoB occurred upon removal of brefeldin A. To characterize the lipoprotein reuptake pathway of degradation, we employed an LDLR mutant defective in constitutive endocytosis and internalization of apoB. This mutant was as effective in reducing apoB secretion as the wild-type LDLR. However, the effect was dependent on apolipoprotein E (apoE) as only the wild-type LDLR, and not the endocytic mutant, reduced apoB secretion in apoE-null cells. Treatment with heparin rescued a pool of apoB in cells expressing the endocytic mutant, indicating that reuptake of VLDL via apoE still occurs with this mutant. Finally, an LDLR mutant defective in binding apoB but not apoE reduced apoB secretion in an apoE-dependent manner. Together, these data suggest that the LDLR directs apoB to degradation in a post-ER compartment. Furthermore, the reuptake mechanism of degradation occurs via internalization of apoB through a constitutive endocytic pathway and apoE through a ligand-dependent pathway.     

Misfolded proteins traffic from the endoplasmic reticulum (ER) due to ER export signals

Most misfolded secretory proteins remain in the endoplasmic reticulum (ER) and are degraded by ER-associated degradation (ERAD). However, some misfolded proteins exit the ER and traffic to the Golgi before degradation. Using model misfolded substrates, with or without defined ER exit signals, we found misfolded proteins can depart the ER by continuing to exhibit the functional export signals present in the corresponding correctly folded proteins. Anterograde transport of misfolded proteins utilizes the same machinery responsible for exporting correctly folded proteins. Passive ER retention, in which misfolded proteins fail to exit the ER due to the absence of exit signals or the inability to functionally present them, likely contributes to the retention of nonnative proteins in the ER. Intriguingly, compromising ERAD resulted in increased anterograde trafficking of a misfolded protein with an ER exit signal, suggesting that ERAD and ER exit machinery can compete for binding of misfolded proteins. Disabling ERAD did not result in transport of an ERAD substrate lacking an export signal. This is an important distinction for those seeking possible therapeutic approaches involving inactivating ERAD in anticipation of exporting a partially active protein.     

The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER-associated degradation pathway

The hepatitis B virus precore protein is closely related to the nucleocapsid core protein but is processed distinctly in the cell and plays a different role in the viral cycle. Precore is addressed to the endoplasmic reticulum (ER) through a signal peptide, and the form present in the ER is the P22 protein. P22 is then cleaved in its C-terminal part to be secreted as HBe antigen. In addition, a cytosolic form of 22 kDa less characterized has been observed. Precore gene was shown to be implicated in viral persistence, but until now, the actual protein species involved has not been determined. Our work focuses on the cytosolic form of precore. Using human cells expressing precore and a convenient fractionation assay, we demonstrated that the cytosolic form is identical to the ER form and retrotransported in the cytoplasm through the ER-associated degradation pathway. This cellular machinery translocates misfolded proteins to the cytoplasm, where they are ubiquitinated on lysine residues and degraded by proteasome. We showed that precore escapes proteasome due to its low lysine content and accumulates in the cytosol. The role of this retrotransport was investigated. In the presence of precore, we found a specific redistribution of the Grp78/BiP chaperone protein to cytosol and demonstrated a specific interaction between precore and Grp78/BiP. Altogether, these data support the idea that the hepatitis B virus develops a strategy to take advantage of the ER-associated degradation pathway, allowing distinct subcellular localization and probably distinct roles for the viral precore protein.     

SG2NA is a regulator of endoplasmic reticulum (ER) homeostasis as its depletion leads to ER stress

SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.     

An endoplasmic reticulum (ER) stress-suppressive compound and its analogues from the mushroom Hericium erinaceum

Three new compounds, 3-hydroxyhericenone F (1), hericenone I (2), and hericenone J (3), were isolated from the mushroom Hericium erinaceum. The structures of 1-3 were determined by the interpretation of spectral data. Compound 1 showed the protective activity against endoplasmic reticulum (ER) stress-dependent Neuro2a cell death, however, compounds 2 and 3 did not.     

Grp78 is involved in retention of mutant low density lipoprotein receptor protein in the endoplasmic reticulum

The low density lipoprotein (LDL) receptor is responsible for removing the majority of the LDL cholesterol from the plasma. Mutations in the LDL receptor gene cause the disease familial hypercholesterolemia (FH). Approximately 50% of the mutations in the LDL receptor gene in patients with FH lead to receptor proteins that are retained in the endoplasmic reticulum (ER). Misfolding of mutant LDL receptors is a probable cause of this ER retention, resulting in no functional LDL receptors at the cell surface. However, the specific factors and mechanisms responsible for retention of mutant LDL receptors are unknown. In the present study we show that the molecular chaperone Grp78/BiP co-immunoprecipitates with both the wild type and two different mutant (W556S and C646Y) LDL receptors in lysates obtained from human liver cells overexpressing wild type or mutant LDL receptors. A pulse-chase study shows that the interaction between the wild type LDL receptor and Grp78 is no longer detectable after 2(1/2) h, whereas it persists for more than 4 h with the mutant receptors. Furthermore, about five times more Grp78 is co-immunoprecipitated with the mutant receptors than with the wild type receptor suggesting that Grp78 is involved in retention of mutant LDL receptors in the ER. Overexpression of Grp78 causes no major alterations on the steady state level of active LDL receptors at the cell surface. However, overexpression of Grp78 decreases the processing rate of newly synthesized wild type LDL receptors. This indicates that the Grp78 interaction is a rate-limiting step in the maturation of the wild type LDL receptor and that Grp78 may be an important factor in the quality control of newly synthesized LDL receptors.     

Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low density lipoprotein (apoprotein B-PIII)

Human low-density lipoproteins (LDL) were isolated from single donors by differential centrifugation between densities of 1.020 and 1.050 g/mL. The LDL were reduced and alkylated in 7 M guanidine hydrochloride, and the lipid was removed by multiple extractions in the cold with a mixture of diethyl ether and ethanol. Sedimentation studies on the resultant human apoprotein B (apoprotein B-PI) at low concentrations in 6.00 M guanidine hydrochloride showed a single sharp boundary with a sedimentation coefficient of 2.15 +/- 0.04 S at 25 degrees C, uncorrected for viscosity or density. Diffusion experiments performed in the same solvent at low speeds in the analytical ultracentrifuge gave a D25 = 0.694 +/- 0.043 Fick. Combining these values with an apparent specific volume of 0.703 mL/g yielded a molecular weight of 387 000, indistinguishable from that obtained by sedimentation equilibrium analysis in 7 M guanidine hydrochloride. Similar values were also obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rat very low density lipoproteins (VLDL), isolated from sera of Triton WR1339 treated animals, were used as the source of rat apoprotein B-PIII. The delipidated VLDL were solubilized in sodium dodecyl sulfate, and apoprotein B-PIII was isolated by Sepharose 4B chromatography. With appropriate corrections for density and viscosity, the behavior of rat apoprotein B-PIII was identical, upon analytical ultracentrifugation, in 6 and 7.7 M guanidine hydrochloride, corresponding to sedimentation and diffusion coefficients of 1.47 S and 0.92 Fick, respectively, in 6 M guanidine hydrochloride. These data may be combined to yield a molecular weight of 210 000. Similar values were obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.     

Specific heterodimer formation is a prerequisite for uroplakins to exit from the endoplasmic reticulum

Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER.     

Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein

The binding of native rabbit beta-very low density lipoproteins (beta-VLDL) to the low density lipoprotein receptor-related protein (LRP) requires incubation with exogenous apolipoprotein (apo) E. Inclusion of a mixture of the C apolipoproteins in the incubation inhibits this binding. In the present study, the ability of the individual C apolipoproteins (C-I, C-II, and C-III) to block binding of beta-VLDL to the LRP was examined by measuring cholesteryl ester formation in mutant fibroblasts that lack low density lipoprotein receptors or by measuring binding to the LRP using ligand blotting. In each assay, both apoC-I and apoC-II inhibited binding; apoC-I was the more effective inhibitor. Apolipoprotein C-III had no effect on binding activity, regardless of its sialylation level. Binding of human apoE to rabbit beta-VLDL in the absence or presence of human apoC-I, apoC-II, and monosialo-apoC-III was also determined, by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of these studies are consistent with a mechanism in which exogenous human apoE displaces the endogenous apoE and the beta-VLDL particle becomes enriched with apoE (by 4.2-fold in this study). At this higher apoE content, the beta-VLDL bound to the LRP. Inclusion of apoC-I, apoC-II, or apoC-III in the incubation mixture resulted in a differential displacement of apoE from the beta-VLDL; however, at the concentrations examined, only apoC-I and apoC-II were capable of displacing sufficient apoE to abolish binding to LRP.     

Resistance of a very low density lipoprotein subpopulation from familial dysbetalipoproteinemia to in vitro lipolytic conversion to the low density lipoprotein density fraction

