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Evidence of glucuronidation of the glycation product LW-1: tentative structure and implications for the long-term complications of diabetes

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Abstract

LW-1 is a collagen-linked blue fluorophore whose skin levels increase with age, diabetes and end-stage renal disease (ESRD), and correlate with the long-term progression of microvascular disease and indices of subclinical cardiovascular disease in type 1 diabetes. The chemical structure of LW-1 is still elusive, but earlier NMR analyses showed it has a lysine residue in an aromatic ring coupled to a sugar molecule reminiscent of advanced glycation end-products (AGEs). We hypothesized and demonstrate here that the unknown sugar is a N-linked glucuronic acid. LW-1 was extracted and highly purified from ~99 g insoluble skin collagen obtained at autopsy from patients with diabetes/ESRD using multiple rounds of proteolytic digestion and purification by liquid chromatography (LC). Advanced NMR techniques (1H–NMR, 13C–NMR, 1H-13C HSQC, 1H-1H TOCSY, 1H-13C HMBC) together with LC-mass spectrometry (MS) revealed a loss of 176 amu (atomic mass unit) unequivocally point to the presence of a glucuronic acid moiety in LW-1. To confirm this data, LW-1 was incubated with β-glycosidases (glucosidase, galactosidase, glucuronidase) and products were analyzed by LC-MS. Only glucuronidase could cleave the sugar from the parent molecule. These results establish LW-1 as a glucuronide, now named glucuronidine, and for the first time raise the possible existence of a “glucuronidation pathway of diabetic complications”. Future research is needed to rigorously probe this concept and elucidate the molecular origin and biological source of a circulating glucuronidine aglycone.

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Acknowledgments

We thank the University Hospitals Cleveland Medical Center, Cleveland, OH and the National Disease Research Interchange (NDRI), Philadelphia, PA for providing the skin tissue. We thank Christopher Strauch for assistance with mass spectrometry analyses.

Funding

This study was funded by grants from the NIDDK (R21 DK-79432 to D.R.S., DK101123 to V.M.M.) and JDRF (17–2010-318 to V.M.M.).

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Authors and Affiliations

  1. Department of Pathology, Case Western Reserve University, Wolstein Research Bldg. 5-301, 2103 Cornell Road, Cleveland, OH, 44106, USA

    David R. Sell & Vincent M. Monnier

  2. Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195, USA

    Ina Nemet

  3. School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China

    Zhili Liang

  4. Department of Biochemistry, Case Western Reserve University, Cleveland, OH, 44106, USA

    Vincent M. Monnier

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  1. David R. Sell
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Sell, D.R., Nemet, I., Liang, Z. et al. Evidence of glucuronidation of the glycation product LW-1: tentative structure and implications for the long-term complications of diabetes. Glycoconj J 35, 177–190 (2018). https://doi.org/10.1007/s10719-017-9810-7

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