In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post- Translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid-derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of a1b1 integrin consistent with the modification of collagen IV by hypochlorous (HOCl) and hypobromous acids. Noncollagenous (NC1) hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W28 and W192 are significantly elevated compared with nondiabetic controls. Molecular dynamics simulations predicted a more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/ refolding. Our results suggest that hypohalous acid-derived modifications of renal ECM, and specifically collagen IV networks, contribute to functional protein damage in diabetes.
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Acknowledgments. The authors thank Ms. Parvin Todd, