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. 2015 Mar 27;290(13):8613-22.
doi: 10.1074/jbc.M114.634915. Epub 2015 Feb 2.

Post-translationally abnormal collagens of prolyl 3-hydroxylase-2 null mice offer a pathobiological mechanism for the high myopia linked to human LEPREL1 mutations

David M Hudson  1 , Kyu Sang Joeng  2 , Rachel Werther  1 , Abbhirami Rajagopal  2 , MaryAnn Weis  1 , Brendan H Lee  2 , David R Eyre  3
Affiliations

Post-translationally abnormal collagens of prolyl 3-hydroxylase-2 null mice offer a pathobiological mechanism for the high myopia linked to human LEPREL1 mutations

David M Hudson et al. J Biol Chem. .

Abstract

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2(n/n)) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2(n/n) mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.

Keywords: 3-Hydroxyproline; Animal Model; Collagen; Extracellular Matrix; Mass Spectrometry (MS); Post-translational Modification (PTM); Prolyl 3-Hydroxylase; Sclera.

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Figures

FIGURE 1.
FIGURE 1.
Generation and expression analysis of P3h2-null (P3h2n/n) mice. A, knock-out first allele mice (−, P3h2+/−) were generated using embryonic stem cells obtained from EUCOMM. Conditional wild-type mice (c, P3h2c/+) were generated by removing the lacZ expression cassette, using Rosa26-Flipase. Null mice (n, P3h2n/+) were generated by removing exon 3 using CMV-Cre. Mice were maintained on a C57/BL6J background. P3h2 expression was found throughout multiple tissues of the whole-mount mouse (B) but was localized to the glomeruli of kidney sections (C) as determined by β-galactosidase staining. D, immunoblot analysis using a polyclonal P3h2 antibody against total protein extracts from whole kidneys of wild-type (WT) and P3h2−/− (KO) mice supported the knock-out of protein expression in the P3h2n/n mice.
FIGURE 2.
FIGURE 2.
P3h2 expression in the mouse eye. A, β-galactosidase staining revealed P3h2 expression in the cornea (B) and sclera (C) of mouse eye sections. B, at the anterior of the eye, staining was localized to the corneal epithelium (CE) with no P3h2 expression observed in the corneal stroma (CS). C, at the posterior of the eye, staining was localized exclusively to the sclera (S). The red inset box displays a digitally magnified image of sclera. The black arrows indicate positive staining in sclera. The following abbreviations were used: Ch, choroid; PE, pigment epithelium; and RL, receptor layer.
FIGURE 3.
FIGURE 3.
P3h1 substrate sites in type I collagen from mouse eye scleral tissue. LC-MS profiles of in-gel trypsin digests of the type I collagen α-chains from wild-type and P3h2n/n mouse sclera. A, MS profile of the α1-chain from the P3h2n/n mouse confirms no effect on Pro-986 3-hydroxylation (782.52+). B, MS spectrum from the α2-chain shows significant reduction of 3-hydroxylation at Pro-707 (895.02+) in the P3h2n/n mouse. A similar phenomenon was observed in type I collagen from cornea and tendon (data not shown). The trypsin-digested peptide is shown with P# indicating 3Hyp and P* indicating 4Hyp.
FIGURE 4.
FIGURE 4.
Pro-707 site in α1(I) is a tissue-specific substrate unique to P3h2. LC-MS profiles of in-gel trypsin digests of the collagen α1(I) chain from tendon and sclera of wild-type and P3h2n/n mice. A, wild-type mouse tendon and sclera show 65 and 45% 3-hydroxylation at Pro-707 (929.02+), respectively; B, P3h2n/n mouse tendon and sclera show an almost complete loss of prolyl 3-hydroxylation at Pro-707 (922.02+). The trypsin digested peptide is shown with P# indicating 3Hyp and P* indicating 4Hyp.
FIGURE 5.
FIGURE 5.
