Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/7715
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Type: Journal article
Title: Purification and characterization of recombinant human lysosomal a-mannosidase
Author: Berg, T.
King, B.
Meikle, P.
Nilssen, O.
Tollersrud, O.
Hopwood, J.
Citation: Molecular Genetics and Metabolism, 2001; 73(1):18-29
Publisher: Academic Press Inc Elsevier Science
Issue Date: 2001
ISSN: 1096-7192
1096-7206
Abstract: Lysosomal alpha-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 alpha-mannosidosis. As an initial step toward enzyme replacement therapy for alpha-mannosidosis, the human lysosomal alpha-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). alpha-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 alpha-mannosidase. Generation of monoclonal antibodies against the recombinant enzyme made it possible to develop a single-step immunoaffinity purification procedure for alpha-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 alpha-mannosidase was taken up by cultured alpha-mannosidosis fibroblasts and was trafficked to the lysosomes via the mannose 6-phosphate pathway where it reduced the amounts of stored mannose-containing oligosaccharides.
Keywords: CHO Cells
Fibroblasts
Animals
Humans
Mice
Dimethyl Sulfoxide
Mannosidases
alpha-Mannosidase
Mannosephosphates
Recombinant Proteins
DNA, Complementary
Antibodies, Monoclonal
Culture Media, Conditioned
Electrophoresis, Polyacrylamide Gel
Transfection
Gene Expression Regulation, Enzymologic
Time Factors
Cricetinae
DOI: 10.1006/mgme.2001.3173
Appears in Collections:Aurora harvest
Paediatrics publications

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