Characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli for enzyme replacement therapy of Morquio A disease

Mosquera, Ángela; Rodríguez, Arturo; Soto, Carlos Y.; Leonardi, Felice; Espejo, Ángela J.; Sánchez, Oscar F.; Alméciga-Díaz, Carlos J.; Barrera, Luis Alejandro

doi:10.1016/j.procbio.2012.07.028

Cited by 20 publications

(43 citation statements)

References 32 publications

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“…The purified enzyme showed an optimal pH of 5.5, and was stable for 8 days at 4°C, and up to 6 h in human serum. As expected, this recombinant enzyme was neither taken up by HEK293 cells nor Morquio A skin fibroblasts, suggesting that N-linked oligosaccharides in GALNS are not necessary for producing an active and stable enzyme, but they are for the protein cell uptake [64]. This study provided the first evidence about the stability of a recombinant human sulfatase produced in E. coli BL21(DE3), as well as the effect of glycosylation absence on this type of enzymes.…”

Section: Recombinant N-acetylgalactosamine-6-sulfate Sulfatasesupporting

confidence: 60%

“…These results show for the first time the advantage of sulfatase/SUMF1 co-expression within a yeast expression system. The purified enzyme showed a specific activity of 25.3 nmol h −1 mg −1 , which is higher than the one reported for E. coli BL21(DE3) [64], but lower than that of the enzyme produced in CHO cells [61,70,71]. Nevertheless, as mentioned before the activities are not comparable between microorganisms systems and CHO cells due to substantial differences in the enzyme activity assays.…”

Section: Recombinant N-acetylgalactosamine-6-sulfate Sulfatasementioning

confidence: 56%

“…Furthermore, recombinant GALNS secretion was also promoted by the presence of native GALNS signal peptide, showing the high conservation of the cotranslational transport of extracellular proteins between bacteria and mammals. Under these semi-continuous culture conditions, the intracellular activity was 0.05 nmol h −1 mg −1 , (5-fold higher than that obtained under batch culture conditions), while extracellular activity was up to 6.18 nmol h −1 mg −1 [64]. Then, recombinant GALNS was purified from this conditioned medium through and used to evaluate the effect of temperature and pH, the stability in human serum, and the in-vitro cell uptake (Fig.…”

Section: Recombinant N-acetylgalactosamine-6-sulfate Sulfatasementioning

confidence: 57%

“…Recently ERT for Morquio A disease was approved, which showed a modest improvement in a 6-min walk test (6MWT), and a reduction of urinary KS, after a 24-weeks treatment [62]. As an alternative, recombinant GALNS have been produced in E. coli K12 [11,63,64] and in the methylotrophic yeast P. pastoris [65,66] (Table 1).…”

Section: Recombinant N-acetylgalactosamine-6-sulfate Sulfatasementioning

confidence: 99%

See 3 more Smart Citations

Human recombinant lysosomal enzymes produced in microorganisms

Espejo-Mojica

Alméciga-Díaz

Rodríguez

et al. 2015

Molecular Genetics and Metabolism

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Section: Recombinant N-acetylgalactosamine-6-sulfate Sulfatasesupporting

confidence: 60%

Section: Recombinant N-acetylgalactosamine-6-sulfate Sulfatasementioning

confidence: 56%

Section: Recombinant N-acetylgalactosamine-6-sulfate Sulfatasementioning

confidence: 57%

Section: Recombinant N-acetylgalactosamine-6-sulfate Sulfatasementioning

confidence: 99%

See 2 more Smart Citations

Human recombinant lysosomal enzymes produced in microorganisms

Espejo-Mojica

Alméciga-Díaz

Rodríguez

et al. 2015

Molecular Genetics and Metabolism

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“…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]. Enzyme produced in E. coli does not have N -glycosylation, so that the enzyme can only be taken up into cells by nonspecific endocytosis [109,117]. …”

Section: Therapy and Managementmentioning

confidence: 99%

Mucopolysaccharidosis IVA and glycosaminoglycans

Khan

Alméciga-Díaz

Sawamoto

et al. 2017

Molecular Genetics and Metabolism

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Mucopolysaccharidosis IVA (MPS IVA; Morquio A: OMIM 253000) is a lysosomal storage disease with an autosomal recessive trait caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to accumulation of specific glycosaminoglycans (GAGs): chondroitin-6-sulfate (C6S) and keratan sulfate (KS). C6S and KS are mainly produced in the cartilage. Therefore, the undegraded substrates are stored primarily in cartilage and in its extracellular matrix (ECM), leading to a direct impact on cartilage and bone development, and successive systemic skeletal dysplasia. Chondrogenesis, the earliest phase of skeletal formation, is maintained by cellular interactions with the ECM, growth and differentiation factors, signaling pathways, and transcription factors in a temporal-spatial manner. In patients with MPS IVA, the cartilage is disrupted at birth as a consequence of abnormal chondrogenesis and/or endochondral ossification. The unique skeletal features are distinguished by a disproportional short stature, odontoid hypoplasia, spinal cord compression, tracheal obstruction, pectus carinatum, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. In spite of many descriptions of these unique clinical features, delay of diagnosis still happens. The pathogenesis and treatment of systemic skeletal dysplasia in MPS IVA remains an unmet challenge. In this review article, we comprehensively describe historical aspect, property of GAGs, diagnosis, screening, pathogenesis, and current and future therapies of MPS IVA.

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Human recombinant lysosomal β‐Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay‐Sachs fibroblasts

Espejo-Mojica

Rodríguez-López

et al. 2020

American J of Med Genetics Pt C

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GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the β-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αβ or ββ subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidoses, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant β-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were takenup via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant β-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.

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Characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli for enzyme replacement therapy of Morquio A disease

Cited by 20 publications

References 32 publications

Human recombinant lysosomal enzymes produced in microorganisms

Human recombinant lysosomal enzymes produced in microorganisms

Mucopolysaccharidosis IVA and glycosaminoglycans

Human recombinant lysosomal β‐Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay‐Sachs fibroblasts

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