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Mammalian Lactoferrin Receptors: Structure and Function

哺乳动物乳铁蛋白受体：结构和功能

基本信息

批准号：
7154155
负责人：
BO L LONNERDAL
金额：
$ 21.81万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
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/7154155
关键词：
Antibodies Baculoviruses Bile fluid Binding Biochemical Biological Blocking Antibodies Caco-2 Cells Caveolae Cell Line Cell Proliferation Cells Characteristics Chimera organism Competence Complex Confocal Microscopy Crystallography Development Disease Embryonic Development Endocytosis Exons Exposure to Fc Receptor Gene Silencing Generations Genes Glycoproteins Human Human Cloning Human Milk Immune In Situ Hybridization In Transferrin Infant Intestines Iron Kinetics Knock-out Knockout Mice Lactoferrin Lead Lipopolysaccharides Lobe Mammary gland Mediating Milk Mus Nitric Oxide Oligonucleotide Probes Pancreas Pathway interactions Peptides Physiological Prevention Procedures Property Proteins Receptor Gene Recombinants Reproduction Resolution Restriction Mapping Role Signal Transduction Signal Transduction Pathway Site Small Intestines Staging Structure Surface System Tissues Transfection Transferrin Transgenic Mice Transgenic Mouse Facility Work X ray diffraction analysis X-Ray Diffraction absorption antimicrobial brush border membrane cDNA Probes cell type coated pit combinatorial chemistry cytokine design embryonic stem cell human fetus tissue inhibitor/antagonist lactoferrin receptors mortality neutrophil programs protective effect pup receptor receptor expression receptor structure function reproductive function response uptake vector ward

项目摘要

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(申请人提供)：乳铁蛋白(Lf)是一种80kD的糖蛋白，可以结合两个Fe(111)原子，是母乳中的主要蛋白质，也存在于外分泌物中，如胰液和胆汁，以及中性粒细胞中。Lf被认为具有多种功能，包括在肠道铁吸收、生殖功能、抗菌作用、细胞增殖和免疫功能方面的作用。然而，尽管已经获得了对这类活动的一些支持，但对LF行使这些职能的机制知之甚少。我们先前通过动力学结合研究证明了人小肠中存在LF受体(LFR)。我们最近克隆并表达了人和小鼠的LFR，它们在几个组织中都有表达。转染人肠道细胞后，铁摄取增加，LF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复合体的三级结构，以更好地了解LFR分子的组织及其与LF的相互作用。我们还将使用杆状病毒中表达为与转铁蛋白嵌合体的截断版本(N-叶、C-叶)来确定与LFR结合的LF分子的特定位置。与LFR结合的多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，重点是小肠和小鼠胚胎发育。LF内化的途径将通过特异性抑制涂层凹坑和小窝介导的途径的抑制剂来研究。细胞对LFR介导的LF的反应将通过信号转导途径的微阵列来研究。用多种细胞系研究铁、脂多糖和一氧化氮对LFR表达的影响。我们还将通过使用条件基因敲除小鼠来探索LFR的生物学意义。我们将克隆小鼠LFR基因，并构建一个第一外显子两侧带有两个IoxP位点的靶向载体。然后，我们将使用可诱导的cre转基因小鼠在各种组织中产生LFR基因敲除，如小肠和乳腺，以及发育的不同阶段。我们将把基因敲除的基本特性与野生型进行比较。在基因敲除中可能观察到的现象包括繁殖和免疫能力受损，牛奶中LF水平下降，发育异常，幼崽铁状态降低，以及LF对脂多糖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了解将会增加。