Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

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.

The Role of Galectin-3 in Retinal Degeneration and Other Ocular Diseases: A Potential Novel Biomarker and Therapeutic Target

“…It is a powerful collagenase activator with proteolytic activity to break down a variety of components of the vitreoretinal interface, including Gal3. It has been approved by the European Medicines Agency for the treatment of adult vitreomacular traction and macular holes related to macular traction with a pore size ≤ 400 µm [170][171][172], implying the potential for targeting Gal3 in the treatment of retinal disease.…”

Established tools and emerging trends for the production of recombinant proteins and metabolites in Pichia pastoris

“…Sitedirected mutagenesis was also carried out for proteins of biomedical interest, e.g. to study immunogenic residues of the buckwheat allergen (84), to increase efficacy and stability of ocriplasmin (85) or to strengthen the affinity of a scFv to its target and should be even more facilitated by the high-throughput cloning tools available for P. pastoris now (see section Tools & Techniques).…”