Activation of Human Acid Sphingomyelinase through Modification or Deletion of C-terminal Cysteine

Abstract

One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at ؊20°C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys 629 ). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by Cterminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

TRAIL activates acid sphingomyelinase via a redox mechanism and releases ceramide to trigger apoptosis

“…This notion is consistent with a previous biochemical study (Qiu et al, 2003) indicating that oxidation of purified ASM at cysteine 629 results in stimulation and a disulfide bond-mediated dimerization A central role of ROS for the induction of apoptosis by CD95 has been previously reported (Gulbins et al, 1996;Um et al, 1996) and was recently confirmed by several studies (Reinehr et al, 2005;Yin, 2005). Experiments on hepatocytes demonstrated that an inhibitor of the ASM blocks the release of ROS suggesting that ROS functions downstream of the ASM (Reinehr et al, 2005;Yin, 2005).…”

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

“…However, further details of the stimulation of neutral sphingomyelinase are presently not known. The acid sphingomyelinase is activated by oxidation [43, 50, 72]. It is unknown whether the protein is directly oxidized in vivo or regulated via pathways that are redox-sensitive.…”

“…It is unknown whether the protein is directly oxidized in vivo or regulated via pathways that are redox-sensitive. Scavengers of reactive oxygen intermediates prevent activation of the acid sphingomyelinase by stimuli such as DR5 or Cu 2+ [43, 50, 72]. In vitro studies by Qui et al .…”

“…In vitro studies by Qui et al . [72] on purified acid sphingomyelinase suggest a direct oxidation of the acid sphingomyelinase at cysteine residue 629 with a subsequent copper-promoted dimerisation of the acid sphingomyelinase [72]. The oxidation of cysteine 629 correlated with an activation of the enzyme in vitro .…”

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

“…It has previously been suggested that carboxyl-terminal modification of aSMase might serve as a mechanism to regulate enzyme activity. Qiu et al (8) described a mechanism whereby oxidation, mutation, and/or deletion of the C-terminal Cys 629 resulted in activation of the enzyme. Based on these results the authors postulated that loss of the C-terminal Cys 629 might serve as a "cysteine switch," as has been described for matrix metalloproteinases (9), whereby loss of C-terminal Cys residues favors hydration of Zn 2ϩ thereby promoting enzyme activation (8).…”