In this study, we focus on non-enzymatic glycations (abnormal sugar additions) on collagens in aging tissue. Among the many known cross-links in fibrillar collagens, perhaps the least understood but most speculative pathologically are advanced glycation end-products (AGEs). Normal enzymatic lysyl oxidase-mediated cross- links between individual collagen molecules in a fibril are essential for the strength and integrity of tendons and most musculoskeletal tissues. On the other hand, non-enzymatic glycations are thought to accumulate randomly on aging tissue collagens, with the potential to form abnormal intermolecular cross-links. Glycations and subsequent AGE cross-links in collagens have been linked to limited joint mobility, tissue fragility, and diminished healing capacity, all of which are commonly associated with aging and age-related diseases. Collagen glycations, therefore, not only affect the rapidly aging US population, but also one in three Americans already suffering from prediabetes and hyperglycemia. We propose to 1) identify the main sites of non- enzymatic glycations in type I collagen in aging tendon, and 2) characterize changes in the normal lysyl oxidase-mediated collagen cross-linking associated with non-enzymatic glycations in tendon. Although collagen is a frequently cited substrate for non-enzymatic glycation, the lack of experimental data on any molecular sites of collagen glycation, let alone AGE cross-linking, is striking. Instead, total AGE cross-links in collagen have typically been measured from whole tissue hydrolysates. We propose a paradigm shifting hypothesis that glycations occur preferentially, not randomly, at the helical domain cross-linking lysine residues in tendon type I collagen. These newly identified glycated lysine residues on type I collagen will be compared in aging (young vs. old) human Achilles tendons using targeted ion trap mass spectrometry. We next hypothesize that glycations and AGE products can prevent nascent collagen from enzymatically cross-linking to fibrils in the tendon unit. Changes in the divalent and trivalent collagen cross-link profile will be simultaneously quantitated using an innovative, newly adapted silica hydride based chromatographic approach coupled with established mass spectrometric methods. Age-related glycations at the helical domain cross- linking lysines of type I collagen are predicted to weaken tissues by hindering normal cross-linking as tissues age. Biomechanical testing will be used to correlate increased collagen glycations with changes in tendon material properties. It is essential to locate the primary sites of collagen glycation and their subsequent AGE products before real progress can be made in understanding the specific pathogenic consequences of glycation. These sites could then serve as potential molecular markers of pathology and therapeutic targets. The knowledge gained from this study could help develop targeted therapeutics used to prevent the effects of aging and hyperglycemia in musculoskeletal tissue by preventing abnormal AGE products from forming in collagen.

Public Health Relevance

Aging has many adverse effects on connective tissues, including limited joint mobility, increased risk of injury and impaired healing ability. The random addition of sugar molecules to collagen, a process called protein glycation, is thought to contribute to musculoskeletal complications that are commonly associated with aging. Using the power of mass spectrometry, the goal is to identify the specific sites of glycation on collagen molecules in order to help understand how sugar molecules can negatively affect connective tissues.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Exploratory/Developmental Grants (R21)
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Skeletal Biology Structure and Regeneration Study Section (SBSR)
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Williams, John
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University of Washington
Schools of Medicine
United States
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