人补体调节蛋白MCP、CD59共表达体系的构建及其功能研究

利用双启动子构建含人补体调节蛋白MCP和CD59 cDNA的双顺反子重组表达载体pcDNA3-MCPCD59-DP,以磷酸钙沉淀法转染NIH3T3细胞,用G418筛选获得NIH3T3pcDNA3-MCPCD59-DP转化细胞。PCR实验结果显示人MCP和CD59整合在转化的NIH3T3细胞的染色体上,RT-PCR和Western印迹实验分别从RNA水平和蛋白质水平证实了人MCP和CD59在转化细胞中的共表达。 检测连续传代30次的NIH3T3pcDNA3-MCPCD59-DP结果表明人MCP和CD59基因仍稳定整合在细胞基因组中,并未随着传代而丢失,为稳定的转双基因细胞系。 补体溶破实验表明, pcDNA3-MCPCD59-DP转染细胞由于人MCP和CD59的共表达获得了较MCP或CD59单一表达时更好的保护功效,能有效地保护NIH3T3细胞免受人补体的攻击,从而抑制补体依赖的细胞毒反应的发生。 以上结果表明本研究所构建的双基因重组表达载体实现了人补体调节蛋白基因高效转移和高水平共表达,在克服超急性排斥反应的基因治疗中有潜在的应用价值。     

Co-expression of human complement regulatory proteins DAF and MCP with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects

C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation. Published in Russian in Biokhimiya, 2008, Vol. 73, No.9, pp. 1274–1280.     

人补体调节蛋白DAF、MCP在哺乳动物细胞中的共表达及协同作用研究

利用双启动子构建含人补体调节蛋白DAF和MCP cDNA的双顺反子重组表达载体pcDNA3-DAFMCP-DP, 以磷酸钙沉淀法转染NIH3T3细胞, 用G418筛选获得NIH3T3 pcDNA3-DAFMCP-DP转化细胞。PCR实验结果显示人补体调节蛋白基因DAF和MCP整合在转化的异源细胞的染色体上。RT-PCR和Western blot印迹实验分别从RNA水平和蛋白质水平证实了人补体调节蛋白分子DAF和MCP在细胞系中皆获得同步表达。检测连续传代30次的NIH3T3 pcDNA3-DAFMCP-DP结果表明人DAF和MCP基因仍稳定整合在细胞基因组中, 并未随着传代而丢失, 为稳定的转双基因细胞系。补体依赖的细胞毒反应表明, pcDNA3-DAFMCP-DP转染细胞系由于DAF和MCP的共表达获得较DAF或MCP单一表达时更强的保护能力, 能更好地抑制人补体依赖的细胞毒作用的发生, 保护宿主细胞免受人补体的攻击。以上结果表明, DAF和MCP双基因重组表达载体实现了人补体调节蛋白基因高效转移和高水平共表达, 为获得表达多种人补体调节蛋白的理想供体提供了有效策略。而且共表达的DAF和MCP具有协同效应, 能更有效地阻止补体激活造成的细胞损伤, 在克服超急性排斥反应的基因治疗中具有潜在的临床应用价值。     

H1亚型猪流感病毒血凝素基因在昆虫细胞中的表达及其间接

根据GenBank发表的H1亚型猪流感HA基因序列设计引物,扩增出HA基因片段.将其克隆到pFastBacGP67B杆状病毒载体上,筛选阳性重组转座载体pFastBacGP67B-H1,转化含有杆状病毒穿梭载体(bacmid)的DH10Bac感受态细胞,构建杆状病毒表达载体获得重组转座子(rBacmid-H1),在脂质体介导下转染sf9昆虫细胞,获得重组杆状病毒(rBV-H1),再感染细胞,收获目的蛋白.通过血凝试验、免疫印迹法、免疫组化分析表明该蛋白得到表达,且具有良好的生物学活性.利用表达的蛋白作为猪流感间接ELISA的抗原,初步建立H1亚型猪流感的间接ELISA检测方法,并对内蒙古、辽宁和黑龙江等地送检的93份猪血清进行了检测,阳性率为31.18%,为研制开发快速、准确、简便的H1亚型猪流感鉴别诊断试剂盒奠定基础.     

