Yan Chi

Core Fucosylation of μ Heavy Chains Regulates Assembly and Intracellular Signaling of Precursor B Cell Receptors

Background: Formation of a functional precursor B cell receptor (pre-BCR) is dependent on N-glycosylation of μ-heavy chains (μHC). Results: Lack of core fucosylation of μHC attenuated the interaction between μHC and λ5. Conclusion: Core fucosylation of μHC mediates the assembly of pre-BCR. Significance: Learning how Fut8 regulates the assembly of pre-BCR is crucial for understanding pre-B cell development. α1,6-Fucosyltransferase (Fut8) knock-out (Fut8−/−) mice showed an abnormality in pre-B cell generation. Membrane assembly of pre-BCR is a crucial checkpoint for pre-B cell differentiation and proliferation in both humans and mice. The assembly of pre-BCR on the cell surface was substantially blocked in the Fut8-knockdown pre-B cell line, 70Z/3-KD cells, and then completely restored by re-introduction of the Fut8 gene to 70Z/3-KD (70Z/3-KD-re) cells. Moreover, loss of α1,6-fucosylation (also called core fucosylation) of μHC was associated with the suppression of the interaction between μHC and λ5. In contrast to Fut8+/+ CD19+CD43− cells, the subpopulation expressing the μHC·λ5 complex in the Fut8−/− CD19+CD43− cell fraction was decreased. The pre-BCR-mediated tyrosine phosphorylation of CD79a and activation of Btk were attenuated in Fut8-KD cells, and restored in 70Z/3-KD-re cells. The frequency of CD19lowCD43− cells (pre-B cell enriched fraction) was also reduced in Fut8−/− bone marrow cells, and then the levels of IgM, IgG, and IgA of 12-week-old Fut8−/− mice sera were significantly lower than those of Fut8+/+ mice. Our results suggest that the core fucosylation of μHC mediates the assembly of pre-BCR to regulate pre-BCR intracellular signaling and pre-B cell proliferation.

7-ketocholesteryl-9-carboxynonanoate induced nuclear factor-kappa B activation in J774A.1 macrophages.

AIMS 7-Ketocholesteryl-9-carboxynonanoate (oxLig-1), a lipid moiety of oxidized low-density lipoprotein (oxLDL), has been reported to be a crucial ligand of beta2-glycoprotein I (β(2)-GPI), and plays a potential role in the development of atherosclerosis (AS), however, the role of the sole oxLig-1 in the development of AS remains unclear. MAIN METHODS Expression and phosphorylation levels of several proteins, such as nuclear factor-kappaB (NF-κB), protein kinase C (PKC), IκBα and inter-cellular adhesion molecule 1 (ICAM-1) were determined by Western blot; nuclear localization of NF-κB was studied by immunocytochemistry; NF-κB activation was assayed by electrophoretic mobility shift assay (EMSA); and expressions of genes associated with AS process were investigated by Mouse Atherosclerosis RT(2) Profiler PCR Array assay. KEY FINDINGS The present work indicated that oxLig-1 induced IκBα phosphorylation and results in the nuclear translocation of NF-κB in J774A.1 macrophages. Moreover, oxLig-1-induced NF-κB DNA binding activity was detected by EMSA. Indeed, oxLig-1 led to the activation of PKC prior to activating NF-κB. The treatment of oxLig-1 in J774A.1 macrophages up-regulates the expression of NF-κB target genes including ICAM-1. SIGNIFICANCE OxLig-1 on the oxLDL plays an important role in AS process, as evidenced by the NF-κB activation and up-regulation of genes involved in AS development in oxLig-1 challenged J774A.1 macrophages.

Recombinant Domain V of β2-Glycoprotein I Inhibits the Formation of Atherogenic oxLDL/β2-Glycoprotein I Complexes

Abstractβ2-glycoprotein I (β2-GPI) is a plasma protein that interacts with oxidized low-density lipoproteins (oxLDL) via β2-GPI domain V to form oxLDL/β2-GPI complexes, potential autoantigens promoting atherogenesis in patients with antiphospholipid syndrome (APS). Such a interaction would expose β2-GPI domain I or/and IV, structures recognized by anti-β2-GPI autoantibodies. IgG immune complexes with oxLDL/β2-GPI complexes can interact with macrophages via Fcγ receptor, causing oxLDL/β2-GPI endocytosis and foam cell formation, contributing to atherosclerosis. Here, we use recombinant domain V to study the interaction between oxLDL and β2-GPI and hypothesized that domain V would interfere with this interaction thereby reducing oxLDL macrophage uptake and foam cell formation. The β2-GPI domain V sequence was expressed by using the Pichia pastoris expression system to obtain recombinant domain V of β2-GPI (P.rβ2-GPI DV). ELISA tests demonstrated that P.rβ2-GPI DV interacted with oxLDL via 7-ketocholesteryl-9-carboxynonanoate (oxLig-1), a negatively charged lipid moiety of oxLDL. The ω-carboxyl residue of oxLig-1 is required for the interaction. Serologic tests showed a significant increase in oxLDL and oxLDL/β2-GPI levels in patients with APS (p < 0.05 compared to controls). P.rβ2-GPI DV was able to bind oxLDL in high affinity and competitively inhibited native β2-GPI (nβ2-GPI) binding to free oxLDL as well as to oxLDL from the oxLDL/β2-GPI complexes. These observations suggest that P.rβ2-GPI DV may be used to inhibit the formation of the oxLDL/β2-GPI complexes, a potential approach for reducing foam cell development and mitigating atherogenesis in patients with APS. The present work provides a new effective strategy to prevent the progression of atherothrombotic vascular complications in APS patients.

