Monoclonal antibodies and the characterization of apolipoprotein structure and function

Marcel, Yves L.; Weech, Philip K.; Milthorp, Peter; Tercé, François; Vézina, Camilla; Milne, Ross W.

doi:10.1016/0163-7827(84)90010-9

Cited by 29 publications

(12 citation statements)

References 99 publications

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“…The antibodies used here have been obtained from mice immunized with either apoA-I or HDL (21). Solid-phase radioimmunoassays in the absence of Tween 20 were carried out as described earlier (22).…”

Section: Methodsmentioning

confidence: 99%

Biochemical and Physical Properties of Remnant-HDL₂ and of Preβ₁-HDL Produced by Hepatic Lipase

et al. 1999

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The hepatic lipase acting on triglyceride-rich high-density lipoprotein2 (HDL2) induces the formation of pre beta 1-HDL, leaving a residual alpha-migrating HDL particle that was named "remnant-HDL2" (Barrans, A., Collet, X., Barbaras, R., Jaspard, B., Manent, J., Vieu, C., Chap, H., and Perret, B. (1994) J. Biol. Chem. 269, 11572-11577.]. In this study, these two product particles generated by hepatic lipase were isolated by density gradient ultracentrifugation. Particles were first characterized in terms of chemical composition, density, and mass. The pre beta 1-HDL obtained in vitro contain one to two molecules of apoA-I, associated with phospholipids, and free and esterified cholesterol. When compared to triglyceride-rich HDL2, remnant-HDL2 have lost on average one molecule of apoA-I, 60% of triacylglycerols, and 15% of phospholipids. The estimated composition is concordant with the hypothesis of the splitting of a substrate particle into one pre beta 1-HDL and one remnant-HDL2. Spectroscopic studies were carried out to monitor changes in lipid fluidity upon lipolysis. The fluorescence anisotropy was measured using (1,6)-diphenyl-hexa-(1,3, 5)-triene as a probe, and the degree of order was calculated from electron spin resonance spectra using the 5-nitroxy-derivative of stearic acid. Both approaches showed a decreased lipid fluidity in remnant-HDL2, as compared to triglyceride-rich HDL2. The immunoreactivity of apoA-I toward several monoclonal antibodies was assayed as a reflection of changes of apoA-I conformation. In remnant-HDL2, as compared to triglyceride-rich HDL2, a lower reactivity was noted with the 2G11 antibody, which interacts in the NH2 terminal part of apoA-I. Finally, remnant-HDL2 was clearly different from HDL3 with respect to all of the parameters studied, demonstrating that hepatic lipase does not promote the direct conversion of HDL2 to HDL3. Thus, hepatic lipase produces remnant-HDL2 particles, which display modifications of apoA-I conformation and of fluidity of the lipid environment. This newly described HDL2 subfraction may play a major role in the reverse cholesterol transport.

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Section: Methodsmentioning

confidence: 99%

Biochemical and Physical Properties of Remnant-HDL₂ and of Preβ₁-HDL Produced by Hepatic Lipase

et al. 1999

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“…To examine the abilities of ∆(187-243) and the central deletion mutants to form lipoprotein-like structures, media was obtained from macrophages after incubation with the apolipoproteins, concentrated, and then electrophoresed on an 8 to 25% native gradient gel to determine changes in particle size ( Figure 2). The separated proteins were transferred to nitrocellulose and Western blotted with monoclonal antibodies against apoA-I [2F1, 5F6, and A44 (34,35)]. Figure 2A illustrates the native gradient gel for medium that has not been incubated with cells.…”

Section: Lipid Efflux From Cholesterol-loaded Thp-1 Macrophagementioning

confidence: 99%

Deletion of the C-Terminal Domain of Apolipoprotein A-I Impairs Cell Surface Binding and Lipid Efflux in Macrophage

