C5L2 不再是孤儿受体——进展与展望

C5a Like Receptor2--C5L2,又称G蛋白偶联受体77(GPR77),是 C5aR(CD88)亚家族成员,在氨基酸序列上与C5aR(CD88)有35%的同源性.广泛分布于全身各组织细胞,在粒细胞和未成熟树突状细胞(immature dendritic)及脾脏中有大量的表达.由于长期以来未发现其生理性配体而被称为孤儿受体(orphan receptor).当前大量研究表明C5L2能与C5a,C3a,C4a,去精 C5a(C5a des-Arg74)和去精 C3a(C3a des-Arg77) 结合,且与C5a des-Arg74结合能力比CD88与C5a des-Arg74高10倍.在结构、功能等方面与CD88也有很多相似之处.与CD 88不同的是由于C5L2第三跨膜片断末端的DRY序列上精氨酸被亮氨酸替代,有一个更短的胞内第三环,缺乏丝氨酸/苏氨酸残基,所以不能强有力地与G蛋白偶联,和C5a结合后仅诱导转染细胞发生微弱的磷酸化,不引起细胞内钙的流动及转染细胞脱颗粒;C5L2与配体结合后不发生内化,并具有与配体缓慢的结合与解离的特性.作为过敏毒素受体,C5L2在过敏毒素C5a参与的相关疾病,如脓毒症(sepsis)、急性肺损伤(ALI)、急性呼吸窘迫综合征(ARDS)等的发生、发展以及对G蛋白偶联受体的研究都具有重要的意义.     

C5a受体的作用位点及信号传导

C5aR(CD88)属G蛋白偶联/STR超家族起成员,它至少具两个配体结合位点:一个位于N末端,另一个可能为第二细胞外环上的Glu~(199)。C末端和第三胞浆内环与G蛋白偶联。与C5aR偶联的G蛋白有Giα2、Giα3、G16和Gz,已知涉及的信号传导通路为PI-PLC、Ras/Raf/MAPK、cAMP。C5aR分子及基因结构已阐明,各功能位点、信号传导还有待深入研究。     

补体裂解产物C5a的研究进展

C5a由C5裂解而来,由74个氨基酸构成,是一种潜在的前炎症因子。C5a在羧肽酶的作用下迅速变成C5adesArg,通过G蛋白受体经典的通路与C5aR结合后在天然免疫中和适应性免疫中均发挥着重要的作用,非G蛋白受体通路目前的研究还不明朗。研究发现,C5a是许多自身免疫性疾病的重要病原起始物,所以抑制C5a的释放在未来的治疗中是一个很好的策略和方向。     

肾癌荷瘤小鼠组织中补体 C5a / C5aR 通路活化与肿瘤高凝状态 的关系及其机制研究

目的 研究肾癌荷瘤小鼠组织中补体 C5a / C5aR 通路活化与肿瘤高凝状态的关系及其机制。 方法 收集 2016 年 7 月至 2019 年 10 月于唐山市人民医院收治的 32 例经病理学活检确诊的肾癌患者的癌组织和 癌旁组织。 采用 Western 印迹法检测组织中 C5a / C5aR 通路活化相关蛋白表达情况。 于 BALB/ c-nu / nu 裸鼠 前肢腋窝皮下接种人 ACHN 肾癌细胞株, 接种成功后随机分为干预组、 模型组, 其中干预组尾静脉注射 C5a 联合 C5aR 阻断剂 (C5aR antagonist, C5aRA), 模型组等体积注射生理盐水, 另取 6 只空白小鼠作为对 照组。 观察小鼠肿瘤生长情况, 检测高凝状态相关指标水平。 脱颈处死小鼠, 收集癌组织, 采用 Western 印迹法检测组织中 C5a / C5aR 通路活化相关蛋白表达情况。 结果 两组小鼠肿瘤体积于荷瘤 6 ~ 21 d 差异具 有统计学意义, 且干预组的肿瘤体积明显低于模型组 ( P< 0. 05)。 3 组小鼠于荷瘤第 1 天的血清 D-D、 vWF、 TF 水平差异无统计学意义, 荷瘤第 14 天、 第 21 天干预组、 模型组血清凝血指标水平明显增加, 其 中干预组均明显低于模型组 (P< 0. 05)。 干预组癌组织中 C5aR、 C5b-9、 FGL2、 P38、 p-P38 的表达水平均 明显低于模型组 (P< 0. 05)。 肾癌患者癌组织中 C5aR、 C5b-9、 FGL2、 P38、 p-P38 的表达水平均明显高于 癌旁组织 (P< 0. 05)。 32 例肾癌患者中高凝状态患者 14 例, 非高凝状态患者 18 例, 高凝状态组癌组织中 C5aR、 C5b-9、 FGL2、 P38、 p-P38 的表达水平均明显高于非高凝状态组 (P< 0. 05)。 结论 补体 C5a / C5aR 通路可通过调控 FGL2 的表达而诱导肾癌的高凝状态, 进而参与肾癌的发病过程。     

