P4HA1 and P4HA2 accept both shared and isoenzyme-specific substrate residues in type I collagen to directly and indirectly orchestrate its post-translational modification landscape
Type I collagen, the most abundant protein of vertebrate organisms, is polymerized into a macroscopic-scale fibrillar network, the detailed arrangement of which is controlled by numerous post-translational modifications (PTMs) to determine the biomechanics of connective tissues. Its most frequent post-translational modification, prolyl-4-hydroxylation (P4H), catalyzed by prolyl-4-hydroxylases (P4HAs), occupies about 100 residue positions per procollagen alpha chain and is crucial to stabilize its triple helical building block thermodynamically. Recently, the two main P4HAs, P4HA1 and P4HA2, were found to exhibit mutual substrate specificity based on the nature of the amino acid residue standing N-terminally to the considered substrate residue. Independently, the activities of both enzymes were suggested to influence lysyl residue hydroxylation and glycosylation. To clarify these findings, we performed here a proteome-, tripeptide- and amino acid-scale analysis of prolyl and lysyl modifications of collagens, both in the established ATDC5 cell line and in primary mouse skin fibroblasts after adenovirus-mediated Crispr/Cas9 invalidation of either or both P4ha1 and/or P4ha2 genes. We conclude that both enzymes have no more than 18 and 14 respective specific substrate positions among more than 200 potential P4H sites available on type I collagen alpha chains. Combining procollagen tryptic peptide and tripeptide analysis, we confirm that glutamate localized N-terminally to potential P4H sites determines the substrate specificity of P4HA2. P4HA1-specific sites are instead preceded by phenylalanine, lysine, arginine, asparagine or histidine. Furthermore, we evidence that P4H is not required for prolyl-3-hydroxylation at the so-called A1 site and demonstrate that it reduces lysyl glycosylation at multiple sites in a cell-type-dependent manner. These findings highlight the strategic molecular function of P4HA1 and P4HA2 within the collagen maturation machinery, for, together, steering subtle collagen post-translational modifications.