A quantitative mannose 6-phosphate receptor-based in vitro assay for recombinant human N-acetylgalactosamine-4-sulfatase.
An assay was developed, using two similar formats, to simultaneously measure both the lysosomal targeting receptor binding and enzyme activity of the recombinant human enzyme N-acetylgalactosamine-4-sulfatase. This assay also has potential application for all phosphorylated lysosomal enzymes that contain mannose-6-phosphate residues. The receptor was either purified from fetal bovine sera then adsorbed, or produced in situ by growing and fixing diploid human fibroblast-like cells, to a solid phase. The enzyme substrate was 4-methylumbelliferyl sulfate which fluoresces after cleavage of the sulfate moiety. Both the precursor and mature forms of the recombinant enzyme were used to demonstrate the specificity and usefulness of the assay. The assay is rapid and sensitive and has a wide dynamic range. Association between the receptor and the mannose-6-phosphate residues was abrogated in the presence of a competitive inhibitor, mannose 6-phosphate. However, partial activity was still measured when the mature enzyme was incubated in the presence of mannose 6-phosphate when using the fixed fibroblast format. This would indicate that the recombinant enzymes contain at least one terminal sugar moiety other than mannose 6-phosphate which can recognize receptors on the surface of human fibroblast-like cells. Other possible applications of this assay are also discussed.
Kleinig
M
M
Research and Development Division, CSL Limited, 45 Poplar Road, Parkville, Victoria, 3052, Australia. mkleinig@csl.com.au
Cox
J
J
D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002460:Cell Line; D002803:Chondro-4-Sulfatase / Q000032:analysis / Q000378:metabolism*; D005347:Fibroblasts; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; C019492:4-methylumbelliferyl sulfate; D006923:Hymecromone; D002803:Chondro-4-Sulfatase