title: 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 degrees 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 (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal 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.
authors.affiliation: Cell and Protein Therapeutics R & D Department, Genzyme Corp., Framingham, Massachusetts 01701, USA. huawei.qiu@genzyme.com
authors.forename: Huawei
authors.initials: H
authors.lastname: Qiu
authors.forename: Tim
authors.initials: T
authors.lastname: Edmunds
authors.forename: Jennifer
authors.initials: J
authors.lastname: Baker-Malcolm
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Karey
authors.forename: Scott
authors.initials: S
authors.lastname: Estes
authors.forename: Cordula
authors.initials: C
authors.lastname: Schwarz
authors.forename: Heather
authors.initials: H
authors.lastname: Hughes
authors.forename: Scott M
authors.initials: SM
authors.lastname: Van Patten
mesh_terms: D000818:Animals; D016466:CHO Cells; D002268:Carboxypeptidases / Q000737:chemistry; D043402:Cathepsin A; D003300:Copper / Q000737:chemistry; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D015032:Zinc / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003300:Copper; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A; D015032:Zinc; D003545:Cysteine