Protection of xenogeneic cells from human complement-mediated lysis by the expression of human DAF, CD59 and MCP

CD59 and membrane cofactor protein (MCP, CD46) are widely expressed cell surface glycoproteins that protect host cells from the effect of homologous complement attack. cDNAs encoding human CD59 and MCP cloned from Chinese human embryo were separately transfected into NIH/3T3 cells resulting in the expression of human CD59 and MCP protein on the cell surface. The functional properties of expressed proteins were studied. When the transfected cells were exposed to human serum as a source of complement and naturally occurring anti-mouse antibody, they were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rabbit and guinea pig complement. Human CD59 and MCP can only protect NIH/3T3 cells from human complement-mediated lysis. These results demonstrated that complement inhibitory activity of these proteins is species-selective. The cDNAs of CD59 and MCP were also separately transfected into the endothelial cells (ECs) of the pigs transgenic for the human DAF gene to investigate a putative synergistic action. The ECs expressing both DAF and MCP proteins or both DAF and CD59 proteins exhibited more protection against cytolysis by human serum compared to the cells with only DAF expressed alone.     

Transgenic mice designed to express human α-1,2-fucosyltransferase in combination of human DAF and CD59 to avoid xenograft rejection

The expression of human α-1,2-fucosyltransferase (HT) or complement regulatory proteins has been proved as an strategy to overcome hypercute rejection in discordant xenogeneic organ transplantation. In this study, we examined whether peripheral blood mononuclear cells (PBMCs) from polytransgenic mice expressing the human HT, and complement regulatory proteins (DAF and CD59), can provide more effective protection against xenograft rejection. Transgenic mice were produced by co-injection of gene constructs for human HT, DAF and/or CD59. Flow Cytometry (FCM) was used to screen the positive transgenic mice. PBMCs from transgenic mice were incubated with 15% human serum to evaluate natural antibody binding, complement activation and expression of adhesion molecules. Three transgenes were strongly expressed in PBMCs of transgenic mice, and HT expression significantly reduced expression of the major xenoepitope galactose-α-1,3-galactose (α-Gal). Functional studies with PBMCs showed that co-expression of HT and DAF or CD59 markedly increased their resistance to human serum-mediated cytolysis when compared with single transgenic PBMCs. Moreover, the combined expression of triple transgenes in PBMCs led to the greatest protection against human serum-mediated cytolysis, avoided hyperacute rejection and reduced expression of adhesion molecules. Strong co-expression of triple transgenes was completely protected from xenograft hyperacute rejection and partially inhibited acute vascular rejection. The studies suggest that engineering mice to express triple molecules represents an critical step toward prolonging xenograft survival and might be more suitable for xenotransplantation.     

Transgenic mice designed to express human α-1,2-fucosyltransferase in combination of human DAF and CD59 to avoid xenograft rejection

The expression of human α-1,2-fucosyltransferase (HT) or complement regulatory proteins has been proved as an strategy to overcome hypercute rejection in discordant xenogeneic organ transplantation. In this study, we examined whether peripheral blood mononuclear cells (PBMCs) from polytransgenic mice expressing the human HT, and complement regulatory proteins (DAF and CD59), can provide more effective protection against xenograft rejection. Transgenic mice were produced by co-injection of gene constructs for human HT, DAF and/or CD59. Flow Cytometry (FCM) was used to screen the positive transgenic mice. PBMCs from transgenic mice were incubated with 15% human serum to evaluate natural antibody binding, complement activation and expression of adhesion molecules. Three transgenes were strongly expressed in PBMCs of transgenic mice, and HT expression signifi-cantly reduced expression of the major xenoepitope galactose-α-1,3-galactose (α-Gal). Functional studies with PBMCs showed that co-expression of HT and DAF or CD59 markedly increased their re-sistance to human serum-mediated cytolysis when compared with single transgenic PBMCs. Moreover, the combined expression of triple transgenes in PBMCs led to the greatest protection against human serum-mediated cytolysis, avoided hyperacute rejection and reduced expression of adhesion mole-cules. Strong co-expression of triple transgenes was completely protected from xenograft hyperacute rejection and partially inhibited acute vascular rejection. The studies suggest that engineering mice to express triple molecules represents an critical step toward prolonging xenograft survival and might be more suitable for xenotransplantation.     

Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor.

Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.     

Characterisation of the complement-regulatory proteins decay-accelerating factor (DAF,CD55) and membrane cofactor protein (MCP,CD46) on a human colonic adenocarcinoma cell line

 To avoid destruction by complement, normal and malignant cells express membrane glycoproteins that restrict complement activity. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and protectin (CD59), which are all expressed on colonic adenocarcinoma cells in situ. In this study we have characterised the C3/C5 convertase regulators DAF and MCP on the human colonic adenocarcinoma cell line HT29. DAF was found to be a glycosyl-phosphatidylinositol-anchored 70-kDa glycoprotein. Blocking experiments with F(ab′)₂ fragments of the anti-DAF monoclonal antibody BRIC 216 showed that DAF modulates the degree of C3 deposition and mediates resistance to complement-mediated killing of the cells. The expression and function of DAF were enhanced by tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β). Cells incubated with interferon γ (IFNγ) did not alter their DAF expression. Two MCP forms were expressed, with molecular masses of approximately 58 kDa and 68 kDa, the lower form predominating. MCP expression was up-regulated by IL-1β, but not by TNFα or IFNγ. Expression of DAF and MCP promotes resistance of colonic adenocarcinoma cells to complement-mediated damage, and represents a possible mechanism of tumour escape. Received: 18 July 1995 / Accepted: 4 January 1996     

Overexpression of protectin (CD59) down-modulates the susceptibility of human melanoma cells to homologous complement

The clinical efficacy of therapeutic complement (C)-activating monoclonal antibodies (mAb) to melanoma-associated antigens can be impaired by the levels of expression of C-inhibitory molecules on neoplastic cells. Protectin (CD59) is a glycosylphosphatidylinositol (GPI)-anchored cell membrane glycoprotein, acting as terminal regulator of C cascade, which is heterogeneously expressed in melanomas and represents the main restriction factor of C-mediated lysis of melanoma cells. Thus, we investigated whether the overexpression of CD59 could influence the constitutive susceptibility of distinct melanoma cells to homologous C. Infection of CD59-positive Mel 100 and 70-W melanoma cells by a retroviral vector carrying the CD59 cDNA, significantly (P < 0.05) upregulated their constitutive expression of CD59, whereas it did not affect that of additional C-regulatory molecules. Transduced CD59 was entirely GPI-anchored and showed a molecular weight identical to native CD59. Additionally, higher amounts of soluble CD59 were detected in the conditioned media of CD59-transduced melanoma cells compared with parental cells. CD59-transduced melanoma cells, sensitized by the anti-GD3 disialoganglioside mAb R24, were significantly (P < 0.05) less susceptible to homologous C-lysis than were parental cells; this effect was fully reverted by the masking of CD59 with F(ab')(2) fragments of the anti-CD59 mAb YTH53.1. These results provide conclusive evidence demonstrating that absolute levels of CD59 expression regulate the susceptibility to homologous C of specific melanoma cells, and suggest an additional explanation for the poor clinical results obtained with C-activating mAb in the clinical setting.     

突变人CD59分子真核表达体系的构建和功能的初步研究

构建突变人CD59分子(HMCD59)真核表达体系,探讨HMCD59糖基化前后抗补体活性的变化。采用重组PCR定点诱变技术在CD59易于糖基化的位点构建CD59突变基因,克隆入真核表达质粒pALTER-MAX。利用阳离子脂质体将重组质粒和pcDNA3共转染中国仓鼠卵巢细胞(CHO)。G418压力筛选稳定转染细胞克隆,检测筛选HMCD59蛋白高表达株。免疫印迹技术(Western blot)检测出HMCD59蛋白分子量约为20kDa。2,7-二羧乙基-5,6羧基荧光素(BCECF)释放实验初步证实HMCD59具有抗补体活性,糖基化后抗补体活性明显降低,为进一步研究糖尿病血管增殖症的发生机制提供了线索。     

Induction of decay accelerating factor and membrane cofactor protein by resveratrol attenuates complement deposition in human coronary artery endothelial cells

