Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells.

Mallory, Joanne Bednarz; Kushner, Peter J.; Protter, Andrew A.; Cofer, Claire L.; Appleby, Vanessa; Lau, Kenneth; Schilling, James; Vigne, Jean-Louis

doi:10.1016/s0021-9258(18)61338-9

Cited by 40 publications

(4 citation statements)

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“…Properties of Apo A1 Particles. The cells were induced with Zn 2+ and Fe 2+ to activate the metallothein promoter driving apo A1 transcription, and the medium was collected after 16 h. The concentration of apo A1 in the culture medium (12 h) was 3 ( 0.5 µg/mL, comparable to that previously reported (19). The s 20,w of apo A1 was 2.4 ( 0.1, and the SR was 2.6 ( 0.1 nm (Table 1).…”

Section: Resultssupporting

confidence: 74%

“…Much less is known of the steps by which new HDL is formed. Both PL-poor and PL-rich HDL fractions have been purified from the culture medium of hepatocytes (15)(16)(17), intestinal cells (18), and CHO cells stably expressing apo A1 (19). In human Tangier disease, the ATP-binding cassette A1 (ABCA1) transmembrane lipid transporter, responsible for the transfer of PL from cell membranes to circulating HDL, is genetically defective.…”

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confidence: 99%

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Mechanism of Prebeta-HDL Formation and Activation

et al. 2006

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The mechanism of formation of functional high-density lipoprotein (HDL) from secreted lipid-free apolipoprotein A1 (apo A1) was determined using human liver-derived (HepG2) cells, human intestine-derived (CaCO2) cells, and CHO cells stably expressing full-length human apo A1 (CHO-A1 cells). In each cell line, a significant proportion of secreted apo A1 had a Stokes radius of 2.6 nm and was inactive in binding phospholipids (PL) or free cholesterol (FC). Extracellularly, in a reaction dependent on membrane transporter ABCA1, prealpha-migrating 2.6 nm apo A1 was converted to a prebeta-migrating product that was able to bind PL. Both forms were reactive with mAb55201, a monoclonal antibody specific for native plasma lipid-poor (prebeta1) HDL [Nakamura, Y., et al. (2004) Biochemistry 43, 14311-14318]. The physical properties of precursor and product apo A1 suggested that both are monomers, with Stokes radii of 2.6 and 3.6 nm, respectively, consistent with the absence of intermolecular cross-linking of apo A1 in lipid-poor HDL, reported previously. Product but not precursor apo A1 promoted reverse cholesterol transport (RCT) from human aortic smooth muscle cells. These studies suggest an important contribution of secreted lipid-free apo A1 to HDL formation.

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Section: Resultssupporting

confidence: 74%

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confidence: 99%

Mechanism of Prebeta-HDL Formation and Activation

et al. 2006

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“…Cell Culture. CHO cells stably transfected with the human apo A1 gene under control of the metallothein promoter (CHO-A1 cells) were grown until nearly confluent in 3.5 cm plastic wells containing DMEM/Ham's F-12 medium (1/1 v/v) with 10% fetal bovine serum (FBS) (12). To induce apo A1 synthesis, the cells were transferred to the same medium containing 30 µM ZnSO 4 and 30 µM FeSO 4 .…”

Section: Methodsmentioning

confidence: 99%

Bone Morphogenetic Protein-1 (BMP-1) Cleaves Human Proapolipoprotein A1 and Regulates Its Activation for Lipid Binding

Chau

Fielding

2007

Biochemistry

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Apolipoprotein A1 (apo A1), the major protein of high-density lipoprotein, is secreted as a proprotein and then cleaved by an uncharacterized metalloproteinase. Here this enzyme is identified as C-terminal procollagen endoproteinase/bone morphogenetic protein-1 (BMP-1). Studies with recombinant BMP-1, BMP-1 antibody, and BMP-1 siRNA establish this proteinase as the major or only apo A1-converting activity secreted by human liver-derived (HepG2) cells and CHO cells stably expressing human apo A1. BMP-1 stimulates the conversion of newly secreted proapo A1 to its phospholipid- (PL-) binding form. In this way it promotes formation of functional HDL and reverse cholesterol transport, while inhibiting filtration and clearance of uncleaved proprotein. Alpha2-macroglobulin, a protease inhibitor secreted as part of the innate immune response, inhibits BMP-1 activity and blocks the maturation of proapo A1. The decrease in circulating apo A1 levels that is characteristic of the response to inflammation and infection may be mediated, at least in part, via BMP-1 by this novel mechanism.

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“…However, due to the instability of pro-apoA-I, yields were low, and consequently other expression systems were developed (17 -20). In 1987, human apoA-I was successfully expressed from Chinese hamster ovary cells at a concentration of 100 g/ml (21) in condi-Abbreviations: apoA-I, apolipoprotein A-I; DPPC, L-␣ -phosphatidylcholine dipalmitoyl; FRET, fluorescence resonance energy transfer; 5-IAF, 5-iodoacetamido-fluorescein; rHDL, recombinant HDL; 5-TMRIA, tetramethylrhodamine-5-iodoacetamide-dihydroiodide. 1 To whom correspondence should be addressed at the Department of Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. e-mail: msthomas@wfubmc.edu tioned medium.…”

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Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL

Thomas

Pan

et al. 2001

Journal of Lipid Research

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Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester. Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of lipid-bound apoA-I. Herein, we report new methodologies allowing rapid expression, fluorescent labeling, and recombinant HDL (rHDL) preparation for use in apoA-I in fluorescence resonance energy transfer (FRET) studies. Cysteinecontaining mutant forms of human apoA-I were cloned into the pTYB12 vector containing a T7 promoter, a modified self-splicing protein element (intein), and a small affinity tag [chitin binding domain (CBD)]. The fusion proteins were expressed in Escherichia coli , isolated from cell lysates, and bound to a chitin-affinity column. Release of mature human apoA-I was initiated by the addition of DTT, which induced self-cleavage at the COOH terminus of the intein-CBD fusion protein. ApoA-I was further purified by Q-sepharose and then used for fluorescent probe labeling. Discoidal rHDL were then prepared with donor and/ or acceptor labeled apoA-I and characterized with respect to their size, composition and ability to activate LCAT.

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Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells.

Cited by 40 publications

References 44 publications

Mechanism of Prebeta-HDL Formation and Activation

Mechanism of Prebeta-HDL Formation and Activation

Bone Morphogenetic Protein-1 (BMP-1) Cleaves Human Proapolipoprotein A1 and Regulates Its Activation for Lipid Binding

Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL

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