In vitro lipolysis of very low density lipoprotein (VLDL) from normolipidemic and familial dysbetalipoproteinemic plasma by purified bovine milk lipoprotein lipase was studied using the combined single vertical spin and vertical autoprofile method of lipoprotein analysis. Lipolysis of normolipidemic plasma supplemented with autologous VLDL resulted in the progressive transformation of VLDL to low density lipoprotein (LDL) via intermediate density lipoprotein (IDL) with the transfer of the excess cholesterol to high density lipoprotein (HDL). At the end of 60 min lipolysis, 92-96% of VLDL triglyceride was hydrolyzed, and, with this process, greater than 95% of the VLDL cholesterol and 125-I-labeled VLDL protein was transferred from the VLDL to the LDL and HDL density region. When VLDL from the plasma of an individual with familial dysbetalipoproteinemia was substituted for VLDL from normolipidemic plasma, less than 50% of the VLDL cholesterol and 65% of 125I-labeled protein was removed from the VLDL density region, although 84-86% of VLDL triglyceride was lipolyzed. Analysis of familial dysbetalipoproteinemic VLDL fractions from pre- and post-lipolyzed plasma showed that the VLDL remaining in the postlipolyzed plasma (lipoprotein lipase-resistant VLDL) was richer in cholesteryl ester and tetramethylurea-insoluble proteins than that from prelipolysis plasma; the major apolipoproteins in the lipoprotein lipase-resistant VLDL were apoB and apoE. During lipolysis of normolipidemic VLDL containing trace amounts of 125I-labeled familial dysbetalipoproteinemic VLDL, removal of VLDL cholesterol was nearly complete from the VLDL density region, while removal of 125I-labeled protein was only partial. A competition study for lipoprotein lipase, comparing normolipidemic and familial dysbetalipoproteinemic VLDL to an artificial substrate ([3H]triolein), revealed that normolipidemic VLDL is clearly better than familial dysbetalipoproteinemic VLDL in competing for the release of 3H-labeled free fatty acids. The results of this study suggest that, in familial dysbetalipoproteinemic individuals, a subpopulation of VLDL rich in cholesteryl ester, apoB, and apoE is resistant to in vitro conversion by lipoprotein lipase to particles having LDL-like density. The presence of this lipoprotein lipase-resistant VLDL in familial dysbetalipoproteinemic subjects likely contributes to the increased level of cholesteryl ester-rich VLDL and IDL in the plasma of these subjects.     

Apolipoprotein B mediates the capacity of low density lipoprotein to suppress neutrophil stimulation by particulates

Low density lipoprotein (LDL) inhibits phagocytosis of certain negatively charged particulates and also inhibits subsequent cellular secretory and oxidative responses to these particulates. In the present work, we have defined the structural features of LDL involved in this activity. Starch-heptane extraction depleted greater than 95% of neutral lipids but had little effect on the capacity of LDL to inhibit monosodium urate crystal- or polystyrene latex bead-induced neutrophil chemiluminescence (CL). Liposomes containing gamma-palmitoyl-beta-oleoylphosphatidylcholine (PC) with unesterified cholesterol (PC:cholesterol = 2:1), PC and sphingomyelin (PC:sphingomyelin = 2.3:1), or PC alone lacked the capacity to inhibit urate-induced CL. However, incorporation of apoB-100 into liposomes via cholate dialysis rendered them nearly as inhibitory for urate-induced neutrophil CL as LDL on a protein weight basis. Moreover, delipidated apoB-100, containing less than 3% residual phospholipid, inhibited neutrophil responses to urate crystals or latex beads (degranulation and superoxide anion release) in a stimulus-specific manner. Modifications of the lysine residues of apoB (e.g. acetylation) reduced both the capacity of LDL to inhibit urate crystal-induced CL and to bind to urate crystals. The effects of apoB lysine residue modification were reversible, proportional to the extent of modification, and were not attributable to alteration of the net charge of apoB. Thus, the apoB-100 of LDL both mediates and shares the capacity of native LDL to inhibit certain neutrophil responses to particulates.     

Cutting edge: Tapasin is retained in the endoplasmic reticulum by dynamic clustering and exclusion from endoplasmic reticulum exit sites

Tapasin retains empty or suboptimally loaded MHC class I molecules in the endoplasmic reticulum (ER). However, the molecular mechanism of this process and how tapasin itself is retained in the ER are unknown. These questions were addressed by tagging tapasin with the cyan fluorescent protein or yellow fluorescent protein (YFP) and probing the distribution and mobility of the tagged proteins. YFP-tapasin molecules were functional and could be isolated in association with TAP, as reported for native tapasin. YFP-tapasin was excluded from ER exit sites even after accumulation of secretory cargo due to disrupted anterograde traffic. Almost all tapasin molecules were clustered, and these clusters diffused freely in the ER. Tapasin oligomers appear to be retained by the failure of the export machinery to recognize them as cargo.     

Apolipoprotein E mediates binding of normal very low density lipoprotein to heparin but is not required for high affinity receptor binding

The relationship between the cholesteryl ester content of normal human very low density lipoprotein (VLDL) and its ability to bind to apolipoprotein E (apoE), heparin, and the low density lipoprotein (LDL) receptor have been compared. Plasma VLDL were separated by heparin affinity chromatography into two fractions: one with apoE and one without. Both fractions had the same cholesteryl ester content relative to apolipoprotein B (apoB). LDL, on the other hand, had a greater cholesteryl ester content. VLDL were modified by lipolysis to express the ability to bind apoE (Ishikawa, Y., Fielding, C. J., and Fielding, P. E. (1988) J. Biol. Chem. 263, 2744-2749). Lipolyzed VLDL with or without apoE were compared for their ability to bind to heparin or the up-regulated fibroblast LDL receptor. Lipolyzed VLDL bound with the same affinity to the receptor whether or not the particles contained apoE. ApoB, not apoE, appears then to be the important ligand for normal VLDL. On the other hand, modified VLDL without apoE, even though binding to the LDL receptor, did not bind to heparin. These data suggest that apoE mediates heparin binding in normal VLDL, that apoB mediates receptor binding, and that the cholesteryl ester content of VLDL is not a factor in the induction of the ability to bind apoE.