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

Abstract

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 LSMase, which contain several highly conserved zincbinding 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 SMasenegative 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.

The role of ceramide in receptor- and stress-induced apoptosis studied in acidic ceramidase-deficient Farber disease cells

“…It has been recently described that acidic SMase is not exclusively localized in endosomal and lysosomal compartments, but in fact may also be targeted to the plasma membrane, where it is localized in caveolae (Liu and Anderson, 1995;Schissel et al, 1998). It is conceivable that generation of ceramide increases membrane¯uidity and raft formation, thereby facilitating receptor signaling (Holopainen et al, 1998;Zundel et al, 2000).…”

The Role of Sphingolipids and Ceramide in Pulmonary Inflammation in Cystic Fibrosis

“…Sphingomyelinases display peak activities depending on the pH and, thus, were named acid, neutral and alkaline sphingomyelinase. Mammalian cells express one gene for the acid sphingomyelinase, which can be targeted to lysosomes or for secretion into secretory lysosomes depending on the glycosylation pattern of the protein [69, 70]. Mammalian cells express at least two different neutral sphingomyelinases and one alkaline sphingomyelinase [65].…”

Sphingomyelinase, an Enzyme Implicated in Atherogenesis, Is Present in Atherosclerotic Lesions and Binds to Specific Components of the Subendothelial Extracellular Matrix

“…16,17 This gene gives rise to a single mRNA that encodes a single polypeptide that has 2 distinct fates during cellular trafficking: targeting to lysosomes or secretion. 10,18 The lysosomal form (L-SMase) has high-mannose oligosaccharides and an N-terminus slightly foreshortened by a proteolytic clip; the secretory form (S-SMase) has complex sugars and an intact NЈ-terminus. 18 Both forms are zinc-metalloenzymes, although most L-SMase becomes complexed with Zn 2ϩ inside the cell, whereas most S-SMase is secreted without Zn 2 and thus requires physiological concentrations of Zn 2ϩ in the extracellular space for enzymatic activity.…”

“…10,18 The lysosomal form (L-SMase) has high-mannose oligosaccharides and an N-terminus slightly foreshortened by a proteolytic clip; the secretory form (S-SMase) has complex sugars and an intact NЈ-terminus. 18 Both forms are zinc-metalloenzymes, although most L-SMase becomes complexed with Zn 2ϩ inside the cell, whereas most S-SMase is secreted without Zn 2 and thus requires physiological concentrations of Zn 2ϩ in the extracellular space for enzymatic activity. 18 While both forms of this SMase have an acid pH optimum, S-SMase has been shown to hydrolyze the sphingomyelin of certain atherogenic lipoproteins at neutral pH.…”