Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[(32)P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.     

Potentiation of ABCA3 lipid transport function by ivacaftor and genistein

ABCA3 is a phospholipid transporter implicated in pulmonary surfactant homoeostasis and localized at the limiting membrane of lamellar bodies, the storage compartment for surfactant in alveolar type II cells. Mutations in ABCA3 display a common genetic cause for diseases caused by surfactant deficiency like respiratory distress in neonates and interstitial lung disease in children and adults, for which currently no causal therapy exists. In this study, we investigated the effects of ivacaftor and genistein, two potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR), on ABCA3‐specific lipid transport function. Wild‐type (WT) and functional ABCA3 mutations N568D, F629L, G667R, T1114M and L1580P were stably expressed in A549 cells. Three‐dimensional modelling predicted functional impairment for all five mutants that was confirmed by in vitro experiments (all <14% of WT functional activity). Treatment with potentiators rescued the mutants N568D (up to 114% of WT), F629L (up to 47% of WT), and G667R (up to 60% of WT), the latter variation needing higher concentrations of genistein, showing reduced affinity of the potentiator to the mutant protein. Our results present a first proof that functional ABCA3 mutations are rescued by CFTR potentiators, making them a potential therapeutical option for patients suffering from surfactant deficiency due to ABCA3 mutations.     

Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency

The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.     

Cellular localization and trafficking of the human ABCA1 transporter

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.     

Metabolic labelling of choline phospholipids probes ABCA3 transport in lamellar bodies

In the metabolism of pulmonary surfactant, the ATP-binding cassette sub-family A member 3 (ABCA3) is a crucial protein in the formation of the storage compartment for surfactant, the lamellar body (LB), and the transport of phospholipids in it. Mutations in ABCA3 not only disturb surfactant metabolism but also cause chronic interstitial lung diseases. Assays for ABCA3 transport function are needed to investigate pathophysiology of the mutations and treatment options for the patients.We metabolically labeled choline (Cho) head phospholipids with the Cho analogue, propargyl-Cho. The universal incorporation of propargyl-Cho was confirmed by mass spectrometry and labeled lipids were visualized in confocal microscopy by click reaction with an azide fluorophore. After pulse-labeling propargyl-Cho labeled lipids accumulated in ABCA3+ vesicles in a time and concentration dependent manner. When treated with the choline kinase inhibitor MN58b during the first 12 h, the lipids intensity inside ABCA3+ vesicles decreased, whereas intensity was unchanged when treated after 12 h. Miltefosine, a substrate of ABCA3, decreased the incorporation of labeled lipids in ABCA3+ vesicles at all time points. The lipids intensity inside the mutated (p.N568D or p.L1580P) ABCA3+ vesicles was decreased compared to wild type, while the intensity outside of vesicles showed no difference.Propargyl-Cho can metabolically pulse-label Cho phospholipids. Visualization and quantification of fluorescence intensity of the labeled lipids inside ABCA3+ vesicles at equilibrium can specifically assess the transport function of ABCA3.     

Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1(-/-)) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/-) cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/-) MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/-) cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.     

Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells

Background

ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER).

Methods

Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level.

Results

We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling.

Conclusion

Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.     

Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells

Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.     

Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.     

The carboxyterminus of the ATP-binding cassette transporter A1 interacts with a beta2-syntrophin/utrophin complex

Recent work identified ABCA1 as the major regulator of plasma HDL-cholesterol responsible for the removal of excess choline-phospholipids and cholesterol from peripheral cells and tissues. ABCA1 function may depend on the association with heteromeric proteins and to identify these candidates a human liver yeast two-hybrid library was screened with the carboxyterminal 144 amino acids of ABCA1. Beta2-syntrophin was found to interact with ABCA1 and the C-terminal five amino acids of ABCA1 proned to represent a perfect tail for binding to syntrophin PDZ domains. Immunoprecipitation further confirmed the association of ABCA1 and beta2-syntrophin and in addition utrophin, known to couple beta2-syntrophin and its PDZ ligands to the F-actin cytoskeleton, was identified as a constituent of this complex. ABCA1 in the plasmamembrane of human macrophages was found to be partially associated with Lubrol rafts and effluxed choline-phospholipids involve these microdomains. Beta2-syntrophin does not colocalize in these rafts indicating that beta2-syntrophin may participate in the retaining of ABCA1 in cytoplasmic vesicles and for the targeting of ABCA1 to plasmamembrane microdomains when ABCA1 is released from beta2-syntrophin.     

