MOLECULAR BASIS OF SELECTIVE COBALAMIN MALABSORPTION

人类和其他哺乳动物吸收钴胺(Cbl，维生素B12)，一种 必需的微量营养素，通过复杂的相互作用来自饮食 CBL结合蛋白与内源性因子特异性受体Cbl (IFCR)在回肠刷缘上皮细胞中表达。未经治疗的Cbl缺乏症 在人类中威胁生命，最常见的原因是慢性阻塞性肺疾病 吸收。回肠肠上皮细胞转运Cbl的细节如下 未知，但IFCR的关键重要性通过一个 遗传性Cbl吸收障碍称为Imerslund-Grasbeck综合征 在人类身上。近年来，一种犬选择性肠梗阻模型的建立 已经描述了吸收不良，这是唯一已知的IFCR缺陷模型， 这似乎与Imerslund-Grasbeck综合征非常相似。这个 研究建议使用这种独特的疾病模型来促进理解 Cbl吸收和刷状缘上皮细胞的分子水平 受体在健康和疾病状态下的表达。的具体目标 本研究旨在克隆正常犬IFCR基因，鉴定其功能。 在受影响的狗身上引起疾病的IFCR突变，并定义 正常IFCR表达的组织特异性和发育调节。 通过比较IFCR的表达和翻译后的动力学 在正常和受影响的狗组织中的修饰，细胞 IFCR突变的后果将被定义。使用以下技术 分子生物学、蛋白质化学和细胞生物学的研究 旨在检验这样的假设，即存在组织特异性 正常IFCR的翻译后修饰与 异常IFCR在细胞内的处置，且IFCR表达是 在回肠粘膜和近端小管中有不同的表达。最初，一个 将通过体外IFCR试验对犬组织进行调查 配基结合活性和免疫印迹确定有意义的部位 IFCR的表达及其在不同组织中的可能作用 CBL动态平衡。代谢物标记、细胞分级和 免疫沉淀的IFCR将被用来定义细胞 组织中IFCR正常表达和受体缺陷的表型 来自正常和患病犬的外植体和/或细胞系。 从日本血吸虫组织中纯化的IFCR的蛋白质和寡糖研究 将对正常和受影响的狗进行检查，以研究可能的组织 IFCR EFECT翻译后修饰的差异 病犬异常IFCR的细胞内处置。 用标准分子遗传学方法克隆正常IFCR基因 确定基因突变，并检测正常和正常对照的IFCR基因 受影响的狗组织。这种综合方法将阐明以下几个方面 IFCR结构影响回肠和肾脏的功能和表达 刷缘上皮。

Humans and other mammals absorb cobalamin (Cbl, vitamin B12), an essential micronutrient, from diet via a complex interaction of Cbl-binding proteins and a specific receptor for intrinsic factor-Cbl (IFCR) in the ileal brush-border epithelium. Untreated Cbl deficiency is life-threatening in humans and most often results from disorders of Cbl absorption. The details of Cbl transcytosis by ileal enterocytes are unknown, but the pivotal importance of IFCR is exemplified by an inherited disorder of Cbl absorption known as Imerslund-Grasbeck syndrome in humans. Recently, a canine model of selective intestinal Cbl malabsorption has been described, the only known model of an IFCR defect, that appears to be very similar to Imerslund-Grasbeck syndrome. The studies proposed use this unique disease model to advance understanding of the molecular aspects of Cbl absorption and brush-border epithelial receptor expression in health and disease states. The specific aims of these investigations are to clone normal canine IFCR cDNA, to identify the IFCR mutation causing disease in affected dogs, and to define the tissue-specific and developmental regulation of normal IFCR expression. By comparison of the kinetics of IFCR expression and posttranslational modification in normal and affected dog tissues, the cellular consequences of the IFCR mutation will be defined. Using techniques of molecular biology, protein chemistry, and cell biology, studies are designed to examine the hypotheses that there are tissue-specific differences in the post-translational modification of normal IFCR and the intracellular disposition of abnormal IFCR, and that IFCR expression is differently regulated in ileal mucosa and proximal tubules. Initially, a survey of canine tissues will be made by in vitro assay of IFCR ligand-binding activity and immunoblot to determine sites of significant IFCR expression and the possible contributions of different tissues to Cbl homeostasis. Metabolic labelling, cell fractionation, and immunoprecipitation of IFCR will be used to define the cellular phenotypes of normal IFCR expression and the receptor defect in tissue explants and/or cell lines derived from normal and affected dogs. Protein and oligosaccharide studies of IFCR purified from tissues of normal and affected dog' will be done to investigate possible tissue differences in postranslational modifications of IFCR affect intracellular disposition of the abnormal IFCR of affected dogs. Standard molecular genetic approach be used to clone normal IFCR cDNA, to define the genetic mutation, and to examine IFCR mRNA in normal and affected dog tissues. This integrated approach will elucidate aspects of IFCR structure affect function and expression in ileal and renal brush-border epithelia.