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

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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.