Synthesis, intracellular processing, and signal peptide of human apolipoprotein E.

New isoforms of apolipoprotein (apo)C-I and apoC-III have been detected in delipidated fractions from very low density lipoprotein (VLDL) using matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry (MS). The cleavage sites of truncated apoC-III isoforms have also been identified. The VLDL fractions were isolated by fixed-angle single-spin ultracentrifugation using a self-generating sucrose density gradient and delipidated using a newly developed C18 solid phase extraction protocol. Fifteen apoC isoforms and apoE were identified in the MALDI spectra and the existence of the more abundant species was verified by ESI-MS. The relative intensities of the apoCs are closely correlated in three normolipidemic subjects. A fourth subject with type V hyperlipidemia exhibited an elevated apoC-III level and a suppressed level of the newly discovered truncated apoC-I isoform. ApoC-II was found to be particularly sensitive to in vitro oxidation. The dynamic range and specificity of the MALDI assay shows that the complete apoC isoform profile and apoE phenotype can be obtained in a single measurement from the delipidated VLDL fraction.-Bondarenko,

mentioning

confidence: 99%

“…b The common subscript coding for apoC-III was introduced by Schmitz et al (43). c The subscript coding was introduced by Zannis et al (23).…”

mentioning

confidence: 99%

Mass spectral study of polymorphism of the apolipoproteins of very low density lipoprotein

Bondarenko¹,

Cockrill

Watkins

et al. 1999

“…To further specify the serum effects on apoE secretion, re-binding, and degradation, we asked which class of serum lipoproteins might mediate the observed effects. ApoE3, the apoE isoform synthesized by HepG2 cells (12), is able to associate with all types of plasma lipoproteins, although a certain preference for HDL was reported (50). We found that apoE newly secreted by HepG2 cells bound preferentially to LDL particles present in the fasted serum used, but we also observed strong binding to HDL (data not shown).…”

Section: Ratio Between Pools Of Secreted Cell-associated and Degraded Apoe Is Influenced By Apoe Ligands In The Extracellular Mediummentioning

confidence: 66%

“…The secretion of apoE has been studied in some detail, mostly in the human hepatoma cell line HepG2 and in macrophages (11)(12)(13)(14)(15)(16). A considerable amount of newly synthesized apoE is not secreted to the culture medium, but instead remains associated with cells (15)(16)(17), forming a cell-associated pool of apoE.…”

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confidence: 99%

Regulated turnover of a cell surface-associated pool of newly synthesized apolipoprotein E in HepG2 cells

Schmitt¹,

Grand-Perret²

1999

Newly synthesized apolipoprotein E (apoE) is incompletely secreted and partially degraded by hepatocytes. To evaluate current models concerning apoE's role in remnant lipoprotein clearance by the liver, we performed a detailed and quantitative pulse-chase analysis of apoE secretion, subcellular distribution, and proteolytic degradation by the human hepatoma cell line HepG2. Only about 35% of newly synthesized apoE were found to be secreted to the culture medium. As determined by a protease-protection assay, a substantial amount of newly synthesized apoE was transported to the cell surface, constituting more than half of the cellular apoE under steady-state conditions. A subpopulation representing almost 40% of newly secreted apoE was rapidly rebound to the cell surface, indicating a dynamic equilibrium between cell surface and secreted apoE. These pools of newly synthesized apoE were subject to proteolytic turnover that occurred in lysosomes, presumably after re-endocytosis. We found that the proteolytic turnover of cell surface and secreted apoE was regulated by the availability of apoE ligands, being almost completely suppressed by the presence of lipoprotein-containing human serum or isolated lipoproteins, namely LDL and HDL. The characteristics of regulated degradation of cell surface apoE shed new light on potential physiological functions of this pool of apoE. Our results provide evidence that hepatocytes are capable of forming a large pool of cell surface-associated apoE, thereby supporting the previously proposed secretion-recapture model for apoE.-Schmitt, M., and T. Grand-Perret. Regulated turnover of a cell surface-associated pool of newly synthesized apolipoprotein E in HepG2 cells.

“…Apolipoprotein (apo)E is a component of plasma lipoproteins, and plays an important role in the transport of lipids in plasma, both as an exchangeable apolipoprotein and as a ligand for cell surface proteoglycans and receptors of the LDL receptor family (1,2). The secretion of apoE has been studied in detail, particularly in hepatocytes and macrophages, which are major contributors of circulating apoE (3)(4)(5)(6). However, the secretory pathway of newly synthesized apoE is complex and only partly characterized at present.…”

mentioning

confidence: 99%

Dynamic and stable pools of apoE differ functionally at the HepG2 cell surface

Burgess

Marcel

2001

Synthesis of apolipoprotein (apo)E in hepatocytes leads to both secretion and retention in cell surface pools. Inclusion of Brefeldin A to HepG2 cells prompted a rapid decrease of cell surface apoE to about 37% of control values after a 3-h incubation. The t 1/2 for this dynamic pool was estimated to be 15 min. In contrast, a stable fraction of apoE ( t 1/2 Ͼ 20 h) was found in association with the extracellular matrix (ECM). Increased content of apoE on the ECM correlated with decreased binding of VLDL. Decreased apoE on the cell surface correlated with increased binding of VLDL to cells. Collectively, this suggests that glycosaminoglycan-bound apoE can occlude binding sites for apoE-containing lipoproteins on glycosaminoglycans. In solid-phase assays, heparin, suramin, and chondroitin sulfates A and B efficiently inhibited the binding of apoE to heparan sulfate proteoglycans, but were unable to displace apoE from this glycosaminoglycan. Finally, decreasing cell surface apoE with suramin subsequently decreased the apoE content on secreted apoB-containing lipoproteins without affecting the overall secretion of apoE or apoB to the extracellular medium. In summary, cell surface apoE comprises both dynamic fractions, which can be donated to newly secreted lipoproteins, and stable fractions, which may act to minimize the unproductive binding of lipoproteins to the ECM. -Burgess, J. W., and Y. L. Marcel. Dynamic and stable pools of apoE differ functionally at the HepG2 cell surface.