高密度脂蛋白受体及结合蛋白主题词]脂蛋白,高密度;受体;动脉粥样硬化;胆固醇;逆向转运[摘要]高密度脂蛋白受体在脂质代谢中发挥着重要的作用。
最近十年,有关高密度脂蛋白受体及结合蛋白的研究受到了广泛的重视,其中被公认为高密度脂蛋白受体的清道夫受体BÑ及很有可能被确定为受体的高密度脂蛋白结合蛋白2最引人注目。
两者的分子结构、特异性配体及在胆固醇逆向转运中的作用具有较大的差异,进一步探明两者之间的关联可为研究动脉粥样硬化的发病机理及其新的治疗途径提供有力的理论基础。
迄今为止,在不同细胞的表面及细胞内已分离出了多种可与高密度脂蛋白(h i g h density lipo p r o t e i n,HDL)结合的蛋白质,它们具有截然不同的分子结构,分别参与多种生物化学过程的调控。
其中某些蛋白质可特异性识别并以高亲和力与HDL结合,引发下游的生物学, 称之为HDL的特异性受体。
有些蛋白质也可与HDL结合,但不产生或只产生较弱的效应,则称之为HDL的结合蛋白。
至今,研究比较深入的HDL受体及结合蛋白包括清道夫受体BÑ(s c a v e n g e r re-c e p t o r c l a s s Bt y p eÑ,SRBÑ)、高密度脂蛋白结合蛋白(HDLb i n d i n g p r o t e i n, H B)、CD36、v i g i l i n及cubili等,其中只有SRBÑ被公认为HDL受体[1],并对其它几种的结构功能进行深入的研究,以探明其与HDL及相关效应的关系。
1清道夫受体BÑ1.1结构SRBÑ在结构上的同源性隶属于CD36膜蛋白家族成员,在功能上与CD36、SRBÒ同属于B类清道夫受体家族,此类受体存在一免疫优势区域(i m m u n o d o m i n a n t d o m a i n),类似于A类清道夫受体带正电荷的胶原样结构,具有广泛的配体结合特性,可与低密度脂蛋白(l o w d ensityl i p o p r o t e i n,LDL)、修饰的L D L、HDL等结合,却不能与A类受体某些其它的配体如岩藻多糖、多聚鸟苷酸、角叉聚糖等结合。
J o h n s o n 等[2]克隆了大鼠卵巢细胞上mSRBÑ的基因)然后由结构基因推测SRBÑ含有509个氨基酸残基,分子量约为57kDa,包括一个较长的胞外结构域,胞外段上含有8个半胱氨酸和11个潜在的N -糖基化位点,另外还包括两个疏水区和两个胞内结构域(图1)。
两个疏水区参与形成跨膜片段,而胞内段含有潜在的蛋白激酶C(p r o t e i n kinaseC,PKC)和蛋白激酶A(p r o t e i n k i n a s e A,P K A)的丝氨酸磷酸化位点。
在受体蛋白的羧基端有一个亮氨酸拉链区,决定蛋白质二聚体结构的形成;另外还存在一个亲过氧化物酶的序列(PTS1),可与特异的P T S1受体(PTS1导入受体)结合,指导HDL与过氧化物酶结合,使胆固醇发生B-氧化降解。
图1. mSRBÑ示意图. 小鼠SRBÑ含有两个胞浆域、两个疏水的跨膜域和一个胞外域.B a b i t t等[3]用免疫化学法发现,mSRBÑ经N-寡糖链糖基化后,分子量变为82kDa,并且可抵抗蛋白水解酶的消化。
另外,位于跨膜区与胞浆区交界处的半胱氨酸残基可发生脂酰化,有利于SRBÑ与胞浆侧的蛋白质(如胞内的酪氨酸激酶和内皮细胞NO 合酶)相互作用,触发信号的转导。
另外还发现SRBÑ存在于被称作caveolae的膜微凹陷区。
caveolae是细胞膜表面50~100nm的瓶状凹陷,此结构中主要成分为21~24kDa的c a v e o l i n,是一种胆固醇结合蛋白[13]。
c a v e o-l a e在SRBÑ介导的脂质转运中发挥着重要的作用。
1.2 功能及分布1996年,A c t o n等[1]克隆了SRBÑc D N A,将其染到中华仓鼠卵巢c h i n e s e h a m s t e r ovary,CH O)细胞上表达, 利用核素标记HDL中的载脂蛋白和胆固醇,测定受体相关指标及胆固醇酯(c h o l e s t e r o l e s t e r,CE)的摄取,结果显示HDL可与SRBÑ以高亲和力结合。
当细胞与标记配体结合达到平衡后,18%的CE 被细胞摄取,只有0.5%的载脂蛋白与细胞结合。
为了区分CE进入细胞是净转运还是细胞与HDL脂质的交，,又测定了胆固醇含量,发现反应达平衡后转染细胞内胆固醇含量大约增加了20%,与上述结果基本吻合,说明CE选择性摄取是一种净转运过程。
同时证实了SRBÑ是HDL 的特异性受体,介导CE的选择性摄取。
用抗体阻断鼠肾上腺皮质及卵巢细胞上SRBÑ的胞外段,CE的选择性摄取被抑制[4]。
用基因打靶技术使小鼠SRB基因突变,则血浆中HDL浓度升高,肾上腺皮质细胞中中性脂质含量随之下降;而肝脏中SRBÑ表达降低二分之一后,CE的摄取也随之减少[5。
可见SRBÑ是介导肝及类固醇激素生成细胞中选择性摄取CE的受体。
另有证据表明, SRBÑ可介导胆固醇外流,从而在胆固逆向转运过程的前期发挥作用。
J i等[8]发现在用鼠SRBÑ转染后的CH O细胞中胆固醇的外流明显增强。
外流率与受体的表达呈正相关性(r=0.859) 。
此结果在腹腔巨噬细胞、成纤维细胞、肝肿瘤细胞及肾上腺细胞中均得到证实。
SRBÑ在鼠肝、卵巢、肾上腺细胞上高度表达,在睾丸和乳腺细胞上表达相对较,在心脏上几乎无表[1] ,其分布与生理意义基本吻合。
但是,啮齿类动物中HDL携带着循环中大部分的胆固醇,在人体中主要的胆固醇转运体是D L,而不是HDL,如培养的肾上腺细胞在A C T H作用下,LDL的摄取及代谢增加了5~6倍,而HDL中的胆固醇并未参与激素的合成[6]。
High-density lipoprotein receptorand binding proteinMeSH ] lipoprotein , high density ; receptor ; atherosclerosis ; cholesterol ; antiporter[ Abstract ]High-density lipoprotein receptor in lipid metabolism plays an important role. The last decade , about high -density lipoprotein receptor and binding protein has been widely appreciated , which is recognized as the high-density lipoprotein receptor scavenger receptor BÑ and is likely to be identified as the receptor density the most striking lipoprotein -binding protein 2 . Both the molecular structure of specific ligands and in reverse