Low density lipoprotein degradation by rat mast cells. Demonstration of extracellular proteolysis caused by mast cell granules

The interaction between rat serosal mast cells and low density lipoproteins (LDL) was studied in vitro. When rat 125I-LDL was incubated with mast cells, it was bound to a binding site on the mast cell surface but was not internalized by the cells. Even though 125I-LDL was not internalized, its protein component, apolipoprotein B, was rapidly degraded. The proteolytic activity responsible for the degradation of apolipoprotein B was present in the extracellular fluid of mast cells. It could be shown that the degradation was caused entirely by specific cell organelles of mast cells, the granules, which were spontaneously released into the extracellular fluid during preparation and incubation of the cells. In contrast to uncontrolled spontaneous degranulation, a controlled specific degranulation of mast cells can be induced by treating the cells with the compound 48/80. When increasing amounts of 48/80 were added to mast cell suspensions, a dose-dependent release of granules was observed and an increase in the rate of 125I-LDL degradation resulted. The increase in 125I-LDL degradation closely followed the increase in granule release. Thus, a quantitative relationship between the amount of granules present in the extracellular fluid and the amount of degradation of 125I-LDL could be established. The apolipoprotein part of LDL was extensively degraded by isolated mast cell granules. Analysis by polyacrylamide gel electrophoresis showed that upon incubation of LDL with isolated granules, the apolipoprotein B band rapidly disappeared with simultaneous appearance of several low molecular weight bands. The degradation of 125I-LDL by mast cell granules proceeded optimally at neutral pH and at physiological ionic strength. The results show that mast cell granules are able to efficiently degrade LDL in vitro, once released from mast cells into the extracellular fluid.     

Modification of low density lipoproteins by secretory granules of rat serosal mast cells

When low density lipoprotein (LDL) is incubated with granules isolated from rat serosal mast cells, a fraction of LDL is bound to the granule heparin proteoglycan. If incubation is continued at 37 degrees C, the bound LDL, but not the unbound LDL, is degraded by granule neutral proteases. In the early stage of incubation, all the granule-bound LDL can be released by 0.3 M NaCl (the "salt-sensitive" fraction of LDL). With time, an increasing proportion of the granule-bound LDL requires 0.5 M NaCl for release (the "salt-resistant" fraction of LDL). Chemical analysis showed that, on average, 20% of the apolipoprotein B LDL was lost from the salt-sensitive fraction and 60% from the salt-resistant fraction, without any change in the composition of the lipid portion. Electron microscopic analysis disclosed large fused particles of LDL (diameters up to 100 nm) in the highly proteolyzed salt-resistant fraction, but no fused particles could be found in the less proteolyzed salt-sensitive fraction. We conclude that both binding and extensive degradation of LDL by mast cell granules is required for fusion of LDL particles on the granule surface. As compared with native LDL, the mast cell granule-modified LDL particles exhibit (i) increased particle size, (ii) selective loss of protein (apoB), (iii) a decrease in hydrated density, and (iv) stronger ionic interaction between apoB and heparin proteoglycan. The particles resemble the extracellular lipid droplets found in atherosclerotic lesions of both man and animals. Modification of LDL by mast cells may therefore provide a model of how these lipid structures are formed.     

Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan

Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0 degrees C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared with 37 degrees C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with bis (sulfosuccinimidyl) suberate (BS3) covalently cross-linked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC by using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan.     

Release of arylsulfatase A but not B from rat mast cells by noncytolytic secretory stimuli.

The net percentage of release of arylsulfatase activity from purified rat mast cells induced by rabbit anti-rat F(ab')2 was consistently only about 1/3 that of histamine. Isoelectric focusing of the released and residual arylsulfatase activities demonstrated specific release of the A type without B and a net percentage of immunologic release of arylsulfatase A equivalent to that of histamine. When the net percentage of histamine and arylsulfatase A release were nearly maximal (88 and 76%) in response to the calcium ionophore A23187, specific release of arylsulfatase B did not occur. Thus, arylsulfatase A and not B was associated with the secretory granule released from the rat mast cell by reversed anaphylaxis or the calcium ionophore. In contrast, subcellular fractionation of water-lysed mast cells yielded arylsulfatase B with the heparin- and chymase-containing granule fraction and arylsulfatase A in the aqueous fraction comprised of cell sap and granule water eluate. It may be that arylsulfatase B resides in a minor second granule, whereas arylsulfatase A is loosely associated with the predominant secretory granule of the rat mast cell.     

