Roghayyeh Baghban1,2,3,4, Safar Farajnia5,6, Younes Ghasemi7, Reyhaneh Hoseinpoor8, Azam Safary9, Mojtaba Mortazavi10, Nosratollah Zarghami1. 1. Medical Biotechnology Department, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Poostchi Ophthalmology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. 4. Biotechnology Research Center, Tabriz University of Medical Sciences, Daneshgah Ave, Tabriz, Iran. 5. Biotechnology Research Center, Tabriz University of Medical Sciences, Daneshgah Ave, Tabriz, Iran. farajnias@tbzmed.ac.ir. 6. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. farajnias@tbzmed.ac.ir. 7. Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shiraz University of Medical Science, Shiraz, Iran. 8. Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 9. Connective Tissue Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 10. Department of Biotechnology, Institute of Science and High Technology and Environmental Science, Graduate University of Advanced Technology, Kerman, Iran.
Abstract
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.
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