cholesterol transport in the role of a big difference , and further proved for the association between atherosclerosis study the pathogenesis and new therapeutic approaches to provide a strong theoretical basis.So far, in a different cell surface and intracellular been isolated from a wide range with high-density lipoprotein (highdensitylipoprotein, HDL) -binding proteins , they have very different molecular structure , were involved in many biochemicalProcess control . Some of these proteins could be specifically identified and with high affinity binding of HDL , the downstream biological trigger ,Called HDL receptor specificity . Some proteins can be combined with HDL , but do not produce orProduced only weak effects , is called binding protein of HDL . So far, more in-depth study of the HDL receptor and binding protein packageIncluding scavenger receptor BÑ (scavengerre-ceptorclassBtypeÑ, SRBÑ), high density lipoprotein -binding protein (HDLbindingprotein,HB), CD36, vigilin and cubili , etc., which is recognized only SRBÑ HDL receptor [ 1 ] , and several other structural features in-depth study to ascertain its relationship between HDL andrelated effects . A scavenger receptor BÑ1.1 structure SRBÑ structurally homologous CD36 membrane proteins belonging to family members , in function with CD36, SRBÒ belong to the same class B scavenger receptor family , there is an immunological advantage of these receptors area (immunodominantdomain), similar to the class A scavenger receptor positive charge of the collagen -like structure , with a broad ligand binding properties , can be used with low-density lipoprotein (lowdensitylipoprotein, LDL), modified LDL, HDL , etc. with , but a can with some other receptors such as ligands fucoidan , poly guanylic acid , carrageenan and other polysaccharides with . Johnson et al.[ 2 ] Cloning of rat ovarian cells mSRBÑ gene ) and the structural gene presumed SRBÑ 509 amino acid residues with a molecular weight of about 57kDa, including a longer extracellular domain , containing the extracellular segment of eight half cystine and 11 potential N- Glycosylation sites , also includes two hydrophobic region and two intracellular domain ( Figure 1 ) . Two hydrophobic transmembrane segments involved in the formation and intracellular protein kinase containing potential C (proteinkinaseC, PKC) and protein kinase A (proteinkinaseA, PKA) serine phosphorylation sites . In the receptor protein has a carboxy-terminal leucine zipper domain , determine the formation of the protein dimer ; There is also another pro- peroxidase sequences (PTS1), PTS1 with specific receptors (PTS1 import a body ) combined with peroxidase-conjugated HDL guidance to make cholesterol B- oxidative degradation occurs . Figure 1. MSRBÑ schematic mouse SRBÑ contains two cytoplasmic domains,Two hydrophobic transmembrane domain and a extracellular domain . Babitt , etc. [ 3 ] The immunochemical found , mSRBÑ oligosaccharide chains by N- glycosylation , the molecular weight becomes 82kDa, and is resistant to