Dataset Information

Production of recombinant human type III collagen in Physcomitrella

ABSTRACT: We produced a 30 kDa His-tagged human collagen polypeptide in Physcomitrella moss plants.

INSTRUMENT(S):

ORGANISM(S): Physcomitrella Patens Subsp. Patens (moss)

TISSUE(S): Protonema

SUBMITTER: Sebastian Hoernstein

LAB HEAD: Ralf Reski

PROVIDER: PXD071359 | Pride | 2026-02-09

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	F003783.mgf	Mgf
	ORBI-RSLC037310.raw	Raw
	R_No134_Collagen_Tryp_45kDa_HisTrap_F003783.csv	Csv
	R_No134_Collagen_Tryp_45kDa_HisTrap_F003783.dat	Other
	R_No134_Collagen_Tryp_45kDa_HisTrap_F003783.mzid	Mzid

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Publications

Secretion-based production of prolyl-hydroxylated human type III collagen in scalable Physcomitrella photobioreactors.

Bohlender Lennard L LL Parsons Juliana J Mitgau Antonia A Hoernstein Sebastian N W SNW Grigolon Giovanna G Henes Bernhard B Decker Eva L EL Reski Ralf R

Plant cell reports 20260128 2

<h4>Key message</h4>Scalable moss bioreactors enable the production of high-quality recombinant prolyl-hydroxylated human collagen without heterologous P4H expression, offering a sustainable and vegan alternative to conventional collagens derived from animals. Collagens are structural proteins of the extracellular matrix essential for skin elasticity and integrity. They are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, ...[more]

PMID: 41604015

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Project description:The hydroxylation of proline residues to 4-trans-hydroxyproline (Hyp) is a common post-translational protein modification in plants, mediated by prolyl 4-hydroxylases (P4Hs). Hyps predominantly occur in a group of cell wall proteins, the Hydroxyproline-rich glycoproteins (HRGPs), where they are frequently O-glycosylated. While prolyl-hydroxylation and O-glycosylation are important, e.g. for cell wall stability, they are not desirable in plant-made pharmaceuticals. Sequence motifs recognized for prolyl-hydroxylation derived from vascular plants were proposed but did not include data from mosses, such as Physcomitrella. Here, a phylogenetic reconstruction of plant P4Hs identified six P4Hs in four subfamilies in mosses. We analysed the amino acid sequences and structural environments around Hyps in Physcomitrella utilizing 73 Hyp sites in 24 secretory proteins from multiple MS/MS datasets, and found that prolines in close proximity to other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of the Hyp sites were predictable with a combination of previously defined motifs and methods. In our data, AOV (Ala-Hyp-Val) was the most frequent prolyl-hydroxylation pattern. Additionally, short arabinose chains were attached to Hyps in two cell-wall pectinesterases. A combination of 443 AlphaFold structure models and our MS data of peptides with nearly 3000 proline sites found Hyps predominantly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited plant-specific O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a P4H1 single knock-out (KO) Physcomitrella mutant. Quantitative proteomics after isotope labelling with different P4H-KO moss mutants revealed specific changes in the amount of proteins, including HRGPs, and a modified prolyl-hydroxylation pattern from the mutants, suggesting a differential function of the six Physcomitrella P4Hs. Quantitative RT-PCR proved a differential effect of single P4H KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 and a peptide substrate suggested significant amino acids in the active centre of the enzyme that form H-bonds with the peptide substrate, and revealed differences between P4H1 and the other five Physcomitrella P4Hs.