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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/47952
Title: A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation: Requirement for Carboxyl-Terminal Proteolytic Processing
Authors: Jenkins, Russell W.; Idkowiak-Baldys, Jolanta; Simbari, Fabio; Canals, Daniel; Roddy, Patrick; Riner, Clarke D.; Clarke, Christopher J.; Hannun, Yusuf A.
Keywords: Golgi
Intracellular Trafficking
Lysosomal Glycoproteins
Acid Sphingomyelinase
Issue Date: 2011
Publisher: American Society for Biochemistry and Molecular Biology
Citation: Journal of Biological Chemistry
Abstract: Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn2+)-independent lysosomal aSMase (L-SMase) and a Zn2+-dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn2+-activated proenzyme to a mature 65 kDa, Zn2+-independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn2+-dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same Mr as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ΔR608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function.
Publisher version (URL): http://dx.doi.org/10.1074/jbc.M110.155234
URI: http://hdl.handle.net/10261/47952
DOI: 10.1074/jbc.M110.155234
ISSN: 0021-9258
E-ISSN: 1083-351X
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