Collectins and collectin receptors in innate immunity
Collectins and collectin receptors in innate immunity
复制标题
DOI：
10.1111/j.1600-0463.2000.tb05694.x
复制
发表时间：
2000-08
期刊：
影响因子：
2.8
通讯作者：
U. Holmskov
中科院分区：
文献类型：
--
作者：
U. Holmskov
本论文在阅读了9篇文献的基础上，对天然免疫中的凝集素及其受体进行了综述。胶原凝集素是一个蛋白质家族，其中单个链由通过α-卷曲颈区连接到胶原结构域的C型凝集素结构域组成。这些链被组织成三重胶原螺旋并通过位于N末端的半胱氨酸寡聚化。胶原凝集素具有双重功能：一个是特异性结合病原体表面的碳水化合物结构;另一个是随后招募其他细胞和分子来破坏病原体。C型凝集素结构域含有以保守序列模式排列的110-130个氨基酸残基，这允许该结构域折叠成明确定义的三级结构。已描述了五种收集素。肺表面活性蛋白A和D（SP-A和SP-D）主要存在于覆盖肺上皮细胞腔表面的表面活性剂中，但也由胃肠道内衬细胞产生。甘露聚糖结合凝集素（MBL）、连接蛋白和凝集素-43（CL-43）是由肝脏产生的血清蛋白。到目前为止，仅在牛科动物中发现了Convagin和CL-43。胶原凝集素参与先天性非适应性免疫防御。它们与微生物表面碳水化合物结合，诱导聚集，从而阻碍感染性或通过吞噬细胞上的特异性受体介导吞噬作用。在结合微生物碳水化合物后，MBL可以通过新发现的途径激活补体系统，该途径利用两种丝氨酸蛋白酶（MASP-1和MASP-2）来激活补体因子C4和C2。在人类中，由胶原蛋白区域突变引起的低血清MBL浓度与常见的调理素缺陷相关。CL-43是一种新的凝集素，其与甘露聚糖的结合依赖于钙离子，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上其还原态的M（r）为43 kDa。经甘露聚糖-琼脂糖亲和层析、兔抗牛IG-Sepharose-4 B吸附、离子交换层析等分离纯化。CL-43在SDS-PAGE上未还原状态下的表观分子量为120 kDa，在非解离条件下凝胶色谱上洗脱时的表观分子量为750 kDa。氨基酸分析和对胶原酶消化的敏感性表明CL-43是一种聚集蛋白。纯化的CL-43的电子显微镜显示只有棒状单体亚基37.4 nm长。双向凝胶电泳显示CL-43有两种异构体，pI分别为4.9和5.3，对应于CL-43的天然形式和缺少前9个氨基酸残基的截短形式。根据CL-43的N端氨基酸序列设计引物，以牛肝cDNA为模板进行PCR扩增。克隆了CL-43的cDNA，其开放阅读框编码301个氨基酸残基，其中N端28个氨基酸残基，胶原蛋白114个氨基酸残基，颈CRD 159个氨基酸残基。CL-43的氨基酸序列与牛conglutinin的同源性为74%，与牛SP-D的同源性为70%，但其胶原蛋白区域明显短于conglutinin和SP-D。北方印迹分析表明CL-43仅在牛肝脏中合成，在包括肺在内的各种其他牛组织中未检测到信号。在来自绵羊、人、大鼠或小鼠肝脏的mRNA中未检测到交叉杂交信号。由于CL-43和conglomerin仅在牛科成员中检测到，因此这两种蛋白质的祖先基因可能首先来源于SP-D样基因，并且该祖先基因在进化过程中经历了复制。采用固相甘露聚糖作为配体，通过生物素化CL-43的抑制试验分析CL-43的碳水化合物结合特征。（摘要截断）
This thesis is based on nine papers and a review on the collectins and collectin receptors in innate immunity. The collectins are a family of proteins in which the individual chains consist of a C-type lectin domain attached to a collagen domain via an alpha-coiled neck region. The chains are organized into a triple collagen helix and oligomerized through N-terminally located cysteines. The collectins have a dual function: one is to bind specifically to carbohydrate structures on the surface of a pathogen; the other is subsequently to recruit other cells and molecules to destroy the pathogen. The C-type lectin domains contain 110-130 amino-acid residues arranged in a conserved sequence pattern which allows the domain to fold into a well-defined tertiary structure. Five collectins have been described. Lung surfactant proteins A and D (SP-A and SP-D) are mainly found in the surfactant coating the luminal surface of the pulmonary epithelial cells, but are also produced by cells lining the gastrointestinal tract. Mannan-binding lectin (MBL), conglutinin and collectin-43 (CL-43) are serum proteins produced by the liver. Conglutinin and CL-43 have so far only been found in Bovidae. The collectins are involved in innate, nonadaptive immune defense. They bind to microbial surface carbohydrates, inducing aggregation and thereby impeding infectivity or mediating phagocytosis through specific receptors on the phagocytes. After binding microbial carbohydrate, MBL can activate the complement system through a newly discovered pathway which makes use of two serine proteases (MASP-1 and MASP-2) to activate the complement factors C4 and C2. In man, low serum MBL concentrations resulting from mutations in the collagen region are associated with a common opsonic defect. CL-43 was identified as a new collectin by its calcium-dependent binding to mannan and by its M(r) of 43 kDa in the reduced state on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lectin was isolated by affinity chromatography on mannan-Sepharose, absorption with rabbit anti-bovine Ig coupled to Sepharose-4B and ion-exchange chromatography. CL-43 shows an apparent molecular mass of 120 kDa in the unreduced state on SDS-PAGE and elutes with an apparent molecular mass of 750 kDa on gel chromatography under nondissociating conditions. Amino-acid analysis and susceptibility to collagenase digestion indicated that CL-43 was a collectin. Electron microscopy of purified CL-43 revealed only rod-like monomer subunits 37.4 nm long. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms of pI 4.9 and 5.3 respectively, corresponding to the native form of CL-43 and a truncated form which lacks the first 9 amino-acid residues. The N-terminal amino-acid sequence of CL-43 was used to design primers for PCR with a bovine liver cDNA as template. The cDNA of CL-43 was cloned and the open reading frame was found to encode a protein of 301 amino-acid residues, including an N-terminal region of 28 residues, a collagen region of 114 residues and a neck-CRD region of 159 residues. The amino-acid sequence of CL-43 shows 74% identity with bovine conglutinin and 70% identity with bovine SP-D, but the collagen region is considerably shorter than those of conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, no signal being detected in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from ovine, human, rat or mouse liver. Since CL-43 and conglutinin have only been detected in members of the Bovidae, it is probable that an ancestral gene of these two proteins was first derived from a SP-D-like gene and that this ancestral gene underwent duplication during evolution. The carbohydrate binding profile of CL-43 was analyzed by an inhibition assay with biotinylated CL-43, using solid-phase mannan as the ligand. (ABSTRACT TRUNCATED)