Carboxyl-terminal Domain Truncation Alters Apolipoprotein A-I in Vivo Catabolism

Schmidt, Hartmut; Remaley, Alan T.; Stonik, John A.; Ronan, R; Wellmann, Axel; Fairwell, T; Zech, Loren A.; Brewer, H. Bryan; Hoeg, Jeffrey M.

doi:10.1074/jbc.270.10.5469

Cited by 68 publications

(56 citation statements)

References 70 publications

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“…A number of large fragments of apolipoprotein A-I have been observed previously by 2-dimensional electrophoresis suggesting susceptibility of this protein to degradation in vivo [23]. Shortening of apolipoprotein A-I significantly affects its distribution among lipoprotein particles and circulating half-life [24]. All of the protein fragments previously observed by 2-dimensional electophoresis are substantially larger than those described here.…”

Section: Resultscontrasting

confidence: 37%

Diverse range of small peptides associated with high-density lipoprotein

Hortin

Shen

Martin

et al. 2006

Biochemical and Biophysical Research Communications

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High-density lipoproteins (HDL) were examined as potential carriers of small peptides in plasma. HDL purified by density gradient centrifugation was delipidated and fractionated by size-exclusion chromatography under denaturing conditions. By hplc and mass spectrometry, more than 100 peptide components were found in the size range from 1,000 to 5,000 Da. By sequence analysis, peptides were identified as fragments of proteins such as apolipoproteins, fibrinogen, α 1 -proteinase inhibitor, and transthyretin. The results indicate that purified HDL bears a complex range of small peptides. It is unclear whether the peptides have any significant functional role as apolipopeptides, but they may represent a pathway for peptide delivery or scavenging and a significant reservoir of plasma peptides for diagnostic evaluation. KeywordsHigh-density lipoprotein; Apolipoprotein; Peptidomics; Transthyretin; Apolipoprotein A-I; Proteomics Abbreviations HDL, high-density lipoprotein; MALDI TOF MS, matrix-assisted laser desorption time-of-flight mass spectrometry; m/z, mass/charge ratio Lipoprotein particles in the blood circulation have multiple physiological roles as carrier molecules. In addition to the well recognized role of lipoproteins in cholesterol and triglyceride transport [1,2], lipoprotein particles including high-density, low-density, and very low-density lipoproteins (HDL, LDL, and VLDL) serve as carriers for other lipids and for fat-soluble vitamins such as vitamins A, D, E, K, and carotenoids [3,4]. A number of well-characterized proteins are major structural and functional components of HDL, LDL, and VLDL including apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, and E [1]. Other molecules without direct roles in lipoprotein metabolism such as endotoxin [5], tissue factor pathway inhibitor [6], paraoxonase [7], and ghrelin [8] also bind to HDL. The diversity of molecules binding to lipoprotein particles suggests that they can serve as a versatile adsorptive phase, analogous to stationary phases in reverse-phase chromatography. This characteristic led to a hypothesis that lipoproteins might serve as a reservoir for the hundreds of small peptides that circulate in human plasma [9,10]. The complex pattern of plasma peptides contains large amounts of potential information, and some studies suggest this information may be probed for cancer detection or other diagnostic purposes [11,12]. Initial promise of this approach has been slowed by reproducibility and standardization issues [13,14]. Most of the small peptides in plasma or serum now are considered to circulate bound to albumin and subfractionation of binding components may improve analyses [15][16][17]. Here, we examine whether HDL serves as a major carrier of small peptides. Materials and MethodsCalibrators and α-cyano-4-hydroxycinnamic acid and sinapinic acid were from Bruker Daltonics (Billerica, MA HDL was prepared from 400 mL of blood freshly collected in citrate preservative from a healthy donor. Plasma was separated from cells by centrifugation an...

