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Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.
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24695516 ???displayArticle.pmcLink???PMC3973637 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 2. Mass spectra of (GPP)n containing tryptic peptides from adult animal tendons.Full scan spectra from LC-MS profiles of in-gel trypsin digests of α1(I) from human, chicken and xenopus tendon with 6% SDS-PAGE lanes at left (A). MS/MS fragmentation spectrum of the parent ion (1265.33+) from human tendon (B). The sequence is shown with b and y ion breakages. P*, 4Hyp; P#, 3Hyp; K*, Hyl. The cross-linking telopeptide Lys of the human peptide was fully hydroxylated in all posttranslational variants.
Figure 3. Edman N-terminal sequence analysis confirming 3-hydroxyproline in the (GPP)n motif of tendon type I collagen.The (GPP)n-containing tryptic peptides from the α1(I) and α2(I) α-chains of rat tail tendon were recovered as a chromatographic pool and sequenced simultaneously. Sequential phenylthiohydantoin-derivative HPLC chromatograms are shown for sequencer cycles 8-15 (sequencer cycles 1-7 were as predicted from the known sequences of both α-chains). The new residues at each cycle are highlighted in blue for the α1-chain and green for the α2-chain. The 3Hyp residue gave a distinctive double peak as previously reported [18].
Figure 4. Mass spectra showing prolyl 3-hydroxylation distributed throughout the whole fibril.Collagen was solubilized from adult human tendon using SDS extraction (A) and CNBr digestion (B). Lanes of 6% (A) and 12% (B) SDS-PAGE gels are shown to the left. Similar levels of 3Hyp (∼two 3Hyp per α2(I) chain) were observed using each approach. The 12102+ and 12182+ ions in both ion ladders represent unrelated peptides with a 2+ charge (these ions are indicated with φ).
Figure 5. Developmental control of prolyl 3-hydroxylation in tendon.Reduced levels of 3Hyp were observed in fetal tendon relative to adult tissue. MS scan of fetal human Achilles tendon α1(I) with 6% SDS-PAGE lane at left (A). The 1248.43+ ion contains a mix of two peptide posttranslational variants (one 3Hyp and four 4Hyp; and no 3Hyp and five 4Hyp). MS scan of fetal human Achilles tendon α2(I) (B).
Figure 1. Protein sequence alignment of the collagen (GPP)n motif from phylogenetically diverse animals.Conservation of the (GPP)n motif is shown in red for fibrillar collagens from early chordates through amphibians, birds and mammals. Genomic sequences are from Ensembl. Lamprey and ciona collagen sequences are from Petromyzon marinus transcript: COL2A1 ENSPMAT00000009617 and Ciona intestinalis transcript: FCOL1 ENSCINT00000014311, respectively.
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