Apolipoprotein B-100 (Apo-B100) is a major component of low density lipoprotein (LDL). Apo B-100 protein has 4,536 amino acid sequence and these amino acids are classified into peptide groups A to G with subsequent 20 amino acids (P1-P302). The peptide groups were act as immunoglobulin (Ig) antigens which oxidized via malondialdehyde (MDA). The mimetic peptide P1 (EEEMLENVSLVCPKDAT RFK) out of D-group peptides carrying the highest value of IgG antigens were selected for structural studies that may provide antigen specificity. Circular Dichroism (CD) spectra were measured for peptide secondary structure in the range of 190-250 nm. Experimental results show that P1 exhibit partial of ${\beta}-sheet$ and random coil structure. Homonuclear (COSY, TOCSY, NOESY) 2D-NMR experiments were carried out for NMR signal assignments and structure determination for P1. On the basis of these completely assigned NMR spectra and distance data, distance geometry (DG) and Molecular dynamics (MD) were carried out to determine the structures of P1. The proposed structure was selected by comparisons between experimental NOE spectra and back calculated 2D NOE results from determined structure showing acceptable agreement. The total Root-Mean-Square-Deviation (RMSD) value of P1 obtained upon superposition of all atoms was in the range $0.33{\AA}$. The solution state P1 has mixed structure of ${\beta}-sheet$ (Glu[1] to Cys[12]) and random coil (Pro[13] to Lys[20]). These NMR results are well consistent with secondary structure from experimental results of circular dichroism. Structural studies based on NMR may contribute to the studies of atherosclerosis and observed conformational characteristics of apo B-100 in LDL using monoclonal antibodies.
Apolipoprotein B-100 (apo B-100), the main protein component that makes up LDL (Low density lipoprotein), consists of 4,536 amino acids and serves to combine with the LDL receptor. The oxidized LDL peptides by malondialdehyde (MDA) or acetylation in vivo were act as immunoglobulin (Ig) antigens and peptide groups were classified into 7 peptide groups with subsequent 20 amino acids (P1-P302). The biomimetic peptide P99 (KGTYG LSCQR DPNTG RLNGE) out of B-group peptides carrying the highest value of IgM antigens were selected for structural studies that may provide antigen specificity. Circular Dichroism (CD) spectra were measured for peptide secondary structure in the range of 190-260 nm. Experimental results show that P99 has pseudo α-helice and random coil structure. Homonuclear (COSY, TOCSY, NOESY) 2D-NMR experiments were carried out for NMR signal assignments and structure determination for P99. On the basis of these completely assigned NMR spectra and proton distance information, distance geometry (DG) and molecular dynamic (MD) were carried out to determine the structures of P99. The proposed structure was selected by comparisons between experimental NOE spectra and back-calculated 2D NOE results from determined structure showing acceptable agreement. The total Root-Mean-Square-Deviation (RMSD) value of P99 obtained upon superposition of all atoms were in the set range. The solution state P99 has mixed structure of pseudo α-helix and β-turn(Gln[9] to Thr[13]). These NMR results are well consistent with secondary structure from experimental results of circular dichroism. Structural studies based on NMR may contribute to the prevent oxidation studies of atherosclerosis and observed conformational characteristics of apo B-100 in LDL using monoclonal antibodies.
Apolipoprotein B-100 (apo B-100), the main protein component that makes up LDL (Low density lipoprotein), consists of 4,536 amino acids and serves to combine with the LDL receptor. The oxidized LDL peptides by malondialdehyde (MDA) or acetylation in vivo act as immunoglobulin (Ig) antigens and peptide groups were classified into 7 peptide groups with subsequent 20 amino acids (P1-P302). The biomimetic peptide P143 (IALDD AKINF NEKLS QLQTY) out of C-group peptides carrying the highest value of IgG antigens were selected for structural studies that may provide antigen specificity. Experimental results show that P143 has β-sheet in Ile[1]-Asn[9] and α-helice in Gln[16]-Tyr[20] structure. Homonuclear 2D-NMR (COSY, TOCSY, NOESY) experiments were carried out for NMR signal assignments and structure determination for P143. On the basis of these completely assigned NMR spectra and proton distance information, distance geometry (DG) and molecular dynamic (MD) were carried out to determine the structures of P143. The proposed structure was selected by comparisons between experimental NOE spectra and back-calculated 2D NOE results from determined structure showing acceptable agreement. The total Root-Mean-Square-Deviation (RMSD) value of P143 obtained upon superposition of all atoms were in the set range. The solution state P143 has a mixed structure of pseudo α-helix and β-turn(Phe[10] to Glu[12]). These results are well consistent with calculated structure from experimental data of NOE spectra. Structural studies based on NMR may contribute to the prevent oxidation studies of atherosclerosis and observed conformational characteristics of apo B-100 in LDL using monoclonal antibodies.
Hong Ki Min;Jeonghyeon Moon;Seon-Yeong Lee;A Ram Lee;Chae Rim Lee;Jennifer Lee;Seung-Ki Kwok;Mi-La Cho;Sung-Hwan Park
IMMUNE NETWORK
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v.21
no.6
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pp.43.1-43.14
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2021
Group 3 innate lymphoid cells (ILC3), which express IL-22 and IL-17A, has been introduced as one of pathologic cells in axial spondyloarthritis (axSpA). Dyslipidaemia should be managed in axSpA patients to reduce cardiovascular disease, and dyslipidaemia promotes inflammation. This study aimed to reveal the role of circulating ILC3 in axSpA and the impact of dyslipidaemia on axSpA pathogenesis. AxSpA patients with or without dyslipidaemia and healthy control were recruited. Peripheral blood samples were collected, and flow cytometry analysis of circulating ILC3 and CD4+ T cells was performed. The correlation between Ankylosing Spondylitis Disease Activity Score (ASDAS)-C-reactive protein (CRP) and circulating immune cells was evaluated. The effect of oxidized low-density lipoprotein cholesterol (oxLDL-C) on immune cell differentiation was confirmed. AxSpA human monocytes were cultured with with oxLDL-C, IL-22, or oxLDL-C plus IL-22 to evaluate osteoclastogenesis using tartrate-resistant acid phosphatase (TRAP) staining and real-time quantitative PCR of osteoclast-related gene expression. Total of 34 axSpA patients (13 with dyslipidaemia and 21 without) were included in the analysis. Circulating IL-22+ ILC3 and Th17 were significantly elevated in axSpA patients with dyslipidaemia (p=0.001 and p=0.034, respectively), and circulating IL-22+ ILC3 significantly correlated with ASDAS-CRP (Rho=0.4198 and p=0.0367). Stimulation with oxLDL-C significantly increased IL-22+ ILC3, NKp44- ILC3, and Th17 cells, and these were reversed by CD36 blocking agent. IL-22 and oxLDL-C increased TRAP+ cells and osteoclast-related gene expression. This study suggested potential role of circulating IL-22+ ILC3 as biomarker in axSpA. Furthermore, dyslipidaemia augmented IL-22+ ILC3 differentiation, and oxLDL-C and IL-22 markedly increased osteoclastogenesis of axSpA.
Purpose: Hyperglycemia accelerates the formation of advanced glycation end products (AGEs), a group of compounds formed via non-enzymatic glycation/glycoxidation. Type 2 diabetes mellitus (T2DM) is related to oxidative stress, resulting in some overgeneration of AGEs. The accumulation of AGEs in T2DM patients leads to increased inflammation, DNA damage, tissue damage, progression of diabetic microvascular disease, and nephropathy. Heme oxygenase-1 (HO-1) is an intracellular enzyme that catalyzes the oxidation of heme. Expression of HO-1 in the endothelium and in muscle monocytes/macrophages was upregulated upon exposure to reactive oxygen species or oxidized low-density lipoprotein. Cells activated by oxidative stress are reported to release HO-1 in the serum. In the current study, we discuss the oxidative status according to the level of AGEs and the association of HO-1 with AGEs or urinary DNA damage marker in type 2 diabetic Korean patients. Methods: This study enrolled 36 diabetic patients. Subjects were classified into two groups by serum AGEs level (Low AGEs group: < 0.85 ng/mL serum AGEs; High AGEs group: ≥ 0.85 ng/mL serum AGEs). Body composition was measured using bioelectrical impedance analysis. Blood and urinary parameters were measured using commercial kits. Results: No significant differences were observed in the general characteristics and body composition between the two groups. Serum HO-1 concentration was significantly higher in the High AGEs group than in the Low AGEs group. After adjustment of age and gender, a correlation was performed to assess the association between serum HO-1 and serum AGEs or urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG). Our results indicate that serum HO-1 is positively correlated with serum AGEs and urinary 8-OHdG. Conclusion: Taken together, our results indicate that in diabetes patients, a high level of HO-1 is associated with a high concentration of AGEs and 8-OHdG, probably reflecting a protective response against oxidative stress.
It has been proposed that oxidative modification of LDL (oxLDL) plays a significant role in the pathogenicity of atherogenesis. We tested the hypothesis that chitin and chitosan may function as antioxidants with respect to 0.1 mg cholesterol/ml LDL incubated with 5 $\mu$ M Cu$^2$$^{+}$alone or in the P338Dl mouse macrophage system using L-ascorbic acid as a standard classical antioxidant. The degree of oxLDL formation was ascertained by the relative electrophoretic mobility (rEM) in the combination of thiobarbituric acid reactive substances (TBARS) levels, and the cytotoxicity of oxLDL was detected by macrophage viability. The oxLDL uptake and foam cell formation of macrophages were measured by Oil Red O staining. Incubation with Cu$^2$$^{+}$and macrophages increased rEM of LDL and stimulated TBARS formation. Culture of macrophages with LDL in the presence 5 $\mu$ M Cu$^2$$^{+}$induced macrophage death. In cell-free system 200 $\mu$g/ml water-soluble chitosan and chitosan-oligosaccharide blocked oxLDL formation. Water-soluble chitosan and chitosan-oligosaccharide blocked oxLDL formation near-completely relative to L-ascorbic acid, whereas water-soluble chitin and chitin-oligosaccharide had no measurable antioxidant effect. In macrophage system water-soluble chitosan and chitosan-oligosaccharide blocked oxidation of LDL with a significant increase in cell viability, and decreased TBARS in medium. As for the inhibitory effect on macrophage foam cell formation, chitosan and its oligosaccharide, but not watersoluble chitin, revealed the effectiveness. The endothelial expression of lectin-like oxLDL receptor-1 (LOX-1) was tested by Western blot analysis, and chitosan, chitosan-oligosaccharide and chitin-oligosaccharide blocked LOX-1 expression. These results indicate that water-soluble chitosan and its oligosaccharide showed the inhibitory effect on Cu$^2$$^{+}$-induced LDL oxidation of macrophages, and chitosan, chitosan-oligosaccharide and chitin-oligosaccharide had blocking effect on oxLDL receptor expression in the human umbilical vein endothelial system. Thus, water-soluble chitosan and its oligosaccharides possess anti-atherogenic potentials possibly through the inhibition of macrophage LDL oxidation or endothelial oxLDL receptor expression depending on chemical types.l types.
Oxidized low-density lipoprotein (oxLDL) has been shown to modulate transactivations by the peroxisome proliferator activated receptor (PPAR)$\gamma$ and nuclear factor-kappa B (NF$\kappa$B). In this study, the oxLDL signaling pathways involved with the NF$\kappa$B transactivation were investigated by utilizing a reporter construct driven by three upstream NF$\kappa$B binding sites, and various pharmacological inhibitors. OxLDL and its constituent lysophophatidylcholine (lysoPC) induced a rapid and transient increase of intracellular calcium and stimulated the NF-KB transactivation in resting RAW264.7 macrophage cells in an oxidation-dependent manner. The NF$\kappa$B activation by oxLDL or lysoPC was inhibited by protein kinase C inhibitors or an intracellular calcium chelator. Tyrosine kinase or PI3 kinase inhibitors did not block the NF$\kappa$B transactivation. Furthermore, the oxLDL-induced NF$\kappa$B activity was abolished by the PPAR$\gamma$ ligands. When the endocytosis of oxLDL was blocked by cytochalasin B, the NF$\kappa$B transactivation by oxLDL was synergistically increased, while PPAR transactivation was blocked. These results suggest that oxLDL activates NF-$\kappa$B in resting macrophages via protein kinase C- and/or calcium-dependent pathways, which does not involve the endocytic processing of oxLDL. The endocytosis-dependent PPAR$\gamma$ activation by oxLDL may function as an inactivation route of the oxLDL induced NF$\kappa$B signal. Short heterodimer partner (SHP), specifically expressed in liver and a limited number of other tissues, is an unusual orphan nuclear receptor that lacks the conventional DNA-binding domain. In this work, we found that SHP expression is abundant in murine macrophage cell line RAW 264.7 but suppressed by oxLDL and its constituent I3-HODE, a ligand for peroxisome proliferator-activated receptor y. Furthermore, SHP acted as a transcription coactivator of nuclear factor-$\kappa$B (NF$\kappa$B) and was essential for the previously described NF$\kappa$B transactivation by lysoPC, one of the oxLDL constituents. Accordingly, NF$\kappa$B, transcriptionally active in the beginning, became progressively inert in oxLDL-treated RAW 264.7 cells, as oxLDL decreased the SHP expression. Thus, SHP appears to be an important modulatory component to regulate the transcriptional activities of NF$\kappa$B in oxLDL-treated, resting macrophage cells.
