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）代谢对人类的正常发育和生存至关重要。钴胺素（cbl）被细胞内吞摄取后，在转化为活性辅酶甲基钴胺素和腺苷钴胺素之前，必须通过溶酶体转运。在两种极为罕见的先天性cbl代谢缺陷中，即cblF和cblJ，游离的未结合的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LMBRD1（编码溶酶体膜蛋白LMBD1的基因）的突变是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蛋白表达丧失。此外，我们能够证明ABCD4中的突变导致cblJ缺陷。因为无论是ABCD4或LMBD1蛋白显示放射性标记的CBL在体外的运输活性，因为这两种蛋白质似乎没有相互作用，我们建议存在另一个衔接蛋白，这有利于溶酶体CBL运输。在当前项目的继续阶段，我们现在的目标是鉴定这种衔接蛋白，并描述其在溶酶体cbl转运中的功能。我们将用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下拉试验，以确定推定的相互作用伴侣。沉淀蛋白将通过质谱法鉴定。如果我们不能鉴定直接相互作用的蛋白质，我们将对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，以鉴定可能的溶酶体cbl转运刺激物。所鉴定蛋白质的功能作用将通过不同细胞系中辅酶合成测定和cBL转运测定中相应蛋白质的过表达和敲减来表征。我们预期，溶酶体cbl转运机制的鉴定进一步阐明了更常见疾病（包括阿尔茨海默病和帕金森病）的发病机制，因为在这些疾病中，溶酶体转运过程也受损。

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.