7-Ketocholesteryl-9-carboxynonanoate enhances the expression of ATP-binding cassette transporter A1 via CD36

BACKGROUND CD36 signal transductions have been reported by a variety of lipid moiety of oxidized low-density lipoprotein (oxLDL), however, CD36 signal induced by 7-ketocholesteryl-9-carboxynonanoate (oxLig-1), a lipid moiety of oxLDL has not been elucidated. METHODS AND RESULTS OxLig-1 leads to activation and recruitment of Src kinase Fyn, Lyn and caveolin-1 to CD36 in J774A.1 cells, but not in CD36 knockdown cells. The mitogen-activated protein (MAP) kinases c-Jun N-terminal kinase 2 (JNK2) and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are specifically phosphorylated in J774A.1 cells after treatment with oxLig-1 and inhibited by pretreatment of Src inhibitor, AG1879. The expression of ABCA1 is up-regulated by treatment with oxLig-1in J774A.1 cells, and the increased expression of ABCA1 is dramatically down-regulated by pretreatment with pharmacological inhibitors of ERK (PD98059) and JNK (SP600125). CONCLUSIONS The specific CD36 signal induced by oxLig-1 initiated the activation of Fyn, Lyn, caveolin-1, JNK2 and ERK1/2, and resulted in the up-regulation of ABCA1.

7-ketocholesteryl-9-carboxynonanoate enhances ATP binding cassette transporter A1 expression mediated by PPARγ in THP-1 macrophages.

BACKGROUND ATP binding cassette transporter A1 (ABCA1) is a member of the ATP-binding cassette transporter family. It plays an essential role in mediating the efflux of excess cholesterol. It is known that peroxisome proliferator-activated receptor gamma (PPARγ) promoted ABCA1 expression. We previously found 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) upregulated ABCA1 partially through CD36 mediated signals. In the present study, we intended to test if PPARγ signally is involved in the upregulation mediated by oxLig-1. METHODS AND RESULTS First, we docked oxLig-1 and the ligand-binding domain (LBD) of PPARγ by using AutoDock 3.05 and subsequently confirmed the binding by ELISA assay. Western blotting analyses showed that oxLig-1 induces liver X receptor alpha (LXRα), PPARγ and consequently ABCA1 expression. Furthermore, oxLig-1 significantly enhanced ApoA-I-mediated cholesterol efflux. Pretreatment with an inhibitor for PPARγ (GW9662) or/and LXRα (GGPP) attenuated oxLig-1-induced ABCA1 expression. Under PPARγ knockdown by using PPARγ-shRNA, oxLig-1-induced ABCA1 expression and cholesterol efflux in THP-1 macrophages was blocked by 62% and 25% respectively. CONCLUSIONS These observations suggest that oxLig-1 is a novel PPARγ agonist, promoting ApoA-I-mediated cholesterol efflux from THP-1 macrophages by increasing ABCA1 expression via induction of PPARγ.

The lipid moiety 7-ketocholesteryl-9-carboxynonanoate mediates binding interaction of oxLDL to LOX-1 and upregulates ABCA1 expression through PPARγ

