Development of a functional bioassay for arylsulfatase B using the natural substrates of the enzyme.
A functional bioassay has been developed for measuring the intracellular activity of recombinant human arylsulfatase B (rhASB) on its natural glycosaminoglycan (GAG) substrates, dermatan sulfate (DS), and chondroitin sulfate (CS) when the enzyme is taken up into cultured ASB-deficient human fibroblasts (GM00519). The enzyme ASB is a lysosomal exohydrolase, cleaving sulfate from the N-acetylgalactosamine-4-sulfate (GalNAc-4S) residue at the nonreducing terminal of GAG structures. ASB-deficient cells accumulate DS and CS, which may be partially hydrolyzed by other lysosomal hydrolases, with the reactions stopping if a GalNAc-4S residue is reached on the nonreducing end of the oligosaccharide. When rhASB is added to the culture medium, the enzyme is taken up and translocates to the lysosomes and the intracellular DS and CS are depleted, demonstrating that the uptake of rhASB is able to restore lysosomal function in an in vitro cell-based assay. The accumulation and depletion of DS and CS are measured by digesting the residual intracellular DS and CS content with chondroitin ABC lyase and monitoring a characteristic disaccharide digestion product by laser-induced fluorescence-capillary zone electrophoresis (LIF-CZE). In the proposed assay format, GM00519 cells are cultured 5 weeks postconfluence to accumulate DS/CS, followed by incubation with rhASB (1-20 pM) for 5 days, and the CS/DS depletion profiles are compared between samples. The assay measures depletion of DS/CS independently of their molecular size or processing state; in this approach, all DS- and CS-like substances accumulating in the absence of ASB activity are considered to be natural substrates of the enzyme.
Pungor
Erno
E
BioMarin Pharmaceutical Inc., Novato, CA 94949, USA. epungor@bmrn.com
Hague
Charles M
CM
Chen
Ginger
G
Lemontt
Jeffrey F
JF
Dvorak-Ewell
Melita
M
Prince
William S
WS
D019943:Amino Acid Substitution; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000032:analysis*; D003871:Dermatan Sulfate / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005347:Fibroblasts / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D020051:N-Acetylgalactosamine-4-Sulfatase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D003871:Dermatan Sulfate; D002809:Chondroitin Sulfates; D020051:N-Acetylgalactosamine-4-Sulfatase