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

Residues important for hydroxylase function and catalytic mutations rescue the stability of CRTAP.

<p>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 <i>LEPRE1</i> loss of function fibroblasts with WT or H662A mutant <i>LEPRE1</i> 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).</p

A High-Density Genetic Recombination Map of Sequence-Tagged Sites for <i>Sorghum</i>, as a Framework for Comparative Structural and Evolutionary Genomics of Tropical Grains and Grasses

Abstract We report a genetic recombination map for Sorghum of 2512 loci spaced at average 0.4 cM (∼300 kb) intervals based on 2050 RFLP probes, including 865 heterologous probes that foster comparative genomics of Saccharum (sugarcane), Zea (maize), Oryza (rice), Pennisetum (millet, buffelgrass), the Triticeae (wheat, barley, oat, rye), and Arabidopsis. Mapped loci identify 61.5% of the recombination events in this progeny set and reveal strong positive crossover interference acting across intervals of ≤50 cM. Significant variations in DNA marker density are related to possible centromeric regions and to probable chromosome structural rearrangements between Sorghum bicolor and S. propinquum, but not to variation in levels of intraspecific allelic richness. While cDNA and genomic clones are similarly distributed across the genome, SSR-containing clones show different abundance patterns. Rapidly evolving hypomethylated DNA may contribute to intraspecific genomic differentiation. Nonrandom distribution patterns of multiple loci detected by 357 probes suggest ancient chromosomal duplication followed by extensive rearrangement and gene loss. Exemplifying the value of these data for comparative genomics, we support and extend prior findings regarding maize-sorghum synteny—in particular, 45% of comparative loci fall outside the inferred colinear/syntenic regions, suggesting that many small rearrangements have occurred since maize-sorghum divergence. These genetically anchored sequence-tagged sites will foster many structural, functional and evolutionary genomic studies in major food, feed, and biomass crops.</jats:p

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

Micro-Computed Tomography and cortical biomechanical analyses.

<p>3D reconstruction of spines from the <i>Lepre1^H662A/H662A</i> mice compared to wild-type littermates. The <i>Lepre1^H662A/H662A</i> 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 <i>Lepre1^H662A/H662A</i> mice have normal cortical bone but reduced trabecular bone. (N = 10, each genotype).</p