Figure 1.
Residues important for hydroxylase function and catalytic mutations rescue the stability of CRTAP.
Evolutionary trace analysis identified residues important for the function of P3H1 by comparing each residue within the dioxygenase domain family. The highest ranking residues are mapped onto the dioxygenase domain responsible for prolyl 3-hydroxylation (A). These residues include 3 that interact with iron (His590, Asp592, and His662) shown in red and 1 that interacts with 2-oxoglutarate (Arg672) shown in yellow. An alanine substitution was introduced at HIS662 (H662A) to deactivate the hydroxylase function. To test whether the mutant P3H1 was able to rescue the stability of CRTAP, we transduced immortalized LEPRE1 loss of function fibroblasts with WT or H662A mutant LEPRE1 cDNA and assayed for the presence of CRTAP by immunofluorescence and immunoblot. Here we demonstrate that the stability of CRTAP is rescued by immunofluorescence (B) and by immunoblot (C).
Figure 2.
Loss of Prolyl 3-hydroxylation at Pro986 in type I collagen in bone and type II collagen in cartilage.
Upon generation of the Lepre1H662A/H662A 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 Lepre1H662A/H662A and the Lepre1+/+ 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 Lepre1H662A/H662A mice when compared to Lepre1+/+ 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 Lepre1H662A/H662A mice and is similar to what was reported in Crtap−/− mice (C).
Figure 3.
Prolyl 3-hydroxylation at Pro986 in the α2(V) collagen chain.
Mass spectral analysis of Pro986 hydroxylation in tryptic peptides from the α2(V) chain of bone from Lepre1+/+ and Lepre1H662A/H662A 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).
Table 1.
Hydroxylation status at different collagen sites.
Table 2.
Pyridinoline content and telopeptide hydroxylation of bone collagen.
Figure 4.
Lepre1H662A/H662A mice are normal in gross morphology.
Analysis at 3 months by X-ray shows no difference in terms of skeletal patterning between genotypes (A). By growth curve analysis, there is no difference in weight at any time point over 3 months between Lepre1+/+ and Lepre1H662A/H662A mice (B). We assessed rhizomelia by measuring the length of both the femur and tibia and computing the ratio. Comparing Lepre1+/+ to Lepre1H662A/H662A mice, we observed no difference in the length of the femur, tibia, or their ratio (C). N = 10, both genotypes.
Figure 5.
Lepre1H662A/H662A mice have a normal femoral hypertrophic zone.
Since the Lepre1−/− animals showed disorganization of the hypertrophic zone, we assessed the hypertrophic zone (P1) of Lepre1H662A/H662A mice by H&E staining (A) and by specifically marking the hypertrophic zone using an antibody directed against type×collagen (B). The hypertrophic zone of the femur of the Lepre1H662A/H662A mice are indistinguishable from their wild-type littermates (B) and this is confirmed by quantifying the width of the hypertrophic zone in which there is no difference in the width between genotypes (C) (N = 10, both genotypes).
Figure 6.
Micro-Computed Tomography and cortical biomechanical analyses.
3D reconstruction of spines from the Lepre1H662A/H662A mice compared to wild-type littermates. The Lepre1H662A/H662A mice have less trabecular bone as quantified by reduced bone volume (BV/TV), reduced trabecular number (Tb.N), reduced trabecular thickness (Tb.Th), reduced trabecular bone mineral density (BMD), and increased trabecular separation (Tb.Sp). Cortical parameters are similar to wild-type, as quantified by normal cortical BMD or stiffness and force to failure. These results suggest that the Lepre1H662A/H662A mice have normal cortical bone but reduced trabecular bone. (N = 10, each genotype).
Figure 7.
Histomorphometry of Lepre1H662A/H662A mice.
By histomorphometry, we were able to confirm the low trabecular bone mass phenotype in the Lepre1H662A/H662A mice as quantified by decreased bone volume (BV/TV) and trabecular thickness (Tb.Th). We observed no differences in osteoblast and osteoclast parameters, as measured by the number of osteoblasts (N.Ob/BS) and osteoclast surface (OcS/BS). We observed no difference in the kinetic indices of bone formation, as measured by mineral apposition (MAR), mineralizing surface (MS/BS) and bone formation rate (BFR/TV) and in osteoid parameters, as measured by osteoid volume (OV/BV) and osteoid surface (OS/BS) (mean ± SD, N = 9, both genotypes).
Figure 8.
Lepre1H662A/H662A mice have smaller collagen fibril diameter.
Transmission EM analysis of collagen fibrils from skin revealed fibrils more homogenous in size in the Lepre1H662A/H662A 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).
Figure 9.
Lepre1H662A/H662A fibroblast procollagen secretion rate and collagen modification is normal.
Analysis of procollagen secretion by the collagen pulse-chase assay suggests that the procollagen secreted from Lepre1H662A/H662A fibroblasts is similar to Lepre1+/+ fibroblasts (A, B). Additionally, there does not appear to be a decrease in the amount of procollagen secreted from the Lepre1H662A/H662A fibroblasts in comparison to Lepre1+/+ fibroblasts (A, B). These findings are in contrast to that of the Crtap−/− 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 Lepre1+/+(+/+) and Lepre1H662A/H662A (H662A/H662A) fibroblasts (C). These assays were repeated three times.
Table 3.
Table comparing Lepre1H662A/H662A phenotype to established OI mouse models.