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Identification of paracrine neuroprotective candidate proteins by a functional assay-driven proteomics approach

Hauck, Stefanie M.; Gloeckner, Christian J.; Harley, Margaret E.; Schoeffmann, Stephanie; Boldt, Karsten; Ekström, Per LU and Ueffing, Marius (2008) In Molecular & Cellular Proteomics 7(7). p.1349-1361
Abstract
Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell... (More)
Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Toward this goal we first isolated conditioned medium (CM) from primary porcine RMGs cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins toward their identification consists of several fractionation steps combined with tests for biological function. Here we combined 1) size fractionation, 2) ion exchange chromatography, 3) reverse phase liquid chromatography, and 4) mass spectrometry (Q-TOF MS/MS or MALDI MS/MS) for protein identification. As a result of this integrated work flow, the insulin-like growth factor-binding proteins IGFBP5 and IGFBP7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7, significantly increased photoreceptor survival when secreted from HEK293 cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multifactorial with IGFBP5 and CTGF as an integral part of this activity. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Molecular & Cellular Proteomics
volume
7
issue
7
pages
1349 - 1361
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • wos:000257463000012
  • scopus:47849090253
ISSN
1535-9484
DOI
10.1074/mcp.M700456-MCP200
language
English
LU publication?
yes
id
a035ab04-a2b9-4fa1-8903-ce5e6adf3d0d (old id 1256969)
date added to LUP
2008-10-15 09:16:38
date last changed
2017-01-19 15:59:46
@article{a035ab04-a2b9-4fa1-8903-ce5e6adf3d0d,
  abstract     = {Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Toward this goal we first isolated conditioned medium (CM) from primary porcine RMGs cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins toward their identification consists of several fractionation steps combined with tests for biological function. Here we combined 1) size fractionation, 2) ion exchange chromatography, 3) reverse phase liquid chromatography, and 4) mass spectrometry (Q-TOF MS/MS or MALDI MS/MS) for protein identification. As a result of this integrated work flow, the insulin-like growth factor-binding proteins IGFBP5 and IGFBP7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7, significantly increased photoreceptor survival when secreted from HEK293 cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multifactorial with IGFBP5 and CTGF as an integral part of this activity.},
  author       = {Hauck, Stefanie M. and Gloeckner, Christian J. and Harley, Margaret E. and Schoeffmann, Stephanie and Boldt, Karsten and Ekström, Per and Ueffing, Marius},
  issn         = {1535-9484},
  language     = {eng},
  number       = {7},
  pages        = {1349--1361},
  publisher    = {American Society for Biochemistry and Molecular Biology},
  series       = {Molecular & Cellular Proteomics},
  title        = {Identification of paracrine neuroprotective candidate proteins by a functional assay-driven proteomics approach},
  url          = {http://dx.doi.org/10.1074/mcp.M700456-MCP200},
  volume       = {7},
  year         = {2008},
}