Collagen Diversity and Pathobiology in Skeletal Tissues
In this competing renewal of AR036794, we focus on a newly discovered mechanism of regulation of collagen fibril diversity that is important for understanding skeletal tissue differentiation and pathobiology. We have evidence that 3-hydroxproline (3Hyp) residues play a fundamental role in directing the manner of fibrillar collagen supramolecular assembly. The first hint came from our demonstration by mass spectrometry that the single, fully occupied 3Hyp site (P986) near the C-terminus of collagen a1(I) and a1(II) chains fails to be hydroxylated in collagens of the crtap mouse and recessive forms of human osteogenesis imperfecta (O.I.) caused by CRTAP or LEPRE1 mutations. We now have evidence for three classes of 3Hyp site in fibril-forming collagens. For example, a second site in type II collagen is highly hydroxylated in vitreous, meniscus and intervertebral disc but not in hyaline cartilage. The sequence motif of this second site is reproduced at D-periodic intervals in a2(V), with 3Hyp present, and shows similarities to a 3Hyp motif in type IV collagen. Tendon collagen uniquely contains a third type of 3Hyp motif, which we believe is characteristic and functionally important in tendon, ligament and related highly tensile tissues. Under 4 aims, we intend to pursue this concept aggressively since it is central to understanding how different cell types regulate the diversity of heteropolymeric collagen assemblies between different cartilages, bone, tendon and other connective tissues. If correct, it also has concept- changing implications for the field of vertebrate collagen biology. The clinical significance is in providing a molecular basis for understanding processes that cause cartilages and other collagenous tissues of low turnover to degenerate in the adult musculoskeleton in osteoarthritis, disc degeneration and related disorders of collagen framework failure. In addition, a full understanding of the effects of disrupting prolyl 3-hydroxylation in recessive forms of osteogenesis imperfecta we believe will reveal a molecular mechanism for brittle bone common to all forms of O.I. Such findings will also be significant for understanding qualitative changes in bone matrix that add significant risk of osteoporotic fracture in the population as a whole and a potential for novel biomarkers and therapeutic targets.
PUBLIC HEALTH RELEVANCE: The goal is to understand molecular mechanisms that govern the diversity in properties of collagen, the protein that forms the structural framework of all major skeletal tissues in the body including bone, cartilages, tendons and ligaments. Specifically, we aim to define the biochemical pathways that equip bone collagen to mineralize, cartilage to last a lifetime as the bearing surfaces of joints and tendons and ligaments to transmit or restrain high mechanical loads without failing. With this knowledge new targets for therapy and prevention of genetic and acquired human disorders of bones and joints are predicted.
Collagen Cross-Linking in Skeletal Aging and Disease
We are studying how collagen cross-links have evolved to adapt human bone, cartilage and other supporting tissues for their distinctive functions. We have raised the structure which predicts a novel chemistry and critical role for pyrrole cross-links in bone collagen and identified a previously unknown cross-link, arginoline, in cartilage collagen. We propose and will seek to validate a unified theory of oxidative maturation for cross-links in the fibrillar collagens of all tissues. Though the lysyl oxidase cross-linking mechanism was discovered in the 1970s, much is still unknown. Post-translational differences in collagen quality between individuals, notably in the cross-linking chemistry of bone and cartilages, are potential risk factors for osteoporotic fracture and joint failure. They result from cumulative environmental influences rather than a direct genetic basis. We are pursuing this through analyses of bone, cartilage and other skeletal tissue collagens using advanced mass spectrometric protein techniques. The clinical significance is the promise of new molecular targets in the effort to meet the public health challenges of osteoporosis and osteoarthritis. Skeletal tissues depend heavily on the cross-linking of highly specialized collagens for their unique strengths, properties and longevity. The translational aim, therefore, is to seek new molecular targets for therapy and non- invasive biomarkers based on urinary collagen peptides that can index a patient's bone quality, joint cartilage breakdown rate and other measures of skeletal health.
PUBLIC HEALTH RELEVANCE: Using the power of protein mass spectrometry the goal is to complete a basic scientific understanding of how different skeletal collagens are covalently cross-linked. Qualitative differences in bone collagen cross-linking that develop with age and may to be linked to risk of osteoporotic fracture independent of bone density are of particular interest. In parallel with basic tissue studies, collagen fragments in urine are being examined by similar methods as potential non-invasive biomarkers of a patient's bone and joint health.
Protein Biochemisty Core
This Core resource to a program-project is focused on defining inborn protein defects that cause osteochondrodysplasia syndromes. We are determining the protein consequences at the molecular level of mutations in genes that cause defects in cartilage structure. This Protein Biochemistry Core actively collaborates in parallel with all projects by providing analytical data from specialized methods, including protein mass spectrometry, applied to tissue samples, purified proteins and cell-culture products. Both luman material from the International Skeletal Dysplasias Registry and mouse tissue and cellular products rom genetically engineered transgenic strains are being studied. Hypotheses as to the nature and biochemical effects of mutations that cause abnormal skeletal development and adult function are integral to the overall goals and aims of all projects and cores. The focus of the Protein Biochemistry Core is to understand the downstream molecular effects of mutations, both new ones identified by human genetic inkage and mutational analysis, and established mutations still with key questions on their protein pathogenesis and created transgenic defects in mice. An example of the latter is the systemic collagen defect in crtap -/- mice. The clinical significance of core work includes direct benefits to families with the conditions under study through more specific diagnoses. In the long term, through an understanding of the effects of altered gene expression, rational approaches to therapy are possible.