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. 2017 Mar 3;292(9):3877-3887.
doi: 10.1074/jbc.M116.762245. Epub 2017 Jan 23.

P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA

David M Hudson  1 , MaryAnn Weis  2 , Jyoti Rai  2 , Kyu Sang Joeng  3 , Milena Dimori  4 , Brendan H Lee  3 , Roy Morello  4 , David R Eyre  2
Affiliations

P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA

David M Hudson et al. J Biol Chem. .

Abstract

Tandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both Sc65-/- and P3h3-/- skin revealed the same abnormal chain pattern on SDS-PAGE with an overabundance of a γ112 cross-linked trimer. The latter proved to be from native molecules that had intramolecular aldol cross-links at each end. The lysine under-hydroxylation was shown to alter the divalent aldimine cross-link chemistry of mutant skin collagen. Furthermore, the ratio of mature HP/LP cross-links in bone of both P3h3-/- and Sc65-/- mice was reversed compared with wild type, consistent with the level of lysine under-hydroxylation seen in individual chains at cross-linking sites. The effect on cross-linking lysines was quantitatively very similar to that previously observed in EDS VIA human and Plod1-/- mouse tissues, suggesting that P3H3 and/or SC65 mutations may cause as yet undefined EDS variants.

Keywords: bone; collagen; cross-links; endoplasmic reticulum (ER); mass spectrometry (MS); post-translational modification (PTM); skin.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
Generation of P3h3−/− mice. A, the Leprel2 gene was inactivated by homologous recombination in ES cells using BAC technology. Mice were maintained on a C57/BL6J background. B, immunoblot analysis using a polyclonal P3H3 antibody against total protein extracts from whole kidneys of WT and P3h3−/− mice supported the loss of protein in the knock-out mice. Human fetal cartilage extract was loaded as a positive control marker for P3H3. C, representative H&E images of skin sections from P3h3−/− and WT mice.
FIGURE 2.
FIGURE 2.
SDS-PAGE reveals altered cross-linking in skin type I collagen from P3h3−/− and Sc65−/− mice. As visualized by 6% SDS-PAGE, type I collagen from P3h3−/− and Sc65−/− mouse skin acid extracts reproducibly yielded more γ112 relative to WT. Accompanying this increased band intensity in knock-out mice skin is the decrease in β12 relative to WT mice. This shift in banding pattern is not observed in acid extracts of bone and tendon.
FIGURE 3.
FIGURE 3.
Increased intramolecular C-telopeptide aldol cross-links in knock-out skin type I collagen. The γ112 trimer from P3h3−/− mouse skin has a distinct tryptic peptide profile from that of the α1(I) monomer when analyzed using mass spectrometry. A, the LC-MS profile of in-gel trypsin digests of the γ112 band from P3h3−/− mouse skin confirmed the presence of the α1(I) C-telopeptide lysine aldehyde aldol dimer in the γ112 band. B, MS/MS fragmentation spectrum of the parent ion (1724.45+) from the P3h3−/− γ112 band. The trypsin-digested dimeric peptide is shown with P* indicating 4Hyp.
FIGURE 4.
FIGURE 4.
Under-hydroxylation at cross-linking Lys-87 in P3h3−/− and Sc65−/− skin collagen. LC-MS profiles of in-gel trypsin digests of the α1(I) collagen chains from WT, Sc65−/−, and P3h3−/− mouse skin. A, MS profile of α1(I) from WT mouse skin reveals 100% glucosyl-galactosyl-Hyl (460.54+ and 613.63+) at residue α1(I) Hyl87. B, from Sc65−/− mouse skin, Lys-87 is 100% unmodified. Trypsin cleaves after unmodified Lys-87 yielding a smaller tryptic peptide (574.22+). C, from P3h3−/− mouse skin, Lys-87 is predominantly unmodified (80% Lys (574.12+); 20% glucosyl-galactosyl-Hyl (460.44+)). D, MS-MS fragmentation spectrum of the parent ion (574.12+) from P3h3−/− skin α1(I). The b and y ion masses reveal no modifications on α1(I) Lys-87. In-gel trypsin digests typically undergo complete oxidation of methionine residues yielding methionine sulfoxide. The trypsin-generated peptide is shown with P* indicating 4Hyp, M* indicating methionine sulfoxide, and -galglc indicating glucosyl-galactosyl-.
