Purification and characterization of recombinant human lysosomal α-mannosidase

Thomas Berg, Barbara King, Peter J. Meikle, Øivind Nilssen, Ole K. Tollersrud, John J. Hopwood

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Lysosomal α-mannosidase (EC 3.2.1.24) is required in the degradation of the asparagine-linked carbohydrates of glycoproteins. Deficiency of this enzyme leads to the lysosomal storage disorder α mannosidosis. As an initial step toward enzyme replacement therapy for α-mannosidosis, the human lysosomal α-mannosidase cDNA was cloned into the pcDNA 3.1 vector and expressed in Chinese hamster ovary cells. Dimethyl sulfoxide (DMSO) added to the cell culture media to induce growth arrest led to a 4-fold increase in the enzyme production, with an average yield of 3.2 mg L- 1 day- 1 . α-Mannosidase was secreted as an active homodimer of a 130-kDa precursor that was proteolyzed into two polypeptides of 55 and 72 kDa during the subsequent purification of the enzyme. N-terminal sequence analysis of the purified enzyme revealed that the proteolysis occurred close to a cleavage site previously identified in the intracellular form of lysosomal α-mannosidase. Generation of monoclonal antibodies against the recombinant enzyme made it possible to develop a single-step immunoaffinity purification procedure for α-mannosidase. The immunoaffinity-purified enzyme which mainly consisted of the 130-kDa precursor, displayed specific activity and kinetics similar to those of the processed form. Recombinant α-mannosidase was taken up by cultured α-mannosidosis fibroblasts and was trafficked to the lysosomes via the mannose 6-phosphate pathway where it reduced the amounts of stored mannose-containing oligosaccharides.

LanguageEnglish
Pages18-29
Number of pages12
JournalMolecular Genetics and Metabolism
Volume73
Issue number1
DOIs
Publication statusPublished - 1 Jan 2001

Keywords

  • DMSO
  • Enzyme replacement
  • Lysosomal α-mannosidase
  • Monoclonal antibodies
  • Therapy
  • α-mannosidosis

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Endocrinology

Cite this

Berg, T., King, B., Meikle, P. J., Nilssen, Ø., Tollersrud, O. K., & Hopwood, J. J. (2001). Purification and characterization of recombinant human lysosomal α-mannosidase. Molecular Genetics and Metabolism, 73(1), 18-29. https://doi.org/10.1006/mgme.2001.3173
Berg, Thomas ; King, Barbara ; Meikle, Peter J. ; Nilssen, Øivind ; Tollersrud, Ole K. ; Hopwood, John J. / Purification and characterization of recombinant human lysosomal α-mannosidase. In: Molecular Genetics and Metabolism. 2001 ; Vol. 73, No. 1. pp. 18-29.
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Berg, T, King, B, Meikle, PJ, Nilssen, Ø, Tollersrud, OK & Hopwood, JJ 2001, 'Purification and characterization of recombinant human lysosomal α-mannosidase', Molecular Genetics and Metabolism, vol. 73, no. 1, pp. 18-29. https://doi.org/10.1006/mgme.2001.3173

Purification and characterization of recombinant human lysosomal α-mannosidase. / Berg, Thomas; King, Barbara; Meikle, Peter J.; Nilssen, Øivind; Tollersrud, Ole K.; Hopwood, John J.

In: Molecular Genetics and Metabolism, Vol. 73, No. 1, 01.01.2001, p. 18-29.

Research output: Contribution to journalArticle

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AU - King, Barbara

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AB - Lysosomal α-mannosidase (EC 3.2.1.24) is required in the degradation of the asparagine-linked carbohydrates of glycoproteins. Deficiency of this enzyme leads to the lysosomal storage disorder α mannosidosis. As an initial step toward enzyme replacement therapy for α-mannosidosis, the human lysosomal α-mannosidase cDNA was cloned into the pcDNA 3.1 vector and expressed in Chinese hamster ovary cells. Dimethyl sulfoxide (DMSO) added to the cell culture media to induce growth arrest led to a 4-fold increase in the enzyme production, with an average yield of 3.2 mg L- 1 day- 1 . α-Mannosidase was secreted as an active homodimer of a 130-kDa precursor that was proteolyzed into two polypeptides of 55 and 72 kDa during the subsequent purification of the enzyme. N-terminal sequence analysis of the purified enzyme revealed that the proteolysis occurred close to a cleavage site previously identified in the intracellular form of lysosomal α-mannosidase. Generation of monoclonal antibodies against the recombinant enzyme made it possible to develop a single-step immunoaffinity purification procedure for α-mannosidase. The immunoaffinity-purified enzyme which mainly consisted of the 130-kDa precursor, displayed specific activity and kinetics similar to those of the processed form. Recombinant α-mannosidase was taken up by cultured α-mannosidosis fibroblasts and was trafficked to the lysosomes via the mannose 6-phosphate pathway where it reduced the amounts of stored mannose-containing oligosaccharides.

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