Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells

He, Xingxing; Miranda, Sílvia R.P.; Xiong, Xiufang; Dagan, Arie; Gátt, Shimon; Schuchman, Edward H.

doi:10.1016/s0167-4838(99)00069-2

Cited by 91 publications

(99 citation statements)

References 26 publications

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“…If there were significant intercellular transport of ASM, we believe that it would have been detectable by immunohistochemistry, Western blotting, or enzyme activity. That is not to say, however, that secretion of hASM does not occur at all, because it has been amply shown in transfected cells (He et al, 1999). However, it is unclear how efficient this process really is in vivo.…”

Section: Discussionmentioning

confidence: 98%

“…Recombinant ASM purified from the culture medium of Chinese hamster ovary (CHO) cells (He et al, 1999) is rapidly taken up from the circulation 1 of ASM knockout mice primarily into liver and spleen, where it is directed to acidified organelles and can degrade SM (Miranda et al, 2000). This finding strongly suggests that enzyme replacement therapy by intravenous infusion of recombinant ASM is a feasible strategy for the treatment of the peripheral symptoms of Niemann-Pick disease as seen in both types A and B.…”

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confidence: 99%

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Safety Study of Adeno-Associated Virus Serotype 2-Mediated Human Acid Sphingomyelinase Expression in the Nonhuman Primate Brain

et al. 2012

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Niemann-Pick disease is a lysosomal storage disorder resulting from inherited deficiency in acid sphingomyelinase (ASM). Use of adeno-associated virus serotype 2 (AAV2) to deliver human acid sphingomyelinase (hASM) is currently being explored as a means to treat the devastating neurological features of NPD, which are refractory to traditional enzyme replacement therapy. In this study, we evaluated the long-term efficacy and safety of AAV2-hASM after direct infusion into the CNS of nonhuman primates. First, we confirmed the efficacy of AAV2-hASM in naive rats, which exhibited increased ASM expression and enzyme activity after infusion, without evidence of local or systemic toxicity. Next, the model was adapted to naive nonhuman primates (NHPs) with various doses of AAV2-hASM or saline delivered into the brainstem and both thalami. Strikingly, NHPs that received a high dose of AAV2-hASM displayed significant motor deficits that were not seen in lowdose animals in both the short-term (3-month) and long-term (9-month) treatment groups. In treated NHPs, ASM expression and activity were elevated with associated alterations in the sphingolipidomic profile in brain regions transduced with AAV2-hASM. Initial histological analysis indicated marked inflammatory reactions, and immunohistochemical analysis confirmed a robust inflammatory response. Importantly, pronounced upregulation of the chemokine CCL5, a target of ASM-mediated inflammatory signaling, was detected that correlated with the inflammatory response, providing a possible mechanism for hASM-associated toxicity. This study defines dose-dependent and dose-independent toxicities of AAV2-hASM in the naive primate brain, and reveals potential challenges in the design of a clinical trial.

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Section: Discussionmentioning

confidence: 98%

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confidence: 99%

Safety Study of Adeno-Associated Virus Serotype 2-Mediated Human Acid Sphingomyelinase Expression in the Nonhuman Primate Brain

et al. 2012

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“…Identity of Mature L-SMase-Acid SMase has been purified from a variety of different sources including human urine (27)(28)(29), human brain (30), human placenta (7,31,32), overexpression in CHO cells (33), and Sf21 insect cells (34). Importantly, aSMase purified from extracellular sources (e.g.…”

Section: Discussionmentioning

confidence: 99%

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Jenkins

Idkowiak‐Baldys

Simbari

et al. 2011

Journal of Biological Chemistry

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Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn 2؉ )-independent lysosomal aSMase (L-SMase) and a Zn 2؉ -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, Zn 2؉ -activated proenzyme to a mature 65 kDa, Zn 2؉ -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 Zn 2؉ -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 M r as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ⌬R608) 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.

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“…Cells were maintained in polypropylene tubes with 1 ml of RPMI 1640 medium supplemented with 10% fetal calf serum, antibiotics, and fungicide. After approximately 18 hours, purified recombinant ASM (1.0 g/ml, He et al, 1999) was added to one of the suspension culture tubes from each mouse. Superoxide production was measured as described above approximately 54 hours after addition of the enzyme.…”

Section: Superoxide Productionmentioning

confidence: 99%

Analysis of the Lung Pathology and Alveolar Macrophage Function in the Acid Sphingomyelinase–Deficient Mouse Model of Niemann-Pick Disease

Dhami

Gordon

et al. 2001

Laboratory Investigation

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SUMMARY:Types A and B Niemann-Pick disease (NPD) are lipid storage diseases caused by the deficient activity of the lysosomal enzyme, acid sphingomyelinase (ASM). Type B NPD is associated with progressive pulmonary function decline and frequent respiratory infections. ASM knock-out (ASMKO) mice are available as a model for NPD, but the lung pathology in these mice has not been adequately characterized. This study shows that by 10 weeks of age ASMKO mice have a significantly higher number of cells in their pulmonary airspaces than normal mice, consisting primarily of enlarged and often multinucleated macrophages. These mice also have much higher levels of sphingomyelin in their airspaces at 10 weeks of age, and both cell numbers and sphingomyelin concentrations remain elevated until 26 weeks of age. In these older mice an increased number of neutrophils is also seen. The alveolar cell population in the ASMKO mice produces less superoxide when stimulated, but this can be corrected by providing recombinant ASM to the culture media. Elevated levels of the chemokines macrophage inflammatory protein-2 and macrophage inflammatory protein-1␣ were also present in the bronchoalveolar lavage fluid of ASMKO mice, and this correlated with increased production of these chemokines by cultured macrophages and enhanced immunostaining in situ. Also, lung histology showed increased cellularity in the alveolar walls of ASMKO mice, but no evidence of fibrosis. Ultrastructural analysis of the lungs showed that the ASMKO mice have similar pathologic features to human NPD patients, with variable lipid storage evident in type I pneumocytes, endothelial cells, and airway ciliated epithelia. The alveolar macrophage, however, was the most dramatically affected cell type in both mice and humans. These studies indicate that the ASMKO mice can be used as a model to study the lung pathology associated with NPD, and demonstrate that the cellular and biochemical analysis of pulmonary airspaces may be a useful approach to monitoring disease progression and/or treatment. (Lab Invest 2001, 81:987-999).

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Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells

Cited by 91 publications

References 26 publications

Safety Study of Adeno-Associated Virus Serotype 2-Mediated Human Acid Sphingomyelinase Expression in the Nonhuman Primate Brain

Safety Study of Adeno-Associated Virus Serotype 2-Mediated Human Acid Sphingomyelinase Expression in the Nonhuman Primate Brain

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Analysis of the Lung Pathology and Alveolar Macrophage Function in the Acid Sphingomyelinase–Deficient Mouse Model of Niemann-Pick Disease

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