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Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris
Biological Procedures Online ( IF 3.7 ) Pub Date : 2020-12-13 , DOI: 10.1186/s12575-020-00138-0
Roghayyeh Baghban 1, 2, 3, 4 , Safar Farajnia 4, 5 , Younes Ghasemi 6 , Reyhaneh Hoseinpoor 7 , Azam Safary 8 , Mojtaba Mortazavi 9 , Nosratollah Zarghami 1
Affiliation  

Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

中文翻译:

奥卡蓝蛋白降低毕赤酵母蛋白水解和自溶活性的突变分析

Ocriplasmin (Jetrea) 用于治疗有症状的玻璃体黄斑粘连。由于其自溶性质,这种酶在注射入眼后会经历快速失活和有限的活性持续时间。此外,蛋白水解活性会导致光感受器损伤,更严重的情况下可能会导致视力障碍。本研究旨在通过定点诱变减少奥克蓝蛋白的缺点。为了降低第一个变体中奥克蓝蛋白的自溶活性,赖氨酸 156 变为谷氨酸,在第二个变体中为了降低蛋白水解活性,丙氨酸 59 突变为苏氨酸。第三个变体包含这两种突变。野生型和奥克蓝蛋白构建体的三种突变变体的表达在巴斯德毕赤酵母表达系统中进行。对突变体进行了计算机和体外分析,并与野生型进行了比较。ocriplasmin 变体的动力学参数显示,两种具有 K156E 取代的变体比野生型更能抵抗自溶降解。这些变体还表现出降低的 Kcat 和 Vmax 值。Km 值增加,导致自溶和混合变体的催化效率(Kcat/Km 比率)降低。此外,在具有A59T突变的变体中,Kcat和Vmax值与野生型相比有所降低。混合变体显示 Km 值增加最多(几乎 2 倍),并且对底物的酶亲和力降低。因此,结果表明,ocriplasmin 序列的组合突变比单一突变更有效。结果表明,此类变异代表了研究旨在非手术解决玻璃体黄斑粘连的治疗策略的宝贵工具。
更新日期:2020-12-14
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