Human Molecular Genetics Advance Access [Accepted Manuscript] published online on October 21, 2009
Human Molecular Genetics, doi:10.1093/hmg/ddp481
Published by Oxford University Press 2009
Prolyl 3-Hydroxylase 1 and CRTAP are Mutually Stabilizing in the Endoplasmic Reticulum Collagen Prolyl 3-Hydroxylation Complex
1 Bone and Extracellular Matrix Branch, NICHD, NIH, Bethesda, Maryland, 2 Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia
* Corresponding author: Joan C. Marini, MD, PhD, Chief, Bone and Extracellular Matrix Branch, Bldg. 10; Rm 10N260, 9000 Rockville Pike, Bethesda, MD 20892, (T) 301-594-3418, (F) 301-480-3188, (e) oidoc{at}helix.nih.gov
Received July 8, 2009; Revised September 23, 2009; Accepted October 15, 2009
Null mutations in cartilage-associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1/LEPRE1) cause Types VII and VIII OI, respectively, two novel recessive forms of osteogenesis imperfecta with severe to lethal bone dysplasia and overmodification of the type I collagen helical region. CRTAP and P3H1 form a complex with cyclophilin B in the ER which 3-hydroxylates the Pro986 residue of 
1(I) and 1(II) collagen chains. We investigated the interaction of complex components in fibroblasts from types VII and VIII OI patients. Both CRTAP and P3H1 are absent or reduced on Western blots and by immunofluorescence microscopy in cells containing null mutations in either gene. Levels of LEPRE1 or CRTAP transcripts, however, are normal in CRTAP- or LEPRE1-null cells, respectively. Stable transfection of a CRTAP or LEPRE1 expression construct into cells with null mutations for the transfected cDNA restored both CRTAP and P3H1 protein levels. Normalization of collagen helical modification in transfected CRTAP-null cells demonstrated that the restored proteins functioned effectively as a complex. These data indicate that CRTAP and P3H1 are mutually stabilized in the collagen prolyl 3-hydroxylation complex. Cyclophilin B levels were unaffected by mutations in either CRTAP or LEPRE1. Proteasomal inhibitors partially rescue P3H1 protein in CRTAP-null cells. In LEPRE1-null cells, secretion of CRTAP is increased compared to control cells and accounts for 15-20% of the decreased CRTAP detected in cells. Thus, mutual stabilization of P3H1 and CRTAP in the ER collagen modification complex is an underlying mechanism for the overlapping phenotype of type VII and VIII OI.