Oleate-mediated stimulation of apolipoprotein B secretion from rat hepatoma cells. A function of the ability of apolipoprotein B to direct lipoprotein assembly and escape presecretory degradation.

Physiological concentrations of oleate stimulate apolipoprotein (apo) B-containing lipoprotein secretion from HepG2 cells without increasing apoB mRNA levels. The purpose of this study was to determine whether oleate acts by increasing translation of apoB mRNA or through posttranslational effects on the apoB protein. To address the mechanism of oleate-stimulated secretion of apoB, a series of carboxyl terminally truncated apoB constructs was made. Each contained the SV40 early promoter, the apoB 5'-untranslated region, and SV40 polyadenylation signals. Any difference in the response to oleate between endogenous apoB and the proteins encoded by the constructs or between the constructs themselves should thus depend on the protein sequence. Stable transformants were established for each of the constructs in the rat hepatoma cell line McArdle-RH7777. The effect of oleate on secretion of the apoB protein products was determined by labeling with [35S]methionine, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carboxyl-terminal truncation of apoB41 resulted in a loss of the ability of apoB secretion to respond to oleate. Ultracentrifugation of secreted proteins on continuous CsCl gradients from 1.0-1.4 g/ml revealed that this correlated with a decrease in the ability of apoB to be recovered as a buoyant lipoprotein particle. Addition of oleate decreased the densities at which the short forms of apoB secreted as lipoproteins were recovered. Pulse-chase analysis of the secretion of apoB100 and of the truncated proteins revealed that they all underwent rapid posttranslational intracellular degradation. We conclude that oleate has no effect on the translation of apoB mRNA but promotes the secretion of apoB-containing lipoproteins by reducing presecretory degradation of those forms of apoB that can produce buoyant lipoproteins.     

Cooperation between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II

The octapeptide angiotensin II (Ang II) exerts a wide range of effects on the cardiovascular system but has also been implicated in the regulation of cell proliferation, fibrosis, and apoptosis. Ang II is formed by cleavage of Ang I by angiotensin-converting enzyme, but there is also evidence for non-angiotensin-converting enzyme-dependent conversion of Ang I to Ang II. Here we address the role of mast cell proteases in Ang II production by using two different mouse strains lacking mast cell heparin or mouse mast cell protease 4 (mMCP-4), the chymase that may be the functional homologue to human chymase. Ang I was added to ex vivo cultures of peritoneal cells, and the generation of Ang II and other metabolites was analyzed. Activation of mast cells resulted in marked increases in both the formation and subsequent degradation of Ang II, and both of these processes were strongly reduced in heparin-deficient peritoneal cells. In the mMCP-4(-/-) cell cultures no reduction in the rate of Ang II generation was seen, but the formation of Ang-(5-10) was completely abrogated. Addition of a carboxypeptidase A (CPA) inhibitor to wild type cells caused complete inhibition of the formation of Ang-(1-9) and Ang-(1-7) but did not inhibit Ang II formation. However, when the CPA inhibitor was added to the mMCP-4(-/-) cultures, essentially complete inhibition of Ang II formation was obtained. Taken together, the results of this study indicate that mast cell chymase and CPA have key roles in both the generation and degradation of Ang II.     

Identification of sites in apolipoprotein A-I susceptible to chymase and carboxypeptidase A digestion

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe²²⁵) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL₃) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe²²⁹ and Tyr¹⁹² residues were the main cleavage sites. Interestingly, the Phe²²⁵ residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL₃; however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL₃ at only the N-terminus, especially at Phe³³. CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe²²⁵ and Phe²²⁹ residues newly exposed by chymase, but did not cleave Tyr¹⁹². These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).     

Low density lipoprotein binding, internalization, and degradation in human adipose cells.

