Impact of isoform and Cys-thiol modifications of apolipoprotein E on the LRP1-mediated metabolism of triglyceride-rich lipoproteins

doi:10.21203/rs.3.rs-3292146/v1

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Impact of isoform and Cys-thiol modifications of apolipoprotein E on the LRP1-mediated metabolism of triglyceride-rich lipoproteins

https://doi.org/10.21203/rs.3.rs-3292146/v1

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Background

The low-density lipoprotein (LDL) receptor-related protein (LRP)1 participates in the metabolism of apolipoprotein (apo) E-containing lipoproteins; however, the details of its function have not been fully elucidated.

Methods

We investigated the effects of the isoform and modifications of the cysteine (Cys)-thiol of apoE on LRP1-mediated metabolism using a cell-based assay for the interaction between apoE-containing fluorescence-labeled emulsion particles (apoE-F-EP) and human fibroblasts expressing the LRP1 and lacking the LDL receptor.

Results

Among the three isoforms, apoE3-F-EP were most effectively bound to LRP1 and were catabolized. ApoE2-F-EP exhibited the lowest affinity to LRP1 but were significantly catabolized, whereas apoE4-F-EP were sufficiently bound to LRP1 but showed the lowest catabolic capability. Redox modifications of Cys112-thiol and Cys158-thiol had an antagonistic effect on the LRP1-mediated interaction of apoE-F-EP. The Tris (2-carboxyethyl) phosphine-reduction enhanced the binding and suppressed the catabolism of apoE3-F-EP, but had no effect on apoE2-F-EP. Interestingly, the formation of disulfide-linked complexes with apoAII suppressed binding, but enhanced the catabolism of apoE2-F-EP.

Conclusions

Redox modifications of apoE-Cys-thiol may modulate the LRP1-mediated metabolism of apoE2 or apoE3 containing lipoproteins, whereas apoE4, which has no Cys, essentially lacks this function. The failure or deficiency of this regulatory function may be a critical trigger for the development of dyslipidemia and related atherosclerosis.

apoAII

atherosclerosis

human fibroblast

isoelectric point

redox

remnant lipoproteins

Human apolipoprotein (apo) E, a component of plasma lipoproteins comprising 299 amino acids with a molecular weight of 35 kDa, is crucial in lipoprotein metabolism as a ligand for members of the LDL receptor (LDLR) family [1].

The cysteine (Cys)-arginine (Arg) interchanges at residues 112 and/or 158 are the principal characteristics that distinguish the three major isoforms [apoE2 (Cys112/Cys158), apoE3(Cys112/Arg158), and apoE4 (Arg112/Arg158)]. These substitutions affect the structure and function of isoforms and have various biological consequences [2, 3]. In particular, the difference in the binding affinity to LDLR is the most important distinguishing feature among the three isoforms, that is, apoE3 and apoE4 readily bind to LDLR, while apoE2 binds poorly to the receptor [1].

LDLR is involved in the uptake of LDL and remnant lipoproteins (RLP). RLP, which are derived from the lipolytic processing of triglyceride (TG)-rich lipoproteins, are a proatherogenic apoE-containing lipoproteins (apoE-LP), and their accumulation leads to the development of atherosclerosis and various atherosclerotic diseases, such as coronary artery disease [4, 5]. Under normal circumstances, RLP are rapidly cleared from circulation via LDLR, whereas carriers homozygous for apoE2 show defects in RLP clearance through LDLR owing to its inability to bind to LDLR. Hence, the homozygosity of apoE2 is a potent causative factor for type III hyperlipoproteinemia [6], which is characterized by a remarkable increase in RLP. However, in reality, only a few percent of them develop hyperlipoproteinemia [7], because the LDLR-independent pathways compensate for this defect via other members of the LDLR superfamily [8].

