Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

Abstract

Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A.

Production and characterization of a human lysosomal recombinant iduronate‐2‐sulfatase produced inPichia pastoris

“…Screening of the P. pastoris clones was done at 10 and 100 mL . Clones were grown in the YPD medium [yeast extract 1% (w/v); peptone 2% (w/v); dextrose 2% (w/v)] during 48 H at 28 °C and 250 rpm.…”

“…The effect of pH and serum on prIDS stability was assayed as previously reported . To evaluate the effect of pH on prIDS stability, 20 μL of the purified enzyme was incubated with 20 μL of 50 mM sodium citrate (pH 3.0 ± 0.2), or 50 mM sodium acetate pH 4.0, 5.0, or 6.0 ± 0.2, or 50 mM Tris–HCl (pH 7.0 or 8.0 ± 0.2), respectively.…”

“…As an alternative to the production in mammalian cells, a growing number of studies have shown the possibility to produce active and therapeutic forms of lysosomal proteins in microorganisms . For instance, we have reported the production of active forms of human rIDS and N ‐acetylgalactosamine‐6‐sulfate sulfatase (GALNS) and β‐hexosaminidase A, B, and S in E. coli and/or P. pastoris . Furthermore, recombinant α‐glucosidase , α‐galactosidase A , GALNS , and lysosomal acid lipase produced in yeasts have shown successful cellular uptake both in cellular and animal models, and the reduction of the storage material.…”

“…For instance, we have reported the production of active forms of human rIDS and N ‐acetylgalactosamine‐6‐sulfate sulfatase (GALNS) and β‐hexosaminidase A, B, and S in E. coli and/or P. pastoris . Furthermore, recombinant α‐glucosidase , α‐galactosidase A , GALNS , and lysosomal acid lipase produced in yeasts have shown successful cellular uptake both in cellular and animal models, and the reduction of the storage material. Taken together, these results support the feasibility of the use of recombinant proteins produced in yeasts for the development of ERTs for lysosomal storage diseases.…”

Mucopolysaccharidosis IVA and glycosaminoglycans

“…As an alternative, recombinant GALNS have also been produced in E. coli [10,108–111] and in the yeast P. pastoris [112–114]. In all cases, the recombinant enzymes show highest activity at pH 5.0 and are taken-up by cultured fibroblast, chondrocytes, or HEK293 cells in a dose-dependent manner via the mannose-6-phosphate receptor (CHO-produced enzyme) [26,107,115] or mannose receptor (yeast-produced enzyme) [116].…”