Human adipose tissue derives its cholesterol primarily from circulating lipoproteins. To study fat cell-lipoprotein interactions, low density lipoprotein (LDL) uptake and metabolism were examined using isolated human adipocytes. The 125I-labelled LDL (d = 1.025-1.045) was bound and incorporated by human fat cells in a dose-dependent manner with an apparent Km of 6.9 + 0.9 microgram LDL protein/mL and a Vmax of 15-80 microgram LDL protein/mg lipid per 2 h. In time-course studies, LDL uptake was characterized by rapid initial binding followed by a linear accumulation for at least 4 h. The 125I-labelled LDL degradation products (trichloroacetic acid soluble iodopeptides) accumulated in the incubation medium in a progressive manner with time. Azide and F- inhibited LDL internalization and degradation, suggesting that these processes are energy dependent. Binding and cellular internalization of 125I-labelled LDL lacked lipoprotein class specificity in that excess (25-fold) unlabelled very low density lipoprotein (VLDL) (d less than 1.006) and high density lipoprotein (HDL) (d = 1.075-1.21) inhibited binding and internalization of 125I-labelled LDL. On an equivalent protein basis HDL was the most potent. The 125I-labelled LDL binding to an adipocyte plasma membrane preparation was a saturable process and almost completely abolished by a three- to four-fold greater concentration of HDL. The binding, internalization, and degradation of LDL by human adipocytes resembled that reported by other mesenchymal cells and could account for a significant proportion of in vivo LDL catabolism. It is further suggested that adipose tissue is an important site of LDL and HDL interactions.     

Measurement of apolipoprotein A-I in rat high density lipoprotein and in rat plasma by radioimmunoassay.

A double antibody radioimmunoassay (RIA) for rat apolipoprotein A-I is reported. The ApoA-I isolated from delipidated HDL by gel filtration yielded a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), and its amino acid composition resembled that reported by others. ApoA-I was iodinated by lactoperoxidase and the resulting 125I-apoA-I was purified by gel filtration. Up to 93% of 125I-apoA-I was precipitable by antibody and greater than 99% of bound 125I-apoA-I was displaced by "cold" apoA-I. Other rat lopoproteins and apolipoproteins did not react in this system. Human plasma were also not reactive, nor were dog, goat, and sheep plasmas.     

The sites of degradation of purified rat low density lipoprotein and high density lipoprotein in the rat

Low density lipoprotein and high density lipoprotein were isolated from rat serum by sequential ultracentrifugation in the density intervals 1.025-1.050 g/ml and 1.125-1.21 g/ml, respectively. The isolated lipoproteins were radioiodinated using ICl. Low density lipoprotein was further purified by concanavalin A affinity chromatography and concentrated by ultracentrifugation. 95% of the purified low density lipoprotein radioactivity was precipitable by tetramethylurea, while only 4% was associated with lipids. The radioiodinated high density lipoprotein was incubated for 1 h at 4 degrees C with unlabelled very low density lipoprotein, followed by reisolation by sequential ultracentrifugation. Only 3% of the radioactivity was associated with lipids and 90% was present on apolipoprotein A-I. The serum decay curves of labelled and subsequently purified rat low and high density lipoprotein, measured over a period of 28 h, clearly exhibited more than one component, in contrast to the monoexponential decay curves of iodinated human low density lipoprotein. The decay curves were not affected by the methods used to purify the LDL and HDL preparations. The catabolic sites of the labelled rat lipoproteins were analyzed in vivo using leupeptin-treated rats. In vivo treatment of rats with leupeptin did not affect the rate of disappearance from serum of intravenously injected labelled rat low density lipoprotein and high density lipoprotein. Leupeptin-dependent accumulation of radioiodine occurred almost exclusively in the liver after intravenous injection of iodinated low density lipoprotein, while both the liver and the kidneys showed leupeptin-dependent accumulation of radioactivity after injection of iodinated high density lipoprotein.     

Synthesis of prostaglandins and eicosanoids by the mast cell secretory granule

The identification of a non-bilayer phospholipid storage in the secretory granule and the linking of the eicosanoid production with the release of histamine have prompted us to examine whether the secretory granule may also serve as both the source as well as the site of prostaglandin synthesis during exocytosis. By exposing the contents of purified granules to exogenous arachidonic acid at neutral pH, we observed the rapid formation of many eicosanoids. The presence of prostaglandins E2, D2 and F2a were identified. The kinetics of E2 formation was also followed. The localization of the arachidonic acid cascade to the secretory granule explains why the production of eicosanoids is so intimately tied to the process of granule exocytosis.     