The LDLR-related protein (LRP)1, a ubiquitously expressed type I transmembrane protein, is a representative member of the LDLR superfamily [9]. It participates in RLP metabolism [10–12] as well as various biological processes (e.g., inflammation and various signal transductions) [13]. Similar to LDLR, cell surface LRP1 associates with RLP particles by recognizing the apoE embedded in the particles as its ligand and catabolizes RLP in cooperation with cell surface heparan sulfate proteoglycan (HSPG), especially Syndecan1 (SDC1) [14]. In the metabolic process of RLP, the LRP1-HSPG pathway may serve as a backup for the LDLR pathway, and HSPG, rather than LRP1, is regarded as a central player in this pathway [15, 16]. Nevertheless, we wonder whether the contribution of LRP1 is still debatable. Considering the pleiotropic functions of LRP1, we predicted that LRP1 may be essential in RLP metabolism and that its impairment may lead to the development of hyperlipoproteinemia. In addition, several studies have demonstrated that LRP1 is relevant to the pathophysiology of atherosclerosis [12, 13, 17, 18], supporting our notion.

In a series of processes to metabolize lipoproteins, the association between the receptor and its ligand is the most critical step. Ruiz et al. [19] reported that in contrast to LDLR, LRP1 can recognize all apoE isoforms with equal affinity using a solid-phase assay; however, the apoE-isoform specificity of LRP1 has not yet been sufficiently characterized. Cumulative evidence has indicated that LRP1 also contributes to the pathogenesis of Alzheimer’s disease, which shows distinct apoE-isoform-specific differences in its incidence [20, 21], suggesting that LRP1 also discriminates among apoE isoforms. Even if this is not the case, some molecular modifications of apoE may affect its association with LRP1.

The Cys-thiol residue, which is the major characteristic that distinguishes apoE2 and apoE3 from apoE4, is susceptible to post-translational modifications derived from the change in redox status. Multiple lines of evidence suggest that the modulation of Cys-thiol is involved in a variety of physiological functions and in the pathogenesis of various diseases [22–24]. We have recently reported that redox modifications of Cys-thiol in apoE3 or apoE2 molecules significantly affect their physiological functions, especially the serum TG levels, strongly suggesting the possibility that their redox modifications participate in RLP metabolism [25–27].

In the present study, we re-examined the function of LRP1 in the metabolism of TG-rich lipoproteins and whether LRP1 can discriminate among three apoE isoforms by chasing the behavior of apoE containing fluorescence dye-labeled emulsion particles (apoE-F-EP) in a cell culture system using human fibroblasts GM00701, which express high levels of LRP1, but not LDLR and SDC1 [28] (Fig. 1). In addition, we investigated the influence of the redox status of apoE-Cys-thiol on the association of apoE with LRP1 to understand the role of LRP1 in RLP metabolism.

Materials

Recombinant apoE isoforms (apoE2, apoE3, and apoE4) were purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan). Human plasma apoAII was obtained from Athens Research & Technology Inc. (Athens, GA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-apoE polyclonal antibody (HRP-anti-apoE) was purchased from Academy Bio-medical Company Inc. (Houston, TX, USA). Rabbit anti-LRP1 (N-terminal) monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Triolein, Egg phosphatidylcholine (PC), and 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC) were supplied by Avanti Polar Lipids, Inc. (Birmingham, AL). All the other chemicals used were of the highest grade.

Cells and cell culture

Human fibroblast GM00701, supplied by Coriell Institute (Camden, NJ, USA), was maintained in Minimal Essential Medium, Eagle modification with Earle’s balanced salt solution (MEM-Eagle’s, Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 15% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 µg/mL) in 5% CO₂-air at 37°C.

Redox modifications of Cys-thiol of recombinant apoE

Recombinant apoEs were incubated with Tris (2-carboxyethyl) phosphine (TCEP) for 30 min at room temperature, followed by the removal of TCEP by dialysis against phosphate-buffered saline (PBS) using an Xpress Micro Dialyzer (Scienova GmbH, Jena, Deutschland). These reduced apoEs were incubated with N, N, N’, N’-tetramethylazodi-carboxamide (diamide), 2-iodoacetamide (IAM), or iodoacetate (IAA) for 30 min at room temperature or with apoAII for 24 h at 37°C. After incubation, the redox-modified recombinant apoEs were separated from excess agents by dialysis against PBS.

