背景:谷胱甘肽β-合酶(CBS)T236N是一种与吡哆醇无反应性相关的新型突变,这在高半胱氨酸尿症的医学治疗中存在很大的困难。报道的高半胱氨酸尿症患者的严重表型突出了迫切需要理解突变致病性的分子机制以促进疾病的发展。
方法:在本研究中,我们使用多学科的方法来研究细菌表达和纯化的重组CBST236N蛋白的分子特性,我们直接将其与野生型(CBSWT)蛋白进行比较。
结果:我们的数据揭示了p.T236N突变对CBS酶活性的深远影响,与CBSWT蛋白相比显著减少了约96%。圆二色性(CD)实验表明,p.T236N突变没有显著改变蛋白质的二级结构。然而,CD光谱揭示了CBSWT和CBST236N突变蛋白种类的热稳定性的明显差异。此外,化学变性实验进一步强调,CBSWT蛋白表现出比CBST236N突变体更高的热力学稳定性,表明这种突变的不稳定作用。
结论:我们的发现解释了p.T236N突变的致病性,阐明其在严重的高半胱氨酸尿症表型中的作用。这项研究有助于更深入地了解CBS缺乏症,并可能改善受影响个体的针对性治疗策略的开发。
BACKGROUND: Cystathione beta-synthase (CBS) T236N is a novel mutation associated with pyridoxine non-responsiveness, which presents a significant difficulty in the medical treatment of
homocystinuria. Reported severe phenotypes in
homocystinuria patients highlight the urgent requirement to comprehend the molecular mechanisms underlying mutation pathogenicity for the advancement of the disease.
METHODS: In this study, we used a multidisciplinary approach to investigate the molecular properties of bacterially expressed and purified recombinant CBST236N protein, which we directly compared to those of the wild-type (CBSWT) protein.
RESULTS: Our data revealed a profound impact of the p.T236N mutation on CBS enzymatic activity, with a dramatic reduction of ~96% compared to the CBSWT protein. Circular dichroism (CD) experiments indicated that the p.T236N mutation did not significantly alter the secondary structure of the protein. However, CD spectra unveiled distinct differences in the thermal stability of CBSWT and CBST236N mutant protein species. In addition, chemical denaturation experiments further highlighted that the CBSWT protein exhibited greater thermodynamic stability than the CBST236N mutant, suggesting a destabilizing effect of this mutation.
CONCLUSIONS: Our findings provide an explanation of the pathogenicity of the p.T236N mutation, shedding light on its role in severe
homocystinuria phenotypes. This study contributes to a deeper understanding of CBS deficiency and may improve the development of targeted therapeutic strategies for affected individuals.