David R. Eyre

Quantitation of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography

An HPLC method for quantifying the 3-hydroxypyridinium crosslinks of collagen is described. It can be applied to crude hydrolysates of all types of connective tissue. Mineralized tissues can be hydrolyzed directly and analyzed without interference from the mineral ions. The hydroxylysyl (HP) and lysyl (LP) forms of hydroxypyridinium residue were resolved on a reverse-phase C18 column using a gradient of acetonitrile in water and 0.01 M n-heptafluorobutyric acid as an ion-pairing agent. The crosslinking amino acids were accurately quantified down to 2 PM (1 ng) injected, by detecting their natural fluorescence with a spectrofluorometer. Tissues in which hydroxypyridinium crosslinks were plentiful included all forms of cartilage, bone, dentin, ligament, tendon, fascia, intervertebral disc, lung, gut, cervix, aorta, and vitreous humor. Among normal tissues, LP, the minor form of the crosslink, was present in significant amounts relative to HP only in bone and dentin. Both crosslinks were essentially absent from skin, cornea, rat tail tendon, and basement membranes.

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.

Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee.

UNLABELLED Wounds penetrating articular cartilage to bone heal with cartilage described variably as either fibrous or hyaline. In the present study, such repair cartilage was induced in the rabbit for biochemical comparison with normal articular cartilage. The main collagen in the repair tissue after three weeks was type I. By six to eight weeks, type II had become predominant and continued to be enriched up to one year; but type I still persisted as a significant constituent of the repair tissue even after a year, so the repair cartilage never fully resembled normal articular cartilage. From radiochemical analysis, type II was determined to be the major collagen synthesized by the repair tissue after three to four weeks. After six months, the repair cartilage contained more collagen and less hexosamine than control cartilage, suggesting that the fibrous texture that often developed was due to a loss of proteoglycans rather than to a change in the type of collagen. CLINICAL RELEVANCE Procedures capable of inducing the differentiation of authentic articular cartilage to resurface degenerated human joints would be invaluable. Surgical methods, such as drilling through to subchondral bone, are often attempted. It is not known, however, whether the cartilage that forms is true articular cartilage or, for example, fibrocartilage. The present experimental study in rabbits compared the properties of such repair cartilage with those of normal articular cartilage.

CRTAP Is Required for Prolyl 3- Hydroxylation and Mutations Cause Recessive Osteogenesis Imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

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.

Collagen type IX: Evidence for covalent linkages to type II collagen in cartilage

A major site of pyridinoline cross‐linking in bovine type IX collagen was traced to a tryptic peptide derived from one of the molecules HMW chains. This peptide gave two amino acid sequences (in ratio) consistent with it being a three‐chained structure. The major sequence matched exactly that of the C‐telopeptide of type II collagen from the same tissue. A second HMW chain that contained pyridinoline cross‐links also gave two amino‐terminal sequences, one from its own amino terminus, the other matching exactly the N‐telopeptide cross‐linking sequence of type II collagen. We conclude that type IX collagen molecules are covalently cross‐linked in cartilage to molecules of type II collagen, probably at fibril surfaces.

Homozygosity for a Missense Mutation in SERPINH1, which Encodes the Collagen Chaperone Protein HSP47, Results in Severe Recessive Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is characterized by bone fragility and fractures that may be accompanied by bone deformity, dentinogenesis imperfecta, short stature, and shortened life span. About 90% of individuals with OI have dominant mutations in the type I collagen genes COL1A1 and COL1A2. Recessive forms of OI resulting from mutations in collagen-modifying enzymes and chaperones CRTAP, LEPRE1, PPIB, and FKBP10 have recently been identified. We have identified an autosomal-recessive missense mutation (c.233T>C, p.Leu78Pro) in SERPINH1, which encodes the collagen chaperone-like protein HSP47, that leads to a severe OI phenotype. The mutation results in degradation of the endoplasmic reticulum resident HSP47 via the proteasome. Type I procollagen accumulates in the Golgi of fibroblasts from the affected individual and a population of the secreted type I procollagen is protease sensitive. These findings suggest that HSP47 monitors the integrity of the triple helix of type I procollagen at the ER/cis-Golgi boundary and, when absent, the rate of transit from the ER to the Golgi is increased and helical structure is compromised. The normal 3-hydroxylation of the prolyl residue at position 986 of the triple helical domain of proalpha1(I) chains places the role of HSP47 downstream from the CRTAP/P3H1/CyPB complex that is involved in prolyl 3-hydroxylation. Identification of this mutation in SERPINH1 gives further insight into critical steps of the collagen biosynthetic pathway and the molecular pathogenesis of OI.

