STRUCTURAL BASIS OF VITAMIN B12 (COBALAMIN) DEFICIENCY

哺乳动物蛋白质转钴胺素II （TCII）负责通过血液运输维生素B12（更准确地称为钴胺素）。维生素B12被摄入并通过肠道回肠细胞进入血液，在那里它被转钴胺素II携带到呈现特定TCII/钴胺素表面受体的细胞。在受体介导的内吞作用后，维生素需要进一步修饰，才能采用细胞质蛋氨酸合成酶（甲基- b12）和线粒体甲基丙二醇辅酶a变化酶（5'-脱氧腺苷- b12）两种酶所需的活性辅因子结构。TCII基因突变引起常染色体隐性遗传病，在婴儿期出现巨幼细胞性贫血。转钴胺素II与钴胺素结合的三维结构将用x射线晶体学解决，并将提供人类遗传疾病的分子解释。肠道转运蛋白作为一种内在因子，与TCII具有高度的同源性，也将通过同源性建模或直接结构测定在结构上进行研究。具体目的：1)利用x射线衍射技术解析转钴胺素II的三维结构，研究维生素B12转运生理中断的结构基础。2)确定TCII和钴胺素之间的关键相互作用，并绘制与识别TCII/钴胺素复合物的特定细胞表面受体相互作用的蛋白质表面。3)基于与TCI的高氨基酸序列一致性，建立了内在因子（IF）、触觉蛋白（haptocorrin）和转钴胺素（TCI）的模型。

The mammalian protein transcobalamin II (TCII) is responsible for the transport of vitamin B12 (more accurately termed cobalamin) through the bloodstream. Vitamin B12 is ingested and passed through gut ileal cells to the bloodstream where it is carried by transcobalamin II to cells presenting a specific TCII/cobalamin surface receptor. After receptor mediated endocytosis the vitamin requires further modification before adopting an active cofactor structure required for two enzymes, cytoplasmic methionine synthase (methyl-B12) and mitochondrial methylmalonyl CoA mutase (5'-deoxyadenosyl-B12). Genetic mutants of TCII cause an autosomal recessive disorder that presents itself in infancy with megaloblastic anemia. The 3-dimensional structure of transcobalamin II bound to cobalamin will be solved using X-ray crystallography, and will provide a molecular explanation of a human genetic disorder. The intestinal transport protein, intrinsic factor, shares high homology with TCII and will also be pursued structurally, either by homology modeling or direct structure determination. Specific Aims: 1) Solve the three-dimensional structure of transcobalamin II by X-ray diffraction techniques, and investigate the structural basis for the physiological disruption of vitamin B12 transport. 2) Determine the critical interactions between TCII and cobalamin, and map the surface of the protein which interacts with the specific cell-surface receptor that recognises the TCII/cobalamin complex. 3) Produce a model of intrinsic factor (IF), haptocorrin and transcobalamin I (TCI) based on the high amino acid sequence identity with TCII.