Abstract
Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.
Highlights
Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues
P3H1 forms a complex with cartilage-associated protein (CRTAP) and cyclophilin B (CypB), and this complex was shown to 3-hydroxylate proline residue 986 of the ␣1-chain of type I collagen [8, 11]
The lack of 3-hydroxylation of proline residue 986 in type I collagen extracted from P3H1 null tissues clearly demonstrates that P3H1 is solely responsible for the modification of this site
Summary
P3H, prolyl 3-hydroxylase; CRTAP, cartilage-associated protein; CypB, cyclophilin B; 3-Hyp, 3-hydroxyproline; 4-Hyp, 4-hydroxyproline; FWHM, full width of half-maximum; MS/MS, tandem mass spectrometry. Posttranslational Modifications in Mouse Type I Collagen the A4 site (position 470). The ␣1-chain of type I collagen was shown to only contain a 3-hydroxyproline at the A1 site, which was 96 –99% occupied. The ␣2-chain of type I collagen was 80% 3-hydroxylated at the A3 site. The degree of glycosylation varies among different collagens [24, 25]. For type I collagen, the degree of glycosylation varies between different tissues. We see only small differences in the fibril diameter of newborn mice tail tendons as compared with the severe disorganization found in adult P3H1 null mice, implicating the lateral growth of fibrils in the phenotype
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