FIGURE 5.
FIGURE 5.
Complete loss of hydroxylation at cross-linking Lys-930 in P3h3−/− skin collagen. LC-MS profiles of peptides containing the helical domain Lys-930 cross-linking site from collagen α1(I) prepared by bacterial collagenase digestion of total dermal collagen. A, MS profile from P3h3+/− mouse skin reveals 100% lysyl hydroxylation at residue 930 (472.76+, 567.05+, and 708.44+). B, in P3h3−/− mouse skin, Lys-930 is 100% unmodified (467.46+, 560.65+, and 700.44+). Identical profiles were found at this site in skin, bone, and tendon from both P3h3−/− and Sc65−/− mice.
FIGURE 6.
FIGURE 6.
Altered divalent cross-link structure (Lys-87 to C-telopeptide) from P3h3−/− mouse skin. LC-MS profiles of type I collagen divalent cross-linking structures prepared by bacterial collagenase digestion of sodium borohydride-treated skin. A, MS profile of a fully glycosylated (glucosyl-galactosyl-) reduced aldimine structure from P3h3+/− mouse skin (721.26+, 865.15+, 1081.14+, and 1440.63+). B, from P3h3−/− mouse skin, two structures were identified in equal amounts. The same fully glycosylated reduced aldimine found in P3h3+/− (721.26+, 865.15+, 1081.14+, and 1440.63+) plus a non-glycosylated reduced aldimine (664.56+, 797.05+, 995.84+, and 1327.13+). C, MS/MS fragmentation spectrum of the parent ion (7975+) from P3h3−/− confirm the mass of the lysine variant from mutant skin. Molecular ions from the lysine and glycosylated hydroxylysine variants are distinguished in red and black, respectively.
FIGURE 7.
FIGURE 7.
Altered divalent cross-link structure (Lys-930 to N-telopeptide) from P3h3−/− mouse skin. LC-MS profiles of type I collagen divalent cross-linking structures prepared by bacterial collagenase digestion of sodium borohydride-treated skin. A, MS profile of a hydroxylated reduced aldimine structure (hydroxylysinonorleucine) from P3h3+/− mouse skin (801.06+, 960.95+, 1200.94+, and 1600.13+). B, from P3h3−/− mouse skin, only the non-hydroxylated lysine variant (lysinonorleucine) was recovered (795.76+, 954.65+, 1192.64+, and 1589.43+). C, MS/MS fragmentation spectrum of the parent ion (7955+) from P3h3−/− mouse skin.
FIGURE 8.
FIGURE 8.
Model speculating how collagen cross-link formation is affected in P3h3−/− and Sc65−/− mouse skin. Under normal conditions, LH1 catalyzes the hydroxylation of helical lysines 87 and 930; and LH2 catalyzes the hydroxylation of the N- and C-telopeptide lysines (A). In the fibril, collagen molecules are spatially arranged such that intermolecular cross-link placement is optimal (B). In WT skin, fully glycosylated Hyl87 preferentially forms an intermolecular aldimine cross-link with a C-telopeptide lysine aldehyde (C). In the P3h3−/− and Sc65−/− mouse tissues the LH1 substrates are under-hydroxylated and subsequently under-glycosylated, which alters collagen cross-linking chemistry (D). From mutant skin the results are consistent with the C-telopeptide lysine aldehydes preferentially forming intramolecular aldol cross-links (as opposed to intermolecular aldols). We predict that under normal conditions the presence of the disaccharide on Hyl87 favors aldimine formation with a single C-telopeptide aldehyde and hinders intramolecular aldol formation with a second α1(I) C-telopeptide from the same molecule as the first one, so favoring intermolecular interactions. The net effect of under-hydroxylated Lys-87 then would be fewer stable aldol intermolecular cross-links within and between fibrils.
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