Journal of the Korea Academia-Industrial cooperation Society
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v.20
no.8
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pp.477-484
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2019
Several types of scavenger receptors, including the Collectin-Placenta 1 (CL-P1) receptorthat is present in mammals, are molecules that are expressed on the surfaces of endothelial cells, macrophages and smooth muscle cells. These molecules are cell surface glycoproteins that can be conjugated to oxidized low density lipoprotein (oxLDL). Among these molecules, the effect of quercetin on CL-P1 activation has been confirmed. Quercetin is known as an antioxidant that stops oxidation because it acts to remove free radicals that are responsible for the oxidation reaction. In this study, fragments from the transcription start site of the mouse CL-P1 gene promoter to the -500th base were cloned using DNA polymerase. These fragments were then introduced into macrophage like RAW264.7 cells and fibroblast-like NIH3T3 cells to study the effect of quercetin on the CL-P1 gene expression. As a result, we found that bases ranging from -250 to -350 in the anterior part where gene expression starts are important for producing CL-P1 protein. Among them, the DNA mutation experiments we performed confirmed that the E2F binding sites are critical for producing the CL-P1 protein? In addition, when quercetin was added to the RAW264.7 culture medium, which was a culture of adherent cells, observedthe phenomenon of the cells falling off from the surface of the culture container.
Ahn, Youngsook;Regu, Geberea manuel Meron;Oh, Eun Kyoung;Kwon, Oran
Journal of Nutrition and Health
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v.50
no.3
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pp.225-235
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2017
Purpose: This study aimed to investigate the potential of freeze-dried persimmon powder (Diospyros kaki Thumb.) to protect against dyslipidemia induced by a high-fat/cholesterol diet (HFD) in a rat model. Methods: Fifty Wistar rats were randomly divided into five groups: normal control (NC), high-fat/cholesterol control (HC), tannin in HFD (HT, 1% of diet), immature persimmon in HFD (HI, 7% of diet), and mature persimmon in HFD (HM, 7% of diet). Tannin was used as a positive control. Biochemical, molecular, and histopathological changes were observed in the blood and liver. Results: We confirmed that a high fat/cholesterol diet successfully induced dyslipidemia, which was characterized by significantly altered lipid profiles in the plasma and liver. However, oxidized low-density lipoprotein levels, histopathological damage in the liver, and hepatic triglyceride levels were significantly reduced in all HT, HI, and HM groups compared to those in the HF group. In contrast, plasma apolipoprotein B level was significantly reduced only in the HT and HM groups, whereas reduction of the LDL-C level was detected only in the HI group. Although HF-induced sterol regulatory element-binding protein (SREBP) gene expression was significantly reduced in all treated groups, downstream gene expression levels varied among the different groups; significant reduction of fatty acid synthase (FAS) and 3-hydroxy-3-methylglutaryl-CoA (HMGCR) gene expression was detected only in the HI group, whereas cholesterol $7{\alpha}$-hydroxylase (CYP7A1) gene expression was significantly elevated only in the HM group. Conclusion: Taken together, the data suggest that protection of LDL oxidation and hepatic lipogenesis might be, at least partly, attributed to tannin in persimmons. However, the identified mechanisms varied up to the maturation stage of persimmon. In the case of immature persimmon, modulation of FAS and HMGCR gene expression was prominent, whereas in the case of mature persimmon, modulation of CYP7A1 gene expression was prominent.
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