Clinical or ATPase domain mutations in ABCD4 disrupt the interaction between the vitamin B12-trafficking proteins ABCD4 and LMBD1
Clinical or ATPase domain mutations in ABCD4 disrupt the interaction between the vitamin B12-trafficking proteins ABCD4 and LMBD1
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DOI：
10.1074/jbc.m117.784819
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发表时间：
2017-07-14
影响因子：
4.8
通讯作者：
Baumgartner, Matthias R.
中科院分区：
文献类型：
--
作者：
Fettelschoss, Victoria;Burda, Patricie;Baumgartner, Matthias R.
维生素B-12（钴胺素（Cbl）），以辅因子形式甲基-Cbl和腺苷-Cbl，分别是必需酶甲硫氨酸合酶和甲基丙二酰-CoA还原酶的功能所需的。Cbl通过受体介导的蛋白质结合的Cbl的内吞作用进入哺乳动物细胞，随后通过溶酶体将游离Cbl输出到胞质溶胶并进一步加工成这些辅因子形式。整合膜蛋白LMBD 1和ABCD 4是Cbl的溶酶体释放所需的，并且基因LMBRD 1和ABCD 4中的突变导致钴胺素代谢障碍cblF和cblJ。我们报告了一例新的（第五例）cblJ疾病患者，在7日龄时出现喂养不良、张力减退、甲基丙二酸尿症和血浆同型半胱氨酸升高，并在ABCD 4基因中携带突变c.1667_1668delAG [p.Glu556Glyfs*27]和c.1295G > A [p.Arg432Gln]。Cbl辅因子形式在来自该患者的成纤维细胞中减少，但可以通过ABCD 4或出乎意料地LMBD 1的过表达来拯救。使用灵敏的活细胞FRET测定，我们证明了ABCD 4和LMBD 1之间的选择性相互作用，当ABCD 4携带患者突变p.Arg432Gln或p.Asn141Lys或人工突变破坏ATP酶结构域时，相互作用降低。最后，我们发现ABCD 4溶酶体靶向依赖于LMBD 1的共表达和相互作用。这些数据拓宽了cblJ缺乏症的患者和突变谱，建立了一种灵敏的活细胞测定法来检测LMBD 1-ABCD 4相互作用，并证实了这种相互作用对于ABCD 4和钴胺素辅因子合成的适当细胞内靶向的重要性。
Vitamin B-12 (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes LMBRD1 and ABCD4 result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G > A [p.Arg432Gln] in the ABCD4 gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis.