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Tissue-regulated collagen hydroxylation at GEP/GDP motifs mediated by P4HA2

ABSTRACT: Collagen, the most abundant organic compound of vertebrate organisms, is a supramolecular protein-made polymer. Details of its subtle post-translational maturation largely determine the mechanical properties of connective tissues. Its assembly requires massive, heterogeneous prolyl-4-hydroxylation (P4H), catalyzed by Prolyl-4-hydroxylases (P4HA1-3), providing thermostability to its elemental, triple helical building block. So far, there was no evidence of tissue-specific regulation of P4H, nor of a differential substrate repertoire of P4HAs. Here, the positional P4H profiles of collagen extracted from bone, skin, and tendon were compared, revealing lower hydroxylation of most GEP/GDP motifs in the tendon, across homeotherms. P4HA2 mRNA was found low in tendon. Invalidation of P4HA2, unlike decreased, generalized P4H activity, in the ATDC5 cellular model of collagen assembly, mimicked the tendon-related P4H profile. Therefore, P4HA2 has a better ability than other P4HAs to hydroxylate GEP/GDP motifs and the differential expression of P4HAs in tissues dictates the positional P4H profile of collagen, which participates in determining tissue specificities of its assembly.

INSTRUMENT(S): autoflex TOF/TOF

ORGANISM(S): Gallus Gallus (ncbitaxon:9031) Mus Musculus (ncbitaxon:10090)

SUBMITTER: vincourt

PROVIDER: MSV000091002 | MassIVE | Tue Jan 03 05:27:00 GMT 2023

SECONDARY ACCESSION(S): PXD039221

REPOSITORIES: MassIVE

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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.

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Tissue-regulated collagen hydroxylation at GEP/GDP motifs mediated by P4HA2

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