Preparation of F-EP and apoE-E-EP

F-EP were prepared using the method described by Rensen et al. [29] with a small modification. Briefly, the mixture of triolein, egg PC, and NBD-PC with a molar ratio of 3.5:1.0:0.05 was dried under nitrogen steam to a thin film on the wall of a round-bottomed flask, and desiccated overnight using an evaporator. The dried lipid film was dispersed in 10 mM Tris-HCl, pH 7.4, containing 100 mM NaCl (LP-buffer) and sonicated for 30 min at 55°C. Homogeneous F-EP were obtained by removing the contaminated liposomes using an ultracentrifuge. To form the complex of apoE and F-EP (apoE-F-EP), the prepared F-EP were incubated with various redox-modified recombinant apoEs for 30 min at 37°C.

Cell surface binding-assay and clearance/degradation-assay

For the cell surface binding assay, GM00701 cells at approximately 80% confluence were plated on a 48-well culture plate and rinsed with 20 mM HEPES, pH7.4, containing 150 mM NaCl and 2 mM CaCl₂ (HEPES buffer), followed by incubation with F-EP or apoE-F-EP for 3 h at 4°C. After washing away unbound F-EP with cold HEPES buffer, the cells were solubilized in RIPA buffer with a protease inhibitor cocktail (FUJIFILM Wako Pure Chemical). The fluorescence intensities of NBD in the whole-cell extracts were measured at excitation and emission wavelengths of 490 nm and 530 nm, respectively, using a fluorescence spectrophotometer (Molecular Devices Japan Co. Tokyo, Japan) and normalized to total cellular protein concentration, as determined using the BCA protein assay kit (Thermo Fisher Scientific Inc.). For inhibition experiments, the cells were incubated with F-EP or apoE-F-EP in the presence or absence of 5.0 mg/mL lactoferrin (FUJIFILM Wako Pure Chemical), 100 µg/mL anti-LRP1 (N-terminal) antibody, or cells were pretreated with a 2.5-U/mL heparinase cocktail containing heparinase I, II, and III (Sigma-Aldrich Co.), followed by incubation with F-EP or apoE-F-EP. The fluorescence intensities of the cells were also visualized using a fluorescence microscope and were quantified using Image J 1.45 software from the National Institute of Health.

For the clearance and degradation assays, rinsed cells were incubated with F-EP or apoE-F-EP in 5% CO₂-air for 3 h at 37°C. To estimate the clearance of F-EP, we measured the fluorescence intensities of F-EP or apoE-F-EP remaining in conditioned media before and after 3 h of culture and calculated the clearance rate of F-EP (%) (i) by dividing the difference in the fluorescence intensity between 0 h and 3 h (ii) by the fluorescence intensity of 0 h. Furthermore, we measured the total cellular (extracellular + intracellular) fluorescence intensities (iii) after 3 h of culture. Briefly, the rinsed cells were directly treated with RIPA buffer, and the solubilized proteins were diluted with LP-buffer at a ratio of 1:4 (w/w). The fluorescence intensities of the whole-cell extracts were measured and normalized, as described above. The intracellular degradation rate of F-EP was calculated using the following formula: [(ii – iii) × 100 / (ii)] × (i) (%).

Analysis of Isoelectric point

The isoelectric points of TCEP-treated recombinant apoE2 and apoE3 were analyzed using isoelectric focusing (IEF) and immunoblotting methods, as previously described [30].

Statistical methods

Data are presented as medians (25–75% quartiles). Differences between two groups were assessed using the Mann-Whitney U test. Results from three or more groups were analyzed by the Kruskal–Wallis test with the Steel–Dwass post hoc test. The statistical significance was set at p < 0.05.

ApoE-dependent binding of emulsion particles with the cell surface LRP1

First, to investigate the interaction of apoE-containing EP with GM00701 cells, we incubated the cells with F-EP or apoE-F-EP, and measured the fluorescence intensity in each whole-cell extract. The fluorescence intensity in the presence of apoE was approximately three-fold higher than that in the absence of apoE (p < 0.01) (Fig. 2A). The fluorescence intensity was proportional to the concentration of apoE, indicating that F-EP was bound to cells via apoE in a dose-dependent manner (Supplementary Fig. 1). The induction levels exhibited good reproducibility at final concentration of a 2.0-mg/dL apoE; hence, this concentration was used for subsequent experiments. Reflecting the results obtained by a fluorescence spectrophotometer, the cells in the presence of apoE showed stronger fluorescence intensities than those in the absence of apoE when visualized by fluorescence microscopy (Fig. 2B).

