MOLECULAR BASIS OF SELECTIVE COBALAMIN MALABSORPTION

人类和其他哺乳动物吸收钴胺素（Cbl，维生素B12）， 必需的微量营养素，从饮食通过一个复杂的相互作用， Cbl结合蛋白及其特异性受体 （IFCR）在回肠刷状缘上皮。 未经治疗的Cbl缺乏症是 在人类中危及生命，最常见的是由Cbl 吸收 回肠肠细胞的Cbl转胞吞的细节是 未知的，但IFCR的关键重要性是由一个 称为Imerslund-Grasbeck综合征的Cbl吸收遗传性疾病 在人类身上。 最近，一种选择性肠道Cbl的犬模型， 已经描述了吸收不良，这是IFCR缺陷的唯一已知模型， 与伊-格二氏综合征非常相似 的 提出的研究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 在健康和疾病状态下的受体表达。 的具体目标 本研究旨在克隆正常犬IFCR cDNA， IFCR突变导致患病犬的疾病，并定义 正常IFCR表达的组织特异性和发育调节。 通过比较IFCR表达和翻译后动力学 在正常和受影响的狗组织中， 将定义IFCR突变的后果。 使用的技术 分子生物学，蛋白质化学和细胞生物学，研究是 旨在检验存在组织特异性 正常IFCR的翻译后修饰和 异常IFCR细胞内处置，且IFCR表达是 在回肠粘膜和近端小管中有不同的调节。 最初 通过IFCR体外测定对犬组织进行调查 配体结合活性和免疫印迹以确定显著的 IFCR表达和不同组织对IFCR表达的可能贡献 Cbl体内平衡。 代谢标记，细胞分级，和 IFCR的免疫沉淀将用于确定细胞 组织中正常IFCR表达和受体缺陷的表型 外植体和/或来源于正常和患病狗的细胞系。 从组织中纯化的IFCR的蛋白和寡糖研究 正常和受影响的狗将进行调查可能的组织 IFCR翻译后修饰的差异影响 受影响犬的异常IFCR的细胞内处置。 标准分子遗传学方法可用于克隆正常IFCR cDNA， 确定基因突变，并检查IFCR mRNA在正常和 受影响的狗组织。 这一综合办法将阐明 IFCR结构影响回肠和肾脏中的功能和表达 刷状缘上皮

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.