The Cellular Trafficking and Zinc Dependence of Secretory and Lysosomal Sphingomyelinase, Two Products of the Acid Sphingomyelinase Gene

• Published in: The Journal of biological chemistry 1998-07, Vol.273 (29), p.18250-18259
• Main Authors: Schissel, S L, Keesler, G A, Schuchman, E H, Williams, K J, Tabas, I
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Arteriosclerosis - enzymology; CHO Cells; Cricetinae; Cytoplasmic Granules - enzymology; Hexosaminidases - metabolism; Lysosomes - enzymology; Molecular Sequence Data; Signal Transduction; Sphingomyelin Phosphodiesterase - genetics; Sphingomyelin Phosphodiesterase - metabolism; Zinc - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.29.18250

The acid sphingomyelinase (ASM) gene, which has been implicated in ceramide-mediated cell signaling and atherogenesis, gives rise to both lysosomal SMase (L-SMase), which is reportedly cation-independent, and secretory SMase (S-SMase), which is fully or partially dependent on Zn 2+ for enzymatic activity. Herein we present evidence for a model to explain how a single mRNA gives rise to two forms of SMase with different cellular trafficking and apparent differences in Zn 2+ dependence. First, we show that both S-SMase and L-SMase, which contain several highly conserved zinc-binding motifs, are directly activated by zinc. In addition, SMase assayed from a lysosome-rich fraction of Chinese hamster ovary cells was found to be partially zinc-dependent, suggesting that intact lysosomes from these cells contain subsaturating levels of Zn 2+ . Analysis of Asn-linked oligosaccharides and of N-terminal amino acid sequence indicated that S-SMase arises by trafficking through the Golgi secretory pathway, not by cellular release of L-SMase during trafficking to lysosomes or after delivery to lysosomes. Most importantly, when Zn 2+ -dependent S-SMase was incubated with SMase-negative cells, the enzyme was internalized, trafficked to lysosomes, and became zinc-independent. We conclude that L-SMase is exposed to cellular Zn 2+ during trafficking to lysosomes, in lysosomes, and/or during cell homogenization. In contrast, the pathway targeting S-SMase to secretion appears to be relatively sequestered from cellular pools of Zn 2+ ; thus S-SMase requires exogeneous Zn 2+ for full activity. This model provides important information for understanding the enzymology and regulation of L- and S-SMase and for exploring possible roles of ASM gene products in cell signaling and atherogenesis.