Next, to confirm whether this interaction could be attributed to the association between apoE on F-EP and cell surface LRP1, we measured the fluorescence intensity of the cells incubated with apoE-F-EP in the presence or absence of lactoferrin or anti-LRP1 antibody, which are the inhibitors of LRP1. The fluorescence intensity in the presence of lactoferrin or anti-LRP1 antibody, significantly decreased by approximately 19% (p < 0.01) or 22% (p < 0.01) of that in the absence of lactoferrin or LRP1 antibody, respectively (Fig. 2A).

Furthermore, to examine the effect of residual SDC1 in GM00701 cells, we treated cells with heparinase followed by incubation with apoE-F-EP. The fluorescence intensity of heparinase-treated cells decreased by approximately 89% compared to that of untreated cells, but the difference was not statistically significant (Fig. 2A).

Effect of apoE isoform on the LRP1-mediated binding of F-EP

To examine the effect of apoE isoforms on the binding of F-EP to GM00701 cells, we prepared apoE2-, apoE3-, or apoE4-F-EP and apoE-free F-EP (negative control, NC) and then incubated each of them with cells at 4°C for 3 h, which were pretreated with or without heparinase. The fluorescence intensity was then measured in each whole-cell extract. Regardless of heparinase pretreatment, the ability of apoE-F-EP to bind to cells differed significantly among the three apoE isoforms (Fig. 3A). In both cases with and without heparinase-pretreatment, apoE3-F-EP had a higher ability for binding to cells when compared with that of apoE2-F-EP (1.9-fold, p < 0.01, for binding to unpretreated cells; 1.7-fold, p < 0.05 for binding to pretreated cells). The binding ability of apoE3-F-EP was 1.3-fold higher than that of apoE4-F-EP; however, the difference was not statistically significant. The binding ability of apoE2-F-EP was also higher than that of the respective NC (approximately 2.2-fold, p < 0.01, for binding to both unpretreated and pretreated cells). However, regardless of heparinase-pretreatment, the binding ability of apoE2-F-EP was approximately 0.5-fold (p < 0.01 for binding to unpretreated cells; p < 0.05 for binding to pretreated cells) and 0.7-fold (p < 0.05) lower than that of apoE3- or apoE4-F-EP, respectively, and was the lowest among the three apoE isoforms containing F-EP. These differences among apoE isoforms were also observed under a fluorescence microscope (Fig. 3B). The fluorescence intensity of the whole-cell extract, which was prepared from cells incubated with NC, was significantly higher than the cellular autofluorescence (p < 0.05), indicating that a portion of F-EP bound to the cells, not through the apoE-LRP1 interaction.

Effect of apoE isoform on the LRP1-mediated clearance and degradation of apoE-F-

EP

To investigate the effect of apoE isoforms on LRP1-mediated clearance and degradation of F-EP, the prepared apoE-free (NC), apoE2-, apoE3-, and apoE4-F-EP were individually incubated with GM00701 cells for 3 h in 5% CO₂-air at 37°C. The clearance rates of both apoE2- and apoE3-F-EP were approximately 19%, which was 1.6-fold higher than that of the NC (p < 0.05). Although not statistically significant, the clearance rate of apoE4-F-EP was 0.8-fold lower than those of apoE2- and apoE3-F-EP (Fig. 4A). Similarly, the degradation rates of apoE2- and apoE3-F-EP were approximately 15% (76% of the cleared F-EP) and 2.0-fold higher than that of NC (p < 0.01). The degradation rate of apoE4-F-EP was approximately 0.5-fold lower than those of apoE2- and apoE3-F-EP (p < 0.05) (Fig. 4B). Unexpectedly, the clearance and degradation of NC were observed, indicating that a portion of F-EP was taken up into the cells even in the absence of apoE. We confirmed that apoE-F-EP could be cleared from the culture medium and intracellularly degraded via LRP1 by LRP1 antibody inhibition experiments using apoE3-F-EP (Supplementary Fig. 2).

