Mass spectra of tryptic peptides containing the A3 site in chicken fibrillar collagens.

<p>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.</p

Isolated collagen species from chicken and xenopus on 6% SDS-PAGE.

<p>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.</p

Chicken and xenopus sequences and locations of 3Hyp residues in A-clade and B-clade collagen α-chains.

<p>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.</p

Mass spectra of tryptic peptides containing the A1 site from chicken and xenopus α1(III).

<p>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.</p

Protein sequence alignment of the collagen (GPP)_n motif from phylogenetically diverse animals.

<p>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 <i>Petromyzon marinus</i> transcript: COL2A1 ENSPMAT00000009617 and <i>Ciona intestinalis</i> transcript: FCOL1 ENSCINT00000014311, respectively.</p

Mass spectra showing prolyl 3-hydroxylation distributed throughout the whole fibril.

<p>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).</p

Developmental control of prolyl 3-hydroxylation in tendon.

<p>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).</p

Mass spectra of (GPP)_n containing tryptic peptides from adult animal tendons.

<p>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.</p

Summary of 3Hyp occupancy in the (GPP)_n of type I and II collagen α-chains.

<p>The table shows the average number of 3Hyp residues per (GPP)_n motif with the percentage of α-chains containing at least one 3Hyp residue per (GPP)_n given in parentheses. The percentage of each posttranslational variant was determined based on the ratio of the heights of the m/z peaks. For example, the human tendon α1(I) (GPP)_n tryptic peptide, TGDAGPV<b>GPPGPPGPPGPPGPP</b>SAGFDFSFLPQPPQE<b>K</b>, was found to be a mix of eight distinct molecular species giving a hydroxylation (±16 Da) ladder, each representing a posttranslational variant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093467#pone-0093467-g002" target="_blank">Figure 2</a>). The molecular location of the each hydroxylated residue (3Hyp, 4Hyp and Hyl) was determined using MS/MS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093467#pone.0093467.s001" target="_blank">Figure S1</a>). The C-terminal lysine was predominantly hydroxylated in all Achilles tendons. In this scroll, the 1270.7³⁺ m/z (peptide species containing four 3Hyp residues and five 4Hyp) represents 9% of the total population and the other variations are as follows: 1265.9³⁺ (three 3Hyp residues and five 4Hyp, 10%); 1260.5³⁺ (three 3Hyp residues and four 4Hyp, 13%); 1254.6³⁺ (two 3Hyp residue and four 4Hyp, 19%); 1249.6³⁺ (one 3Hyp residue and four 4Hyp, 16%); 1244.1³⁺ (no 3Hyp residues and four 4Hyp, 18%); 1238.6³⁺ (no 3Hyp residues and three 4Hyp residue, 10%); 1233.1³⁺ (no 3Hyp residues and two 4Hyp residue, 5%). From these percentages, the average number of 3Hyp residues was estimated per α-chain. In this example the calculation is (4×9%)+(3×10%)+(3×13%)+(2×19%)+(1×16%)  =  mean content of 1.6 3Hyp per α1(I) from human tendon. The 3Hyp content in mouse tendon type I collagen was observed to vary markedly with animal age, in the range between one and two 3Hyp residues per (GPP)_n as indicated in the table.</p

Identification of <i>LEPRE1</i> mutations.

<p>A: A partial sequence of PCR product of Patient II-3 is shown. Compound heterozygous frame shift mutations (c.484delG, p.A162LfsX22 and c.2155dupC, p.E719RfsX11) are indicated by arrows. The mutations have been confirmed by the subsequent sequencing of subcloned products of normal and mutant alleles. B: Schematic presentation of the positions of the mutation. <i>LEPRE1</i> cDNA encodes the tetratricopeptide repeat domain (four black regions), the Prolyl/Lysyl/hydroxylase domain (green region), and the KDEL ER- retrieval motif (red region). <i>LEPRE1</i> with a p.E719RfsX11 change results in the lack of only the KDEL ER-retrieval sequence, whereas other functional domains remain intact.</p