Mammalian Lactoferrin Receptors: Structure and Function

哺乳动物乳铁蛋白受体：结构和功能

• 批准号: 6727905
• 负责人: BO L LONNERDAL
• 金额: $ 25.1万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727905
• 关键词: X ray crystallography; age difference; cell line; cell surface receptors; clathrin; combinatorial chemistry; embryo /fetus; endocytosis; gene targeting; genetically modified animals; in situ hybridization; iron; iron metabolism; laboratory mouse; lactoferrin; lipopolysaccharides; mammalian embryology; mature animal; microarray technology; nitric oxide; nucleic acid probes; protein localization; protein structure function; receptor binding; receptor expression; transferrin receptor

DESCRIPTION (provided by applicant): Lactoferrin (Lf), an 80 kD glycoprotein that can bind two atoms of Fe(lll), is a major protein in human milk, and also present in exocrine secretions, e.g. pancreatic fluid and bile, and in neutrophils. Lf has been suggested to have several functions, including a role in intestinal Fe absorption, reproductive function, antimicrobial action, cellular proliferation and immune competence. However, although some support for such activities have been obtained, little is known about the mechanisms by which Lf exerts these functions. We previously showed the existence of Lf receptors (LfR) in human small intestine by kinetic binding studies. We have recently cloned and expressed human and mouse LfR, which are expressed in several tissues. Transfection of human intestinal cells showed increased uptake of iron and of Lf. In the proposed project we intend to determine the tertiary structure of LfR and the Lf-LfR complex by X-ray diffraction to better understand the organization of the LfR molecule and its interaction with Lf. We will also determine the specific site of the Lf molecule that binds to the LfR by using truncated versions (N-lobe, C-lobe) expressed as chimeras with transferrin in baculovirus. Peptides binding to the LfR will be generated by combinatorial chemistry. Oligonucleotide probes will be used for in situ hybridization and an LfR antibody will be used for immunostaining of human and mouse tissues, with particular emphasis on the small intestine and mouse embryonic development. Pathways of Lf internalization will be studied by inhibitors specifically inhibiting coated pits and caveolae-mediated pathways, respectively. Cellular responses to Lf mediated by the LfR will be studied by microarrays for signal transduction pathways. The effect of Fe, lipopolysaccharide (LPS) and nitric oxide on LfR expression will be studied by using several cell lines. We will also explore the biological significance of the LfR by using a conditional knockout mouse. We will clone the mouse LfR gene and construct a targeting vector in which the first exon is flanked by two Iox P sites. We will then use inducible cre transgenic mice to produce LfR knockouts in various tissues, such as small intestine and mammary gland, as well as at various stages of development. Basic properties of the knockouts will be compared with those of the wild type. Possible observations in the knockouts will be impaired reproduction and immune competence, decreased levels of Lf in milk, developmental abnormalities, lower Fe status of pups and diminished protective effect of Lf against LPS-caused mortality. Overall, our understanding of the physiological significance of Lf and its receptor will be increased.

描述（由申请人提供）：乳铁蛋白 (Lf) 是一种 80 kD 的糖蛋白，可以结合两个 Fe(III) 原子，是人乳中的主要蛋白质，也存在于外分泌物中，例如乳汁中。胰液和胆汁，以及中性粒细胞。 Lf 被认为具有多种功能，包括肠道铁吸收、生殖功能、抗菌作用、细胞增殖和免疫能力。然而，尽管已经获得了对此类活动的一些支持，但人们对 Lf 发挥这些功能的机制知之甚少。我们之前通过动力学结合研究证明了人类小肠中 Lf 受体 (LfR) 的存在。我们最近克隆并表达了人和小鼠的 LfR，它们在多种组织中表达。人类肠道细胞的转染显示铁和 Lf 的摄取增加。在拟议的项目中，我们打算通过 X 射线衍射确定 LfR 和 Lf-LfR 复合物的三级结构，以更好地了解 LfR 分子的组织及其与 Lf 的相互作用。我们还将通过使用在杆状病毒中表达为与转铁蛋白的嵌合体的截短版本（N-叶、C-叶）来确定与 LfR 结合的 Lf 分子的特定位点。与 LfR 结合的肽将通过组合化学产生。寡核苷酸探针将用于原位杂交，LfR 抗体将用于人类和小鼠组织的免疫染色，特别是小肠和小鼠胚胎发育。 Lf 内化途径将通过分别特异性抑制包被凹坑和小凹介导途径的抑制剂来研究。将通过信号转导途径的微阵列研究 LfR 介导的细胞对 Lf 的反应。将使用几种细胞系研究 Fe、脂多糖 (LPS) 和一氧化氮对 LfR 表达的影响。我们还将通过使用条件敲除小鼠来探索 LfR 的生物学意义。我们将克隆小鼠 LfR 基因并构建一个靶向载体，其中第一个外显子两侧有两个 Iox P 位点。然后，我们将使用可诱导的 cre 转基因小鼠在各种组织（例如小肠和乳腺）以及各个发育阶段产生 LfR 敲除。敲除基因的基本特性将与野生型的基本特性进行比较。敲除中可能观察到的结果是繁殖和免疫能力受损、牛奶中 Lf 水平降低、发育异常、幼仔的 Fe 状态较低以及 Lf 对 LPS 引起的死亡的保护作用减弱。总体而言，我们对Lf及其受体的生理意义的理解将会增加。