Effect of apoE-Cys-thiol modification on the LRP1-mediated binding of apoE-

F-EP

Using the cell surface binding assay system, we examined whether redox modifications of apoE-Cys-thiol affected the binding of F-EP. The reduction of Cys-thiol with TCEP significantly enhanced the binding ability of apoE3-F-EP to cells by approximately 1.5-fold (p < 0.01) but had no effect on those of apoE2- and apoE4-F-EP (Fig. 5A). Disulfide bond formation by the treatment s treated with diamide (Supplementary Fig. 3A) slightly enhanced the binding ability of apoE3-F-EP to cells by approximately 1.3-fold (p < 0.05); however, the binding ability of apoE2- and apoE4-F-EP was unaffected (Fig. 5B). In contrast, the formation of heterodimers through disulfide bonds by treatment with apoAII (Supplementary Fig. 3B) significantly reduced the binding ability of apoE2-F-EP to cells by approximately 0.5-fold (p < 0.01), but not those of apoE3- and apoE4-F-EP, (Fig. 5C). The IAM-alkylation of Cys-thiol decreased the binding ability of apoE3-F-EP to cells by approximately 0.5-fold (p < 0.01) (Fig. 5D), whereas sodium iodoacetate (IAA)-alkylation of Cys-thiol enhanced it by approximately 1.3-fold (p < 0.05) (Fig. 5E). Although statistically significant, the effects of IAM- and IAA-alkylation on the binding of apoE2-F-EP to cells were similar to those of apoE3-F-EP. However, neither alkylation reaction affected the binding of apoE4-F-EP to cells. We also confirmed that modification of Cys-thiol had no significant impact on the interaction of apoE with F-EP (Supplementary Fig. 4).

Effect of apoE-Cys-thiol modification on the LRP1-mediated clearance and degradation of apoE-F-EP

We examined whether the redox modifications of apoE-Cys-thiol affected the LRP1-mediated clearance and degradation of F-EP. The TCEP reduction suppressed the clearance and degradation of apoE3-F-EP by approximately 0.8-fold and 0.7-fold (p < 0.05), respectively, but did not significantly affect apoE2- and apoE4-F-EP (Fig. 6A, B). Despite the apoE isoform, diamide-treatment had no effect on the clearance and degradation of apoE-F-EP (Fig. 6C, D). The treatment with apoAII significantly enhanced the clearance and degradation of apoE2-F-EP by approximately 2.4-fold (p < 0.01) and 1.8-fold (p < 0.05), respectively, but had no significant effect on those of apoE3- and apoE4-F-EP (Fig. 6E, F). IAM-alkylation enhanced the clearance and degradation of apoE2-F-EP by approximately 1.6-fold (p < 0.05) and 2.1-fold (p < 0.01), respectively, and those of apoE3-F-EP by approximately 1.4-fold and 1.6-fold (p < 0.05), respectively (Fig. 6G, H). IAA-alkylation also enhanced the clearance and degradation of apoE3-F-EP by approximately 1.5-fold (p < 0.05) and 1.6-fold (p < 0.05), respectively. In contrast, although not statistically significant, IAA-alkylation suppressed the clearance and degradation of apoE2-F-EP (Fig. 6I, J). The effects of the apoE-Cys-thiol redox modifications on the binding, clearance, and degradation of apoE2- and apoE3-F-EP are summarized in Table 1.

Isoelectric point of the reduced-apoE2 and apoE3

To investigate the effect of the electric charge of Cys-thiol on the interaction between apoE and LRP1, we conducted IEF using TCEP-treated apoE2 and apoE3. Most of the untreated apoE3 existed in dimeric form (Fig. 7, lane 3), whereas untreated apoE2 existed both in its oligomeric and reduced monomeric forms, whose Cys-thiols may be mainly protonated forms (thiols or thiol + thiolate) [(-SH)² or (-SH) + (-S⁻)] and subtly unprotonated forms (thiolates) [(-S⁻)²] (lane 1). The TCEP-treatment completely reduced oligomeric apoE2 into monomers (lane 2) and mostly, but not completely, reduced dimeric apoE3 into monomers (lane 4). Under reducing conditions, the unprotonated (negatively charged acidic) form of one or both Cys-thiols and the protonated form were predominant for apoE2 and apoE3, respectively.

