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

完整的维生素B12（钴胺素）代谢是正常人类发育和生存所必需的。在细胞内吞后，在转化为其活性辅酶甲基钴胺素和腺苷钴胺素之前，钴胺素（cbl）必须通过溶酶体运输。在cblF和cblJ两种极其罕见的先天性cbl代谢错误中，游离的未结合的cbl在溶酶体中积累，阻碍了cbl辅酶的合成。在之前的研究中，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。在当前DFG资助期间，我们发现患者中存在的LMBRD1突变导致LMBD1蛋白表达缺失。此外，我们能够证明ABCD4的突变导致了cblJ缺陷。由于ABCD4和LMBD1蛋白在体外均未显示出放射性标记的cbl的转运活性，而且这两种蛋白似乎不相互作用，因此我们提出存在另一种促进溶酶体cbl转运的衔接蛋白。在当前项目的继续阶段，我们现在的目标是鉴定这种接头蛋白，并表征其在溶酶体cbl运输方面的功能。我们将对LMBD1和ABCD4抗体偶联蛋白A和G进行免疫沉淀研究，并在患者和对照成纤维细胞中进行拉下试验，以确定可能的相互作用伙伴。沉淀蛋白将用质谱法鉴定。如果我们无法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运输的可能刺激物。鉴定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转运试验中各自蛋白质的过表达和低表达来表征。我们预计，对溶酶体cbl转运机制的鉴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.