Wayne A. Cabral

New perspectives on osteogenesis imperfecta

A new paradigm has emerged for osteogenesis imperfecta as a collagen-related disorder. The more prevalent autosomal dominant forms of osteogenesis imperfecta are caused by primary defects in type I collagen, whereas autosomal recessive forms are caused by deficiency of proteins which interact with type I procollagen for post-translational modification and/or folding. Factors that contribute to the mechanism of dominant osteogenesis imperfecta include intracellular stress, disruption of interactions between collagen and noncollagenous proteins, compromised matrix structure, abnormal cell–cell and cell–matrix interactions and tissue mineralization. Recessive osteogenesis imperfecta is caused by deficiency of any of the three components of the collagen prolyl 3-hydroxylation complex. Absence of 3-hydroxylation is associated with increased modification of the collagen helix, consistent with delayed collagen folding. Other causes of recessive osteogenesis imperfecta include deficiency of the collagen chaperones FKBP10 or Serpin H1. Murine models are crucial to uncovering the common pathways in dominant and recessive osteogenesis imperfecta bone dysplasia. Clinical management of osteogenesis imperfecta is multidisciplinary, encompassing substantial progress in physical rehabilitation and surgical procedures, management of hearing, dental and pulmonary abnormalities, as well as drugs, such as bisphosphonates and recombinant human growth hormone. Novel treatments using cell therapy or new drug regimens hold promise for the future.

Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta

A recessive form of severe osteogenesis imperfecta that is not caused by mutations in type I collagen has long been suspected. Mutations in human CRTAP (cartilage-associated protein) causing recessive bone disease have been reported. CRTAP forms a complex with cyclophilin B and prolyl 3-hydroxylase 1, which is encoded by LEPRE1 and hydroxylates one residue in type I collagen, α1(I)Pro986. We present the first five cases of a new recessive bone disorder resulting from null LEPRE1 alleles; its phenotype overlaps with lethal/severe osteogenesis imperfecta but has distinctive features. Furthermore, a mutant allele from West Africa, also found in African Americans, occurs in four of five cases. All proband LEPRE1 mutations led to premature termination codons and minimal mRNA and protein. Proband collagen had minimal 3-hydroxylation of α1(I)Pro986 but excess lysyl hydroxylation and glycosylation along the collagen helix. Proband collagen secretion was moderately delayed, but total collagen secretion was increased. Prolyl 3-hydroxylase 1 is therefore crucial for bone development and collagen helix formation.

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates.

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The “cell interaction domain” is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The “matrix interaction domain” may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.

Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The probands collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought.

Null mutations in LEPRE1 and CRTAP cause severe recessive osteogenesis imperfecta

Classical osteogenesis imperfecta (OI) is a dominant genetic disorder of connective tissue caused by mutations in either of the two genes encoding type I collagen, COL1A1 and COL1A2. Recent investigations, however, have generated a new paradigm for OI incorporating many of the prototypical features that distinguish dominant and recessive conditions, within a type I collagen framework. We and others have shown that the long-sought cause of the recessive form of OI, first postulated in the Sillence classification, lies in defects in the genes encoding cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1/LEPRE1). Together with cyclophilin B (PPIB), CRTAP and P3H1 comprise the collagen prolyl 3-hydroxylation complex, which catalyzes a specific posttranslational modification of types I, II, and V collagen, and may act as a general chaperone. Patients with mutations in CRTAP or LEPRE1 have a lethal to severe osteochondrodystrophy that overlaps with Sillence types II and III OI but has distinctive features. Infants with recessive OI have white sclerae, undertubulation of the long bones, gracile ribs without beading, and a small to normal head circumference. Those who survive to childhood or the teen years have severe growth deficiency and extreme bone fragility. Most causative mutations result in null alleles, with the absence or severe reduction of gene transcripts and proteins. As expected, 3-hydroxylation of the Pro986 residue is absent or severly reduced, but bone severity and survival length do not correlate with the extent of residual hydroxylation. Surprisingly, the collagen produced by cells with an absence of Pro986 hydroxylation has helical overmodification by lysyl hydroxylase and prolyl 4-hydroxylase, indicating that the folding of the collagen helix has been substantially delayed.