GM00701 cells are suitable for analyses that focus on LRP1 functions, because the cells highly express LRP1, but not LDLR and SDC1 [28]. In the present study, we also designed experiments by utilizing these benefits of the cells, following the previous study [28]. In contrast to previous findings, our data showed that significant LRP1-mediated cellular interaction of apoE-F-EP still occurred, even when the effects of slightly expressed SDC1 and other HSPGs were eliminated by heparinases. As demonstrated by several studies [16, 28, 31], we also believe that SDC1 plays a critical role in the clearance of apoE containing very low-density lipoproteins (apoE-VLDL). In fact, we confirmed that SDC1 high-express transfectants, obtained by transient transfection of full-length human SDC1 cDNA into GM00701 cells, exhibited more effective binding of apoE-EP compared to non-transfected cells (our unpublished data). Nevertheless, although the catabolic capacity of apoE-LP would not be too large, our present data suggested that LRP1 in fibroblasts, even if alone, has the potential to compensate for the lack of SDC1 or LDLR. The difference in the animal species of apoE-LP used for analysis may be the most likely factor causing the discrepancy between the previous and our data; that is, Wilsie et al. [28] used rabbit apoE-VLDL, whereas we used human recombinant apoE containing F-EP. One limitation of our study was the use of artificially synthetic F-EP; hence, we will need to conduct the further experiments using human serum RLP, obtained from subjects with different apoE isoforms. Nevertheless, we believe that the use of the same ligand species as the receptor allowed us to more adequately observe the apoE-LRP1 interaction.

The effect of the apoE-isoform on LRP1-mediated metabolism of apoE-LP in the systemic circulation has not been fully studied, although β-amyloid clearance via LRP1 expressed in the brain is known to be apoE-isoform-dependent [21, 32, 33]. Unlike the report by Ruiz et al. [19], we demonstrated that the binding of apoE-F-EP to LRP1 was distinctly different among the three isoforms. Although apoE2-F-EP showed the lowest binding ability at 4°C, it appeared to be sufficient compared to that of apoE2-LP with LDLR described in the literature [1]. Interestingly, under physiological conditions, the LRP1-mediated catabolic capability of apoE2-F-EP was comparable with that of apoE3-F-EP. In addition, contrary to our expectations, apoE4-F-EP, but not apoE2-F-EP, were difficult to catabolize, with a capability almost equal to that of apoE-free F-EP. Although the exact mechanism of apoE-free F-EP uptake by cells is unclear, contaminated liposomes that were not completely removed during the preparation of F-EP may be taken up by cells based on their cell-membrane fusion property [34]. Hence, all prepared apoEs-F-EP could be predicted to contain liposomes to some extent. Considering this effect, the internalization of apoE4-LP is likely to be inhibited by the binding of apoE4 with LRP1. Altenburg et al. [35] previously demonstrated that the high affinity of apoE4 for LDLR enhances the sequestration of VLDL remnants on the cell surface but delays their internalization and consequently accelerates the production of cholesterol-rich RLP. Similar to this prior finding, a decline in the catabolism of apoE4-F-EP may be attributed to a delay in its clearance, associated with the high affinity of apoE4 to LRP1. In contrast, the limited interaction between apoE2 and LRP1 may rather enhance the clearance of F-EP. This idea could provide a convincing explanation for the difference in the effect of LRP1-mediated interaction between apoE2 and apoE4, but does not explain why apoE3, which has an equal or higher affinity for LRP1 than apoE4, can efficiently catabolize F-EP. These discussions imply that some additional factors may affect the LRP1-mediated metabolism of apoE-LP.