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Section: Resultscontrasting

confidence: 37%

Diverse range of small peptides associated with high-density lipoprotein

Hortin

Shen

Martin

et al. 2006

Biochemical and Biophysical Research Communications

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“…Alternatively, reduced phospholipid binding affinity may account for this observation as this property associates with a greater fractional catabolic rate among some apoA-I mutants (50). Injection of a 4-fold greater dose of the ⌬7-43 and ⌬7-65 apoA-I Ad5 resulted in plasma levels of these proteins similar to Wt apoA-I (Fig.…”

Section: The N Terminus Of Apoa-i Is Essential For Hdl Maturationmentioning

confidence: 98%

The N-terminal Globular Domain and the First Class A Amphipathic Helix of Apolipoprotein A-I Are Important for Lecithin:Cholesterol Acyltransferase Activation and the Maturation of High Density Lipoprotein in Vivo

Scott¹,

McManus²,

Franklin³

et al. 2001

Journal of Biological Chemistry

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To investigate the role of the N terminus of apolipoprotein A-I (apoA-I) in the maturation of high density lipoproteins (HDL), two N-terminal mutants with deletions of residues 1-43 and 1-65 (referred to as ⌬1-43 and ⌬1-65 apoA-I) were studied. In vitro, these deletions had little effect on cellular cholesterol efflux from macrophages but LCAT activation was reduced by 50 and 70% for the ⌬1-43 and ⌬1-65 apoA-I mutants, respectively, relative to wild-type (Wt) apoA-I. To further define the role of the N terminus of apoA-I in HDL maturation, we constructed recombinant adenoviruses containing Wt apoA-I and two similar mutants with deletions of residues 7-43 and 7-65 (referred to as ⌬7-43 and ⌬7-65 apoA-I, respectively). Residues 1-6 were not removed in these mutants to allow proper cleavage of the pro-sequence in vivo. Following injection of these adenoviruses into apoA-I-deficient mice, plasma concentrations of both ⌬7-43 and ⌬7-65 apoA-I were reduced 4-fold relative to Wt apoA-I. The N-terminal deletion mutants, in particular ⌬7-65 apoA-I, were associated with greater proportions of pre␤-HDL and accumulated fewer HDL cholesteryl esters relative to Wt apoA-I. Wt and ⌬7-43 apoA-I formed predominantly ␣-migrating and spherical HDL, whereas ⌬7-65 apoA-I formed only pre␤-HDL of discoidal morphology. This demonstrates that deletion of the first class A amphipathic ␣-helix has a profound additive effect in vivo over the deletion of the globular domain alone (amino acids 1-43) indicating its important role in the production of mature ␣-migrating HDL. In summary, the combined in vitro and in vivo studies demonstrate a role for the N terminus of apoA-I in lecithin:cholesterol acyltransferase activation and the requirement of the first class A amphipathic ␣-helix for the maturation of HDL in vivo. High density lipoproteins (HDL)1 transport cholesterol from peripheral tissues to the liver in a process known as reverse cholesterol transport (1). This pathway is accepted as a primary mechanism by which HDL exert their anti-atherogenic effects. Nascent HDL are secreted by hepatocytes, liberated from chylomicrons during triglyceride lipolysis, and are derived from HDL remodeling by hepatic lipase and cholesteryl ester transfer protein. The importance of phospholipid transfer protein (PLTP) in the lipidation of this nascent HDL pool has recently emerged, because PLTP-deficient mice exhibit defective phospholipid transfer from triglyceride-rich lipoproteins to HDL, reduced HDL levels, and increased HDL catabolism (2). Efflux of cholesterol and phospholipids from cells provide nascent HDL with lipid constituents. This step is important for steady-state concentrations of HDL, because efflux is a ratelimiting step in HDL maturation as heterozygous mutations in the ATP binding cassette transporter A1 (ABCA1) can cause familial HDL deficiency (3). The combined actions of PLTP and ABCA1 generate larger discoidal pre␤ 2 and pre␤ 3 -HDL from the nascent HDL pool, which are converted to spherical ␣-migrating HDL by the actions of lecithin:...

