Purification and characterization of recombinant human lysosomal alpha-mannosidase.
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.
Berg
T
T
Departments of Medical Genetics and Medical Biochemistry, University Hospital of Tromsø, 9037 Tromsø, Norway. thomasb@fagmed.uit.no
King
B
B
Meikle
P J
PJ
Nilssen Ø
Tollersrud
O K
OK
Hopwood
J J
JJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008360:Mannosephosphates / Q000494:pharmacology; D008361:Mannosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D043323:alpha-Mannosidase
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D008360:Mannosephosphates; D011994:Recombinant Proteins; C027693:mannose-6-phosphate; D008361:Mannosidases; D043323:alpha-Mannosidase; D004121:Dimethyl Sulfoxide