The post-translational redox modifications of Cys-thiols affect are known to affect various biological activities [12, 13, 17, 18]. Based on this evidence, we assumed that modifications of the Cys-thiols of apoE2 and apoE3 would participate in the regulation of the LRP1-mediated pathway. We demonstrated that the protonation (conversion to thiol form) of apoE3-Cys by the TCEP-reduction enhanced the binding of apoE3-F-EP with LRP1 but decreased its catabolism. This result may also be attributed to a delay in the clearance of apoE3-F-EP associated with the increasing affinity of apoE3 to LRP1, as in the case of apoE4. Unlike apoE3-F-EP, the TCEP-reduction subtly lowered the LRP1-mediated binding of apoE2-F-EP, suggesting that Cys112- and Cys158-thiol may have different effects on the interaction of apoE2 with LRP1. Innerarity et al. [36] demonstrated that the defective LDLR binding ability of apoE2 could be attributed to the effect of Cys158. We previously demonstrated that Cys158 of apoE2 is more susceptible to redox modifications than Cys112 of apoE2 or apoE3 [27]. Based on these facts, we hypothesized that the present data of the decreased binding of apoE2-F-EP to LRP1 may be ascribed to the effect of Cys158 in apoE2. Furthermore, we hypothesized that redox modifications of Cys-thiols in apoE2 or apoE3 might determine the efficiency of LRP1-mediated metabolism of apoE-LP.

It is well known that a diamide-treatment prominently promotes the homodimer formation [24]. Although no significant changes in both clearance and degradation capability were observed, a diamide-treatment enhanced the binding of apoE3-F-EP but not of apoE2-F-EP with LRP1, suggesting that homodimer formation via Cys112 may have an effect on the process of LRP1-mediated metabolism of apoE3-LP. We presume that the differences between apoE2 and apoE3 may be attributed to the mutually antagonistic properties of Cys112 and Cys158; consequently, the effect of Cys112 may be canceled by that of Cys158. This idea is also supported by the prominent effect of TCEP reduction and IAA- and IAM-alkylation on the LRP1-mediated binding of apoE3-EP, but not of apoE2-EP.

The presence of Cys112 and/or Cys158 also allows apoE2 and apoE3 to form heterooligomers, such as the apo (E-AII) and apo (AII-E2-AII) complexes [37, 38]. We showed that apoAII-incubation had a more pronounced effect on the LRP1-mediated interaction of apoE2-F-EP, compared to diamide treatment. Based on our speculation above, this result may be attributed to the effect of Cys158-modification outweighing that of Cys112-modification. The fact that apoAII incubation decreased the binding but enhanced the clearance and degradation of apoE2-F-EP suggests that the inhibitory effect on the binding of apoE2 with LRP1 by the formation of apoE2-heterooligomers via Cys158 may facilitate the LRP1-mediated internalization of apoE2-F-EP, in contrast to the effect of apoE4 or a reduced-apoE3. Homozygous possession of apoE2 is a necessary but not sufficient condition for the manifestation of type III hyperlipoproteinemia [7, 39]. The apoAII containing VLDL is just a small pool of their particles; however, it has been suggested that apoAII functions as a regulator of VLDL metabolism by spontaneously transferring apoAII from high-density lipoproteins, which serves as a plasma reservoir of apoAII, to the VLDL particles [40]. Taken together, our findings led us to assume that in patients with apoE2 homozygotes, the accumulation of RLP may be accelerated by conditions that chronically suppress the formation of apo (E2-AII) or apo (AII-E2-AII) complex (such as oxidative stress, which irreversibly modifies the apoE2-Cys-thiol), and that such a disorder may be one of triggers for the development of type III and other types of hyperlipoproteinemia.

Innerarity et al. [36] demonstrated that the change in Cys158 in apoE2 to basicity by cysteamine treatment restores its binding to LDLR. Considering this evidence, the fact that apoAII-incubated apoE2 had a more prominent effect on the LRP1-mediated interaction compared to diamide-incubated apoE2 suggests that, in addition to the position of modified Cys, the differences in their acid-base properties of modifying molecules, may also affect the LRP1-mediated metabolic pathway. In fact, the isoelectric point of apoAII (4.9-5.0) is known to be more acidic than that of apoE2 (5.7) [41]. This speculation also led us to hypothesize that the interaction of monomeric apoE2 or apoE3 with LRP1 can be determined by the change in the electric charge of Cys-thiol according to its redox status. Indeed, IEF patterns showed that a TCEP-reduced apoE2 exists predominantly in a relatively negatively charged acidic thiolate form compared to a TCEP-reduced apoE3, indicating that Cys158 may be more difficult to convert into the thiol form than Cys112. This may explain why the TCEP reduction had no effect on the binding of apoE2-F-EP to cells, but enhanced that of apoE3-F-EP to cells. In brief, apoE2-LRP1 binding may also be antagonistically modulated by acid-base changes of Cys112- and Cys158-thiol, and the thiolate form of Cys158 may have a negative effect on apoE2-LRP1 interactions.

