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. 2013 Aug 23;288(34):24742-52.
doi: 10.1074/jbc.M113.464156. Epub 2013 Jul 16.

Posttranslational modifications in type I collagen from different tissues extracted from wild type and prolyl 3-hydroxylase 1 null mice

Elena Pokidysheva  1 , Keith D Zientek, Yoshihiro Ishikawa, Kazunori Mizuno, Janice A Vranka, Nathan T Montgomery, Douglas R Keene, Tatsuya Kawaguchi, Kenji Okuyama, Hans Peter Bächinger
Affiliations

Posttranslational modifications in type I collagen from different tissues extracted from wild type and prolyl 3-hydroxylase 1 null mice

Elena Pokidysheva et al. J Biol Chem. .

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.

Keywords: Collagen; Hydroxylysine; Hydroxyproline; Post translational modification; Prolyl 3-hydroxylase 1; Protein synthesis; Tendon; glycosyl hydroxylysine.

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Figures

FIGURE 1.
FIGURE 1.
Analysis of the A1 site (Pro-986) of the α1-chain of type I collagen. A–C, the α1-chain of type I collagen extracted from tendon (A), skin (B), and bone (C) was analyzed by mass spectrometry. The occurrences of the tryptic peptides DGLNGLOGPIGPOGPR (mass = 1548 Da) and DGLNGLOGPIGZOGPR (mass = 1564 Da) are shown from wild type and P3H1 null mice. O is 4(R)Hyp, and Z is 3(S)Hyp. Panel A was previously published in Ref. .
FIGURE 2.
FIGURE 2.
Analysis of the A3 site (Pro-707) of the α1-chain of type I collagen. A–C, the α1-chain of type I collagen extracted from tendon (A), skin (B), and bone (C) was analyzed by mass spectrometry. The occurrences of the tryptic peptides VGPOGPSGNAGPOGPOGPVGK and VGZOGPSGNAGPOGPOGPVGK are shown from wild type and P3H1 null mice. D, MS/MS spectra of these peptides of the α1-chain of type I collagen are shown. The Y19 + 2H ion shows a mass difference of 8 daltons, corresponding to the hydroxylation of Pro-707 (A3 site). Sequencing identifies this hydroxylation as 3-hydroxyproline. E, N-terminal sequencing cycles for the A3 site are shown. 3-Hyp residues are highlighted to confirm the presence in the cycle. BL, base line; ST, standards; the cycle numbers are indicated (2–4). O is 4(R)Hyp, and Z is 3(S)Hyp.
FIGURE 3.
FIGURE 3.
Analysis of the A3 site (Pro-707) of the α2-chain of type I collagen. A–C, the α2-chain of type I collagen extracted from tendon (A), skin (B), and bone (C) was analyzed by mass spectrometry. The occurrences of the tryptic peptides TGPOGPSGIAGPOGPOGAAGK and TGZOGPSGIAGPOGPOGAAGK are shown from wild type and P3H1 null mice. D, MS/MS spectra of the TGZOGPSGIAGPOGPOGAAGK and TGPOGPSGIAGPOGPOGAAGK peptides of the α2-chain of type I collagen are shown. The Y19 + 2H ion shows a mass difference of 8 daltons, corresponding to the hydroxylation of Pro-707 (A3 site). E, N-terminal sequencing identifies this hydroxylation as 3-hydroxyproline. 3-Hyp residues are highlighted to confirm the presence in the cycle. BL, base line; ST, standards; the cycle numbers are indicated (2–4). O is 4(R)Hyp, and Z is 3(S)Hyp.
FIGURE 4.
FIGURE 4.
Schematic representation of the A1 and A3 3-hydroxylation ratios of type I collagen from skin, tendon and bone of wild type and P3H1 null mice. White circles indicate no 3-hydroxylation, and black circle indicates full 3-hydroxylation. Partial 3-hydroxylation is indicated by blackened quarters.
FIGURE 5.
FIGURE 5.
Graphical representation of real-time quantitative PCR analysis of adult mouse bone, skin, and tendon. Data are represented as the -fold change differences. A shows relative target gene expression to GAPDH signal. B shows -fold change expression of P3H2 and P3H3 genes in P3H1 null relative to wild type control tissues. All experiments were performed in triplicate, and mean values are represented on each graph. P3H1 gene expression is represented by the black boxes, P3H2 is in white, and P3H3 is in gray. Error bars indicate S.D.
FIGURE 6.
FIGURE 6.
MS spectra of trypsin digested mouse tendon type I collagen. A, the peptide GNDGAVGAAGPPGPTGPTGPPGFPGAVGAK(174)GEAGPQGAR is shown for P3H1 null and wild type extracts from tendon. The relative abundance of glycosylated peptide in collagen extracted from P3H1 null mice is much higher. B, MS/MS data for the glycosylated peptide GNDGAVGAAGPPGPTGPTGPPGFPGAVGAK(174)GEAGPQGAR. Spectra labeled 1, 2, and 3 show the partially fragmented 3+ charge state precursor ions for the glucosyl galactosyl, galactosyl, and non-sugar-containing peptides of Lys-174 respectively. Spectra labeled 1a, 2a, and 2c show an expanded view of the previous peptides at the low mass region containing the same identifying fragments for all three peptides.
FIGURE 7.
FIGURE 7.
MS spectra of trypsin digested mouse tendon type I collagen purified by hydrazide extraction. Only the glycosylated variants of the peptide GNDGAVGAAGPPGPTGPTGPPGFPGAVGAK(174)GEAGPQGAR are observed by this method. The relative abundance of the two forms of glycosylation from one extract can be compared, but not the ratio between two extracts.
FIGURE 8.
FIGURE 8.
s2 dependence of FWHMs for diffraction peaks of D-stagger. The FWHMs of low angle meridional x-ray diffraction peaks were estimated by fitting with pseudo Voigt peak function. The black closed circles represent the FWHM from diffraction of wild type mouse tendon, whereas the red open circles represent that of P3H1 null mouse tendon. Error bars indicate S.D.
FIGURE 9.
FIGURE 9.
Electron micrographs of tendon fibrils of wild type and P3H1 null mice. A and B, tendon fibrils of wild type (A) and P3H1 null (B) mice at P5 in cross sections and longitudinal sections and the distribution of measured diameters from the cross sections underneath. The length of the bars in the micrographs corresponds to 100 nm. C and D, tendon fibrils of adult wild type (C) and P3H1 null mice (D) in cross sections and longitudinal sections. The length of the bars corresponds to 500 nm. E and F, the diameter distribution from cross sections is given for P5 (E) and for adult mice (F). Panels C and D are similar to fields published previously, and panel F is taken from Fig. 2E in Ref. .
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