人C5a过敏毒素拮抗剂的分子设计及其活性检测

目的：从蛋白质结构与功能的关系出发,探讨C5aR与其配体的C5a的结合位。方法：按分子设计理论,采用亲水性方案寻找C5aR胞外区高亲水性区域,Fmoc方案人工合成C5aR第9－30位氨基酸基序（P22肽）,经高效液相色谱纯化,毛细管电泳鉴定。结果：合成多肽（P22）的纯度为95.19％,每次缩合的平均效率为99.78％;能与anti-C5aR McAb（S5／1,Serotic公司）有效地结合,酶联OD490极显著高于同浓度对照多肽C20;还可被配体rh-C5a(10ng/ml)明显抑制而降低其酶联OD值（P＜0.05）,此外10ng/mlP22还可抑制rhC5a所致U937细胞胞浆Ca^2 升高（P＜0.01）。结论：从C5a分子中寻找C5a结合位基序,可拮抗C5a的过敏毒素作用,为治疗C5a及其相关疾病进行新型的药物设计。     

人视网膜前膜组织及培养的人RPE细胞上C5a受体的表达

目的：检测手术中取出的增生性玻璃体视网膜(PVR)病变患者的视网膜前膜(ERM)组织及培养的人视网膜色素上皮细胞(RPE)中的C5a受体(C5aR)的表达及分布，阐明C5a受体在发生PVR的病理过程中可能的作用。方法：手术取出ERM组织，常规制备石腊切片。另取新鲜人眼球，常规分离、培养人RPE细胞。以小鼠抗人C5aR单克隆抗体作为检测抗体。对PVR患者的FRM组织及培养的人RPE细胞做细胞免疫化学ABC染色，检测C5aR的表达。结果：PVR患者的ERM组织及培养的人RPE细胞上均有C5aR的表达：结论：C5a作为一种前炎性因子，与RPE细胞上的C5aR结合后，可能介导PVR的发生，即PVR的发生可能与C5a有关。     

C5aR1促进神经干细胞增殖作用的实验研究

目的研究C5aR1在新生小鼠脑内及体外培养神经干细胞内的表达规律,探索C5aR1对神经干细胞增殖调节的作用。方法以免疫组化方法检测在新生小鼠脑内及体外培养的神经干细胞内C5aR1的表达;培养C5aR基因敲除小鼠来源的神经干细胞,同时利用C5aR拮抗剂处理正常小鼠来源的神经干细胞,分别观察神经干细胞的增殖状态,分析C5aR1对神经干细胞增殖的调节作用。结果 1)在新生小鼠脑内,C5aR1主要表达于海马和室下区等神经干细胞分布区域;2)C5aR1与nestin共表达于体外培养的神经干细胞内;3)C5aR特异性拮抗肽下调体外培养的神经干细胞内nestin表达并抑制其增殖;C5aR1基因敲除,神经干细胞的成球能力明显降低。结论 C5aR1在脑内以及神经干细胞内均有表达,对神经干细胞的增殖具有促进作用,其具体机制还有待进一步的实验研究。     