Rapid degradation of neurotensin by stimulated rat mast cells

A RIA towards neurotensin (NT) using C-terminal- and N-terminal-specific antisera was used to study degradation of this tridecapeptide by isolated rat mast cells. Incubation of NT (10 μM) with peritoneal or pleural mast cells resulted in a rapid loss of NT immunoreactivity (iNT), as measured by C-terminal-directed antiserum, with little effect on N-terminal iNT. The rate of the reaction was faster with pleural cells (T_1/2, 30 s) than with peritoneal cells (T_1/2, 180 s) and was > 10-fold slower in the presence of metabolic poisons. The enzyme(s) involved is most likely released from the cells during secretion, as NT was degraded by media conditioned by compound 48/80-stimulated mast cells 40–60 times faster than by media from unstimulated cells. This degradation by conditioned media was concentration dependent, pH dependent, and temperature sensitive. HPLC analyses indicated a near stoichiometric conversion of NT to NT(1–12) (66%) and NT(1–11) (34%) after incubation for 10–30 s with conditioned media. By 30 min only NT(1–11) and NT(1–10) were present. Phenanthroline (1 mM), an inhibitor of carboxypeptidase, prevented the loss of C-terminal iNT and the generation of NT(1–12) and NT(1–11). While NT(1–12) was effective in releasing histamine from mast cells in vitro and increasing vascular permeability in vivo, NT(1–11) was not. These results suggest that carboxypeptidase-like enzyme(s) could modulate the level and form of NT-related peptides in various states involving activation of mast cells.     

Different apolipoprotein B breakdown patterns in models of oxidized low density lipoprotein.

Low density lipoprotein (LDL) oxidation is characterized by alterations in biological properties and structure of the lipoprotein particles, including breakdown and modification of apolipoprotein B (apoB). We compared apoB breakdown patterns in different models of minimally and extensively oxidized LDL using Western blotting techniques and several monoclonal and polyclonal antibodies. It was found that copper and endothelial cell-mediated oxidation produced a relatively similar apoB banding pattern with progressive fragmentation of apoB during LDL oxidation, whereas malondialdehyde (MDA)- and hydroxynonenal (HNE) -modified LDL produced an aggregated apoB. It is conceivable that apoB fragments present in copper and endothelial cell oxidized LDL lead to the exposure on the lipoprotein surface of different protein epitopes than in aggregated MDA-LDL and HNE-LDL. Although all models of extensively oxidized LDL led to increased lipid uptake in macrophages, mild degrees of oxidation interfered with LDL uptake in fibroblasts and extensively oxidized LDL impaired degradation of native LDL in fibroblasts. We suggest that in order to improve interpretation and comparison of results, data obtained with various models of oxidized LDL should be compared to the simpliest and most reproducible models of 3 h and 18 h copper-oxidized LDL (apoB breakdown) and MDA-LDL (apoB aggregation) since different models of oxidized LDL have significant differences in apoB breakdown and aggregation patterns which may affect immunological and biological properties of oxidized LDL.     

Low and high density lipoprotein metabolism in primary cultures of hepatic cells from normal and apolipoprotein E knockout mice.

Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism by mediating the binding of apoE-containing lipoproteins to receptors. The role of hepatic apoE in the catabolism of apoE-free lipoproteins such as low density lipoprotein (LDL) and high density lipoprotein-3 (HDL(3)) is however, unclear. We analyzed the importance of hepatic apoE by comparing human LDL and HDL(3) metabolism in primary cultures of hepatic cells from control C57BL/6J and apoE knockout (KO) mice. Binding analysis showed that the maximal binding capacity (Bmax) of LDL, but not of HDL(3), is increased by twofold in the absence of apoE synthesis/secretion. Compared to control hepatic cells, LDL and HDL(3) holoparticle uptake by apoE KO hepatic cells, as monitored by protein degradation, is reduced by 54 and 77%, respectively. Cleavage of heparan sulfate proteoglycans (HSPG) by treatment with heparinase I reduces LDL association by 21% in control hepatic cells. Thus, HSPG alone or a hepatic apoE-HSPG complex is partially involved in LDL association with mouse hepatic cells. In apoE KO, but not in normal hepatic cells, the same treatment increases LDL uptake/degradation by 2.4-fold suggesting that in normal hepatic cells, hepatic apoE increases LDL degradation by masking apoB-100 binding sites on proteoglycans. Cholesteryl ester (CE) association and CE selective uptake (CE/protein association ratio) from LDL and HDL(3) by mouse hepatic cells were not affected by the absence of apoE expression. We also show that 69 and 72% of LDL-CE hydrolysis in control and apoE KO hepatic cells, respectively, is sensitive to chloroquine revealing the importance of a pathway linked to lysosomes. In contrast, HDL(3)-CE hydrolysis is only mediated by a nonlysosomal pathway in both control and apoE KO hepatic cells. Overall, our results indicate that hepatic apoE increases the holoparticle uptake pathway of LDL and HDL(3) by mouse hepatic cells, that HSPG devoid of apoE favors LDL binding/association but impairs LDL uptake/degradation and that apoE plays no significant role in CE selective uptake from either human LDL or HDL(3) lipoproteins.     

High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver.

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.     

Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B

A method has been described for the measurement of apoB concentration and specific activity in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) during metabolic studies. For measurement of specific activity, apoB was separated from other apolipoproteins and lipids by precipitation in, and subsequent washing with, isopropanol. For determination of apoB concentration, equal volumes of lipoprotein and isopropanol were mixed, and the protein content of the apoB precipitate was measured by the difference between total lipoprotein protein and the protein measured in the supernatant. Evidence that there was no apoB solubilization in isopropanol and that precipitated apoB was virtually free of soluble apolipoproteins was obtained by electrophoresis. Lipid contamination of the apoB precipitate was less than 1%. The methods were applicable to VLDL, intermediate density lipoprotein (IDL), and LDL from normolipemic patients with protein concentrations between 187 micrograms/ml and 1898 micrograms/ml. The data obtained using isopropanol were highly correlated with those using tetramethylurea, and recoveries of apoB were similar. Furthermore, the isopropanol method has been demonstrated to yield consistent data for apoB specific activities in a study of VLDL, IDL, and LDL metabolism.     

Tryptase in rat mast cells: properties and inhibition by various inhibitors in comparison with chymase

Previous studies with trans-4-(guanidinomethyl)cyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a trypsin inhibitor, strongly suggested the involvement of a trypsin-like protease in histamine release from mast cells induced by various secretagogues (Takei, M., Matumoto, T., Endo, K. & Muramatu, M. (1988) Agents and Actions, in press; Takei, M., Matumoto, T., Ito, T., Endo, K. & Muramatu, M.; Takei, M., Matumoto, T., Endo, K. & Muramatu, M. and Takei, M., Matumoto, T., Urashima, H., Endo, K. & Muramatu, M., unpublished results). Two serine proteases, chymase (Benditt, E.F. & Arase, M. (1959) J. Exp. Med. 110, 451-460) and tryptase Kido, H., Fukusen, N. & Katunuma, N. (1985) Arch. Biochem. Biophys. 239, 436-443) were demonstrated in rat peritoneal mast cells. Both enzymes were purified and the effects of inhibitors for trypsin and chymotrypsin on these proteases were examined. The trypsin-like protease was found in saline extract and purified by successive chromatographies on Sephadex G-100 and DEAE-cellulose columns. The molecular mass of this protease was apparently 120,000 Da. This protease showed maximal activity at pH 7.1 and was named pH 7 tryptase. Chymase was obtained from 1.5M NaCl extract. pH 7 Tryptase markedly hydrolysed Boc-Phe-Ser-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec among the various substrates containing arginyl and lysyl bonds but did not cleave Tos-Arg-OMe. Tos-Lys-CH2Cl and diisopropylfluorophosphate strongly inhibited this protease. Various inhibitors for trypsin inhibited pH 7 tryptase, and those for chymotrypsin inhibited chymase. Among the esters of GMCHA examined, GMCHA-OPhBut most strongly and competitively inhibited pH 7 tryptase but it had no effect on chymase.