Kyoko Takata

Protective effect of lipoproteins containing apoprotein A-I on Cu2+ - catalyzed oxidation of human low density lipoprotein

Two apoprotein A‐I (apoA‐I)‐containing lipoproteins, one containing apoA‐I and apoA‐II (LpA‐I/A‐II) and the other containing only apoA‐I (LpA‐I), were examined for their effect on Cu2+‐mediated oxidation of low density lipoprotein (LDL). The presence of LpA‐I or LpA‐I/A‐II prevented LDL oxidation when assessed by the electrophoretic mobility, apoprotein B fragmentation and amounts of thiobarbituric acid‐reactive substances. The protection of LDL oxidation by these lipoproteins was effective for up to 6 h, with LpA‐I being more active than LpA‐I/A‐II. Results from these in vitro model experiments raise a possibility that LpA‐I mayplay a role in protecting LDL from Cu2+ ‐mediated oxidation.

Scavenger receptor-mediated recognition of maleylated albumin and its relation to subsequent endocytic degradation

Rat sinusoidal liver cells take up maleylated bovine serum albumin (maleyl-BSA) and its demaleylated form (demaleyl-BSA) by scavenger receptor-mediated endocytosis. Cellular binding of maleyl-BSA and demaleyl-BSA and its quantitative relation to subsequent intracellular degradation were investigated. The binding affinities of these ligands were almost equal whereas the number of binding sites for maleyl-BSA was more than twice as large than that for demaleyl-BSA. However, no difference was observed in their endocytic degradation. The amounts of maleyl-BSA degraded were proportional to those bound to the cell surface up to a certain level. However, a further increase in cell-bound ligands did not affect the degradation of maleyl-BSA. Several polyanions such as fucoidin and dextran sulfate of Mr = 5000 inhibited the binding of maleyl-BSA but did not affect its degradation. In contrast, acetylated or oxidized low density lipoprotein had virtually no effect on cellular binding of maleyl-BSA but exhibited profound effects on its intracellular degradation. Similar results were obtained with rat peritoneal macrophages. Based on these data, we would propose that two binding sites are involved in the receptor-mediated ligand recognition; one is coupled to subsequent endocytic degradation, and the other serves as a binding site for polyanionic compounds.

Microquantification of cholesterol and cholesteryl esters in rat peritoneal macrophages by reverse-phase high-performance liquid chromatography

A simple and rapid method for the microquantification of cholesterol and cholesteryl esters by reverse-phase high performance liquid chromatography has been established. Comparison of elution patterns of authentic cholesterol and cholesteryl esters revealed that a mu Bondasphere reverse-phase C8 (300-A) column was more suitable than a corresponding reverse-phase C4 or C18 column in terms of rapidity and sensitivity. Recovery of cholesterol and cholesteryl esters from a C8 column was greater than 98% when determined either by radioactive cholesterol and cholesteryl oleate or by cholesteryl heptadecanoate. The sensitivity of the quantification ranged from 5 ng to 50 micrograms for both cholesterol and cholesteryl esters. This method was applied to determination of cellular cholesterol and cholesteryl esters of rat peritoneal macrophages. Lipid extracts of these cells were found to contain 38.01 +/- 2.60 micrograms of cholesterol and 3.18 +/- 0.36 micrograms of cholesteryl esters per milligram of cell protein. When the cells were loaded with cholesteryl esters by incubation for 24 h with various concentrations of acetylated low-density lipoprotein, a cellular level of cholesteryl esters showed a dose-dependent increase and reached a maximal level of 106.60 +/- 3.05 micrograms/mg cell protein. Thus, the present method is useful for the microquantification of cholesterol and cholesteryl esters from lipid extracts of biological samples.

Scavenger receptor of human monocytic leukemia cell line (THP-1) and murine macrophages for nonenzymatically glycosylated proteins

Long-term incubation of proteins with glucose undergo a series of nonenzymatic reactions to form advanced glycosylation end product (AGE) with fluorescence and brown color. The receptor for AGE-proteins was demonstrated in murine macrophages (Vlassara et al. (1985) Proc. Natl. Acad. Sci. USA 82. 5588). Our recent study with rat macrophages revealed that the receptor also recognized proteins modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, indicating its close identity to a scavenger receptor for aldehyde-modified proteins (Takata, K. et al. (1988) J. Biol. Chem. 263. 14819). This notion was tested in the present study with human monocytic leukemia cell line (THP-1 cells), human monocyte macrophages and murine peritoneal macrophages. Endocytic uptake of AGE-proteins and aldehyde-modified proteins was inhibited in a cross-competitive fashion. The receptor activities of THP-1 cells for AGE-albumin and aldehyde-modified proteins were induced synchronously by phorbol 12-myristate 13-acetate. Furthermore, upon reduction by NaBH4 of the Schiff base formed between proteins and glucose or aldehydes, no ligand activity was generated. However, once the ligand activity was generated, NaBH4 was no longer effective for the ligand activity. Thus, a structure in common between AGE-proteins and aldehyde-modified proteins may be crucial for recognition by the human macrophage receptor.