dt_2-cAMP,IFN-γ和PMA对U937胞浆Ca~(2 )和酪氨酸激酶的影响

目的探讨dt2 cAMP ,PMA和IFN γ对U937细胞C5aR表达的影响 ,以及rhC5a刺激受dt2 cAMP ,PMA和IFN γ分化的U937细胞后 ,其胞浆Ca2 浓度的变化情况。方法用流式细胞仪分析胞膜C5aR和胞浆蛋白酪氨酸激酶的表达 ;荧光发光法测定胞浆Ca2 浓度的变化。结果dt2 cAMP ,PMA和IFN γ等可不同程度地上调C5aR表达。用0.5mmol/L的dt2 cAMP就足以上调U937细胞C5aR分子 ,10～20nmol/L浓度范围的PMA对C5aR表达的上调没有明显差异 ,IFN γ也可上调U937细胞上C5aR的表达。此外 ,rhC5a刺激受dt2 cAMP ,PMA和IFN γ分化的U937细胞 ,可使其细胞浆Ca2 浓度上升。蛋白酪氨酸激酶(PTK)抑制剂Genistein可明显抑制rhC5a所致U937细胞胞浆Ca2 浓度升高。结论PTK可能参与了C5a C5aR相互作用过程中的信号转导。     

C5aR在慢性移植物抗宿主病中的表达及其作用机制

目的:探讨补体5a(C5a)及其受体(C5aR)在慢性移植物抗宿主病(cGVHD)中的表达及其作用机制。方法:用流式细胞术检测20例cGVHD患者及9例健康供者外周血淋巴细胞中C5aR的表达及CD4~+CD25~+Foxp3~+调节性T细胞(Tregs)在CD4~+T细胞中的比例,并分析两者的相关性;将体外分离培养小鼠脾细胞分为对照组及重组小鼠C5a蛋白(rmC 5a)刺激组,用流式细胞术检测2组Tregs在CD4~+T细胞中的表达比例;另外,提取患者外周血单个核细胞进行体外培养,分为空白对照组及C5aR拮抗剂(C5aRA)组,用流式细胞术检测2组Tregs在CD4~+T细胞中的表达比例。结果:cGVHD患者外周血淋巴细胞表面C5aR的表达明显增多,而Tregs在CD4~+T细胞中的比例明显减少,两者呈显著负性相关(P0.05);体外培养小鼠脾细胞结果显示C5a下调Tregs在CD4~+T细胞中的比例;而体外培养患者外周血单个核细胞显示阻断C5aR可上调Tregs在CD4~+T细胞中的比例。结论:C5a/C5aR可能通过抑制Tregs的分化来介导cGVHD的发生发展。     

补体C5a/C5aR1通路在咪喹莫特诱导小鼠慢性银屑病样皮炎中的作用机制研究

目的建立咪喹莫特(imiquimod, IMQ)诱导小鼠慢性银屑病样皮炎模型, 探讨补体C5a/C5aR1通路在该病理过程中的作用及机制。方法 IMQ涂抹(每2天1次, 共8次)小鼠背部皮肤建立慢性银屑病样皮炎模型。比较BALB/c(C5aR1+/+)及C5aR1基因敲除(C5aR1-/-)小鼠皮炎程度及病理改变;免疫组化(immuohistochemistry, IHC)检测皮损组织表皮细胞增殖(Ki67)、角蛋白K6/K14表达及中性粒细胞浸润(Ly-6G)情况;实时荧光定量PCR(qPCR)检测皮损组织角蛋白K6/K14及炎症因子表达。流式细胞术检测皮损局部炎症细胞浸润及IL-17A应答。结果 IMQ可诱导银屑病皮炎典型表型, 如银屑、红斑及皮肤增厚。HE染色提示表皮角质细胞异常增殖、棘皮症、微脓肿、炎症细胞浸润及真皮毛细血管异常增生。与C5aR1+/+小鼠比较, C5aR1-/-小鼠皮炎表型明显减轻, 角质细胞异常增殖、角蛋白K6/K14表达及中性粒细胞浸润显著降低;qRCR检测同样发现C5aR1-/-小鼠角蛋白K6/K14、炎症因子(IFN-γ、MIP-1α、IL-1β和TN...     

