转铁蛋白受体及其在药物运输中的作用

血脑屏障的存在阻止了中枢神经系统疾病许多潜在治疗药物的通过.近年来主要利用脑毛细血管内皮细胞膜中的转运蛋白,如转铁蛋白受体、胰岛素受体等,将外源药物与这些受体的特异性抗体相连,通过受体介导的内吞作用将药物转运到脑组织中.转铁蛋白受体在抗癌药物定向运输及恶性肿瘤细胞基因治疗中的研究已经处于临床阶段.     

脑内的铁,转铁蛋白及转铁蛋白受体

脑铁异常增高可能参与脑神经变性疾病的发生发展。这一发现使得脑铁代谢成为近年广为关注和研究较为广泛的领域。本文综述了这一领域某些方面的目前认识。包括：（１）脑铁分布及功能;（２）铁转铁蛋白及转铁蛋白受体在脑内的合成与分布;（３）脑铁摄取和运输。此外,对铁与某些金属离子,转的蛋白和转铁蛋白受体与脑神经变性疾病的关系,以及转铁蛋白受体内吞在生物大分子跨血脑屏障运输中的作用也作了简要讨论。     

转铁蛋白受体——肿瘤的标志物和靶向治疗

转铁蛋白受体不仅介导细胞内铁的摄取和参与细胞生长调节,而且能够在肿瘤细胞表面高度表达,被认为是一种有效的肿瘤标志物,广泛应用于各类恶性肿瘤的靶向治疗。利用转铁蛋白或转铁蛋白受体的单克隆抗体与转铁蛋白受体的特异性结合作用,通过受体介导的内吞机制实现化疗药物、蛋白毒素或治疗性基因等的细胞摄入。随着对转铁蛋白受体的深入研究,靶向转铁蛋白受体的肿瘤药物将在基因治疗和跨血脑屏障运输等多方面得到进一步应用。     

转铁蛋白受体2及其功能与相关疾病

转铁蛋白受体2(transferrin receptor 2, TfR2) 是最近发现的一种重要铁代谢蛋白.研究显示它不仅是一种介导肝脏细胞铁摄取的主要蛋白,而且在调节小肠铁吸收方面起着极其关键的作用,是控制肝脏铁调素合成和释放的关键成分.已经证实,TfR2基因突变是遗传性血色素沉着病的重要原因之一.     

利用噬菌体表面展示技术筛选转铁蛋白黏附肽的研究

为了筛选转铁蛋白黏附肽,应用噬菌体表面展示技术经过三轮生物淘选,成功地从随机七肽库中得到黏附转铁蛋白的重组噬菌体克隆,经过相对亲和力常数测定和DNA测序得到4个转铁蛋白黏附肽的序列。实验中以回收率和选择比为操作参数,对淘选进行了优化,并发展了一种基于噬菌体滴度的相对亲和力常数测定方法。转铁蛋白受体是一种有效的肿瘤标记物,利用转铁蛋白为载体可以实现药物靶向运输,因此转铁蛋白黏附肽将是重组蛋白质药物连接转铁蛋白的有用标签。     

猪血清转铁蛋白含单一铁结合部位的半分子的制备、鉴定和受体结合能力

用胰蛋白酶水解铁饱和的猪血清转铁蛋白 ,可同时获得含单一铁结合部位的N端和C端半分子。比较了猪血清转铁蛋白及其N端和C端半分子与人胎盘细胞膜转铁蛋白受体的结合能力 ,其受体结合能力依次为 :猪血清转铁蛋白 >C端半分子 >N端半分子     

转铁蛋白结构与功能的研究——鸭血清转铁蛋白含单一铁结合部位的结构域的制备和鉴定

分离纯化了鸭血清转铁蛋白、分子量78,000,N-末端为Ala,不能与人胎盘转铁蛋白受体结合。鸭血清转铁蛋白用胰蛋白酶酶解可以同时得到两个含单一铁结合部位的结构域,分别来自分子的N-端和C-端区域。获得的鸭血清转铁蛋白N-端结构域分子量为33,200,C-端结构域分子量为34,900。     

