Mammalian Lactoferrin Receptors: Structure and Function

描述（由申请人提供）：乳铁蛋白（Lf）是一种可结合两个Fe（III）原子的80 kD糖蛋白，是人乳中的主要蛋白质，也存在于外分泌分泌物中，例如胰腺液和胆汁，以及中性粒细胞中。Lf具有多种功能，包括在肠道铁吸收、生殖功能、抗菌作用、细胞增殖和免疫能力中的作用。然而，虽然这些活动得到了一些支持，但对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人小肠中存在Lf受体（LfR）。我们最近克隆并表达了人和小鼠LfR，其在几种组织中表达。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嵌合体的截短形式（N端半叶、C端半叶）来确定与LfR结合的Lf分子的特异性位点。结合LfR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抗体将用于人和小鼠组织的免疫染色，特别强调小肠和小鼠胚胎发育。Lf内化的途径将分别通过特异性抑制包被小窝和小窝介导的途径的抑制剂来研究。将通过用于信号转导途径的微阵列研究由LfR介导的对Lf的细胞应答。铁，脂多糖（LPS）和一氧化氮对LfR表达的影响将通过使用几种细胞系进行研究。我们还将通过使用条件性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，并构建一个靶向载体，其中第一个外显子两侧是两个Iox P位点。然后，我们将使用诱导型cre转基因小鼠在各种组织中产生LfR敲除，如小肠和乳腺，以及在发育的各个阶段。将敲除的基本性质与野生型的基本性质进行比较。在基因敲除中可能观察到生殖和免疫能力受损、乳汁中Lf水平降低、发育异常、幼仔铁水平降低以及Lf对LPS引起的死亡的保护作用减弱。总的来说，我们对Lf及其受体的生理意义的理解将增加。

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.