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J. Biol. Chem., Vol. 275, Issue 36, 28316-28325, September 8, 2000
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From the The sequence similarity with bacterial neutral
sphingomyelinase resulted in the isolation of putative mammalian
counterparts and, subsequently, identification of similar molecules in
a number of other eukaryotic organisms. Based on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and performed site-directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization. Modification of histidine residues and the substrate protection experiments demonstrated the
presence of reactive histidine residues within the active site. Site
directed mutagenesis suggested an essential role in catalysis for two
histidine residues (His-136 and His-272), which are conserved in all
sequences. Mutations of two additional histidines (His-138 and
His-151), conserved only in eukaryotes, resulted in reduced neutral
sphingomyelinase activity. In addition to sphingomyelin, the enzyme
also hydrolyzed lysophosphatidylcholine. Exposure to an oxidizing
environment or modification of cysteine residues using several specific
compounds also inactivated the enzyme. Site-directed mutagenesis of
eight cysteine residues and gel-shift analysis demonstrated that these
residues did not participate in the catalytic reaction and suggested
the involvement of cysteines in the formation/breakage of disulfide
bonds, which could underlie the reversible inactivation by the
oxidizing compounds. Cellular localization studies of a series of
deletion mutants, expressed as green fluorescent protein fusion
proteins, demonstrated that the transmembrane region contains
determinants for the endoplasmic reticulum localization.
Structural Requirements for Catalysis and Membrane Targeting
of Mammalian Enzymes with Neutral Sphingomyelinase and Lysophospholipid
Phospholipase C Activities
ANALYSIS BY CHEMICAL MODIFICATION AND SITE-DIRECTED
MUTAGENESIS*
,,,
Cancer Research Campaign Centre for Cell and
Molecular Biology, Chester Beatty Laboratories, Institute of Cancer
Research, Fulham Road, London SW3 6JB, United Kingdom and the
¶ Laboratoire de Biochimie, INSERM U466, CHU Rangueil,
31403 Toulouse Cedex 4, France
*
This work was supported by the Wellcome Trust and the Cancer
Research Campaign.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Cancer Research
Campaign Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, Fulham Road, London SW3
6JB, United Kingdom. Tel.: 44-207-352-8133; Fax: 44-207-352-3299; E-mail: matilda@icr.ac.uk.
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