Abstract
Type I collagen is the main structural protein in vertebrates and undergoes extensive post-translational modification (PTM) during biosynthesis. Prolyl-3-hydroxylase 1 (P3H1) catalyzes collagen prolyl-3-hydroxylation and functions as a collagen chaperone. Loss of P3H1 causes osteogenesis imperfecta, and P3H1 is consistently upregulated in idiopathic pulmonary fibrosis. However, the full impact of P3H1 deficiency on the collagen biosynthesis machinery, including PTMs, is not known. Here, we comprehensively investigated the consequences of P3H1 deficiency in two independent models: type I collagen from P3H1 KO mouse tail tendon and type I collagen from primary human lung fibroblasts following P3H1 knockdown. Using amino acid analysis, high-resolution tandem mass spectrometry for site-specific PTM and quantification, and gene expression analysis, we show that P3H1 deficiency profoundly disrupts the collagen PTM network. Amino acid analysis revealed global overmodification of prolines and lysines. Site-resolved tandem mass spectrometry analysis confirmed the P3H1-dependent 3-hydroxyproline site COL1A1-P1153 and demonstrated widespread increases in prolyl-3-hydroxylation, prolyl-4-hydroxylation, and lysyl modification in P3H1-deficient tendon. In both models, prolyl-4-hydroxylation frequency was increased at multiple sites, indicating that loss of P3H1 alters local modification kinetics and/or collagen chain accessibility, thereby rapidly promoting prolyl-4-hydroxylation. P3H1 deficiency also led to compensatory increases of P3H2 and P3H3 protein levels. Gene expression analyses revealed selective upregulation of collagen biosynthetic enzymes at the transcript level, including P4ha2 and Lh2 in mouse tendon and P3H2 in human fibroblasts, suggesting feedback mechanisms linking perturbation of collagen biosynthesis to nuclear transcriptional control. Taken together, this study emphasizes the essential role of P3H1 in collagen quality control.