In vitro evaluation of hydrolytic activity of two recombinant Nacetylglucosaminidases as potential therapeutic tools for mucopolysaccharidosis type IIIB

Deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU; E.C. 3.2.1.50) leads to the accumulation of the glycosaminoglycan (GAG) heparan sulfate in the cellular lysosomes of different organs, generating the neurodegenerative disease mucopolysaccharidosis III-B (MPS IIIB). Although recently there is no approved treatment for this disease, enzyme replacement therapy (ERT) has represented an alternative. Some years ago, studies have been focused in the obtaining of efficient recombinant enzymes in non-conventional mammalian expression systems as yeasts. Our investigations have proven the effectiveness of Komagataella phaffii GS115 as an expression system for the production of recombinant lysosomal enzymes. K. phaffii has been genetically modified by deletion in OCH1 gene, to obtain recombinant enzymes with humanized glycosylations. In this study two rhNAGLU were produced in both Komagataella phaffii GS115 and NRRLY::ΔOCH1, and were used to evaluate their ability to be uptaken by two different neuronal cell lines and MPS IIIB fibroblasts. Hydrolysis of accumulated glycosaminoglycans was evaluated in MPS-IIIB cells. The production was carried out by methanol induction of AOX1 promoter at different production scales. rhNAGLU were purified by either ionic or affinity chromatography. All processes, including cellular uptake, were followed by fluorescent activity measurement. Total glycosaminoglycans quantitation were followed by 1,9-dimethylmethylene blue technique. The rhNAGLU production in K. phaffii NRRLY-11430/ΔOCH1 was 1.8-fold higher than obtained with the native strain (GS115). In in vitro assays, both proteins were endocyted showing interesting intracellular activity values of up to 52.08 U/mg and 38.3 U/mg in MPS III-B cells, respectively. Finally, a significant decrease in total glycosaminoglycans was observed after 6 h of incubation with the stimuli, indicating lysosomal activity in rhNAGLU-treated cells. These results showed the potential of both strains as alternative platform to produce recombinant NAGLU as therapeutic tools for the development of an ERT to MPS IIIB.