Characterization of human acid sphingomyelinase puri¢ed from the media of overexpressing Chinese hamster ovary cells Xingxuan He a , Silvia R.P. Miranda a , Xiaoling Xiong a , Arie Dagan c , Shimon Gatt c , Edward H. Schuchman a;b; * a Department of Human Genetics, Box 1498, Mount Sinai School of Medicine, 1425 Madison Avenue, Room 14-20A, New York, NY 10029, USA b Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA c Department of Biochemistry, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel Received 14 December 1998; received in revised form 11 March 1999; accepted 11 March 1999 Abstract A rapid purification method was developed to isolate milligram quantities of human acid sphingomyelinase from the media of overexpressing Chinese hamster ovary cells. The purified, recombinant enzyme (rhASM) had physical and kinetic characteristics that were consistent with those reported for the non-recombinant enzyme, including an acidic pH optimum and sensitivity to sulfhydryl reducing reagents and the zinc specific chelator, 1,10-phenanthroline. A novel assay using fluorescently conjugated sphingomyelin was developed to explore the substrate binding properties of rhASM. Substrate binding required a fatty acid chain length of at least six carbons and the presence of the phosphocholine headgroup on sphingomyelin. Substrate binding also required an acidic pH, and was inhibited by pretreatment of the enzyme with sulfhydral reducing reagents or 1,10-phenanthroline. rhASM was rapidly internalized by cultured skin fibroblasts from Niemann-Pick disease (NPD) patients, and V50% of this uptake was dependent on the mannose 6-phosphate receptor system. Studies using FITC-labeled rhASM revealed that by 1 h the internalized enzyme was localized to acidic compartments and could degrade sphingomyelin, the first demonstration that a lysosomal sphingolipid hydrolase can be fluorescently labeled and retain its biological activity. Intravenous injection of rhASM into ASM knock-out mice showed that the t 1=2 in the plasma was less than 5 min, and that the majority of the injected enzyme was taken up by the liver, followed by the spleen. Thus, these studies lay the foundation for future structure/function investigations of ASM, further investigations into this enzyme's role in ceramide mediated signal transduction, and the evaluation of enzyme replacement therapy for NPD using the mouse model. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Lysosomal enzyme ; Niemann-Pick disease ; Knock-out mice ; Enzyme therapy ; Sphingomyelinase 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4838(99)00069-2 Abbreviations : ASM, acid sphingomyelinase ; ASMKO, acid sphingomyelinase knock-out mice ; bSmase, bacterial sphingomyelinase ; CHO, Chinese hamster ovary cells ; DTT, dithiothreitol ; Endo H, endoglycosylase H ; GC, glucosyl ceramide ; LR-SPM, lissamine rhodamine conjugated sphingomyelin ; L-SMase, intracellular, lysosomal sphingomyelinase ; M6P, mannose 6-phosphate ; NCM, nitro- cellulose membrane ; NPD, Niemann-Pick disease ; PNG-F, peptide-N-glycanase F ; rhASM, recombinant human acid sphingomyelinase ; S-SMase, secreted acid sphingomyelinase * Corresponding author. Fax : +1-212-849-2447 ; E-mail : schuchman@msvax.mssm.edu Biochimica et Biophysica Acta 1432 (1999) 251^264 www.elsevier.com/locate/bba