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Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis

Wu, Jun LU ; Hansen, GH; Nilsson, Åke LU and Duan, Rui-Dong LU (2005) In Biochemical Journal 386. p.153-160
Abstract
Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme... (More)
Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme activity. Immunogold labelling showed that the wild-type enzyme was mainly located in the plasma membrane, whereas the C-terminal domain-truncated enzyme was released into the medium. Deglycosylation blocked the release of the enzyme that accumulated in endosome-like structures. The enzyme activity was also decreased by mutations of the residues forming the putative metalbinding sites and the active core. Substitution of the active core sequence with that of NPP or mutation of T75 in the core abolished the enzyme activity against sphingomyel in but failed to render the enzyme NPP active. Our results indicate that alk-SMase activity is severely affected by defective N-glycosylation and structural alterations of the putative metal-binding sites and the predicted active core. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
phosphodiesterase, nucleotide, mutation, glycosylation, alkaline sphingomyelinase, cancer
in
Biochemical Journal
volume
386
pages
153 - 160
publisher
Portland Press Limited
external identifiers
  • pmid:15458386
  • wos:000227232900017
  • scopus:14244267514
ISSN
0264-6021
DOI
10.1042/BJ20041455
language
English
LU publication?
yes
id
2378b503-830d-4173-a2df-48246514768f (old id 252116)
alternative location
http://www.biochemj.org/bj/386/0153/3860153.pdf
date added to LUP
2007-08-13 16:52:33
date last changed
2017-05-21 04:18:20
@article{2378b503-830d-4173-a2df-48246514768f,
  abstract     = {Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme activity. Immunogold labelling showed that the wild-type enzyme was mainly located in the plasma membrane, whereas the C-terminal domain-truncated enzyme was released into the medium. Deglycosylation blocked the release of the enzyme that accumulated in endosome-like structures. The enzyme activity was also decreased by mutations of the residues forming the putative metalbinding sites and the active core. Substitution of the active core sequence with that of NPP or mutation of T75 in the core abolished the enzyme activity against sphingomyel in but failed to render the enzyme NPP active. Our results indicate that alk-SMase activity is severely affected by defective N-glycosylation and structural alterations of the putative metal-binding sites and the predicted active core.},
  author       = {Wu, Jun and Hansen, GH and Nilsson, Åke and Duan, Rui-Dong},
  issn         = {0264-6021},
  keyword      = {phosphodiesterase,nucleotide,mutation,glycosylation,alkaline sphingomyelinase,cancer},
  language     = {eng},
  pages        = {153--160},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis},
  url          = {http://dx.doi.org/10.1042/BJ20041455},
  volume       = {386},
  year         = {2005},
}