Mutations in the Gene Encoding the RER Protein FKBP65 Cause Autosomal-Recessive Osteogenesis Imperfecta

Osteogenesis imperfecta is a clinically and genetically heterogeneous brittle bone disorder that results from defects in the synthesis, structure, or posttranslational modification of type I procollagen. Dominant forms of OI result from mutations in COL1A1 or COL1A2, which encode the chains of the type I procollagen heterotrimer. The mildest form of OI typically results from diminished synthesis of structurally normal type I procollagen, whereas moderately severe to lethal forms of OI usually result from structural defects in one of the type I procollagen chains. Recessively inherited OI, usually phenotypically severe, has recently been shown to result from defects in the prolyl-3-hydroxylase complex that lead to the absence of a single 3-hydroxyproline at residue 986 of the alpha1(I) triple helical domain. We studied a cohort of five consanguineous Turkish families, originating from the Black Sea region of Turkey, with moderately severe recessively inherited OI and identified a novel locus for OI on chromosome 17. In these families, and in a Mexican-American family, homozygosity for mutations in FKBP10, which encodes FKBP65, a chaperone that participates in type I procollagen folding, was identified. Further, we determined that FKBP10 mutations affect type I procollagen secretion. These findings identify a previously unrecognized mechanism in the pathogenesis of OI.

Quantitative analysis of types I and II collagens in human intervertebral discs at various ages.

Abstract The molecular species of collagen in the human intervertebral disc have been identified and measured quantitatively by assay for specific peptides produced by digestion of whole tissue with cyanogen bromide. As previously found in the pig, the annulus fibrosus of lumbar intervertebral discs contained two genetically distinct molecular types of collagen, types I and II, whereas predominantly type II collagen was found in the nucleus pulposus. In each tissue the proportion of hydroxylysine residues in the total collagen that were glycosylated gave support to these findings, even though the contents of hydroxylysine alone provided an unreliable index of molecular type. The annulus fibrosus of human lumbar discs as a whole contained a considerably higher proportion of type II collagen (50–65% of total collagen) than did pig annulus fibrosus (20% of total collagen). No significant variations with age were found in the relative proportions of types I and II collagens in the annuli fibrosi from lumbar discs of individuals aged 5, 16, 59 and 66 years. At all ages more than 85% of the collagen in the nucleus pulposus was type II. In comparison, using the same procedure in the semi-lunar meniscus of the knee, which is classified as fibrocartilage, at least 95% of the collagen was type I, whereas more than 95% of that in young or old human articular cartilage was type II. The distributions of each type of collagen were assessed in a radial section of the annulus of T12/L1 of a 5 year old spine. Their relative proportions varied inversely and smoothly from being almost all type I collagen at the outer edge of the annulus to only type II on reaching the nucleus pulposus. This distribution already shown in pig annulus fibrosus appears to be a characteristic structural feature of this tissue.

Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis

OBJECTIVE Osteoarthritis (OA) is a chronic and slowly progressive disease for which biomarkers may be able to provide a more rapid indication of therapeutic responses to therapy than is currently available; this could accelerate and facilitate OA drug discovery and development programs. The goal of this document is to provide a summary and guide to the application of in vitro (biochemical and other soluble) biomarkers in the development of drugs for OA and to outline and stimulate a research agenda that will further this goal. METHODS The Biomarkers Working Group representing experts in the field of OA biomarker research from both academia and industry developed this consensus document between 2007 and 2009 at the behest of the Osteoarthritis Research Society International Federal Drug Administration initiative (OARSI FDA initiative). RESULTS This document summarizes definitions and classification systems for biomarkers, the current outcome measures used in OA clinical trials, applications and potential utility of biomarkers for development of OA therapeutics, the current state of qualification of OA-related biomarkers, pathways for biomarker qualification, critical needs to advance the use of biomarkers for drug development, recommendations regarding practices and clinical trials, and a research agenda to advance the science of OA-related biomarkers. CONCLUSIONS Although many OA-related biomarkers are currently available they exist in various states of qualification and validation. The biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two general categories, those that will allow targeting of subjects most likely to either respond and/or progress (prognostic value) within a reasonable and manageable time frame for a clinical study (for instance within 1-2 years for an OA trial), and those that provide early feedback for preclinical decision-making and for trial organizers that a drug is having the desired biochemical effect. As in vitro biomarkers are increasingly investigated in the context of specific drug treatments, advances in the field can be expected that will lead to rapid expansion of the list of available biomarkers with increasing understanding of the molecular processes that they represent.