Membrane Topology and Intracellular Processing of Cyclin M2 (CNNM2)
Membrane Topology and Intracellular Processing of Cyclin M2 (CNNM2)
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DOI：
10.1074/jbc.m112.342204
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发表时间：
2012-04-20
影响因子：
4.8
通讯作者：
Hoenderop, Joost G. J.
中科院分区：
文献类型：
--
作者：
de Baaij, Jeroen H. F.;Stuiver, Marchel;Hoenderop, Joost G. J.
最近，细胞周期蛋白M2 （CNNM2）基因的突变被确定为严重低镁血症的病因。在肾脏中，CNNM2是一种基底侧表达蛋白，主要表达于远曲小管。远曲小管的跨细胞镁（Mg2+）重吸收是Mg2+排泄到尿液之前的最后一步，从而通过严格调节的机制微调其最终排泄。本研究旨在了解CNNM2的结构，并表征其翻译后修饰。在这里，利用分子内表位和免疫细胞化学进行的膜拓扑研究表明，CNNM2具有细胞外N端和细胞内C端。这表明其中一个预测的跨膜区域可能是可重入的。通过同源性建模，我们证明了在患者中发现的功能丧失突变干扰了细胞内胱硫氨酸-合成酶结构域的潜在ATP结合。此外，还详细揭示了CNNM2的细胞加工途径。在内质网中，信号肽酶复合物可切割出约64个氨基酸的n端信号肽。诱变筛选表明，CNNM2在Asn-112残基处糖基化，稳定了CNNM2在质膜上的作用。有趣的是，共免疫沉淀研究证明，CNNM2a与较小的同种异构体CNNM2b形成异源二聚体。这些关于CNNM2结构和加工的新发现可能有助于阐明CNNM2在肾内Mg2+重吸收中的生理作用。
Recently, mutations in the cyclin M2 (CNNM2) gene were identified to be causative for severe hypomagnesemia. In kidney, CNNM2 is a basolaterally expressed protein with predominant expression in the distal convoluted tubule. Transcellular magnesium (Mg2+) reabsorption in the distal convoluted tubule represents the final step before Mg2+ is excreted into the urine, thus fine-tuning its final excretion via a tightly regulated mechanism. The present study aims to get insight in the structure of CNNM2 and to characterize its post-translational modifications. Here, membrane topology studies using intramolecular epitopes and immunocytochemistry showed that CNNM2 has an extracellular N terminus and an intracellular C terminus. This suggests that one of the predicted transmembrane regions might be reentrant. By homology modeling, we demonstrated that the loss-of-function mutation as found in patients disturbs the potential ATP binding by the intracellular cystathionine beta-synthase domains. In addition, the cellular processing pathway of CNNM2 was exposed in detail. In the endoplasmic reticulum, the signal peptidase complex cleaves off a large N-terminal signal peptide of about 64 amino acids. Mutagenesis screening showed that CNNM2 is glycosylated at residue Asn-112, stabilizing CNNM2 on the plasma membrane. Interestingly, co-immunoprecipitation studies evidenced that CNNM2a forms heterodimers with the smaller isoform CNNM2b. These new findings on CNNM2 structure and processing may aid to elucidate the physiological role of CNNM2 in Mg2+ reabsorption in the kidney.