人转铁蛋白受体及其特异性抗体的制备

目的:从胎盘中提取转铁蛋白受体并获得抗转铁蛋白受体的抗体。方法:人新鲜胎盘组织被破碎后,用去污剂TritonX-100裂解细胞膜,释放膜蛋白。利用膜蛋白中的转铁蛋白受体能与铁-转铁蛋白复合物特异性结合的特性对其进行亲和纯化。对纯化得到的目的蛋白,经脱盐后进行ELISA及肽质量图谱分析,证明为所需的转铁蛋白受体后,以其包被免疫管,从全合成人源噬菌体抗体库中筛选抗体。结果:从人源噬菌体抗体库中筛选到5个能够与转铁蛋白受体特异性结合的噬菌体单链抗体。结论:以人源转铁蛋白受体为抗体,可从全人源噬菌体抗体库中筛选到其特异性的抗体。     

24p3/NGAL介导的铁转运途径

生物体内存在另一转铁途径。脂笼蛋白(lipocalin)家族的成员24p3／NGAL介导铁向细胞内转运,并在具有酸性环境的核内体(endosome)中与铁解离,进而调节铁蛋白(ferritin,Fn)基因和转铁蛋白受体-1(transferrin receptor-1,TfR-1)基因的表达。24p3／NGA转铁途径在亚细胞水平上与转铁蛋白(tansferrin,Tf)类似但相互独立。在胚肾发育过程中,24p3／NGAL与Tf介导的铁转运途径为不同时期的原始肾上皮细胞生长与分化所必需。深入研究24p3／NGAL转铁途径的分子机制及与Tf的异同有十分重要的意义。     

十二指肠上皮细胞转铁蛋白受体表达调控的免疫组织化学观察与铁吸收机制的探讨

转铁蛋白受体（ＴｆＲ）在细胞的铁转运方面起重要作用。本研究用免疫组织化学法比较铁缺乏（甲组）、铁过剩（乙组）与对照组（丙组）Ｗｉｓｔａｒ系大鼠活体十二指肠上皮细胞ＴｆＲ的表达调控及其在铁吸收过程中的意义。结果表明：甲组十二指肠上皮细胞内ＴｆＲ表达明显强于雨组,乙组ＴｆＲ表达最弱。可见ＴｆＲ的表达在一定范围内随体内铁含量的增高或降低而减弱或增强,受铁状态的负调节。丙组ＴｆＲ主要位于细胞基底部,游离面ＴｆＲ表达不明显。甲组细胞ＴｆＲ表达增强,基底部可见线状ＴｆＲ高强度表达。乙组虽ＴｆＲ表达明显减弱,但在细胞基底部仍可见线状ＴｆＲ表达。提示十二指肠上皮细胞基底部有ＴｆＲ介导的铁转运,而铁从上皮的游离面进入细胞内并非由ＴｆＲ介导。三组动物小肠粘膜固有层中均可见ＴｆＲ阳性的巨噬细胞。在铁过剩组、此巨噬细胞数量增加,且ＴｆＲ表达未受明显抑制。认为铁过剩时,粘膜固有层的巨噬细胞内可贮存过量的铁,减少其对实质细胞、组织的损伤。     

Inactivation of the murine Transferrin Receptor 2 gene using the Cre recombinase: loxP system

Transferrin Receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 lead to type 3 hemochromatosis in humans. We have developed mice with a targeted deletion of TfR2. The Cre-recombinase:loxP system used to create the mice allows both full deletion and tissue-specific deletion of TfR2. The development of these mice will provide new models for type 3 hemochromatosis and assist in determining the role of TfR2 in iron metabolism.     

