Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues

Erica P Homan,Caressa Lietman,Elda M Munivez,Olivier Lichtarge,Ming-Ming Jiang,Brian Dawson,Yuqing Chen,Rhonald Lua,Dobrawa Napierala,Terry K Bertin,Jennifer Lennington,Roy Morello,Francesco Ramirez,Mary Ann Weis,John Hicks,Brendan H L Lee,Ingo Grafe,David Eyre

doi:10.1371/journal.pgen.1004121

Abstract

Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1H662A). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.

Highlights

Dominant mutations in the type I procollagen genes, COL1A1 and COL1A2, account for the majority of patients with Osteogenesis Imperfecta (OI) (#166200, #166210, #166220, #259420, #259440, #610682, #610915, #610967, #610968, #613848, #613849, #613982, #614856, #615066), the disorder can be inherited in an autosomal recessive manner [1]
Since alanine is a non-bulky, uncharged amino acid that can mimic the secondary structure of many other amino acids, we opted for this substitution rather than a glutamate substitution to preserve the structural integrity of prolyl 3-hydroxylase 1 (P3H1)
We found that the mutant expression construct converting H662 to alanine (P3H1H662A) was the most effective at rescuing cartilage associated protein (CRTAP) compared with the others or the un-transduced LEPRE1 loss of function cells

Summary

Introduction

Dominant mutations in the type I procollagen genes, COL1A1 and COL1A2, account for the majority of patients with Osteogenesis Imperfecta (OI) (#166200, #166210, #166220, #259420, #259440, #610682, #610915, #610967, #610968, #613848, #613849, #613982, #614856, #615066), the disorder can be inherited in an autosomal recessive manner [1]. A mutation in cartilage associated protein (CRTAP) (*605497) was first identified by Morello et al in a class of patients with recessive OI [2]. Other clade A fibrillar collagens, such as collagen type II, are hydroxylated. Mutations in both leucine and proline enriched proteoglycan (LEPRE1), encoding P3H1, and peptidylprolyl isomerase b (PPIB), encoding CYPB, were identified in other patients with recessive forms of OI [6,7,8,9,10,11,12,13]. Mutations in additional genes have since been identified in recessive OI, revealing novel mechanisms of disease through alterations in posttranslational collagen modification, trafficking and signaling

Methods

Results

Conclusion