Cell uptake evaluation of human recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in Pichia pastoris

Mucopolysaccharidosis type IVA (MPS IV A, Morquio syndrome type A) is a lysosomal disease produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). It is characterized by the lysosomal accumulation of keratan- and chondroitin-6-sulfate. Although enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells, the methylotrophic yeast Pichia pastoris might represent an alternative to reduce the cost of the ERT or to produce proteins with improved stability, pharmacokinetic, or pharmacodynamics profiles. Here, we report the results of cell uptake and intracellular traffic for the recombinant GALNS produced in P. pastoris. Recombinant GALNS was produced at bioreactor scale (1,7 L) and purified from the extracellular fraction through ion-exchange and size-exclusion chromatography. Purified protein was added to the media of human skin fibroblast and HEK293 cells cultures, at concentrations of 10 and 50 nM. Cell uptake assay was carried out at 37 and 4 °C, and in the absence or presence of methyl a-D-mannopyranoside or mannose-6-phosphate (M6P). Recombinant enzyme was monitored by using fluorogenic substrate 4MU-Gal-6S and fluorophore labeling. It was observed recombinant GALNS was internalized into the cell, without any additional protein or host modification. Internalization of the recombinant GALNS was reduced at 4°C, suggesting that this is process mediated by an endocytosis pathway. Inhibition assay showed that recombinant GALNS has different glycosylation patterns. These results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio syndrome type A.