Edited by Xiao-Fan WangTandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both The collagen family of proteins has evolved into many different genes and gene translational products each with variably regulated post-translational modifications (1, 2). Even within a single genetic type of collagen, the post-translational quality of the protein is known to be regulated with a high degree of tissue specificity (3, 4). The functional significance of this post-translational variability is still not fully understood, although for fibril-forming collagens the number, placement, and chemistry of covalent intermolecular cross-links seem to be critically important regulators of tissue material properties and function (5-7).In the last decade, new insights on the significance of a relatively rare collagen modification, prolyl 3-hydroxylation, came from the discovery that recessive forms of osteogenesis imperfecta (OI) 2 are caused by biallelic mutations in prolyl 3-hydroxylase 1 (P3H1; Lepre1), CRTAP (Leprel3), or CypB (PPIB) (8). These proteins, which form a P3H1 enzyme complex, act on nascent collagen chains in the ER (9). Mutations in at least 6 further genes that encode either enzymes (e.g. PLOD2 encoding lysyl hydroxylase-2 (LH2) (10)) or chaperones (e.g. FKBP10 encoding FKBP65 (11, 12)), needed for collagen modification, folding, transport, and normal mineralization, have been shown to cause OI variants. We have gained insights on the disease-causing mechanisms by analyzing tissue collagens from OI patients and mouse models of OI. For example, P3H1, CRTAP, and PPIB mutations all cause telopeptide lysine overhydroxylation (5), whereas PLOD2 or FKBP10 mutations cause telopeptide lysine under-hydroxylation (10 -12). A common effect from all these mutations is an altered collagen cross-linking chemistry. These findings suggest an interplay in the ER between the prolyl 3-hydroxylation complex and the lysyl hydroxylation machinery.Human mutations in P3H2 (Leprel1) have been shown to cause the eye disorder high myopia (13-15). Two different P3H2 knock-out mice have so far been generated by separate laboratories, the first was embryonic lethal (16), whereas the second had no obvious phenotype (17). Mass spectral analysis of...

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis.

Respiratory disease is a leading cause of mortality in patients with osteogenesis imperfecta (OI), a connective tissue disease that causes severely reduced bone mass and is most commonly caused by dominant mutations in type I collagen genes. Previous studies proposed that impaired respiratory function in OI patients was secondary to skeletal deformities; however, recent evidence suggests the existence of a primary lung defect. Here, we analyzed the lung phenotype of Crtap knockout (KO) mice, a mouse model of recessive OI. While we confirm changes in the lung parenchyma that are reminiscent of emphysema, we show that CrtapKO lung fibroblasts synthesize type I collagen with altered posttranslation modifications consistent with those observed in bone and skin. Unrestrained whole body plethysmography showed a significant decrease in expiratory time, resulting in an increased ratio of inspiratory time over expiratory time and a concomitant increase of the inspiratory duty cycle in CrtapKO compared with WT mice. Closed-chest measurements using the forced oscillation technique showed increased respiratory system elastance, decreased respiratory system compliance, and increased tissue damping and elasticity in CrtapKO mice compared with WT. Pressure-volume curves showed significant differences in lung volumes and in the shape of the curves between CrtapKO mice and WT mice, with and without adjustment for body weight. This is the first evidence that collagen defects in OI cause primary changes in lung parenchyma and several respiratory parameters and thus negatively impact lung function.

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Members of the Leprecan family of proteins include enzymes, prolyl 3-hydroxylase 1 (P3h1), P3h2 and P3h3, and non-enzymatic proteins, Crtap and Sc65. Mutations in CRTAP and LEPRE1 (encoding P3H1) have been associated with human disease such as recessive osteogenesis imperfecta, however, the function of Sc65 which is closely related and highly homologous to Crtap is unknown. Sc65 has been described as a synaptonemal complex protein, a nucleolar protein, and a cytoplasmic adapter protein. In light of its high sequence similarity with Crtap, an endoplasmic reticulum (ER)-associated protein, and the importance of post-translational modifications such as collagen prolyl 3-hydroxylation in bone metabolism, we hypothesized that Sc65 was an ER-resident protein that would have an important role in bone homeostasis. In this study, we demonstrate that Sc65 is a previously unrecognized ER protein, and that it does not localize in the nucleus of somatic cells. Moreover, Sc65 is expressed and functional during skeletal development since loss of Sc65 results in a progressive osteopenia that affects both trabecular and cortical bone. Bone loss is due to increased bone resorption mediated by a non-cell autonomous effect on osteoclasts. Therefore, Sc65, like its related family member Crtap, is an important modulator of bone homeostasis, acting as a negative regulator of osteoclastogenesis.