Strukturelle und funktionelle Analysen zum humanen LMBD1-Mangel und Aufklärung der molekularen Ursache einer neuen Cbl-Komplementationsgruppe

完整的维生素B12(钴胺)代谢对人类的正常发育和生存至关重要。钴胺(CBL)在内吞进入细胞后，在转化为其活性辅酶甲钴胺和腺苷钴胺之前，必须通过溶酶体转运。在cblF和cblJ这两种极为罕见的CBL代谢先天缺陷中，游离CBL在溶酶体中积累，CBL辅酶合成受阻。在以前的研究中，我们证明了编码溶酶体膜蛋白LMBD1的基因LMBRD1的突变是导致CBL代谢的cblF缺陷的原因。在目前的DFG资助期间，我们发现患者中存在的LMBRD1突变导致了LMBD1蛋白表达的丧失。此外，我们能够证明ABCD4的突变是导致cblJ缺陷的原因。由于ABCD4和LMBD1蛋白在体外都不显示放射性标记CBL的转运活性，而且这两种蛋白似乎不相互作用，我们提出了另一种接头蛋白的存在，它促进了溶酶体CBL的转运。在当前项目的继续阶段，我们现在的目标是鉴定这个接头蛋白，并表征其与溶酶体CBL运输相关的功能。我们将用结合蛋白A和G的LMBD1和ABCD4抗体进行免疫沉淀研究，并在患者和对照成纤维细胞中进行下拉试验，以确定可能的相互作用伙伴。沉淀蛋白质将通过质谱学进行鉴定。如果我们不能确定直接相互作用的蛋白质，我们将对来自患者和对照成纤维细胞的溶酶体提取物进行比较蛋白质组分析，以确定溶酶体CBL运输的可能刺激因素。识别的蛋白质的功能作用将通过在不同细胞系的辅酶合成试验和CBL转运试验中各自的蛋白质的过度表达和击倒来表征。我们预计，溶酶体CBL转运机制的确定将进一步阐明包括阿尔茨海默病和帕金森病在内的更常见疾病的发病机制，因为在这些疾病中，溶酶体转运过程也受到损害。

Intact vitamin B12 (cobalamin) metabolism is essential for normal human development and survival. After endocytotic uptake into the cell and before conversion to its active coenzymes methylcobalamin and adenosylcobalamin, cobalamin (cbl) has to be transported through lysosomes. In two extremely rare inborn errors of cbl metabolism named cblF and cblJ, free unbound cbl accumulates in lysosomes and cbl coenzyme synthesis is hampered. In previous studies, we demonstrated that mutations in LMBRD1, a gene encoding for the lysosomal membrane protein LMBD1, are responsible for the cblF defect of cbl metabolism. During the current DFG funding period we showed that the LMBRD1 mutations present in the patients resulted in a loss of LMBD1 protein expression. Furthermore, we were able to prove that mutations in ABCD4 are causing the cblJ defect. Because neither the ABCD4 nor the LMBD1 protein shows transport activity for radioactively labeled cbl in vitro and because both proteins do not seem to interact, we propose the existence of another adaptor protein, which facilitates lysosomal cbl transport. In the continuation phase of the current project we now aim to identify this adaptor protein and to characterize its function with respect to lysosomal cbl transport. We will perform immunoprecipitation studies with LMBD1 and ABCD4 antibodies coupled to protein A and G and pulldown assays in patient and control fibroblasts to identify a putative interaction partner. The precipitating proteins will be indentified by mass spectrometry. If we fail to identify a directly interacting protein, we will perform a comparative proteome analysis of lysosomal extracts derived from patient and control fibroblasts to identify possible stimulators of lysosomal cbl transport. The functional role of the identified proteins will be characterized by overexpression and knockdown of the respective proteins in coenzyme synthesis assays and in cbl transport assays in different cell lines. We anticipate that the identification of the mechanism of lysosomal cbl transport sheds further light on the pathogenesis of more common disorders including Alzheimer and Parkinson disease, since also in these disorders lysosomal transport processes are impaired.