The cellular trafficking and zinc dependence of secretory and lysosomal sphingomyelinase, two products of the acid sphingomyelinase gene.
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 Zn2+ 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 Zn2+ 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 Zn2+. 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 Zn2+-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 Zn2+ 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 Zn2+; thus S-SMase requires exogeneous Zn2+ 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.
Schissel
S L
SL
Department of Anatomy & Cell Biology, Columbia University, New York, New York 10032, USA.
Keesler
G A
GA
Schuchman
E H
EH
Williams
K J
KJ
Tabas
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001161:Arteriosclerosis / Q000201:enzymology; D016466:CHO Cells; D006224:Cricetinae; D003594:Cytoplasmic Granules / Q000201:enzymology*; D006596:Hexosaminidases / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D013108:Sphingomyelin Phosphodiesterase; D006596:Hexosaminidases; D015032:Zinc