Characterization of human acid sphingomyelinase purified from the media ofoverexpressing Chinese hamster ovary cells

A rapid purification method was developed to isolate milligram quantities o f human acid sphingomyelinase from the media of overexpressing Chinese hams ter ovary cells. The purified, recombinant enzyme (rhASM) had physical and kinetic characteristics that were consistent with those reported for the no n-recombinant enzyme, including an acidic pH optimum and sensitivity to sul fhydryl reducing reagents and the zinc specific chelator, 1,10-phenanthroli ne. A novel assay using fluorescently conjugated sphingomyelin was develope d to explore the substrate binding properties of rhASM. Substrate binding r equired a fatty acid chain length of at least six carbons and the presence of the phosphocholine headgroup on sphingomyelin. Substrate binding also re quired an acidic pH, and was inhibited by pretreatment of the enzyme with s ulfhydral reducing reagents or 1,10-phenanthroline. rhASM was rapidly inter nalized by cultured skin fibroblasts from Niemann-Pick disease (NPD) patien ts, and similar to 50% of this uptake was dependent on the mannose 6-phosph ate receptor system. Studies using FITC-labeled rhASM revealed that by 1 h the internalized enzyme was localized to acidic compartments and could degr ade sphingomyelin, the first demonstration that a lysosomal sphingolipid hy drolase can be fluorescently labeled and retain its biological activity. In travenous 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 e nzyme was taken up by the liver, followed by the spleen. Thus, these studie s lay the foundation for future structure/function investigations of ASM, f urther investigations into this enzyme's role in ceramide mediated signal t ransduction, and the evaluation of enzyme replacement therapy for NPD using the mouse model. (C) 1999 Elsevier Science B.V. All rights reserved.