ApoE4 is a well-known representative risk factor for various atherosclerotic diseases and Alzheimer’s disease [3]; however, the precise underlying mechanisms are still obscure. We showed that apoE4-F-EP was hard to catabolize via LRP1. This result provides an evidence that apoE4 has a detrimental effect on the LRP1-mediated lipoprotein metabolism. Furthermore, given the possibility that redox modifications of Cys-thiols may function as on-off switches for the LRP1-mediated metabolism of apoE2- or apoE3-LP, apoE4, which has no Cys residue, intrinsically lacks such a regulatory system. This may be the reason for the pathological effects of apoE4.

We demonstrated that LRP1 in fibroblasts contributes to TG-rich lipoprotein metabolism in an apoE-isoform-dependent manner and that Cys-thiols of apoE2 and apoE3 and their redox modifications affect this metabolic pathway. The lack or disorder of this regulatory system, depending on the redox-responsive Cys-thiol switch, may lead to the accumulation of RLP and the consequent development of dyslipidemia and its related atherosclerosis.

apo: apolipoprotein

apoE-F-EP: apoE-containing fluorescence-labeled emulsion particles

Arg: arginine

Cys: cysteine

HRP: horseradish peroxidase

HSPG: heparan sulfate proteoglycan

IAA: iodoacetate

IAM: 2-iodoacetamide

IEF: isoelectric focusing

LDL: low-density lipoprotein

LDLR: low-density lipoprotein receptor

LP: lipoprotein

LRP: low-density lipoprotein receptor-related protein

NBD-PC: 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] hexanoyl}-sn-

glycero-3-phosphocholine

NC: negative control

PBS: phosphate-buffered saline

PC: phosphatidylcholine

RLP: remnant lipoprotein

SDC1: syndecan1

TCEP: Tris (2-carboxyethyl) phosphine

TG: triglyceride

VLDL: very low-density lipoprotein

Availability of data and materials

The data that support the findings of this study are available from the corresponding author, [KY], upon reasonable request.

Acknowledgments

not applicable

Funding

This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 21K07310).

Authors and Affiliations

Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu

University, Matsumoto, Japan.

Hiroto Matsuura, Shogo Akahane, Takahiro Kaido, Tomu Kamijo and Kazuyoshi Yamauchi

Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.

Takahiro Kaido and Tomu Kamijo

Department of Laboratory Medicine, University of Yamanashi Hospital, Yamanashi, Japan.

Kenta Sakamoto

Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.

Kazuyoshi Yamauchi

Contributions

KY designed the present project. HM, SA, TK, TK and KY carried out experiments and analyzed experimental data. HM wrote this manuscript. KY reviewed and commented on drafts of the manuscript. All authors approved the final version of this manuscript.

Corresponding authors

Correspondence to Kazuyoshi Yamauchi

Ethical approval and consent to participate

not applicable

Consent for publication

not applicable

Conflict of interests

The authors declare no conflict of interest associated with this　manuscript. The research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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No competing interests reported.

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Impact of isoform and Cys-thiol modifications of apolipoprotein E on the LRP1-mediated metabolism of triglyceride-rich lipoproteins

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Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and Methods

Materials

Cells and cell culture

Redox modifications of Cys-thiol of recombinant apoE

Preparation of F-EP and apoE-E-EP

Cell surface binding-assay and clearance/degradation-assay

Analysis of Isoelectric point

Statistical methods

Results

ApoE-dependent binding of emulsion particles with the cell surface LRP1

Effect of apoE isoform on the LRP1-mediated binding of F-EP

Effect of apoE isoform on the LRP1-mediated clearance and degradation of apoE-F-

EP

Effect of apoE-Cys-thiol modification on the LRP1-mediated binding of apoE-

F-EP

Effect of apoE-Cys-thiol modification on the LRP1-mediated clearance and degradation of apoE-F-EP

Isoelectric point of the reduced-apoE2 and apoE3

Discussion

Conclusions

Abbreviations

Declarations

References

Table 1

Additional Declarations

Supplementary Files

Status:

Version 1