Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain
Luisa N. Pimentel-Vera,
Alexander Rodríguez-López,
Angela J. Espejo-Mojica,
Aura María Ramírez,
Carolina Cardona,
Luis H. Reyes,
Shunji Tomatsu,
Thapakorn Jaroentomeechai,
Matthew P. DeLisa,
Oscar F. Sánchez,
Carlos J. Alméciga-Díaz
Affiliations
Luisa N. Pimentel-Vera
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, D.C., 110231, Colombia
Alexander Rodríguez-López
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, D.C., 110231, Colombia; Dogma Biotech, Bogotá, D.C., 110111, Colombia
Angela J. Espejo-Mojica
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, D.C., 110231, Colombia; Dogma Biotech, Bogotá, D.C., 110111, Colombia
Aura María Ramírez
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, D.C., 110231, Colombia
Carolina Cardona
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, D.C., 110231, Colombia; Grupo de Investigaciones Biomédicas y de Genética Humana Aplicada GIBGA, Facultad de Ciencias de la Salud, Universidad de Ciencias Aplicadas y Ambientales U.D.C.A, Bogotá, D.C., Colombia
Luis H. Reyes
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, D.C., 110231, Colombia; Grupo de Diseño de Productos y Procesos (GDPP), Department of Chemical and Food Engineering, Universidad de los Andes, Bogotá, D.C., Colombia
Shunji Tomatsu
Nemours Children's Health, Wilmington, DE, 19803, USA; Faculty of Arts and Sciences, University of Delaware, Newark, DE, 19716, USA; Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, 501-1193, Japan; Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA, 19144, USA
Thapakorn Jaroentomeechai
Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
Matthew P. DeLisa
Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA; Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA; Cornell Institute of Biotechnology, Cornell University, Ithaca, NY, USA
Oscar F. Sánchez
Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, USA
Carlos J. Alméciga-Díaz
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, D.C., 110231, Colombia; Corresponding author. Institute for the Study of Inborn Errors of Metabolism, Faculty of Sciences, Pontificia Universidad Javeriana, Cra 7 No. 43-82 Building 54, Room 305A. Bogotá, D.C., Colombia.
Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.