多天线糖链对运铁蛋白与受体结合及内吞的研究

应用系列凝集素柱层析法（伴刀豆球蛋白,小扁豆凝集素,欧曼陀罗凝集素）分别从正常人血清及孕妇血清中提纯含有二天线无核心岩藻糖复杂型糖链的运铁蛋白及含有多天线无核心岩藻糖复杂型糖链的运铁蛋白。与正常的含有二天线糖链的运铁蛋白相比,含有多天线糖链的运铁蛋白与ＳＭＭＣ－７７２１细胞膜表面的运铁蛋白受体的亲和力下降,但最大结合量不变,此外,其在内吞过程中于细胞膜上的停留时间延长。结果表明糖链结构改变对运铁蛋白的功能有影响。     

Structural and functional studies on the stalk of the transferrin receptor

Transferrin (Tf) is an iron carrier protein that consists of two lobes, the N- and C-lobes, which can each bind a Fe³⁺ ion. Tf binds to its receptor (TfR), which mediates iron delivery to cells through an endocytotic pathway. Receptor binding facilitates iron release from the Tf C-lobe, but impedes iron release from the N-lobe. An atomic model of the Tf-TfR complex based on single particle electron microscopy (EM) indicated that receptor binding is indeed likely to hinder opening of the N-lobe, thus interfering with its iron release. The atomic model also suggested that the TfR stalks could form additional contacts with the Tf N-lobes, thus potentially further slowing down its iron release. Here, we show that the TfR stalks are unlikely to make strong interactions with the Tf N-lobes and that the stalks have no effect on iron release from the N-lobes of receptor-bound Tf.     

Transferrin uptake in Trypanosoma cruzi is impaired by interference on cytostome-associated cytoskeleton elements and stability of membrane cholesterol, but not by obstruction of clathrin-dependent endocytosis

Transferrin uptake by Trypanosoma cruzi epimastigotes occurs mainly through the cytostome/cytopharynx. Here, we present evidences for the association of sterol-rich membrane domains with the transferrin endocytic site. Assays using pharmacological treatments to disrupt clathrin-coated pits and hinder caveolae formation showed no association between transferrin uptake and clathrin-dependent endocytosis, but indicated that cholesterol stability in membrane domains is essential for the endocytosis of transferrin. Furthermore, it was observed a connection between the integrity of cytoskeleton elements at the cytopharynx and the function of the cytostome. Our data show that T. cruzi epimastigotes depend on a specialized pathway for transferrin uptake, which is cholesterol-dependent, clathrin-independent, and closely associated with the structural stability of the cytostome/cytopharynx cytoskeleton.     

Transferrin and ferritin endocytosis and recycling in guinea-pig reticulocytes

Transferrin and ferritin endocytosis and exocytosis by guinea-pig reticulocytes were studied using incubation with pronase at 4 degrees C to distinguish internalized and membrane-bound protein. Internalization of both transferrin and ferritin occurred in a time- and temperature-dependent fashion. Transferrin endocytosis was more rapid than that of ferritin. Transferrin binding to receptors was not altered, but transferrin endocytosis was decreased in the presence of ferritin. Iron accumulation from transferrin was inhibited by ferritin to a greater extent than could be accounted for by the decreased rate of endocytosis. In pulse-chase experiments, almost all of the transferrin was released intact from reticulocytes, but only about 50% of the total internalized ferritin was released, of which 85% was intact. The endocytosis of transferrin by rabbit reticulocytes was 2- to 2.5-times faster than guinea-pig reticulocytes. These data suggest that ferritin and transferrin are internalized by receptor-mediated endocytosis, possibly involving the same coated pits and vesicles, but that the proteins are recycled only partly in common.     

Transferrin and tooth morphogenesis: Retention of transferrin by mouse embryonic teeth in organ culture

Transferrin is the only serum protein that is required for the early morphogenesis of mouse embryonic teeth in organ culture. Transferrin is able to support tooth morphogenesis and dental cell differentiation by stimulating cell proliferation. Its role in this process is restricted exclusively to iron transport, which takes place by receptor-mediated endocytosis of iron-loaded transferrin. A lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone (PIH), can replace transferrin and support tooth morphogenesis in organ culture. We studied the effects of these two iron transporters on cell proliferation in tooth germs during culture. We found that Fe-PIH and transferrin stimulate proliferation to a similar extent in early cap-stage teeth of 14-day mouse embryos, but have no effect on cell proliferation in bell-stage teeth of 16-day mouse embryos. Day-16 teeth undergo morphogenesis in unsupplemented chemically defined medium, whereas transferrin or Fe-PIH is needed for the morphogenesis of day-14 teeth. Although the need for exogenous iron-transport molecules is lost with advancing development, the level of mitotic activity is still fairly high in bell-stage teeth. The abundant binding of transferrin in areas of active cell proliferation in bell-stage teeth also suggests that transferrin is still needed and used for the transport of iron into proliferating cells. Transferrin is not degraded by the process of receptor-mediated endocytosis. After releasing iron into a cell, transferrin is returned to the extracellular space and is reused. We therefore studied whether the transferrin needed by bell-stage teeth could be adequately supplied by endogenous transferrin synthesized or stored in tissue explants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transferrin-Directed Internalization and Cycling of Transferrin Receptor 2

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1) but has distinct functions from TfR1 in iron homeostasis. In keeping with its proposed role in iron sensing, previous studies showed that TfR2 has a short half-life and that holo-Tf stabilizes TfR2 by redirecting it from a degradative pathway to a recycling pathway. In this study, we characterized how the endocytosis, recycling and degradation of TfR2 relates to its function and differs from TfR1. TfR2 endocytosis was adaptor protein-2 (AP-2) dependent. Flow cytometry analysis showed that TfR1 and TfR2 utilized the same endocytic pathway only in the presence of holo-Tf, indicating that holo-Tf alters the interaction of TfR2 with the endocytic machinery. Unlike TfR1, phosphofurin acidic cluster sorting protein 1 (PACS-1) binds to the cytoplasmic domain of TfR2 and data suggest that PACS-1 is involved in the TfR2 recycling. Depletion of TSG101 by siRNA or expression of a dominant negative Vps4 inhibited TfR2 degradation, indicating that TfR2 degradation occurs through a multivesicular body (MVB) pathway. TfR2 degradation is not mediated through ubiquitination on the single lysine (K31) in the cytoplasmic domain or on the amino terminal residue. No ubiquitination of TfR2 by HA-ubiquitin was detected, indicating a lack of direct TfR2 ubiquitination involvement in its degradation.     

Nano-vectors for efficient liver specific gene transfer

Recent progress in nanotechnology has triggered the site specific drug/gene delivery research and gained wide acknowledgment in contemporary DNA therapeutics. Amongst various organs, liver plays a crucial role in various body functions and in addition, the site is a primary location of metastatic tumor growth. In past few years, a plethora of nano-vectors have been developed and investigated to target liver associated cells through receptor mediated endocytosis. This emerging paradigm in cellular drug/gene delivery provides promising approach to eradicate genetic as well as acquired diseases affecting the liver. The present review provides a comprehensive overview of potential of various delivery systems, viz., lipoplexes, liposomes, polyplexes, nanoparticles and so forth to selectively relocate foreign therapeutic DNA into liver specific cell type via the receptor mediated endocytosis. Various receptors like asialoglycoprotein receptors (ASGP-R) provide unique opportunity to target liver parenchymal cells. The results obtained so far reveal tremendous promise and offer enormous options to develop novel DNA-based pharmaceuticals for liver disorders in near future.     

Endocytosis of a Functionally Enhanced GFP-Tagged Transferrin Receptor in CHO Cells

The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81±3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.     

Transferrin protein and iron uptake by cultured hepatocytes

The binding and uptake of ⁵⁹Fe-loaded ³H-labelled rat transferrin by cultured rat hepatocytes was investigated. At 4°C, there is no evidence for a specific binding of transferrin which could be related to the association of neo-synthesized transferrin with plasma membrane receptors. At 37°C, iron uptake is much more important than transferrin uptake; it proceeds linearly over the time of incubation, is largely proportional to the extracellular transferrin concentration, and is compatible with uptake by fluid phase endocytosis. The difference observed between iron and transferrin uptake implies the existence of a mechanism allowing the reutilization of transferrin after iron delivery.