Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain

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.

• Autores: Ramirez, Aura Maria; Cardona Ramírez, Carolina; Reyes, Luis; Tomatsu, Shunji; Jaroentomeechai, Thapakorn; DeLisa, Matthew; Sanchez, Oscar; Alméciga-Díaz, Carlos Javier
• Tipo de recurso: Article of investigation
• Fecha de publicación: 2024
• Institución: Universidad de Ciencias Aplicadas y Ambientales U.D.C.A
• Repositorio: Repositorio Institucional UDCA
• Idioma: eng
• OAI Identifier: oai:repository.udca.edu.co:11158/5975
• Acceso en línea: https://repository.udca.edu.co/handle/11158/5975; https://doi.org/10.1016/j.heliyon.2024.e32555
• Palabra clave: 610 - Medicina y salud::616 - Enfermedades; Mucopolisacaridosis; Escherichia coli; Acetilgalactosamina; N-linked glycosylation
• Rights: openAccess
• License: https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode.es