Aim: Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), a specific membrane receptor for oxidized low‐density lipoprotein (oxLDL), plays a crucial role in atherosclerosis progression. The aim of this study was to elucidate the role of 7‐ketocholesteryl‐9‐carboxynonanoate (oxLig‐1), a lipid component of oxLDL, in the binding of oxLDL to LOX‐1 and to determine whether oxLig‐1 binding to LOX‐1 is involved in the upregulation of ABCA1 expression. Main methods: OxLig‐1 binding to LOX‐1 was analysed by AutoDock 4.2.6 and confirmed by fluorescence immunocytochemistry and enzyme‐linked immunosorbent assays (ELISAs). LOX‐1, LXR&agr; and ABCA1 protein expression induced by oxLig‐1 or methylated oxLig‐1 was measured by western blotting. In addition, PPAR&ggr; activation was investigated using a dual‐luciferase reporter system. Furthermore, the signalling cascade involved in oxLig‐1‐induced ABCA1 expression was assessed using inhibitors for PPAR&ggr; and LXR&agr; and specific siRNAs against LOX‐1, PPAR&ggr; and LXR&agr;. Key findings: Docking, fluorescence immunocytochemistry and ELISA analyses showed that oxLig‐1 bound LOX‐1 and that the &ohgr;‐carboxyl group was critical for this binding. Moreover, oxLig‐1, but not methylated oxLig‐1, increased LOX‐1, LXR&agr;, and ABCA1 expression. Luciferase reporter assays indicated that oxLig‐1 activated PPAR&ggr; in the presence of LOX‐1. Additionally, the inhibitor and siRNA experiments showed that oxLig‐1 triggered the PPAR&ggr;‐LXR&agr; signalling pathway, leading to upregulation of ABCA1, and that this process required the participation of LOX‐1. Significance: Our observations indicate that oxLig‐1 is a critical epitope of oxLDL that mediates the binding of oxLDL to LOX‐1 and initiates PPAR&ggr; signal transduction to upregulate the expression of ABCA1.

Recombinant domain V of β2-glycoprotein I inhibits the formation of a 7-ketocholesteryl-9-carboxynonanoate and β2-glycoprotein I complex

Our prior study has been reported the formation of the oxidized low-density lipoprotein (oxLDL)/β(2)-glycoproteinI (β(2)-GPI)/autoantibody complex facilitated the antiphospholipid syndrome (APS) process. The domain V of β(2)-GPI binds to the negatively charged molecules, e.g. 7-ketochoresteryl-9-caboxynonanoate (oxLig-1) derived from the oxLDL and mediates the interaction between oxLDL and β(2)-GPI. In the present study, the oxLig-1/β(2)-GPI/anti-β(2)-GPI Ab (WB-CAL-1) model was established. The recombinant domain V of β(2)-GPI (rβ(2)-GPI DV) expressed in Escherichia coli competitively inhibits the interaction between β(2)-GPI and oxLig-1 in the enzyme-linked immunoassay. Moreover, the rβ(2)-GPI DV significantly inhibits the formation of the oxLig-1/β(2)-GPI/autoantibody complex in an APS patient. The present work suggests a novel possibility that rβ(2)-GPI DV could be used to inhibit the formation of oxLDL/β(2)-GPI/autoantibody complex, and give us a hint for the development of new therapeutic strategies to prevent the APS process.

The ω-carboxyl group of 7-ketocholesteryl-9-carboxynonanoate mediates the binding of oxLDL to CD36 receptor and enhances caveolin-1 expression in macrophages

CD36 signal transduction modulates the uptake of oxidized low-density lipoprotein (oxLDL) and foam cell formation. We previously observed that 7-ketocholesteryl-9-carboxynonanoate (oxLig-1), the lipid moiety of oxLDL, activates the CD36-Src-JNK/ERK1/2 signalling pathway. In this study, we assessed the role of the ω-carboxyl group in the binding of oxLig-1 to CD36 and investigated whether the binding of the ω-carboxyl group to CD36 triggers CD36-mediated signalling, thereby resulting in the upregulation of caveolin-1 expression. Our results showed that oxLig-1 bound to CD36 and that the ω-carboxyl group was critical for this binding. Furthermore, immunoprecipitation and Western blot analyses showed that interaction between the ω-carboxyl group of oxLig-1 and CD36 triggered intracellular Src-JNK/ERK1/2 signal transduction. Moreover, the binding of the ω-carboxyl group to CD36 induced caveolin-1 expression and translocation to the membrane in macrophages. Additionally, inhibitors of Src, JNK and ERK and siRNA targeting CD36 and NF-κB significantly suppressed the enhanced caveolin-1 expression induced by oxLig-1. In conclusion, these observations suggest that oxLig-1 is a critical epitope of oxLDL that mediates the binding of oxLDL to CD36 and activates downstream Src-JNK/ERK1/2-NF-κB signal transduction, resulting in upregulation of caveolin-1 expression in macrophages.

