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Screening and purification of bioactive peptides with potential to activate the cholecystokinin receptor type 1

(2013)
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Abstract
Obesity is a world-wide health problem with tremendous health care costs. Weight maintenance is a complex system in which different mechanisms are involved. One of these mechanisms involves the cholecystokinin receptor type 1 (CCK1R). The CCK1R is a GPCR (G-protein coupled receptor) localized in the gastrointestinal tract that induces a feeling of satiety upon activation by its natural hormone cholecystokinin (CCK). Bioactive peptides, which can be released from food protein, can mimic the effect of CCK and have an influence on satiety. Such peptides could be used as a satiating ingredient in the development of new functional foods for the prevention and treatment of obesity. We set up a cell-based bioassay in 96 well-plates to screen for such bioactive peptides that can activate the CCK1R, based on the fluorescent visualization of a Ca-flux when the receptor is activated. Fluorescence measurements were done using a plate reader and a confocal microscope and the assay was benchmarked with CCK-8S (sulfated octapeptide), JMV-180 and lorglumide. The confocal microscope appeared to be the preferred measuring device when complex samples had to be measured, as measurements with the plate reader could easily be biased by background fluorescence of the sample. Screening of different food protein hydrolysates showed that some food protein hydrolysates , such as soy protein hydrolysates, possess significant CCK1R activity. The peptides in the active soy protein hydrolysates were separated by use of size exclusion chromatography, the CCK1R activity of the resulting fractions was re-evaluated and significant in vitro CCK1R activity was found. The effect on food intake of the active fractions with a physiological relevant molecular weight was evaluated in vivo with rats, but no significant effect could be measured. The amino acid sequences of the peptides present in some promising fractions was identified, however it remained not possible to identify which particular peptide(s) accounted for the CCK1R activity as more than 100 peptides were still present in the fractions. Hence, a highly-selective tool to extract and identify the active peptides was necessary. Therefore, a first onset was made to incorporate the CCK1R into NABBs (nanoscale apo-lipoprotein bound bilayer particles), a unique native-like bilayer membrane system for incorporation of GPCRs, as such nanoparticles could be used as an affinity-selection-mass spectrometry technique to identify CCK1R-binding peptides. Functional incorporation of the CCK1R in NABBs was shown by binding with a fluorescent labeled CCK analog.
Keywords
calcium flux, bioactive peptides, soy protein hydrolysate, NABBs, cholecystokinin, cholecystokinin receptor type 1, gel filtration chromatography, affinity chromatography, fluo 4 am

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Citation

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MLA
Staljanssens, Dorien. Screening and Purification of Bioactive Peptides with Potential to Activate the Cholecystokinin Receptor Type 1. Ghent University. Faculty of Bioscience Engineering, 2013.
APA
Staljanssens, D. (2013). Screening and purification of bioactive peptides with potential to activate the cholecystokinin receptor type 1. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Chicago author-date
Staljanssens, Dorien. 2013. “Screening and Purification of Bioactive Peptides with Potential to Activate the Cholecystokinin Receptor Type 1.” Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
Chicago author-date (all authors)
Staljanssens, Dorien. 2013. “Screening and Purification of Bioactive Peptides with Potential to Activate the Cholecystokinin Receptor Type 1.” Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
Vancouver
1.
Staljanssens D. Screening and purification of bioactive peptides with potential to activate the cholecystokinin receptor type 1. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2013.
IEEE
[1]
D. Staljanssens, “Screening and purification of bioactive peptides with potential to activate the cholecystokinin receptor type 1,” Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium, 2013.
@phdthesis{4129242,
  abstract     = {{Obesity is a world-wide health problem with tremendous health care costs. Weight maintenance is a complex system in which different mechanisms are involved. One of these mechanisms involves the cholecystokinin receptor type 1 (CCK1R). The CCK1R is a GPCR (G-protein coupled receptor) localized in the gastrointestinal tract that induces a feeling of satiety upon activation by its natural hormone cholecystokinin (CCK). Bioactive peptides, which can be released from food protein, can mimic the effect of CCK and have an influence on satiety. Such peptides could be used as a satiating ingredient in the development of new functional foods for the prevention and treatment of obesity.
We set up a cell-based bioassay in 96 well-plates to screen for such bioactive peptides that can activate the CCK1R, based on the fluorescent visualization of a Ca-flux when the receptor is activated. Fluorescence measurements were done using a plate reader and a confocal microscope and the assay was benchmarked with CCK-8S (sulfated octapeptide), JMV-180 and lorglumide. The confocal microscope appeared to be the preferred measuring device when complex samples had to be measured, as measurements with the plate reader could easily be biased by background fluorescence of the sample. Screening of different food protein hydrolysates showed that some food protein hydrolysates , such as soy protein hydrolysates, possess significant CCK1R activity. 
The peptides in the active soy protein hydrolysates were separated by use of size exclusion chromatography, the CCK1R activity of the resulting fractions was re-evaluated and significant in vitro CCK1R activity was found. The effect on food intake of the active fractions with a physiological relevant molecular weight was evaluated in vivo with rats, but no significant effect could be measured. The amino acid sequences of the peptides present in some promising fractions was identified, however it remained not possible to identify which particular peptide(s) accounted for the CCK1R activity as more than 100 peptides were still present in the fractions. Hence, a highly-selective tool to extract and identify the active peptides was necessary. Therefore, a first onset was made to incorporate the CCK1R into NABBs (nanoscale apo-lipoprotein bound bilayer particles), a unique native-like bilayer membrane system for incorporation of GPCRs, as such nanoparticles could be used as an affinity-selection-mass spectrometry technique to identify CCK1R-binding peptides. Functional incorporation of the CCK1R in NABBs was shown by binding with a fluorescent labeled CCK analog.}},
  author       = {{Staljanssens, Dorien}},
  isbn         = {{9789059896451}},
  keywords     = {{calcium flux,bioactive peptides,soy protein hydrolysate,NABBs,cholecystokinin,cholecystokinin receptor type 1,gel filtration chromatography,affinity chromatography,fluo 4 am}},
  language     = {{eng}},
  pages        = {{XXI, 178}},
  publisher    = {{Ghent University. Faculty of Bioscience Engineering}},
  school       = {{Ghent University}},
  title        = {{Screening and purification of bioactive peptides with potential to activate the cholecystokinin receptor type 1}},
  year         = {{2013}},
}