Research for properties of the expressed recombinant human serum cholinesterase variant

我们从人群中随机选择了血清BCHE活性低的组和略低的DN和FN组，发现了BCHE L330I杂合子。一名63岁的日本男子在健康检查中显示血清BCHE活性较低。排除了农药中毒和严重肝功能障碍所致的继发性低胆碱酯酶血症。表型分析显示，地布卡因数量(DN)减少，特别是低氟数量(Fn)，类似于FS表型。虽然在高加索人中发现了由T243M和G390V引起的耐氟基因，但在日本人中没有发现这些突变。在他的BCHE基因中发现了一种纯合子错义突变，即988位核苷酸T到A的颠换。该突变导致330密码子(L339I)亮氨酸被异亮氨酸取代，提示异常的糖尿病肾病和纤维蛋白原是由BCHE L330I突变引起的，还是存在其他遗传或修饰因素。将人胎肾细胞分泌的重组BCHE(L339I)与重组野生型(普通基因)BCHE和正常血清BCHE进行比较。JCRB9068、、ATCC、CRL1573)与纯化的pRcCMV-BCHE重组表达载体进行遗传转化，结果表明，BCHE L330I突变导致酶活性降低，DN和FN降低，说明BCHE、Leu330氨基酸替换为ILE，确实导致了DN和FN的异常。

We have found individuals heterozygous for BCHE L330I from a low serum BCHE activity group and slightly low DN and FN group selected randomly from the population. A 63-year-old Japanese man showed low serum BCHE activity on health examination. Secondary hypocholinesterasemia due to agricultural chemical poisoming and severe hepatic dysfunction were ruled out. The phenotyping analysis revealed a reduced dibucaine number (DN) and an esxpecially low fluoride number (FN), similar to an FS phenotype. Although the fluoride-resistant gene has been reported to be caused by T243M and G390V in Caucasian, we could not find these mutations in Japanese containing this case. A homozygousmissense mutation, a T to A transversion at nucleotide 988, was identified in his BCHE gene. This mutation resulted in the replacement of leusine by isoleucine at codon 330 (L339I).It is important to demonstrate whether the abnormal DN and FN are originated from the BCHE L330I mutation alone or there are some other genetic or modifying factors. DN and FN of recombinant BCHE (L339I) secreted by human fetal kidney cells were compared to recombinant wild-type (usual gene) BCHE and normal serum BCHE.The individual nucleotide substitution characterizing the BCHE L330I variant was introduced into the pRcCMV plasmid containing usual BCHE using PCR.By expression of the recombinant BCHE protein in human fetal kidney cells (293 ; JCRB9068、ATCC CRL1573) which were transformed with purified pRcCMV-BCHE plasmid, we showed that the BCHE L330I variant resulted in low enzymatic activity and low DN and FN.These results showed this amino acid substitution of BCHE,Leu330 to Ile, really caused the abnormal DN and FN.We conclude the BCHE L330I mutation is a fluoride-resistant gene, a Japanese type fluoride-resistant gene.