P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA

Abstract

Edited by Xiao-Fan WangTandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both The collagen family of proteins has evolved into many different genes and gene translational products each with variably regulated post-translational modifications (1, 2). Even within a single genetic type of collagen, the post-translational quality of the protein is known to be regulated with a high degree of tissue specificity (3, 4). The functional significance of this post-translational variability is still not fully understood, although for fibril-forming collagens the number, placement, and chemistry of covalent intermolecular cross-links seem to be critically important regulators of tissue material properties and function (5-7).In the last decade, new insights on the significance of a relatively rare collagen modification, prolyl 3-hydroxylation, came from the discovery that recessive forms of osteogenesis imperfecta (OI) 2 are caused by biallelic mutations in prolyl 3-hydroxylase 1 (P3H1; Lepre1), CRTAP (Leprel3), or CypB (PPIB) (8). These proteins, which form a P3H1 enzyme complex, act on nascent collagen chains in the ER (9). Mutations in at least 6 further genes that encode either enzymes (e.g. PLOD2 encoding lysyl hydroxylase-2 (LH2) (10)) or chaperones (e.g. FKBP10 encoding FKBP65 (11, 12)), needed for collagen modification, folding, transport, and normal mineralization, have been shown to cause OI variants. We have gained insights on the disease-causing mechanisms by analyzing tissue collagens from OI patients and mouse models of OI. For example, P3H1, CRTAP, and PPIB mutations all cause telopeptide lysine overhydroxylation (5), whereas PLOD2 or FKBP10 mutations cause telopeptide lysine under-hydroxylation (10 -12). A common effect from all these mutations is an altered collagen cross-linking chemistry. These findings suggest an interplay in the ER between the prolyl 3-hydroxylation complex and the lysyl hydroxylation machinery.Human mutations in P3H2 (Leprel1) have been shown to cause the eye disorder high myopia (13-15). Two different P3H2 knock-out mice have so far been generated by separate laboratories, the first was embryonic lethal (16), whereas the second had no obvious phenotype (17). Mass spectral analysis of...

Re-evaluation of lysyl hydroxylation in the collagen triple helix: lysyl hydroxylase 1 and prolyl 3-hydroxylase 3 have site-differential and collagen type-dependent roles in lysine hydroxylation

“…To confirm that the expression of P3H3 protein was abolished, Western blotting was performed using a whole kidney lysate and the protein signal corresponding to MW of P3H3 (79 kDa) was absent in the P3H3 null lysate (Figure 1C). As previously reported [18], P3H3 null mice were also viable and we did not observe any obvious growth or skeletal phenotypes by growth curves and X-ray images, respectively (Figure 1D and E). LH1 null mice were generated and characterized previously [26].…”

“…The α1 and α2 chain of type I collagen contain multiple lysyl hydroxylation sites in their triple helical sequences [30–32]. A previous report of P3H3 null mice [18] only demonstrated lysyl hydroxylation sites on lysine-87 (K87) in both the α1 and α2 chain. These residues at the amino-terminus of triple helical domain are important for crosslink formation in the ECM [18].…”

“…A previous report of P3H3 null mice [18] only demonstrated lysyl hydroxylation sites on lysine-87 (K87) in both the α1 and α2 chain. These residues at the amino-terminus of triple helical domain are important for crosslink formation in the ECM [18]. We determined the occupancy of PTMs at individual lysyl hydroxylation sites between WTs, P3H3 and LH1 nulls in tendon and skin (Figure 5, Table 3 and Table 4).…”

“…To achieve this, we compared the levels of overall lysyl hydroxylation and PTM occupancy at individual sites between collagens extracted from P3H3 or LH1 null mice. If P3H3 is a chaperone required for LH1 activity as recently suggested [18], then both P3H3 and LH1 null mice should cause similar defects in lysine hydroxylation. Moreover we analyzed different collagens from different tissues to test the hypothesis that LH1 is a collagen type-specific enzyme [22–25], and to understand if P3H3 might contribute to this differential activity.…”

“…Interestingly, the function of prolyl 3-hydroxylase 3 (P3H3) is controversial. It has been suggested that this protein has no prolyl hydroxylase activity and instead acts as a chaperone for lysyl hydroxylase 1 (LH1) [18]. LHs hydroxylate specific lysine residues in both the collagenous and telopeptide regions, and the three isoforms (LH1, 2 and 3) have been proposed to play specific roles based on collagen sequences [16].…”