The C terminus of the human C5a receptor (CD88) is required for normal ligand-dependent receptor internalization

The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild-type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist-induced internalization was analyzed on HEK293 cells co-transfected with C5aR and the pertussis toxin-resistant G protein alpha subunit, Gα16. Staurosporine-sensitive agonist-dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA-induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester-induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin-sensitive or -resistant G protein-dependent signal transduction had no effect on ligand-induced internalization.     

Functions of C5a receptors

The split product of the complement protein, C5, is C5a and is an extremely potent pro-inflammatory peptide that interacts with two C5a receptors, C5aR and C5L2, present on surfaces of phagocytes as well as other cell types. The former is a well-established receptor that initiates G-protein-coupled signaling via mitogen-activated protein kinase pathways. Its in vivo blockade greatly reduces inflammatory injury. Much less is known about C5L2, occupancy of which by C5a does not initiate increased intracellular Ca²⁺. There are numerous conflicting reports suggesting that C5L2 is a “default receptor” that attenuates C5a-dependent biological responses by competing with C5aR for binding of C5a. However, there are other reports suggesting that C5L2 plays an active, positive role in inflammatory responses. Better definition of C5L2 is needed if its in vivo blockade, along with C5aR, is to be considered in complement-dependent inflammatory diseases.     

中和C5a过敏毒素的分子设计研究

目的 从蛋白质结构与功能的关系出发,探讨Ｃ５ａＲ与其配体Ｃ５ａ的结合位。方法 按分子设计理论,采用亲水性方案寻打Ｃ５ａ胞外区高亲水性区域,Ｆｍｏｃ方案人工合成Ｃ５ａ第９～３０位氨基酸基序（Ｐ２２肽）,经高压液相色谱纯化,毛细管电泳鉴定。结果合成多肽（Ｐ２２）的纯度为９５．１９％,每次缩合的平均效率为９９．７８％;能与ａｎｔｉ－Ｃ５ａＲ ＭｃＡｂ（Ｓ５／Ⅰ,Ｓｅｒｏｔｉｃ公司）有效地结合,酶联ＯＤ４     

Expression of complement C3, C5, C3aR and C5aR1 genes in resting and activated CD4+ T cells

Complement activation is traditionally thought to occur in the extracellular space. However, it has been suggested that complement proteins are activated and function at additional locations. T cells contain intracellular stores of C3 and C5 that can be cleaved into C3a and C5a and bind to intracellular receptors, which have been shown to be of vital importance for the differentiation and function of these cells. However, whether the origin of the complement proteins located within T cells is derived from endogenous produced complement or from an uptake dependent mechanism is unknown.The presence of intracellular C3 in T cells from normal donors was investigated by fluorescence microscopy and flow cytometry. Moreover, mRNA expression levels of several genes encoding for complement proteins with primary focus on C3, C3aR, C5 and C5aR1 during resting state and upon activation of CD4⁺ T cells were investigated by a quantitative PCR technique. Furthermore, the gene expression level was evaluated at different time points.We confirmed the presence of intracellular C3 protein in normal T-cells. However, we could not see any increase in mRNA levels using any activation strategy tested. On the contrary, we observed a slight increase in C3 and C5aR1 mRNA only in the non-activated T-cells compared to the activated T cells, and a decrease in the activated T-cells at different incubation time points.Our results show that there is a baseline intracellular expression of the complement C3, C5, C3aR and C5aR1 genes in normal CD4⁺ T cells, but that expression is not increased during T-cell activation, but rather down regulated. Thus, the pool of intracellular complement in CD4⁺ T cells may either be due to accumulated complement due low-grade expression or arise from the circulation from an uptake dependent mechanism, but these possibilities are not mutually exclusive.     

