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The expression system affects the binding affinity between p75NTR and proNGF.

Mami Hino1, Masayuki Nakanishi1, Hiroshi Nomoto1

  • 1Laboratory of Biochemistry, School of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime, 790-8578, Japan.

Biochemistry and Biophysics Reports
|April 10, 2024
PubMed
Summary

Pro-nerve growth factor (proNGF) exhibits dual effects on neurons, influenced by its binding affinity to p75 neurotrophin receptor (p75NTR). This study compared recombinant proNGF and p75NTR affinities across bacterial, insect, and mammalian systems, revealing significant differences likely due to post-translational modifications.

Keywords:
Binding affinityGlycoformProtein expression systemp75NTRproNGF

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Pro-nerve growth factor (proNGF) is a precursor to nerve growth factor (NGF) with opposing effects on neuronal cells.
  • The biological activity of proNGF is determined by the balance of its interactions with TrkA and p75 neurotrophin receptor (p75NTR).
  • Understanding the binding affinity of proNGF to p75NTR is crucial for deciphering its signaling pathways.

Purpose of the Study:

  • To compare the binding affinity of recombinant proNGF to p75NTR across different expression systems (bacterial, insect, mammalian).
  • To investigate the influence of expression system and potential post-translational modifications on proNGF-p75NTR interactions.
  • To elucidate the factors affecting the differential signaling of proNGF.

Main Methods:

  • Recombinant proNGF and extracellular domain of p75NTR were expressed in bacterial, insect, and mammalian cell systems.
  • Binding affinities between recombinant proNGF and p75NTR were measured using consistent experimental methods.
  • Analysis included SDS-PAGE, glycosidase treatments, and lectin binding to assess post-translational modifications, particularly glycosylation.

Main Results:

  • Insect and mammalian p75NTR bound native mature NGF with higher affinity for the insect receptor.
  • Recombinant proNGF from all three systems exhibited neuritogenic activity in PC12 cells.
  • Insect p75NTR demonstrated higher binding affinity for proNGF compared to mammalian p75NTR, with a preference for insect-derived proNGF.
  • Binding affinity varied: insect p75NTR bound insect proNGF > mammalian proNGF > bacterial proNGF.
  • Mammalian p75NTR showed no such preference for proNGF source.
  • Differences in mobility on SDS-PAGE and reactivity with glycosidases/lectins indicated variations in post-translational modifications, likely glycosylation.

Conclusions:

  • The affinity of proNGF to p75NTR is significantly influenced by the expression system used for protein production.
  • Post-translational modifications, especially glycosylation, play a critical role in modulating the binding affinity and potentially the biological activity of recombinant proNGF and p75NTR.
  • These findings highlight the importance of considering protein source and modifications when studying protein-ligand interactions in neurotrophin signaling.