Tissue-specific collagen hydroxylation at GEP/GDP triplets mediated by P4HA2

Collagen, the most abundant organic compound of vertebrate organisms, is a supramolecular, protein-made polymer. Details of its 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 post-translational modifications of collagen extracted from bone, skin, and tendon were compared, revealing lower hydroxylation of most GEP/GDP triplets, together with fewer other residue positions along collagen chains, in the tendon. This regulation is mostly conserved in two distant homeotherm species, mouse and chicken. The comparison of detailed P4H patterns in both species suggests a dual mechanism of specificity. P4ha2 expression is low in tendon and its genetic invalidation in the ATDC5 cellular model of collagen assembly specifically mimics the tendon-related P4H profile. Therefore, P4HA2 has a better ability than other P4HAs to hydroxylate the corresponding residue positions. Its local expression participates in determining the P4H profile, a novel aspect of the tissue specificities of collagen assembly. Data availabilityProteomics data are available via ProteomeXchange with the identifier PXD039221. Reviewer account details: login: MSV000091002_reviewer password: P4HA2tendon.