May 3, 2011;
Insights on the evolution of prolyl 3-hydroxylation sites from comparative analysis of chicken and Xenopus fibrillar collagens.
Recessive mutations that prevent 3-hydroxyproline formation in type I collagen have been shown to cause forms of osteogenesis imperfecta. In mammals, all A-clade collagen chains with a GPP sequence at the A1 site (P986), except α1(III), have 3Hyp at residue P986. Available avian, amphibian and reptilian type III collagen sequences from the genomic database (Ensembl) all differ in sequence motif from mammals at the A1 site. This suggests a potential evolutionary distinction in prolyl 3-hydroxylation between mammals and earlier vertebrates. Using peptide mass spectrometry, we confirmed that this 3Hyp site is fully occupied in α1(III) from an amphibian, Xenopus laevis, as it is in chicken. A thorough characterization of all predicted 3Hyp sites in collagen types I, II, III and V from chicken and xenopus revealed further differences in the pattern of occupancy of the A3 site (P707). In mammals only α2(I) and α2(V) chains had any 3Hyp at the A3 site, whereas in chicken all α-chains except α1(III) had A3 at least partially 3-hydroxylated. The A3 site was also partially 3-hydroxylated in xenopus α1(I). Minor differences in covalent cross-linking between chicken, xenopus and mammal type I and III collagens were also found as a potential index of evolving functional differences. The function of 3Hyp is still unknown but observed differences in site occupancy during vertebrate evolution are likely to give important clues.
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Figure 1. Isolated collagen species from chicken and xenopus on 6% SDS-PAGE.A, chicken collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction ; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. B, xenopus collagens; lane 1, acid soluble fraction; lane 2, neutral 1.0 M salt soluble fraction; lane 3, acid 1.2 M salt fraction; lane 4, neutral salt insoluble fraction; lane 5, reduced neutral salt insoluble fraction. β11(I) and β12(I) are cross-linked α1-α1 and α1-α2 chain dimers, respectively.
Figure 2. Mass spectra of tryptic peptides containing the A1 site from chicken and xenopus α1(III).Comparison of full scan spectra from LC-MS profiles of in-gel trypsin digests of the homologous sequence in the α1(III) chain from A, chicken; and B, xenopus. C, MS/MS analysis of the 3Hyp containing tryptic peptide from xenopus.
Figure 3. Mass spectra of tryptic peptides containing the A3 site in chicken fibrillar collagens.Full scan spectra from LC-MS profiles of in-gel trypsin digests of α1(I) from chicken bone, A; α2(I) from chicken skin, B; α1(II) from chicken cartilage, C; and α2(V) from chicken skin, D. The degree of hydroxylation at P707 is estimated through a correlation with the relative abundance of the representative ions. P*, 4Hyp; P#, 3Hyp.
Figure 4. Chicken and xenopus sequences and locations of 3Hyp residues in A-clade and B-clade collagen α-chains.D-periodic spacing is evident between the 3Hyp residues at sites A4 and A3 and between sites A3 and A2 in the A-clade triple helical procollagen molecule; and between sites B3 and B2 in the B-clade triple helical procollagen molecule. GPP sequences containing potential 3Hyp sites are shown in bold with occupied sites underlined. Percentage of 3Hyp occupancy relative to the unmodified sequence is indicated. Absence of the GPP sequence is indicated with n/a.