the proteolytic enzyme digestion. Further , in the transmembrane region and cytoplasmic region of the cysteine residues at the junction of fat may occur acylated with cytoplasmic side SRBÑ beneficial proteins (such as tyrosine kinases and intracellular endothelial NOSynthase ) interaction , trigger signal transduction . SRBÑ also found present in a film called micro depressions caveolae .caveolae membrane surface is 50 ~ 100nm bottle -shaped depression , this structure is mainly composed of 21 ~ 24kDa of caveolin, a cholesterol -binding protein [ 13 ] . caveo-lae in SRBÑ mediated lipid transport plays an important role. 1.2 Functional and distribution in 1996 , Acton , etc. [ 1 ] cloned SRBÑcDNA, it was transfected into Chinese hamster ovary chinesehamsterovary, CHO) cells expressedUse of radionuclide labeled apolipoprotein of HDL and cholesterol were measured receptor-related indicators and cholesterol ester (cholesterolester, CE) uptake showed that HDL can be used with SRBÑ with high affinity . When the cell with the labeled ligand binding equilibrium is reached , 18% of CE uptake by cells , only 0.5% of the cell binding apolipoprotein . In order to distinguish CE is a net transport into the cell or cellsCross HDL lipids , and cholesterol content was measured and found that the reaction reached equilibrium transfected cells increased cholesterol content of about20% , basically consistent with the above results , indicating that CE selective uptake is a net transfer process . HDL is also confirmed SRBÑThe specific receptor mediated selective uptake of CE . Rat adrenal cortex with blocking antibodies and ovarian cells SRBÑ extracellularSegment , CE selective uptake was inhibited [ 4 ] . Using gene targeting in mice SRB gene mutation , the plasma HDL levels riseHigh adrenocortical cells in neutral lipid content fall ; while reduced expression of liver SRBÑ after half , CE uptake is also reduced[ 5 . Visible SRBÑ is mediated by the liver and steroid hormone -producing cells in the selective uptake of CE receptors. There is also evidence thatSRBÑ be mediated cholesterol efflux , and thus reverse cholesterol transport process in the early stage of play. Ji et al [ 8 ] found that with the mouseSRBÑ transfected CHO cells significantly enhanced outflow of cholesterol . Outflow rate and receptor expression was positively correlated (r = 0.859). This results in peritoneal macrophages , fibroblasts , liver cancer cells and adrenal cells were confirmed . SRBÑ in the rat liver, ovary , adrenal cells, is highly expressed in the testis and relatively expressed on breast cells in the heart of almost no table [1 ]Their distribution is basically consistent with the physiological significance . However , in rodents HDL carries most of the cholesterol circulating in the humanThe main body of cholesterol transporter is DL, but not HDL, such as adrenal cells cultured under the action of ACTH , LDL uptake and metabolic 5-6 times increased , while HDL cholesterol was not involved in the synthesis of hormones [ 6 ] .。