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Title

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation: Requirement for Carboxyl-Terminal Proteolytic Processing

AuthorsJenkins, Russell W.; Idkowiak-Baldys, Jolanta; Simbari, Fabio ; Canals, Daniel; Roddy, Patrick; Riner, Clarke D.; Clarke, Christopher J.; Hannun, Yusuf A.
KeywordsGolgi
Intracellular Trafficking
Lysosomal Glycoproteins
Lysosomes
Sphingolipid
Acid Sphingomyelinase
Desipramine
Issue Date2011
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry
AbstractAcid 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
URIhttp://hdl.handle.net/10261/47952
DOI10.1074/jbc.M110.155234
ISSN0021-9258
E-ISSN1083-351X
Appears in Collections:(IQAC) Artículos
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