The involvement of complement activation in various forms of cardiovascular disease renders it an important factor for disease progression and therapeutic intervention. The protective effect of resveratrol against cardiovascular disease via moderate red wine consumption has been established but the exact mechanisms are still under investigation. The current study utilised human coronary artery endothelial cells (HCAECs) in order to assess the extent to which the protective effect of resveratrol, at concentrations present in red wine, can be attributed to the upregulation of complement regulatory proteins through heme-oxygenase (HO)-1 induction. Resveratrol at concentrations as low as 0.001 μΜ increased HO-1 expression as well as membrane cofactor protein (MCP, CD46) and decay-accelerating factor (DAF, CD55) expression with no-effect on CD59. Silencing of HO-1 expression by HO-1 siRNAs abrogated both DAF and MCP protein expression with no effect on CD59. Resveratrol-mediated induction of DAF and MCP reduced C3b deposition following incubation of HCAECs with 10% normal human serum or normal rat serum as a source of complement. Incubation of HCAECs, with either a DAF blocking antibody or following transfection with HO-1 siRNAs, in the presence of 10% normal rat serum increased C3b deposition, indicating that both DAF and HO-1 are required for C3b reduction. These observations support a novel mechanism for the protective effect of resveratrol against cardiovascular disease and confirm the important role of HO-1 in the regulation of the complement cascade.     

Targeting of functional antibody-decay-accelerating factor fusion proteins to a cell surface

Recombinant soluble complement inhibitors hold promise for the treatment of inflammatory disease and disease states associated with transplantation. Targeting complement inhibitors to the site of complement activation and disease may enhance their efficacy and safety. Data presented show that targeting of decay-accelerating factor (DAF, an inhibitor of complement activation) to a cell surface by means of antibody fragments is feasible and that cell-targeted DAF provides significantly enhanced protection from complement deposition and lysis compared with soluble untargeted DAF. An extracellular region of DAF was joined to an antibody combining site with specificity for the hapten dansyl, at the end of either C(H)1 or C(H)3 Ig regions. The recombinant IgG-DAF chimeric proteins retained antigen specificity and bound to dansylated Chinese hamster ovary cells. Both soluble C(H)1-DAF and C(H)3-DAF were effective at inhibiting complement-mediated lysis of untargeted Chinese hamster ovary cells at molar concentrations within the range reported by others for soluble DAF. However, when targeted to a dansyl-labeled cell membrane, C(H)1-DAF was significantly more potent at inhibiting complement deposition and complement-mediated lysis. Cell-bound C(H)1-DAF also provided cells with protection from complement lysis after removal of unbound C(H)1-DAF. Of further importance, the insertion of a nonfunctional protein domain of DAF (the N-terminal short consensus repeat) between C(H)1 and the functional DAF domain increased activity of the fusion protein. In contrast to C(H)1-DAF, C(H)3-DAF was not significantly better at protecting targeted versus untargeted cells from complement, indicating that a small targeting vehicle is preferable to a large one. We have previously shown that for effective functioning of soluble complement inhibitor CD59, binding of CD59 to the cell surface close to the site of complement activation is required. Significantly, such a constraint did not apply for effective DAF function.     

Complement-dependent enhancement of CD8+ T cell immunity to lymphocytic choriomeningitis virus infection in decay-accelerating factor-deficient mice

Decay-accelerating factor (DAF, CD55) is a GPI-anchored membrane protein that regulates complement activation on autologous cells. In addition to protecting host tissues from complement attack, DAF has been shown to inhibit CD4+ T cell immunity in the setting of model Ag immunization. However, whether DAF regulates natural T cell immune response during pathogenic infection is not known. We describe in this study a striking regulatory effect of DAF on the CD8+ T cell response to lymphocytic choriomeningitis virus (LCMV) infection. Compared with wild-type mice, DAF knockout (Daf-1(-/-)) mice had markedly increased expansion in the spleen of total and viral Ag-specific CD8+ T cells after acute or chronic LCMV infection. Splenocytes from LCMV-infected Daf-1(-/-) mice also displayed significantly higher killing activity than cells from wild-type mice toward viral Ag-loaded target cells, and Daf-1(-/-) mice cleared LCMV more efficiently. Importantly, deletion of the complement protein C3 or the receptor for the anaphylatoxin C5a (C5aR) from Daf-1(-/-) mice reversed the enhanced CD8+ T cell immunity phenotype. These results demonstrate that DAF is an important regulator of CD8+ T cell immunity in viral infection and that it fulfills this role by acting as a complement inhibitor to prevent virus-triggered complement activation and C5aR signaling. This mode of action of DAF contrasts with that of CD59 in viral infection and suggests that GPI-anchored membrane complement inhibitors can regulate T cell immunity to viral infection via either a complement-dependent or -independent mechanism.     

Development of adenovirus vectors encoding rat complement regulators for use in therapy in rodent models of inflammatory diseases

C activation has been implicated in the pathogenesis of numerous inflammatory human diseases and disease models. A therapy based on C inhibition might therefore be of benefit to reduce inflammation and ameliorate disease. C inhibition in vivo can be accomplished by the delivery of soluble recombinant C regulators either systemically or directly to a target site, but effects are transitory. We have developed a strategy for the efficient delivery of the membrane-bound rat C inhibitors, CD59, Crry, and decay-accelerating factor (DAF), using replication-deficient adenovirus vectors with the intention of treating rat models of disease in which C is implicated. The adenovirus recombinants(RAd), RAdCD59, RAdCrry, and RAdDAF, respectively, have been tested for expression and function of the transgene in vitro. Infection of human fetal foreskin fibroblasts resulted in high levels of expression of each of the rat inhibitors. The constructs were also tested for inhibition of rat C-mediated cell lysis and C3b deposition. In a cell lysis assay, each inhibited to varying degrees of efficiency in the order RAdCD59 = RAdDAF > RAdCrry. In a C3b deposition assay, RAdDAF caused a greater reduction in C3b deposition than RAdCrry and RAdCD59 was ineffective. These agents, individually or in combination, provide the tools for testing the effects of prolonged inhibition of C at a target site on the progress of experimental models of disease.     

Characterization of mouse DAF on transfectant cells using monoclonal antibodies which recognize different epitopes

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay-accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI-DAF) and a transmembrane form (TM-DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI-DAF or TM-DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI-DAF transfectant cells and established seven unique mAbs (RIKO-1-7). Western blotting analysis using RIKO-3, which reacts with both GPI-DAF and TM-DAF, and RIKO-4, which is an anti-GPI-DAF specific mAb, indicated that GPI-DAF was expressed on erythrocytes, spleen and testis, and that TM-DAF was expressed only in testis.     

Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis

To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement inhibitors. Decay-accelerating factor (DAF, CD55) is a GPI-linked membrane complement regulator that is widely expressed in mammalian tissues including the kidney. DAF inhibits the C3 convertase of both the classical and alternative pathways. Although DAF deficiency contributes to the human hematological syndrome paroxysmal nocturnal hemoglobinuria, the relevance of DAF in autoimmune tissue damage such as immune glomerulonephritis remains to be determined. In this study, we have investigated the susceptibility of knockout mice that are deficient in GPI-anchored DAF to nephrotoxic serum nephritis. Injection of a subnephritogenic dose of rabbit anti-mouse glomerular basement membrane serum induced glomerular disease in DAF knockout mice but not in wild-type controls. When examined at 8 days after anti-glomerular basement membrane treatment, DAF knockout mice had a much higher percentage of diseased glomeruli than wild-type mice (68.8 +/- 25.0 vs 10.0 +/- 3.5%; p < 0.01). Morphologically, DAF knockout mice displayed increased glomerular volume (516 +/- 68 vs 325 +/- 18 x 10(3) microm(3) per glomerulus; p < 0.0001) and cellularity (47.1 +/- 8.9 vs 32.0 +/- 3.1 cells per glomerulus; p < 0.01). Although the blood urea nitrogen level showed no difference between the two groups, proteinuria was observed in the knockout mice but not in the wild-type mice (1.4 +/- 0.7 vs 0.02 +/- 0.01 mg/24 h albumin excretion). The morphological and functional abnormalities in the knockout mouse kidney were associated with evidence of increased complement activation in the glomeruli. These results support the conclusion that membrane C3 convertase inhibitors like DAF play a protective role in complement-mediated immune glomerular damage in vivo.