Functional identification of the stable transfection C5aR cell line Molt-4

The complement C5 anaphylatoxin receptor is a member of the seven transmembrane-spanning G protein-coupled receptor superfamily that signals through Gcxi and Gtz16. C5aR is mostly expressed on neutrophils, macrophages and endothelial cells. C5a and C5aR interaction plays an important role in numerous biological effects such as in vivo cytokine storm which results in inflammatory damage. Considering the limitation of collection of human peripheral blood neutrophils and their short half life, the stably transfected cell line for studying the biological effects of C5aR is needed. In this study, we transfected C5aR gene into Molt-4 cell line and examined the function of ectopic C5aR. Our results showed stable expression of the C5aR in Molt-4 cell line and their interaction with human C5a induced ERKI/2 phosphorylation, Ca＋＋ influx. This stable transfected cell line may provide a useful tool for studying signal pathways related to C5a and C5aR interplay and antibody development specific for C5aR. Cellular ＆ Molecular Immunology.     

短发夹状RNA基因沉默补体受体C5aR及抑制LPS诱导的肾脏上皮细胞凋亡

目的:探讨短发夹状RNA基因沉默补体受体C5aR及抑制LPS诱导的肾脏上皮细胞凋亡的作用效果。方法:构建针对大鼠补体受体C5aR基因编码区的短发夹状RNA(shRNA)真核表达载体质粒pRNAT-U6.1-C5aR shRNA,采用电穿孔的方法转染RK3E细胞,经G418筛选后,形成稳定的表达C5aR shRNA的细胞系。实验分为3组,①正常对照组:未转染的RK3E细胞;②阴性对照组:转染空载体pRNAT-U6.1的RK3E细胞系;③实验组:转染C5aR shRNA的RK3E细胞系。经脂多糖(LPS)孵育12小时后,流式细胞仪检测各组细胞凋亡率,RT-PCR检测mRNA的表达,γ计数仪测定125I标记的C5a与RK3E的结合情况。结果:与正常对照组和阴性对照组相比,实验组的细胞凋亡率显著降低(P0.01),C5aR mRNA水平显著降低(P0.01),125I标记的C5a与RK3E结合活性显著下降。结论:针对C5aR的特异性短发夹RNA可以明显引起靶基因的沉默,进而抑制LPS诱导的肾脏上皮细胞凋亡的发生。     

Novel insights in localization and expression levels of C5aR and C5L2 under native and post-transplant conditions in the kidney

AimsThe complement system, and especially C5a, plays an important role in the pathophysiology of renal diseases and post-transplant renal injury. The two receptors for C5a are C5a receptor (C5aR) and C5a-like-receptor-2 (C5L2). Only renal C5aR expression has been reported, although exact localization and alterations in expression after transplantation are unknown.Materials and resultsRenal C5aR and C5L2 expression and localization were analyzed immunohistochemically. C5aR and C5L2 expression was analyzed in human kidney biopsies obtained from living donors and patients suffering from acute tubular necrosis, acute cellular and vascular rejection or IF/TA.C5aR was expressed in the thick ascending limb of Henle's loop and first part of the distal convoluted tubule (DCT). Under inflammatory conditions, C5aR was de novo expressed in proximal tubuli. C5L2 was expressed in the kidney and localized to DCT1, DCT2 and connecting tubule. Persistent distal tubular expression of both receptors was demonstrated after renal transplantation.ConclusionsThis study shows distinct renal expression patterns for C5aR and C5L2. Our findings suggest a functional role for renal C5L2 rather than being a C5a decoy receptor. Future studies focusing on renal C5a–C5aR interaction should take differential C5aR and C5L2 expression into account, alongside abundant C5aR expression on infiltrating cells.