Immune Response to Liposome-Associated Recombinant SEF21 Following Oral Immunization in Chickens

SUMMARY. In order to generate Salmonella enterica serovar Enteritidis fimbriae antigens (rSEF21), the intact region encoding SEF21 was amplified from Salmonella Enteritidis by PCR and subcloned into a prokaryotic expression vector pET-28a(+) to yield pET-28a(+)-SEF21. The rSEF21 protein was highly expressed and purified by nickel affinity chromatography. Liposome-associated rSEF21 was prepared for oral immunization to seek protective efficacy for intestinal infection with Salmonella Enteritidis. Evidence of IgA and IgG responses were found in the intestinal tracts and in the sera of a group of chickens immunized. Two weeks after the booster immunization, the chickens were challenged orally with 2 × 106 colony-forming units of live Salmonella Enteritidis, and fecal samples were examined for bacterial excretion from the intestinal tract. Significantly less fecal excretion of bacteria was observed in immunized chickens for 4 wk after challenge. The numbers of bacteria in the intestinal contents (cecum and rectum) were also significantly lower in immunized chickens than in unimmunized controls. Therefore, oral immunization with liposome-associated rSEF21 elicits both systemic and mucosal antibody responses, leading to a reduction in bacterial colonization in the intestinal tract and excretion of Salmonella Enteritidis in the feces. RESUMEN. Respuesta inmune contra una proteína recombinante de Salmonella enterica serovar Enteritidis SEF21 asociada a liposomas, después de la inmunización oral en pollos. Con el fin de generar antígenos fimbriales de Salmonella enterica serovar Enteritidis (rSEF21), se amplificó mediante PCR la región intacta de codificación SEF21 a partir de Salmonella Enteritidis y se subclonó en un vector de expresión procariótica pET-28a (+) para producir pET-28a (+)-SEF21. La proteína rSEF21 fue altamente expresada y se purificó mediante cromatografía de afinidad con níquel. Se preparó la proteína rSEF21 asociada a liposomas para la inmunización oral para determinar la eficacia protectora contra la infección intestinal con Salmonella Enteritidis. Se encontró evidencia de las respuestas de IgA e IgG en los tractos intestinales y en los sueros de un grupo de pollos inmunizados. Dos semanas después de la inmunización de refuerzo, los pollos fueron desafiados oralmente con 2 × 106 unidades formadoras de colonias de Salmonella Enteritidis viva, y las muestras de heces fueron examinadas para la excreción de bacterias en el tracto intestinal. Se observó una excreción fecal de bacterias significativamente menor en pollos vacunados durante cuatro semanas después del desafío. Los números de bacterias en los contenidos intestinales (el ciego y el recto) también fueron significativamente menores en los pollos inmunizados en comparación con los controles no vacunados. Por lo tanto, la inmunización oral con la proteína rSEF21 asociada con liposomas provoca respuestas de anticuerpos sistémicos y en las mucosas, dando lugar a una reducción de la colonización bacteriana en el tracto intestinal y a la excreción de Salmonella Enteritidis en las heces.

Recombinant domain V of beta(2)-Glycoprotein I inhibits oxldl-induced TF expression in macrophages

OBJECTIVE To investigate if lectin-like oxidised low density lipoprotein receptor is implicated in oxidised low density lipoprotein induced up regulation of tissue factor and whether recombinant domain V of beta (2)-Glycoprotein I expressed in Pichia pastoris inhibits the binding of oxidised and lectin-like low density lipoprotein. METHODS The expression of tissue factor and lectin-like oxidised low density lipoprotein receptor was detected using Western blot methods. Small interference ribonucleic acid of lectin-like oxidised low density lipoprotein receptor was used to block lectin-like oxidised low density lipoprotein receptor expression. Flow cytometry was used to test the effect of beta (2)-Glycoprotein I expressed in Pichia pastoris on the binding of oxidised low density lipoprotein with lectin-like oxidised low density lipoprotein receptor by using the lectin-like oxidised low density lipoprotein receptor-expressing 293T cells. RESULTS Oxidised low density lipoprotein at 5-10 g/mL increased tissue factor and lectin-like oxidised low density lipoprotein receptor expression, whereas 20-50 g/mL oxidised low density lipoprotein attenuated tissue factor expression. Inhibiting lectin-like oxidised low density lipoprotein receptor expression by small interference ribonucleic acid of lectin-like oxidised low density lipoprotein receptor impaired oxidised low density lipoprotein-induced tissue factor over expression in macrophages. Pretreatment with beta (2)-Glycoprotein I expressed in Pichia pastoris led to a strong inhibition of tissue factor and lectin-like oxidised low density lipoprotein receptor expression in a dose-dependent manner in macrophages. Flow cytometry analysis showed that beta (2)-Glycoprotein I expressed in Pichia pastoris attenuated the interaction of oxidised low density lipoprotein with lectin-like oxidised low density lipoprotein receptor in lectin-like oxidised low density lipoprotein receptor-expressing 293T cells. CONCLUSIONS Lectin-like oxidised low density lipoprotein receptor was implicated in the expression of tissue factor induced by oxidised low density lipoprotein, and beta (2)-Glycoprotein I expressed in Pichia pastoris inhibited oxidised low density lipoprotein-induced tissue factor and lectin-like oxidised low density lipoprotein receptor expression, at least in part, via inhibition of the interaction between oxidised low density lipoprotein and lectin-like oxidised low density lipoprotein receptor.