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Identification of proteins released by mammalian cells that mediate DNA internalization through proteoglycan-dependent macropinocytosis

Wittrup, Anders LU ; Sandgren, Staffan LU ; Welch, Johanna LU ; Welinder, Charlotte LU ; Gustavsson, Niklas LU ; Mörgelin, Matthias LU and Belting, Mattias LU (2007) In Journal of Biological Chemistry 282(38). p.27897-27904
Abstract
Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the, molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA. The apparent paradox was explained by the action of DNA-transporting proteins present in conditioned medium. Complexes between these proteins and DNA require proteoglycans for cellular entry. Mass spectrometry analysis... (More)
Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the, molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA. The apparent paradox was explained by the action of DNA-transporting proteins present in conditioned medium. Complexes between these proteins and DNA require proteoglycans for cellular entry. Mass spectrometry analysis of cell medium components identified several proteins previously shown to associate with DNA and to participate in membrane transport of macromolecular cargo. The major pathway for proteoglycan-dependent DNA uptake was macropinocytosis, whereas caveolae-dependent and clathrin-dependent pathways were not involved, as determined by using caveolin-1 knock-out cells, dominant-negative constructs for dynamin and Eps15, and macropinocytosis- disruptive drugs, as well as confocal fluorescence co-localization studies. Importantly, a significant fraction of internalized DNA was translocated to the nucleus for expression. Our results provide novel insights into the mechanism of DNA uptake by mammalian cells and extend the emerging role of proteoglycans in macromolecular transport. (Less)
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Contribution to journal
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published
subject
keywords
Animals, Biological Transport, CHO Cells, Cell Nucleus, Cricetinae, Cricetulus, Culture Media, DNA, Humans, Mass Spectrometry, Microscopy, Confocal, Microscopy, Fluorescence, Pinocytosis, Polymers, Proteoglycans, Journal Article, Research Support, Non-U.S. Gov't
in
Journal of Biological Chemistry
volume
282
issue
38
pages
8 pages
publisher
ASBMB
external identifiers
  • wos:000249455600038
  • scopus:34948813707
ISSN
1083-351X
DOI
10.1074/jbc.M701611200http://dx.doi.org/10.1074/jbc.M701611200
language
English
LU publication?
yes
id
84521489-4ad8-4604-b23e-ac1f85800edc (old id 656064)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=17623661&dopt=Abstract
date added to LUP
2007-12-19 09:19:25
date last changed
2017-07-02 03:42:02
@article{84521489-4ad8-4604-b23e-ac1f85800edc,
  abstract     = {Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the, molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA. The apparent paradox was explained by the action of DNA-transporting proteins present in conditioned medium. Complexes between these proteins and DNA require proteoglycans for cellular entry. Mass spectrometry analysis of cell medium components identified several proteins previously shown to associate with DNA and to participate in membrane transport of macromolecular cargo. The major pathway for proteoglycan-dependent DNA uptake was macropinocytosis, whereas caveolae-dependent and clathrin-dependent pathways were not involved, as determined by using caveolin-1 knock-out cells, dominant-negative constructs for dynamin and Eps15, and macropinocytosis- disruptive drugs, as well as confocal fluorescence co-localization studies. Importantly, a significant fraction of internalized DNA was translocated to the nucleus for expression. Our results provide novel insights into the mechanism of DNA uptake by mammalian cells and extend the emerging role of proteoglycans in macromolecular transport.},
  author       = {Wittrup, Anders and Sandgren, Staffan and Welch, Johanna and Welinder, Charlotte and Gustavsson, Niklas and Mörgelin, Matthias and Belting, Mattias},
  issn         = {1083-351X},
  keyword      = {Animals,Biological Transport,CHO Cells,Cell Nucleus,Cricetinae,Cricetulus,Culture Media,DNA,Humans,Mass Spectrometry,Microscopy, Confocal,Microscopy, Fluorescence,Pinocytosis,Polymers,Proteoglycans,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  month        = {09},
  number       = {38},
  pages        = {27897--27904},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Identification of proteins released by mammalian cells that mediate DNA internalization through proteoglycan-dependent macropinocytosis},
  url          = {http://dx.doi.org/10.1074/jbc.M701611200http://dx.doi.org/10.1074/jbc.M701611200},
  volume       = {282},
  year         = {2007},
}