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目的:进一步确认核转录因子NF-κB受体激动剂(RANK)蛋白中调节破骨细胞(OC)分化的特异性基序(motif),为阐明OC分化机制提供理论依据。方法:分别突变RANK蛋白膜内部分第533-540之间的8个氨基酸(突变前氨基酸序列为DIIVVYVS,突变后氨基酸序列为ELLAAFAA),用定点突变方法构建8个由肿瘤坏死因子受体1(TNFR1)和RANK跨膜部分和膜内部分共同组成的TNFR1/RANK突变嵌合体(TNFR1/RANK-533-TNFR1/RANK-540),每1个突变体在其RANK膜内部分含1个突变氨基酸。用包装细胞plat E分别将各突变体包装成逆转录病毒,通过逆转录病毒感染分别将这些突变体转染到TNFR1/TNFR2基因敲除小鼠的骨髓巨噬细胞(BMM)中。用TNF-浕和单核细胞集落刺激因子(M-CSF)刺激、观察转染哪种突变体的BMM不能诱导OC形成,不能诱导OC形成的突变体所包含的突变前氨基酸就是RANK调节OC分化的关键氨基酸,多个关键氨基酸组成的片段就是RANK特异性motif。结果:转染TNFR1/RANK-533、TNFR1/RANK-539和TNFR1/RANK-540的BMM均能分化成OC,表明氨基酸D533、V539、S540突变后对OC分化无影响;转染TNFR1/RANK-534的BMM有极少数能分化成OC,表明氨基酸I534突变后对OC分化有部分影响;而转染TNFR1/RANK-535、TNFR1/RANK-536、TNFR1/RANK-537和TNFR1/RANK-538的BMM均不能分化成OC,表明氨基酸I535、V536、V537和Y538突变后对OC分化起关键影响。结论:I534、I535、V536、V537和Y538这5个氨基酸组成的片段(534-IIVVY-538)可能就是RANK调节OC分化的特异性motif。
OBJECTIVE: To further confirm the specific motif regulating the osteoclast differentiation in nuclear factor-kappa B receptor (RANK) protein and provide a theoretical basis for elucidating the mechanism of OC differentiation. Methods: The 8 amino acids (the amino acid sequence before mutation was DIIVVYVS and the amino acid sequence was ELLAAFAA after mutation) of 533-540 of the RANK protein were mutated respectively, and the 8 amino acid sequences of tumor necrosis factor receptor 1 TNFR1) and the TNFR1 / RANK mutant chimera (TNFR1 / RANK-533-TNFR1 / RANK-540), which are composed of the transmembrane part and the membrane part of RANK. Each mutant contains 1 mutant amino acid in its RANK membrane . Each mutant was packaged as a retrovirus with the packaging cell plat E, respectively, and these mutants were respectively transfected into bone marrow macrophages (BMMs) of TNFR1 / TNFR2 knockout mice by retroviral infection. With TNF-浕 and monocyte colony-stimulating factor (M-CSF) stimulation, to see which mutant transfection BMM can not induce OC formation, can not induce OC formation of mutant pre-mutation amino acids that RANK regulate OC differentiation The key amino acids, a number of key amino acid fragments is the RANK specific motif. Results: The BMM of TNFR1 / RANK-533, TNFR1 / RANK-539 and TNFR1 / RANK-540 all differentiated into OC, indicating that mutation of D533, V539 and S540 had no effect on OC differentiation. 534 had very few BMMs that could differentiate into OC, suggesting that mutated amino acid I534 partially affected OC differentiation. However, transfection of TNFR1 / RANK-535, TNFR1 / RANK-536, TNFR1 / RANK-537 and TNFR1 / RANK-538 BMM did not differentiate into OC, indicating that mutations in amino acids I535, V536, V537 and Y538 have a key effect on OC differentiation. CONCLUSION: The 5 amino acids fragment (534-IIVVY-538) of I534, I535, V536, V537 and Y538 may be the specific motif for RANK regulating OC differentiation.