KINETICS OF B12, INTRINSIC FACTOR AND OTHER PROTEINS

维生素B12（钴胺素，Cbl）是人体（和哺乳动物）中的必需辅因子。 哺乳动物）代谢中同型半胱氨酸的酶促甲基化 甲硫氨酸的生物合成，以及甲硫氨酸的分子重排。 在人（和动物）细胞中甲基丙二酰辅酶A转化为琥珀酰辅酶A 新陈代谢. 缺乏这种维生素会导致严重的巨幼细胞性 贫血和神经精神疾病。 两个综合生理 为了向组织提供足够的Cbl，需要一些过程。 的 首先是肠道吸收，这一过程是由胃介导的 内因子（IF）与Cbl在上小 并将其作为IF-Cbl转运到回肠末端，在那里结合 与上皮粘膜微绒毛膜上的IF受体结合 并通过内吞作用内化。 回肠粘膜内 细胞，释放的Cbl单向移动到门静脉血， 似乎与血浆转运蛋白转钴胺素II（TCII）结合。 TCII介导第二个基本生理过程，即转运 血浆中的维生素，作为TCII-Cbl复合物， 在那里它与细胞质膜上的特异性TCII受体结合 然后通过受体介导的内吞作用内化。 这一复杂过程的扰动导致Cbl缺乏， 肠吸收受损（缺乏IF或肠 吸收不良）和缺乏TCII。 因此，长期目标 该项目的目的是：i）研究 ii）描述TCII缺乏的先天性疾病; iii）阐明TCII和综合框架在促进 CBL的肠道吸收。 为了实现这些目标，我们将描述的基因组顺式元件， TCII基因（我们已经克隆）和细胞质反式激活 调节TCII的组成型和组织特异性表达的因子， 并确定导致TCII基因突变的原因 三种先天性异常TCII表达形式。 这些基因研究 TCII的研究应该确定在细胞中上调TCII合成的因素， 自身免疫性疾病，B细胞增殖和一些癌症， 也下调TCII合成，如在一些受试者中观察到的。 为了实现第三个目标，我们将克隆表达在 豚鼠回肠的回肠粘膜。 我们将首先净化分析 通过使用IF-Cbl的亲和色谱法测定受体的量 亲和基质，然后从N-末端获得氨基酸序列 和内部肽，并使用衍生的寡核苷酸来筛选 从豚鼠回肠粘膜mRNA制备的cDNA文库。 的cDNA 将提供有关受体一级结构的信息， 跨膜结构域和功能，并将用于克隆和 鉴定IF-receptor基因。 这些研究的IF受体和 分子水平上的TCII可以阐明TCII在 Cbl的吸收并阐明Cbl吸收受损的病因 这可能会导致Cbl缺乏症， 内在因素

Vitamin B12 (cobalamin, Cbl) is an essential cofactor in human (and mammalian) metabolism for the enzymatic methylation of homocysteine in the biosynthesis of methionine, and for the molecular rearrangement of methylmalonyl-CoA to succinyl-CoA in human (and animal) cellular metabolism. A deficiency of this vitamin results in severe megaloblastic anemia and neuropsychiatric disorders. Two integrated physiological processes are required for sufficient Cbl to be provided to tissues. The first is intestinal absorption, and this process is mediated by gastric intrinsic factor (IF) which complexes with Cbl in the upper small intestine and transports it as IF-Cbl to the distal ileum where it binds to an IF receptor on the microvillus membranes of the epithelial mucosa and is internalized by a process of endocytosis. Within the ileal mucosal cell, the released Cbl moves unidirectionally to the portal blood where it appears bound to the plasma transport protein, transcobalamin II (TCII). TCII mediates the second essential physiologic process, which is transport of the vitamin in the plasma, as the TCII-Cbl complex, to the tissues where it binds to a specific TCII receptor on the plasma membrane of cells and is then internalized by receptor mediated endocytosis. Perturbations of this complex process that lead to Cbl deficiency result from impaired intestinal absorption (lack of IF or intestinal malabsorption) and lack of TCII. Accordingly, the long-term objectives and aims of this project are: i) to study the regulation of expression of TCII; ii) to characterize the congenital disorders of TCII deficiency; iii) to elucidate the integrated function of TCII and IF in facilitating the intestinal absorption of Cbl. To achieve these aims, we will characterize the genomic cis elements of the TCII gene (which we have cloned) and the cytoplasmic trans-active factors that regulate constitutive and tissue specific expression of TCII, and identify the mutations of the TCII gene that are responsible for the three congenital forms of abnormal TCII expression. These genetic studies of TCII should identify the factors that up-regulate synthesis of TCII in autoimmune diseases, B-cell proliferations and some cancers, and which may also down-regulate TCII synthesis as observed in some subjects. To achieve the third objective we will clone the IF receptor expressed on the ileal mucosa of guinea pig ileum. We will first purify analytical amounts of the receptor by affinity chromatography using an IF-Cbl affinity matrix and then obtain an amino acid sequence from the N-terminus and internal peptides and use the derived oligonucleotides to screen a cDNA library prepared from the mRNA of guinea pig ileal mucosa. The cDNA will provide information about the primary structure of the receptor, the transmembrane domain(s) and function, and will be useful to clone and characterize the IF-receptor gene. Such studies of the IF-receptor and TCII at the molecular level can clarify the role of TCII in the assimilation of Cbl and clarify the etiology of impaired Cbl absorption that can result in Cbl deficiency in the absence of deficiency of intrinsic factor.