Huang Yan, Sirkowski Erich E, Stickney John T, Scherer Steven S
Department of Neurology, The University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
J Neurosci. 2005 Aug 3;25(31):7111-20. doi: 10.1523/JNEUROSCI.1319-05.2005.
Mutations in GJB1, the gene encoding the gap junction protein connexin32 (Cx32), cause the X-linked form of Charcot-Marie-Tooth disease, an inherited demyelinating neuropathy. The C terminus of human Cx32 contains a putative prenylation motif that is conserved in Cx32 orthologs. Using [3H]mevalonolactone ([3H]MVA) incorporation, we demonstrated that wild-type human connexin32 can be prenylated in COS7 cells, in contrast to disease-associated mutations that are predicted to disrupt the prenylation motif. We generated transgenic mice that express these mutants in myelinating Schwann cells. Male mice expressing a transgene were crossed with female Gjb1-null mice; the male offspring were all Gjb1-null, and one-half were transgene positive; in these mice, all Cx32 was derived from expression of the transgene. The mutant human protein was properly localized in myelinating Schwann cells in multiple transgenic lines and did not alter the localization of other components of paranodes and incisures. Finally, both the C280G and the S281x mutants appeared to "rescue" the phenotype of Gjb1-null mice, because transgene-positive male mice had significantly fewer abnormally myelinated axons than did their transgene-negative male littermates. These results indicate that Cx32 is prenylated, but that prenylation is not required for proper trafficking of Cx32 and perhaps not even for certain aspects of its function, in myelinating Schwann cells.
编码间隙连接蛋白连接蛋白32（Cx32）的GJB1基因突变会导致X连锁型夏科-马里-图思病，这是一种遗传性脱髓鞘性神经病变。人Cx32的C末端包含一个假定的异戊二烯化基序，该基序在Cx32直系同源物中是保守的。通过[3H]甲羟戊酸内酯（[3H]MVA）掺入实验，我们证明野生型人连接蛋白32在COS7细胞中可以发生异戊二烯化，这与预计会破坏异戊二烯化基序的疾病相关突变形成对比。我们构建了在髓鞘形成雪旺细胞中表达这些突变体的转基因小鼠。将表达转基因的雄性小鼠与雌性Gjb1基因敲除小鼠杂交；雄性后代均为Gjb1基因敲除小鼠，其中一半为转基因阳性；在这些小鼠中，所有Cx32均来源于转基因的表达。突变的人蛋白在多个转基因品系的髓鞘形成雪旺细胞中定位正确，并且没有改变结旁区和切迹其他成分的定位。最后，C280G和S281x突变体似乎都“挽救”了Gjb1基因敲除小鼠的表型，因为转基因阳性雄性小鼠的异常髓鞘化轴突明显少于其转基因阴性的雄性同窝小鼠。这些结果表明，在髓鞘形成雪旺细胞中，Cx32会发生异戊二烯化，但异戊二烯化对于Cx32的正确运输可能并非必需，甚至对于其某些功能方面可能也不是必需的。
