Generalized Connective Tissue Disease in Crtap-/- Mouse

Mutations in CRTAP (coding for cartilage-associated protein), LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1]) or PPIB (coding for Cyclophilin B [CYPB]) cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation. Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition to the expected loss of proline 986 3-hydroxylation in α1(I) and α1(II) chains, there was also loss of 3Hyp at proline 986 in α2(V) chains. In contrast, at two of the known 3Hyp sites in α1(IV) chains from Crtap-/- kidneys there were normal levels of 3-hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa. These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical proline sites within clade A (types I, II, and V) collagen chains. Loss of this activity leads to a multi-systemic connective tissue disease that affects bone, cartilage, lung, kidney, and skin

Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues

<div><p>Mutations in the genes encoding cartilage associated protein (<i>CRTAP</i>) and prolyl 3-hydroxylase 1 (P3H1 encoded by <i>LEPRE1</i>) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by <i>PPIB</i>), 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 (<i>Lepre1^H662A</i>). 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.</p></div

<i>Lepre1^H662A/H662A</i> fibroblast procollagen secretion rate and collagen modification is normal.

<p>Analysis of procollagen secretion by the collagen pulse-chase assay suggests that the procollagen secreted from <i>Lepre1^H662A/H662A</i> fibroblasts is similar to <i>Lepre1^+/+</i> fibroblasts (A, B). Additionally, there does not appear to be a decrease in the amount of procollagen secreted from the <i>Lepre1^H662A/H662A</i> fibroblasts in comparison to <i>Lepre1^+/+</i> fibroblasts (A, B). These findings are in contrast to that of the <i>Crtap^−/−</i> fibroblasts, which have an increase in the rate of procollagen secretion (A). Collagen modification was assessed using the collagen steady-state assay. We observed no difference in the migration pattern of procollagen and collagen isolated from <i>Lepre1^+/+</i>(+/+) and <i>Lepre1^H662A/H662A</i> (H662A/H662A) fibroblasts (C). These assays were repeated three times.</p

Loss of Prolyl 3-hydroxylation at Pro986 in type I collagen in bone and type II collagen in cartilage.

<p>Upon generation of the <i>Lepre1^H662A/H662A</i> mice, we confirmed the stability of both P3H1 and CRTAP by western blot using protein isolated from mouse calvaria (A, experiments repeated 3 times). Comparing protein isolated from the <i>Lepre1^H662A/H662A</i> and the <i>Lepre1^+/+</i> mice, we found no differences in the levels of P3H1 and CRTAP when compared to γ-Tubulin. Analysis of prolyl 3-hydroxylation of Pro986 on the α(1) chain of type I collagen in bone using mass spectrometry demonstrates complete loss of 3-hydroxylation in the <i>Lepre1^H662A/H662A</i> mice when compared to <i>Lepre1^+/+</i> littermates (B). Similarly, analysis of the Pro986 site on the α1 chain of type II collagen in cartilage demonstrates a reduction to 9% 3-hydroxylation in the <i>Lepre1^H662A/H662A</i> mice and is similar to what was reported in <i>Crtap^−/−</i> mice (C).</p

Prolyl 3-hydroxylation at Pro986 in the α2(V) collagen chain.

<p>Mass spectral analysis of Pro986 hydroxylation in tryptic peptides from the α2(V) chain of bone from <i>Lepre1^+/+</i> and <i>Lepre1^H662A/H662A</i> mice (A and B respectively) shows a marked reduction in hydroxylation at this site. The MS/MS fragmentation patterns shown in C and D identified the 765.8²⁺ peptide and its 3-hydroxylated version 773.9²⁺. A portion (40%) of the latter ion was also found by MS/MS to be contributed by a version lacking 3-Hyp but containing 4-Hyp at P978 (taken into account in the 3-Hyp quantitation).</p

<i>Lepre1^H662A/H662A</i> mice have smaller collagen fibril diameter.

<p>Transmission EM analysis of collagen fibrils from skin revealed fibrils more homogenous in size in the <i>Lepre1^H662A/H662A</i> mice as compared to the wild-type littermates. Additionally, the collagen fibril diameters are slightly smaller, as quantified by a slight increase in the proportion of smaller diameter collagen fibrils. (bar = 100 nm). (N = 3,150 collagen diameters measured per animal, p<0.05).</p