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Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases.

Christine Rempfer1,2, Sebastian N W Hoernstein1, Nico van Gessel1

  • 1Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104 Freiburg, Germany.

Computational and Structural Biotechnology Journal
|July 18, 2024
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Summary

This study identifies six moss prolyl-4-hydroxylases (P4Hs) and reveals hydroxylation patterns in plant proteins. P4H1 knockout significantly reduced O-glycosylation in moss-produced erythropoietin (EPO).

Keywords:
BiopharmaceuticalErythropoietinO-glycosylationPlant-made pharmaceuticalPosttranslational protein modificationProlyl-hydroxylation

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Area of Science:

  • Plant biochemistry
  • Protein modification
  • Enzymology

Background:

  • Prolyl-4-hydroxylases (P4Hs) mediate proline hydroxylation, crucial for plant glycoproteins but undesirable in pharmaceuticals.
  • Existing proline hydroxylation motifs lack data from mosses like Physcomitrella.

Purpose of the Study:

  • To identify moss P4Hs and characterize proline hydroxylation patterns in Physcomitrella.
  • To investigate the role of P4Hs in O-glycosylation of plant-made pharmaceuticals like erythropoietin (EPO).

Main Methods:

  • Phylogenetic reconstruction to identify six moss P4Hs.
  • Mass spectrometry analysis of 73 hydroxyprolines (Hyps) in 24 secretory proteins.
  • AlphaFold modeling and quantitative proteomics on P4H knockout mutants.
  • Quantitative RT-PCR to analyze gene expression.

Main Results:

  • Proline hydroxylation preferentially occurred near other prolines, alanine, serine, threonine, and valine (AOV motif most frequent).
  • 95% of Hyps were predictable using established methods; hydroxylation occurred on protein surfaces in disordered regions.
  • Moss-produced EPO showed O-glycosylation, significantly reduced in a p4h1 knockout mutant.
  • P4H knockouts led to specific protein amount changes and modified hydroxylation patterns, indicating differential P4H functions.

Conclusions:

  • Moss P4Hs exhibit specific hydroxylation preferences and functions.
  • Targeting P4H1 can reduce undesirable O-glycosylation in plant-made pharmaceuticals.
  • Differential gene expression suggests compensatory mechanisms among P4H genes.