The ATP-binding cassette transporter 1 mediates lipid efflux from Sertoli cells and influences male fertility

The liver X receptor/retinoid X receptor (LXR/RXR)-regulated gene ABCA1 effluxes cellular cholesterol and phospholipid to apolipoprotein A1 (apoA1), which is the rate-limiting step in high-density lipoprotein synthesis. The RXR pathway plays a critical role in testicular lipid trafficking, and RXRbeta-deficient male mice are sterile and accumulate lipids in Sertoli cells. Here, we demonstrate that ABCA1 mRNA and protein are abundant in Sertoli cells, whereas germ cells express little ABCA1. LXR/RXR agonists stimulate ABCA1 expression in cultured Sertoli MSC1 and Leydig TM3 cell lines. However, Sertoli TM4 cells lack ABCA1, and TM4 cells or primary Sertoli cells cultured from ABCA1(-/-) mice both fail to efflux cholesterol to apoA1. Expression of exogenous ABCA1 restores apoA1-dependent cholesterol efflux in Sertoli TM4 cells. In vivo, ABCA1-deficient mice exhibit lipid accumulation in Sertoli cells and depletion of normal lipid droplets from Leydig cells by 2 months of age. By 6 months of age, intratesticular testosterone levels and sperm counts are significantly reduced in ABCA1(-/-) mice compared with wild-type (WT) controls. Finally, a 21% decrease (P = 0.01) in fertility was observed between ABCA1(-/-) males compared with WT controls across their reproductive lifespans. These results show that ABCA1 plays an important role in lipid transport in Sertoli cells and influences male fertility.     

Association of ABCA1 with syntaxin 13 and flotillin-1 and enhanced phagocytosis in tangier cells

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.     

Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1

To explore the functional interactions between apoA-I and ABCA1, we correlated the cross-linking properties of several apoA-I mutants with their ability to promote cholesterol efflux. In a competitive cross-linking assay, amino-terminal deletion and double amino- and carboxy-terminal deletion mutants of apoA-I competed effectively the cross-linking of WT (125)I-apoA-I to ABCA1, while the carboxy-terminal deletion mutant apoA-I[Delta(220-243)] competed poorly. Direct cross-linking of WT apoA-I, amino-terminal, and double deletion mutants of apoA-I to ABCA1 showed similar apparent K(d) values (49-74 nM), whereas the apparent K(d) values of the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] were increased 3-fold. Analysis of several internal deletions and point mutants of apoA-I showed that apoA-I[Delta(61-78)], apoA-I[Delta(89-99)], apoA-I[Delta(136-143)], apoA-I[Delta(144-165)], apoA-I[D102A/D103A], apoA-I[E125K/E128K/K133E/E139K], apoA-I[L141R], apoA-I[R160V/H162A], and WT apoA-I had similar ABCA1-mediated lipid efflux, and all competed efficiently the cross-linking of WT (125)I-apoA-I to ABCA1. WT apoA-I and ABCA1 could be cross-linked with a 3 A cross-linker. The WT apoA-I, amino, carboxy and double deletion mutants of apoA-I showed differences in the cross-linking to WT ABCA1 and the mutant ABCA1[W590S]. The findings are consistent with a direct association of different combinations of apoA-I helices with a complementary ABCA1 domain. Mutations that alter ABCA1/apoA-I association affect cholesterol efflux and inhibit biogenesis of HDL.     

Human ABCA3, a product of a responsible gene for abca3 for fatal surfactant deficiency in newborns, exhibits unique ATP hydrolysis activity and generates intracellular multilamellar vesicles

ABCA3 is highly expressed at the membrane of lamellar bodies in alveolar type II cells, in which pulmonary surfactant is stored. ABCA3 gene mutations cause fatal surfactant deficiency in newborns. We established HEK293 cells stably expressing human ABCA3 and analyzed the function. Exogenously expressed ABCA3 is glycosylated and localized at the intracellular vesicle membrane. ABCA3 is efficiently photoaffinity labeled by 8-azido-[alpha(32)P]ATP, but not by 8-azido-[gamma(32)P]ATP, when the membrane fraction is incubated in the presence of orthovanadate. Photoaffinity labeling of ABCA3 shows unique metal ion-dependence and is largely reduced by membrane pretreatment with 5% methyl-beta-cyclodextrin, which depletes cholesterol. Electron micrographs show that HEK293/hABCA3 cells contain multivesicular, lamellar body-like structures, which do not exist in HEK293 host cells. Some fuzzy components such as lipids accumulate in the vesicles. These results suggest that ABCA3 shows ATPase activity, which is induced by lipids, and may be involved in the biogenesis of lamellar body-like structures.     

