A poorly understood collagen post-translational modification has been 3-prolyl-hydroxylation(P3H) converting proline to 3-hydroxy proline (3-Hyp). It occurs uniquely in the fibrillar collagens,i.e., types I, II and III collagen, at only one proline position, but abundantly in other collagens suchas network collagens, i.e., type IV collagen, where it occurs in up to 10% of residues. This begs thequestion of whether 3-Hyp residues may serve divergent biological functions in different settingsand tissues. In recent data, we have identified a novel protein, CRTAP or Cartilage AssociatedProtein, that is representative of the Leprecanfamily of proteins recently reported to contain aconserved 2-oxoglutarate dioxygenase domain that is found in collagen 4-prolyl-hydroxylases,Hypoxic Inducible Factor (HIF) 4-prolyl-hydoxylases (PDHs), and lysyl hydroxylases (PLODs).Moreover, Leprecan or P3H1 has collagen 3-prolyl-hydroxylase activity in vitro implicating thisfamily of genes as the long sought after PSH's. By combining human and mouse genetic, andproteomic approaches, we show (see Preliminary Studies C.2.) that loss of Crtap in mice causes anosteochondrodysplasia characterized by short stature, kyphosis, and severe osteoporosis.Moreover, this phenotype is biochemically associated with conversion of the single 3-hydroxy-proline to proline in the triple helical domain of types I and II collagen. CRTAP can bind P3H1 andis required for P3H activity in vivo. Finally, we found that loss of CRTAP is associatedwithrecessive osteogenesis imperfecta types VII and ll/lll in humans. These data raise importantquestions that we will address in our Specific Aims. 1) Are the phenotypic features of Crtap loss of function due solely to loss of 3-prolyl-hydroxylation of fibrillar collagens and are there redundant functions with P3H1? 2) Whatare the consequences of 3-Hyp loss in cartilage and bone on cellular differentiation andfunction, and collagen biosynthesis? 3)What is the full human clinical spectrum associatedwith loss of 3-prolyl-hydroxylation of fibrillar collagens? By combining mouse and humanstudies, this proposal will interact directly with Projects I, II, and the Protein Biochemistry Core toanswer these questions.
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