et al. 1999

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The contribution of the amphipathic alpha-helices of apoA-I toward lipid efflux from human skin fibroblasts and macrophage was examined. Four apoA-I mutants were designed, each by deletion of a pair of predicted adjacent helices. Three mutants lacked two consecutive central alpha-helices [Delta(100-143), Delta(122-165), and Delta(144-186)], whereas the final mutant lacked the C-terminal domain [Delta(187-243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Delta(187-243) exhibited a marked reduction in its ability to promote either cholesterol or phospholipid efflux from THP-1 macrophages. This mutant also demonstrated a decreased ability to bind lipids and to form lipoprotein complexes. In contrast, the four mutants and apoA-I equally supported cholesterol efflux from fibroblasts, albeit with a reduced capacity when compared to macrophages. Delta(187-243) bound poorly to the macrophage cell surface when compared to apoA-I, and competitive binding studies with the central domain and C-terminal deletions mutants showed that only Delta(187-243) did not compete effectively with [(125)I]apoA-I. Omission of PMA during cholesterol loading enhanced cholesterol efflux to both apoA-I (1.5-fold) and the C-terminal deletion mutant (2.5-fold). Inclusion of the Sandoz ACAT inhibitor (58-035) during loading and, in the absence of PMA, increased and equalized cholesterol efflux to apoA-I and Delta(187-243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Delta(187-243) to the THP-1 cell surface. Overall, these results show that cholesterol efflux from cells such as fibroblasts does not require any specific sequence between residues 100 and 243 of apoA-I. In contrast, optimal cholesterol efflux in macrophages requires binding of the C-terminal domain of apoA-I to a cell surface-binding site and the subsequent translocation of intracellular cholesterol to an efflux-competent pool.

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“…Rabbit polyclonal anti-human apoA-I antibody was purchased from Calbiochem. Monoclonal antibodies directed against human apoA-I (a combination of 4H1 (against the extreme N terminus) and 5F6 (against the central region)) were described previously (23) and biotinylated with sulfo-NHS-biotin from Pierce. Streptavidin-horseradish peroxidase conjugate and protein G-Sepharose were obtained from Amersham Biosciences.…”

Section: Methodsmentioning

confidence: 99%

Differential Regulation of ATP Binding Cassette Protein A1 Expression and ApoA-I Lipidation by Niemann-Pick Type C1 in Murine Hepatocytes and Macrophages

Wang

Franklin

Sundaram

et al. 2007

Journal of Biological Chemistry

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Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr␣ mRNA is not changed, and Lxr␣ target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [35 S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate. Niemann-Pick type C (NPC)2 disease is an autosomal lipid storage disease resulting from mutations in either the Npc1 (95% of families) or Npc2 genes (1-3). It is characterized by progressive hepatosplenomegaly and neurodegeneration, leading to premature death (4, 5). The exact function of both proteins remains unknown, but there is much evidence that they facilitate the transport of lipids, primarily cholesterol, from late endosome to the Golgi apparatus, endoplasmic reticulum (ER), mitochondria, and plasma membrane (6, 7). Thus the storage involves the accumulation of unesterified cholesterol, sphingolipids, and other lipids within the endosomal/lysosomal compartments of cells in almost all body tissues (8). Impaired cholesterol traffic in NPC disease cells prevents the normal down-regulation of endogenous cholesterol synthesis and the low density lipoprotein (LDL) receptor (9, 10), through the interruption of normal cholesterol regulation by sterol regulatory element-binding protein at the ER and the interruption of generation of LDL cholesterol-derived oxysterols in the mitochond...

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Monoclonal antibodies and the characterization of apolipoprotein structure and function

Cited by 29 publications

References 99 publications

Biochemical and Physical Properties of Remnant-HDL₂ and of Preβ₁-HDL Produced by Hepatic Lipase

Biochemical and Physical Properties of Remnant-HDL₂ and of Preβ₁-HDL Produced by Hepatic Lipase

Deletion of the C-Terminal Domain of Apolipoprotein A-I Impairs Cell Surface Binding and Lipid Efflux in Macrophage

Differential Regulation of ATP Binding Cassette Protein A1 Expression and ApoA-I Lipidation by Niemann-Pick Type C1 in Murine Hepatocytes and Macrophages

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Monoclonal antibodies and the characterization of apolipoprotein structure and function

Cited by 29 publications

References 99 publications

Biochemical and Physical Properties of Remnant-HDL2 and of Preβ1-HDL Produced by Hepatic Lipase

Biochemical and Physical Properties of Remnant-HDL2 and of Preβ1-HDL Produced by Hepatic Lipase

Deletion of the C-Terminal Domain of Apolipoprotein A-I Impairs Cell Surface Binding and Lipid Efflux in Macrophage

Differential Regulation of ATP Binding Cassette Protein A1 Expression and ApoA-I Lipidation by Niemann-Pick Type C1 in Murine Hepatocytes and Macrophages

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Product

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Biochemical and Physical Properties of Remnant-HDL₂ and of Preβ₁-HDL Produced by Hepatic Lipase

Biochemical and Physical Properties of Remnant-HDL₂ and of Preβ₁-HDL Produced by Hepatic Lipase