title: P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA.
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 bothSc65-/-andP3h3-/-skin revealed the same abnormal chain pattern on SDS-PAGE with an overabundance of a γ112cross-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 bothP3h3-/-andSc65-/-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 andPlod1-/-mouse tissues, suggesting that P3H3 and/or SC65 mutations may cause as yet undefined EDS variants.
authors.affiliation: From the Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, Washington 98195, dmhudson@uw.edu.
authors.forename: David M
authors.initials: DM
authors.lastname: Hudson
authors.affiliation: From the Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, Washington 98195.
authors.forename: MaryAnn
authors.initials: M
authors.lastname: Weis
authors.affiliation: From the Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, Washington 98195.
authors.forename: Jyoti
authors.initials: J
authors.lastname: Rai
authors.affiliation: the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, and.
authors.forename: Kyu Sang
authors.initials: KS
authors.lastname: Joeng
authors.affiliation: the Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Milena
authors.initials: M
authors.lastname: Dimori
authors.affiliation: the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, and.
authors.forename: Brendan H
authors.initials: BH
authors.lastname: Lee
authors.affiliation: the Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Roy
authors.initials: R
authors.lastname: Morello
authors.affiliation: From the Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, Washington 98195.
authors.forename: David R
authors.initials: DR
authors.lastname: Eyre
mesh_terms: D000818:Animals; D001011:Aorta / Q000378:metabolism; D001324:Autoantigens / Q000235:genetics*; D001842:Bone and Bones / Q000378:metabolism; D002853:Chromatography, Liquid; D003094:Collagen / Q000737:chemistry*; D003315:Cornea / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003804:Dentin / Q000378:metabolism; D004195:Disease Models, Animal; D004535:Ehlers-Danlos Syndrome / Q000235:genetics* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D006801:Humans; D006900:Hydroxylation; D008239:Lysine / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012867:Skin / Q000378:metabolism
chemical_list: D001324:Autoantigens; D003432:Cross-Linking Reagents; C000609794:SC65 protein, mouse; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase; C030606:proline, 2-oxoglutarate 3-dioxygenase; D008239:Lysine
keywords: bone; collagen; cross-links; endoplasmic reticulum (ER); mass spectrometry (MS); post-translational modification (PTM); skin