Intracellular accumulation of cholesteryl esters suppresses production of lipopolysaccharide-induced interleukin 1 by rat peritoneal macrophages

Interleukin 1 (IL-1) is a major cytokine of macrophages secreted by several stimulants such as lipopolysaccharide (LPS). Macrophages are known to possess the scavenger receptor for acetylated low density lipoprotein (acetyl-LDL) and maleylated albumin. In the present study we determined effects of these ligands on LPS-induced IL-1 production by rat peritoneal macrophages. These ligands themselves did not induce IL-1 production. However, upon short incubation with acetyl-LDL, LPS-induced IL-1 production was significantly suppressed. The extent of the suppression was proportional to cellular cholesteryl esters. Thus, intracellular accumulation of cholesteryl esters might be responsible for suppression of LPS-induced IL-1 production.

Scavenger receptor for malondialdehyde-modified high density lipoprotein on rat sinusoidal liver cells

We report here the presence of a membrane-associated receptor which mediates endocytic uptake of malondialdehyde-modified high density lipoprotein (MDA-HDL) on sinusoidal liver cells. Binding of [125I]MDA-HDL to the cells was followed by internalization and degradation in lysosomes. The binding and lysosomal degradation of [125I]MDA-HDL were effectively inhibited by unlabeled MDA-HDL and acetyl-HDL. However, formaldehyde-treated serum albumin or low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by sinusoidal liver cells, did not affect the binding of [125I]MDA-HDL to the cells. These results indicate that a receptor for MDA-HDL is described as a distinct member among the scavenger receptors for chemically modified proteins.

Receptor-mediated endocytosis of aldehyde-modified proteins by sinusoidal liver cells

Formaldehyde-treated serum albumin (f-Alb) is known to be endocytosed by sinusoidal lever cells via a receptor-mediated mechanism. The receptor purified from rat livers exhibited a molecular weight of 125,000, consisting of two glycoprotein components with molecular weights of 53,000 and 30,000, respectively. Experiments using antireceptor antibody demonstrated that the f-Alb receptor is distinct from the receptor that mediates endocytotic uptake of acetylated low-density lipoprotein, but they share a common property of being inhibited by several polyanions, suggesting that polyanion-sensitivity might play an important role in the scavenger function of simusoidal liver cells. Studies on the ligand specificity of this receptor revealed that a covalent modification by formaldehyde of a limited number of lysine residues in albumin has led to the formation of a receptor-recognition domain(s). Furthermore, in addition to formaldehyde, the ligand activity was also generated with albumin modified by other aliphatic aldehydes, such as glycoaldehyde and glyceraldehyde. This phenomenon was extended to several proteins other than albumin. These data suggest therefore that the f-Alb receptor originally described as being specific for albumin modified by formaldehyde may play a general role as a scavenger receptor for aldehyde-modified proteins.

Scavenger receptor-mediated recognition of chemically modified lipoproteins by macrophages.

Scavenger receptor-mediated endocytosis of chemically modified-low density lipoprotein (LDL) by macrophages or macrophage-derived cells has been intensively studied from the potential link to atherosclerosis. Taking the advantage of the fact that maleyl albumin is also endocytosed via the same receptor, we have investigated the ligand specificity of this receptor by using rat peritoneal macarophages and sinusoidal liver cells. Maleylation of albumin of >50% of its lysine residues resulted in a threshold increase in the ligand activity, whereas demaleylation of maleyl-albumin to 20% did not affect the ligand activity, suggesting that maleylation of selective lysine amino groups of albumin may lead to the formation of the domain structure required for the receptor recognition. To further minimize the ligand domain, five albumin peptides isolated from cyanogen bromide-cleaved albumin were maleylated and determined for their ligand activity. The result demonstrated that albumin peptides with 102 amino acids but not with 37 amino acids gained the ligand activity upon maleylation, suggesting that generation of the ligand activity does not require a whole molecule, but rather, the domain itself might be regional. We next compared maleylalbumin with demaleyl-albumin in their cellular binding and endocytic degradation. Amounts of intracellular degradation of maleyl-albumin were proportionally increased up to a certain level. However, a further increase in cell surface-bound ligands did not affect the subsequent intracellular degradation. Based on these data, we would propose the ‘two binding sites’ model, where two binding sites on surface membranes are involved in the scavenger receptor-mediated ligand recognition; one is specific for the ligand domain and coupled to subsequent intracellular degradation and the other serves a binding site for polyanionic compounds.