Mammalian Lactoferrin Receptors: Structure and Function

哺乳动物乳铁蛋白受体：结构和功能

• 批准号: 6994422
• 负责人: BO L LONNERDAL
• 金额: $ 24.43万
• 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/6994422
• 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），一种可以结合Fe两个原子（LLL）的80 kD糖蛋白，是人乳中的主要蛋白质，也存在于外分泌分泌中，例如。胰液和胆汁，以及中性粒细胞。已建议LF具有多种功能，包括在肠道吸收，生殖功能，抗菌作用，细胞增殖和免疫能力中的作用。但是，尽管已经获得了对此类活动的一些支持，但对于LF发挥这些功能的机制知之甚少。我们先前通过动力学结合研究表明了人类小肠中LF受体（LFR）的存在。我们最近克隆并表达了人和小鼠LFR，它们在几个组织中表达。人肠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射线衍射来确定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。我们还将通过使用截短版本（N-Lobe，C-Lobe）在杆状病毒中用嵌合蛋白表示的LF分子的特定位点，该特定位点与LFR结合。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，特别强调了小肠和小鼠胚胎发育。 LF内在化的途径将通过特异性抑制坑和小窝介导的途径进行研究。通过微阵列研究信号转导途径，将研究由LFR介导的LF的细胞反应。 Fe，脂多糖（LPS）和一氧化氮对LFR表达的影响将通过使用多种细胞系进行研究。我们还将使用条件敲除小鼠探索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，其中第一个外显子侧面是两个IOX P位点。然后，我们将使用诱导的CRE转基因小鼠在各种组织中产生LFR敲除，例如小肠和乳腺以及发育的各个阶段。将敲除的基本特性与野生型的基本特性进行比较。敲除的可能观察结果将受到繁殖和免疫能力的损害，LF的LF水平降低，发育异常，幼崽的FE状态降低以及LF对LPS引起的死亡率的保护作用降低。总体而言，我们对LF及其受体的生理意义的理解将会增加。