A novel role for protein phosphatase 2A in the dopaminergic regulation of Na,K-ATPase

Stimulation of dopaminergic type 1 (D(1)) receptors increases lung edema clearance by regulating Na,K-ATPase function in the alveolar epithelium. We studied the role of serine/threonine protein phosphatases in the Na,K-ATPase regulation by D(1) agonists in A549 cells. We found that low doses of the type 1/2A protein phosphatase inhibitor okadaic acid as well as SV40 small t antigen transiently transfected into A549 cells prevented the D(1) agonist-induced increase in Na,K-ATPase activity and translocation from intracellular pools to the plasma membrane. This was associated with a rapid and transient increase in protein phosphatase 2A activity. We conclude that D(1) stimulation regulates Na,K-ATPase activity by promoting recruitment of Na,K-ATPases from intracellular pools into the basolateral membranes of A549 cells via a type 2A protein phosphatase.     

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol

We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1^-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1^-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1^-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1^-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1^-M/-M macrophages. Abca1^-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts.     

Intracellular lipidation of newly synthesized apolipoprotein A-I in primary murine hepatocytes

Hepatocytes, which are the main site of apolipoprotein (apo)A-I and ATP-binding cassette transporter A1 (ABCA1) expression, are also the main source of circulating high density lipoprotein. Here we have characterized the intracellular lipidation of newly synthesized apoA-I, in primary hepatocytes cultured with [3H]choline to label choline-phospholipids, low density lipoprotein-[3H]cholesterol to label the cell surface, or [3H]mevalonate to label de novo synthesized cholesterol. Phospholipidation of apoA-I is significant and most evident in endoplasmic reticulum (ER) and medial Golgi, both in the lumen and on the membrane fractions of the ER and medial Golgi. In the presence of cycloheximide, endogenous apoA-I is substantially phospholipidated intracellularly but acquires some additional lipid after export out of the cell. In cells labeled with low density lipoprotein-[3H]cholesterol, intracellular cholesterol lipidation of apoA-I is entirely absent, but the secreted apoA-I rapidly accumulates cholesterol after secretion from the cell in the media. On the other hand, de novo synthesized cholesterol can lipidate apoA-I intracellularly. We also showed the interaction between apoA-I and ABCA1 in ER and Golgi fractions. In hepatocytes lacking ABCA1, lipidation by low density lipoprotein-cholesterol was significantly reduced at the plasma membrane, phospholipidation and lipidation by de novo synthesized sterols were both reduced in Golgi compartments, whereas ER lipidation remained mostly unchanged. Therefore, the early lipidation in ER is ABCA1 independent, but in contrast, the lipidation of apoA-I in Golgi and at the plasma membrane requires ABCA1. Thus, we demonstrated that apoA-I phospholipidation starts early in the ER and is partially dependent on ABCA1, with the bulk of lipidation by phospholipids and cholesterol occurring in the Golgi and at the plasma membrane, respectively. Finally, we showed that the previously reported association of newly synthesized apoA-I and apoB (Zheng, H., Kiss, R. S., Franklin, V., Wang, M. D., Haidar, B., and Marcel, Y. L. (2005) J. Biol. Chem. 280, 21612-21621) occurs after secretion at the cell surface.     

Induction of cell size vesicles from human lymphoma cell lines and their application to drug carriers

Sodium butyrate (NaB) induced the membrane enclosed cell size vesicles from several IgM producing cell lines. We considered the application of the cell-derived vesicles (CDVs) to drug delivery system (DDS) using the lung cancer specific IgM producing AE6 cell line. Microscopic observation showed that the DiI fluorescence labeled AE6 vesicles were incorporated into the lung cancer cell line A549. The anticancer drug, actinomycin D (actD), contained in AE6 and Ramos vesicles decreased the A549 cell viability to 46 and 62% of control without actD, respectively. The cytotoxic effect in AE6 vesicles was superior to that in the Ramos vesicles that have the lung cancer non-specific IgM on their surfaces. However, the result of the Ramos vesicles suggests that the surface molecules other than IgM may interact with the A549 cells. In our method for vesicle production, more specific and abundant antibodies mounted vesicles can be generated by transfection of their genes into cells followed by NaB treatment. These suggest that the CDVs may be useful for the development of a drug carrier for DDS.     

Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL₃ additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL₃ reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL₃-mediated lipid efflux.