Tags

Type your tag names separated by a space and hit enter

Isolation and characterisation of a recombinant, precursor form of lysosomal acid alpha-glucosidase.
Eur J Biochem 1995; 234(3):903-9EJ

Abstract

Glycogenosis type II (GSD II, Pompe disease) is an autosomal recessive lysosomal storage disease that results from a deficiency of acid alpha-glucosidase (GAA). Patients with this disorder are unable to break down lysosomal glycogen, which consequently accumulates in the lysosome. To evaluate enzyme replacement therapy for GSD II patients, we have expressed human GAA cDNA in Chinese hamster ovary-K1 cells utilising a vector that places the cDNA under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line that secreted precursor recombinant GAA at approximately 18 mg.l-1.day-1 was identified. The precursor recombinant GAA was purified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters similar to those of GAA purified from human tissues. The partial N-terminal amino acid sequence of recombinant GAA conformed to that derived from the nucleotide sequence of the cloned cDNA. The recombinant enzyme was taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocytosed GAA was localised to the lysosome and showed evidence of intracellular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were cultured in the presence of mannose 6-phosphate.

Authors+Show Affiliations

Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, Australia.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8575451

Citation

Fuller, M, et al. "Isolation and Characterisation of a Recombinant, Precursor Form of Lysosomal Acid Alpha-glucosidase." European Journal of Biochemistry, vol. 234, no. 3, 1995, pp. 903-9.
Fuller M, Van der Ploeg A, Reuser AJ, et al. Isolation and characterisation of a recombinant, precursor form of lysosomal acid alpha-glucosidase. Eur J Biochem. 1995;234(3):903-9.
Fuller, M., Van der Ploeg, A., Reuser, A. J., Anson, D. S., & Hopwood, J. J. (1995). Isolation and characterisation of a recombinant, precursor form of lysosomal acid alpha-glucosidase. European Journal of Biochemistry, 234(3), pp. 903-9.
Fuller M, et al. Isolation and Characterisation of a Recombinant, Precursor Form of Lysosomal Acid Alpha-glucosidase. Eur J Biochem. 1995 Dec 15;234(3):903-9. PubMed PMID: 8575451.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Isolation and characterisation of a recombinant, precursor form of lysosomal acid alpha-glucosidase. AU - Fuller,M, AU - Van der Ploeg,A, AU - Reuser,A J, AU - Anson,D S, AU - Hopwood,J J, PY - 1995/12/15/pubmed PY - 1995/12/15/medline PY - 1995/12/15/entrez SP - 903 EP - 9 JF - European journal of biochemistry JO - Eur. J. Biochem. VL - 234 IS - 3 N2 - Glycogenosis type II (GSD II, Pompe disease) is an autosomal recessive lysosomal storage disease that results from a deficiency of acid alpha-glucosidase (GAA). Patients with this disorder are unable to break down lysosomal glycogen, which consequently accumulates in the lysosome. To evaluate enzyme replacement therapy for GSD II patients, we have expressed human GAA cDNA in Chinese hamster ovary-K1 cells utilising a vector that places the cDNA under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line that secreted precursor recombinant GAA at approximately 18 mg.l-1.day-1 was identified. The precursor recombinant GAA was purified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters similar to those of GAA purified from human tissues. The partial N-terminal amino acid sequence of recombinant GAA conformed to that derived from the nucleotide sequence of the cloned cDNA. The recombinant enzyme was taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocytosed GAA was localised to the lysosome and showed evidence of intracellular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were cultured in the presence of mannose 6-phosphate. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/8575451/Isolation_and_characterisation_of_a_recombinant_precursor_form_of_lysosomal_acid_alpha_glucosidase_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=1995&volume=234&issue=3&spage=903 DB - PRIME DP - Unbound Medicine ER -