Mutations near amino end of alpha 1(I) collagen cause combined osteogenesis imperfecta/Ehlers-Danlos syndrome by interference with N-propeptide processing

Patients with OI/EDS form a distinct subset of osteogenesis imperfecta (OI) patients. In addition to skeletal fragility, they have characteristics of Ehlers-Danlos syndrome (EDS). We identified 7 children with types III or IV OI, plus severe large and small joint laxity and early progressive scoliosis. In each child with OI/EDS, we identified a mutation in the first 90 residues of the helical region of α1(I) collagen. These mutations prevent or delay removal of the procollagen N-propeptide by purified N-proteinase (ADAMTS-2) in vitro and in pericellular assays. The mutant pN-collagen which results is efficiently incorporated into matrix by cultured fibroblasts and osteoblasts and is prominently present in newly incorporated and immaturely cross-linked collagen. Dermal collagen fibrils have significantly reduced cross-sectional diameters, corroborating incorporation of pN-collagen into fibrils in vivo. Differential scanning calorimetry revealed that these mutant collagens are less stable than the corresponding procollagens, which is not seen with other type I collagen helical mutations. These mutations disrupt a distinct folding region of high thermal stability in the first 90 residues at the amino end of type I collagen and alter the secondary structure of the adjacent N-proteinase cleavage site. Thus, these OI/EDS collagen mutations are directly responsible for the bone fragility of OI and indirectly responsible for EDS symptoms, by interference with N-propeptide removal.

Components of the collagen prolyl 3-hydroxylation complex are crucial for normal bone development

Prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP) and cyclophilin B (CyPB) form a complex in the endoplasmic reticulum which is responsible for 3-hydroxylation of a limited number of proline residues in types I, II and V collagens. In this complex, CRTAP serves the role of helper protein, while P3H1 provides the enzymatic activity for the modification. In type I collagen, the major protein of the extracellular matrix of bone, the complex 3-hydroxylates only the α1(I)Pro986 residue. P3H1 and CRTAP each also have independent roles as components of matrix. Furthermore, the two proteins have significant homology with each other. The critical importance of the components of the complex for normal bone development has been revealed by a Crtap knock-out mouse and by infants and children with null mutations of CRTAP and LEPRE1, the gene that encodes P3H1. On a clinical level, defects in the components of the prolyl 3-hydroxylation complex have been shown to be the long-sought cause of severe and lethal recessive osteogenesis imperfecta.

Mapping of SPARC/BM-40/Osteonectin-binding Sites on Fibrillar Collagens

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix αA of the extracellular calcium-binding domain of SPARC and is partially masked by helix αC. Previously, we found that the removal of helix αC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site ∼180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven α2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among α chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.

Absence of FKBP10 in Recessive Type XI Osteogenesis Imperfecta Leads to Diminished Collagen Cross-Linking and Reduced Collagen Deposition in Extracellular Matrix

Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% overmodification. Normal chain incorporation, helix folding, and collagen Tm support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross‐linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. Hum Mutat 33:1589–1598, 2012. Published 2012 Wiley Periodicals, Inc.*

Structural Heterogeneity of Type I Collagen Triple Helix and Its Role in Osteogenesis Imperfecta

We investigated regions of different helical stability within human type I collagen and discussed their role in intermolecular interactions and osteogenesis imperfecta (OI). By differential scanning calorimetry and circular dichroism, we measured and mapped changes in the collagen melting temperature (ΔTm) for 41 different Gly substitutions from 47 OI patients. In contrast to peptides, we found no correlations of ΔTm with the identity of the substituting residue. Instead, we observed regular variations in ΔTm with the substitution location in different triple helix regions. To relate the ΔTm map to peptide-based stability predictions, we extracted the activation energy of local helix unfolding (ΔG‡) from the reported peptide data. We constructed the ΔG‡ map and tested it by measuring the H-D exchange rate for glycine NH residues involved in interchain hydrogen bonds. Based on the ΔTm and ΔG‡ maps, we delineated regional variations in the collagen triple helix stability. Two large, flexible regions deduced from the ΔTm map aligned with the regions important for collagen fibril assembly and ligand binding. One of these regions also aligned with a lethal region for Gly substitutions in the α1(I) chain.