Post-translational similarities between sclera and tendon at the C-terminal (GPP)n motif from type I collagen. LC-MS profiles of in-gel trypsin digests of the collagen α1(I) chain from tendon and sclera of wild-type and P3h2n/n mice. A, MS profiles from mouse tendon and sclera reveal a similar hydroxylation ladder. B, complete loss of prolyl 3-hydroxylation is observed in the α1(I) - digested peptide is shown with P# indicating 3Hyp, P* indicating 4Hyp, and K* indicating Hyl at the C terminus where trypsin cleaves in the C-telopeptide.
FIGURE 6.
FIGURE 6.
Prolyl 3-hydroxylation absent in collagen IV from lens capsule of P3h2n/n mice. LC-MS profiles of in-gel trypsin digests of the collagen α1(IV) chain from the lens capsule of wild-type and P3h2n/n mice. A, MS profile reveals a hydroxylation ladder containing partial prolyl 3-hydroxylation at Pro-602 and Pro-605. B, MS/MS fragmentation spectrum of the parent ion (1149.14+) from mouse lens capsule. The b and y ion breakages establish the added 16 Da on Pro-602. C, complete loss of prolyl 3-hydroxylation is observed at Pro-602 and Pro-605 in α1(IV) from P2h2n/n lens capsule. D, MS/MS fragmentation spectrum of the parent ion (1144.94+) from mouse lens capsule. The b and y ion breakages confirm the loss of 16 Da on Pro-602. The trypsin-digested peptide is shown with P# indicating 3Hyp, P* indicating 4Hyp, and galglc indicating glucosyl-galactosyl; M-1624+ indicates the parent ion with hexose loss after fragmentation.
FIGURE 7.
FIGURE 7.
Tissue-specific hydroxylation patterns in type I collagen from bovine eye tissues. LC-MS profiles of in-gel trypsin digests of the α1(I) collagen chains from bovine sclera and cornea. A, MS profile of α1(I) from bovine cornea reveals no 3-hydroxylation at the (GPP)n. B, MS profile of the α1-chain from bovine scleral type I collagen reveals a similar hydroxylation to that observed in mouse tendon and sclera. The trypsin digested peptide is shown with P# indicating 3Hyp and P* indicating 4Hyp.
FIGURE 8.
FIGURE 8.
Post-translational variances in cross-linking lysines between cornea and sclera type I collagen. A, profile of collagenase-digested whole tissue separated on C8 column (bovine sclera shown). B, LC-MS profile of C8 fraction 28 from bovine sclera reveals no glycosylation at cross-linking α1(I) Lys-87 (502.14+). C, MS/MS fragmentation spectrum of the parent ion (501.84+) from bovine sclera. The b and y ion breakages confirm no glycosylation on α1(I) Hyl87. D, LC-MS profile of C8 fraction 28 from bovine cornea reveals complete glycosylation of cross-linking α1(I) Hyl87 as glucosyl-galactosyl (galglc) (582.84+). E, MS/MS fragmentation spectrum of the parent ion (583.14+) from bovine cornea. The b and y ion breakages reveal gain of 340 Da (glucosyl-galactosyl) on α1(I) Hyl87. The trypsin-digested peptide is shown with K* indicating Hyl, K(*) indicating partial Hyl and galglc indicating glucosyl-galactosyl.
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References

    1. Berg R. A., Prockop D. J. (1973) The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem. Biophys. Res. Commun. 52, 115–120 - PubMed
    1. Ogle J. D., Arlinghaus R. B., Logan M. A. (1962) 3-Hydroxyproline, a new amino acid of collagen. J. Biol. Chem. 237, 3667–3673 - PubMed
    1. Hudson D. M., Eyre D. R. (2013) Collagen prolyl 3-hydroxylation: a major role for a minor post-translational modification? Connect. Tissue Res. 54, 245–251 - PMC - PubMed
    1. Hudson D. M., Weis M., Eyre D. R. (2011) Insights on the evolution of prolyl 3-hydroxylation sites from comparative analysis of chicken and Xenopus fibrillar collagens. PLoS One 6, e19336. - PMC - PubMed
    1. Weis M. A., Hudson D. M., Kim L., Scott M., Wu J. J., Eyre D. R. (2010) Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly. J. Biol. Chem. 285, 2580–2590 - PMC - PubMed

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