Research for properties of the expressed recombinant human serum cholinesterase variant

我们从人群中随机选择低血清BCHE活性组和稍低DN和FN组，发现个体杂合BCHE L330I。一名63岁日本男性体检血清BCHE活性低。排除农药中毒引起的继发性低胆碱酯血症和严重肝功能障碍。表型分析显示二布卡因值（DN）降低，氟值（FN）特别低，与FS表型相似。虽然在白种人中有报道抗氟基因是由T243M和G390V引起的，但在含有本病例的日本人中未发现这些突变。在他的BCHE基因中发现了一个纯合错义突变，即核苷酸988处的T到A翻转。该突变导致密码子330 （L339I）上的亮氨酸被异亮氨酸取代。重要的是要证明异常的DN和FN是由BCHE L330I突变引起的，还是有其他遗传或修饰因素。将人胎肾细胞分泌的重组BCHE （L339I）的DN和FN与重组野生型（正常基因）BCHE和正常血清BCHE进行比较。将表征BCHE L330I变体的单个核苷酸替换引入含有常规BCHE的pRcCMV质粒中。用纯化的pRcCMV-BCHE质粒转染人胎肾细胞（293、JCRB9068、ATCC CRL1573），表达重组BCHE蛋白，结果表明BCHE L330I变体酶活性低，DN和FN值低。这些结果表明，BCHE将Leu330取代为Ile确实导致了DN和FN的异常。我们认为BCHE L330I突变是一种耐氟基因，一种日本型耐氟基因。

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.