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“…Recent studies using truncation mutants of the carboxyl-terminal region demonstrated that the domain corresponding to residues 227-243 was critical in modulating the association of apoA-I with lipoproteins as well as in the in vivo metabolism of apoA-I (40,42). Other investigations show that substitution of the carboxyl-terminal region of apoA-I with a helical domain derived from apoA-II increases its hydrophobicity but does not restore the mutant protein's lipoprotein affinity or its ability to associate with HDL 3 (42).…”

Section: Table II Reaction Kinetics Of Recombinant Hdl Particles Withmentioning

confidence: 99%

“…The carboxyl-terminal region or repeats 9 and 10 (residues 209 -243) of apoA-I has been proposed to be important in cell binding (39), lipoprotein association (40,41,42), LCAT activation (25)(26)(27), and in vivo metabolism of HDL (40,42). Recent studies using truncation mutants of the carboxyl-terminal region demonstrated that the domain corresponding to residues 227-243 was critical in modulating the association of apoA-I with lipoproteins as well as in the in vivo metabolism of apoA-I (40,42).…”

Section: Table II Reaction Kinetics Of Recombinant Hdl Particles Withmentioning

confidence: 99%

Alteration in Apolipoprotein A-I 22-Mer Repeat Order Results in a Decrease in Lecithin:Cholesterol Acyltransferase Reactivity

Sorci‐Thomas

Curtiss

Parks

et al. 1997

Journal of Biological Chemistry

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Apolipoprotein A-I contains eight 22-amino acid and two 11-amino acid tandem repeats that comprise 80% of the mature protein. These repeating units are believed to be the basic motif responsible for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. Computer analysis indicates that despite a fairly high degree of compositional similarity among the tandem repeats, significant differences in hydrophobic and amphipathic character exist. Our previous studies demonstrated that deletion of repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98 -99% reduction of LCAT activation as compared with wild-type apoA-I. To determine the effects of substituting one of these repeats with a more hydrophobic repeat we constructed a mutant apoA-I protein in which residues 143-164 (repeat 6) were replaced with repeat 10 (residues 220 -241). The cloned mutant protein, 10F6 apoA-I, was expressed and purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes prepared at a 100: 5:1 molar ratio of L-␣-dimyristoylphosphatidylcholine: cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of 114 and 108 Å on nondenaturing 4 -30% polyacrylamide gel electrophoresis. L-␣-Dimyristoylphosphatidylcholine 10F6 apoA-I complexes had a 5-6-fold lower apparent V max /apparent K m as compared with wild-type apoA-I containing particles. As expected, monoclonal antibody epitope mapping of the lipid-free and lipid-bound 10F6 apoA-I confirmed that a domain expressed between residues 143 and 165 normally found in wild-type apoA-I was absent. The region between residues 119 and 144 in 10F6 apoA-I showed a marked reduction in monoclonal antibody binding capacity. Therefore, we speculate that the 5-6-fold lower LCAT reactivity in 10F6 compared with wildtype apoA-I recombinant particles results from increased stabilization within the 121-165 amino acid domain due to more stable apoprotein helix phospholipid interactions as well as from conformational alterations among adjacent amphipathic helix repeats.

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Carboxyl-terminal Domain Truncation Alters Apolipoprotein A-I in Vivo Catabolism

Cited by 68 publications

References 70 publications

Diverse range of small peptides associated with high-density lipoprotein

Diverse range of small peptides associated with high-density lipoprotein

The N-terminal Globular Domain and the First Class A Amphipathic Helix of Apolipoprotein A-I Are Important for Lecithin:Cholesterol Acyltransferase Activation and the Maturation of High Density Lipoprotein in Vivo

Alteration in Apolipoprotein A-I 22-Mer Repeat Order Results in a Decrease in Lecithin:Cholesterol Acyltransferase Reactivity

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