Cooperative of Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan.
Department of Veterinary Medicine, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan.
J Vet Med Sci. 2021 Mar 5;83(2):315-321. doi: 10.1292/jvms.20-0390. Epub 2020 Dec 21.
Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.
遗传性高铁血红蛋白血症与烟酰胺腺嘌呤二核苷酸-细胞色素 b5 还原酶 (b5R) 缺乏症有关，是一种在动物中罕见的常染色体隐性遗传病。最近，非同义 b5R 基因 (CYB5R3) 变体已被报道与犬和猫遗传性高铁血红蛋白血症有关。然而，这些非同义变体引起的犬和猫高铁血红蛋白血症的潜在分子机制尚未报道。先前，我们报道了一个博美犬家族遗传性高铁血红蛋白血症，携带 CYB5R3 突变，在 7 号外显子的密码子 194 处发生 A>C 转换，将异亮氨酸突变为亮氨酸 (p.Ile194Leu)。在这项研究中，我们研究了野生型和 Ile194Leu 犬 b5R 的可溶性形式的酶学和结构特性，以表征该错义突变的影响。我们的结果表明，该氨基酸取代并未影响突变酶的动力学特性。圆二色性检测到的野生型和 Ile194Leu b5R 的二级结构显示出相似的模式。然而，突变酶表现出降低的热稳定性和增加的胰蛋白酶水解敏感性。此外，热稳定性和展开测量表明，突变酶对温度依赖性变性比野生型 b5R 更敏感。我们从这些结果得出结论，具有正常酶活性的不稳定突变酶特性与博美犬家族的遗传性高铁血红蛋白血症有关。
