title: 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.
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.|Chemical Department, School of Science, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Rodríguez-López
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Alméciga-Díaz
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Jhonnathan
authors.initials: J
authors.lastname: Sánchez
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Jefferson
authors.initials: J
authors.lastname: Moreno
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Laura
authors.initials: L
authors.lastname: Beltran
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Dennis
authors.initials: D
authors.lastname: Díaz
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Andrea
authors.initials: A
authors.lastname: Pardo
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Aura María
authors.initials: AM
authors.lastname: Ramírez
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Angela J
authors.initials: AJ
authors.lastname: Espejo-Mojica
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Luisa
authors.initials: L
authors.lastname: Pimentel
authors.affiliation: Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Barrera
mesh_terms: D002478:Cells, Cultured; D002811:Chondroitinsulfatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004705:Endocytosis; D004795:Enzyme Stability; D004847:Epithelial Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013429:Sulfatases / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; D002811:Chondroitinsulfatases; C480483:SUMF1 protein, human; D013429:Sulfatases; C102295:GALNS protein, human