Kurian Joseph R, Longlais Brett J, Trepanier Lauren A
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA.
Pharmacogenet Genomics. 2007 Aug;17(8):597-603. doi: 10.1097/FPC.0b013e328011aaff.
We have shown that cytochrome b5 (cyt b5), along with its reductase, NADH cytochrome b5 reductase (b5R), is capable of direct xenobiotic biotransformation. We hypothesized that functionally significant genetic variability in cyt b5 could be found in healthy individuals.
Cyt b5 cDNAs were prepared from peripheral blood mononuclear cells from 63 individuals.
One individual was heterozygous for a sequence variant in cyt b5 (A178G), with a predicted amino acid substitution of T60A. This variant, when expressed in Escherichia. coli, maintained a similar Vmax for the hydroxylamines of sulfamethoxazole, 4-aminobiphenyl, and 2-amino-l-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), compared with wild type cyt b5, with a modestly increased Km (2 to 3.5-fold) for each substrate. When expressed in a mammalian system (HeLa cells), however, T60A was associated with a 70% reduction in cyt b5 protein expression compared with wild type. mRNA expression for both isoforms were comparable in HeLa cells, and translation of these mRNAs in a rabbit reticulocyte lysate system with inhibited proteasomal machinery were also similar. Incubation of these translated enzymes with uninhibited rabbit reticulocyte lysate, however, indicated greater susceptibility of T60A to proteasomal degradation.
These data indicate that a naturally occurring variant in cyt b5, T60A, leads to modestly altered affinity for hydroxylamine substrates and dramatically reduced cyt b5 expression. Work is underway to determine the prevalence of this and other variants in cyt b5 or b5R in a larger population, and to determine the association of such variants with differences in hydroxylamine reduction in vivo.
我们已经证明，细胞色素b5（cyt b5）与其还原酶NADH细胞色素b5还原酶（b5R）能够直接进行外源性生物转化。我们推测，在健康个体中可能存在功能上具有显著意义的cyt b5基因变异。
从63名个体的外周血单核细胞中制备cyt b5 cDNA。
一名个体的cyt b5存在序列变异（A178G），预测氨基酸取代为T60A，呈杂合状态。该变异体在大肠杆菌中表达时，与野生型cyt b5相比，对磺胺甲恶唑、4-氨基联苯和2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶（PhIP）的羟胺类物质保持相似的Vmax，但每种底物的Km适度增加（2至3.5倍）。然而，当在哺乳动物系统（HeLa细胞）中表达时，与野生型相比，T60A导致cyt b5蛋白表达降低70%。两种同工型的mRNA在HeLa细胞中的表达相当，并且在蛋白酶体机制受到抑制的兔网织红细胞裂解物系统中这些mRNA的翻译也相似。然而，将这些翻译后的酶与未抑制的兔网织红细胞裂解物一起孵育表明，T60A对蛋白酶体降解更敏感。
这些数据表明，cyt b5中天然存在的变异体T60A导致对羟胺底物的亲和力适度改变，并显著降低cyt b5表达。目前正在开展工作，以确定该变异体及cyt b5或b5R中其他变异体在更大人群中的流行情况，并确定此类变异体与体内羟胺还原差异之间的关联。
