Carboxyl-Terminal Disulfide Bond of Acid Sphingomyelinase Is Critical for Its Secretion and Enzymatic Function

Abstract

The human acid sphingomyelinase (ASM, EC 3.1.4.12), a lysosomal and secretory protein coded by the sphingomyelin phosphodiesterase 1 (SMPD-1) gene, catalyzes the degradation of sphingomyelin (SM) to ceramide and phosphorylcholine. We examined the structural-functional properties of its carboxyl-terminus (amino acids 462-629), which harbors approximately 1/3 of all mutations discovered in the SMPD-1 gene. We created four naturally occurring mutants (DeltaR608, R496L, G577A, and Y537H) and five serial carboxyl-terminal deletion mutants (N620, N590, N570, N510, and N490). Transient transfection of the His/V5-tagged wild-type and mutant recombinant ASM in Chinese hamster ovary cells showed that all the mutants were normally expressed. Nonetheless, none of them, except the smallest deletion mutant N620 that preserved all post-translational modifications, were found capable of secretion to the medium. Furthermore, only the N620 conserved functional integrity (100% activity of the wild type); all other mutants completely lost the ability to catalyze SM hydrolysis. Importantly, cell surface biotinylation revealed that mutant DeltaR608 transfected CHO cells and fibroblasts from a compound heterozygous Niemann-Pick disease type B (NPD-B) patient (DeltaR608 and R441X) have defective translocation to the plasma membrane. Furthermore, we demonstrated that the DeltaR608 and N590 were trapped in the endoplasmic reticulum (ER) quality control checkpoint in contrast to the wild-type lysosomal localization. Interestingly, while the steady-state levels of ubiquitination were minimal for the wild-type ASM, a significant amount of Lys63-linked polyubiquitinated DeltaR608 and N590 could be purified by S5a-affinity chromatography, indicating an important misfolding in the carboxyl-terminal mutants. Altogether, we provide evidence that the carboxyl-terminus of the ASM is crucial for its protein structure, which in turns dictates the enzymatic function and secretion.

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

“…Secretory Pathway-It is well established that aSMase localizes to the endolysosomal compartment (16,17). However, in ongoing studies utilizing MCF7 stably expressing a C-terminal-tagged V5-aSMase, we observed that V5-aSMase exhibited minimal colocalization with the lysosomal marker LAMP1 (Fig.…”

“…Impaired Processing of NPD aSMase Mutants-The subcellular localization of wild-type or some mutant aSMase has previously been investigated (16,17). Lee et al (17) described the localization of YFP-tagged wild-type and ⌬R608 aSMase, and reported that WT aSMase localized properly to lysosomes, whereas the ⌬R608 exhibited minimal colocalization with LAMP2-positive structures and instead appeared to be trapped in the ER.…”

“…A and B) result from mutations in the SMPD1 gene that encodes aSMase (24). Although some of these mutations affect catalysis directly, the cause of enzyme dysfunction for other aSMase mutants is incompletely understood (16,17). To determine whether C-terminal processing is defective in certain aSMase mutants, the maturation of three C-terminal Niemann-Pick mutants, R600H, R600P, and ⌬R608 (25, 26), was investigated.…”

Roles and regulation of secretory and lysosomal acid sphingomyelinase

“…There are several aspects of this mechanism that require further explanation, including the phosphorylation of the luminal aSMase by the cytosolic protein kinase C δ (PKCδ). Confirmatory studies by Lee et al failed to demonstrate phosphorylation of L-SMase in response to phorbol ester and other agonists [62]; however, the phosphorylation of S-SMase was not investigated nor was the presence of PKCδ, which appears to be the critical PKC isoform required for activation of aSMase.…”

Solving the secretory acid sphingomyelinase puzzle: Insights from lysosome‐mediated parasite invasion and plasma membrane repair

“…Inactive splice variants were proposed to have regulatory and/or dominant-negative effects, but their physiological roles remain largely unknown (Rhein et al, 2012). Consistent with its primary localisation inside lysosomes (Jones, He, Katouzian, Darroch, & Schuchman, 2008;Lee et al, 2007), ASM has a pH optimum of 4.5-5.0 (Schuchman, 2010). ASM also shows a pattern of maturation that is often seen with lysosomal enzymes: C-terminal processing of a 75-kDa proenzyme generates a 65-kDa active form that cofractionates with lysosomes (Jenkins et al, 2011).…”