ECTS2013 Poster Presentations Other diseases of bone and mineral metabolism (48 abstracts)
Abnormal type I collagen glycosylation pattern and cross-linking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta
Wayne Cabral 1 , Irina Perdivara 2 , MaryAnn Weis 3 , Masahiko Terajima 5 , Angela Blissett 1 , Weizhong Chang 1 , Elena Makareeva 4 , Sergey Leikin 4 , David Eyre 3 & Mitsuo Yamauchi 5
1Bone and Extracellular Matrix Branch, NICHD, NIH, Bethesda, MD, USA; 2Laboratory of Structural Biology, NIEHS, NIH, Research Triangle Park, NC, USA; 3Orthopaedic Research Laboratories, University of Washington, Seattle, WA, USA; 4Section on Physical Biochemistry, NICHD, NIH, Bethesda, MD, USA; 5North Carolina Oral Health Institute, University of North Carolina, Chapel Hill, NC, USA.
Introduction: Recessive osteogenesis imperfecta (OI) is caused by mutations in genes encoding proteins involved in post-translational interactions with type I collagen. Types VII–IX OI involve defects in the collagen prolyl 3-hydroxylation complex, which modifies α1(I)Pro986. PPIB encodes CyPB, a complex component with PPIase activity and the major isomerase facilitating collagen folding. We investigated the role of CyPB in collagen post-translational modifications and crosslinking.
Methods: Ppib KO mice were generated using a gene-trap ES cell clone with a β-geo reporter inserted into Ppib intron 1. Type I collagen modifications were analyzed by LC–MS/MS and HPLC. Bone architecture was investigated by micro-CT and DXA.
Results: Ppib transcripts and protein are absent in skin, fibroblasts, femora, and calvarial osteoblasts; only residual (<10%) α1(I)P986 3-hydroxylation is detectable in fibroblast and osteoblast collagen. Although collagen from KO cells has delayed electrophoretic mobility, total collagen 5-lysyl and prolyl 4-hydroxylation was normal, suggesting altered glycosylation in KO. MS analyses indicated that, except for lysyl residues involved in crosslinking, most helical residues of KO FB and OB collagen have increased diglycosylation. Total mature crosslinks (HP+LP) in KO bone were increased 1.5–1.7× vs WT. We detected a 4–5-fold increase in trivalent LP crosslinks (P=0.001), which decreased the HP:LP ratio correspondingly. Total immature cross-links (DHLNL+HLNL) were also significantly increased and the DHLNL:HLNL ratio decreased, implying reduced lysyl hydroxylation of helical crosslink residues in KO bone. Abnormal collagen modification is associated with 70–80% reduction of collagen deposited into KO matrix in culture, associated with smaller long bones with significantly reduced BMD, BV and TbN.
Conclusions: In Ppib KO mice, absence of CyPB delays collagen folding and alters collagen glycosylation patterns in culture; tissue investigations are ongoing to confirm these effects. Altered modification may impair collagen matrix interactions and promote abnormal bone mineralization. Collagen crosslink patterns are shifted to trivalent forms lacking helical Hyl, possibly contributing to decreased matrix deposition and bone strength.
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Bone Abstracts
ISSN 2052-1219 (online)
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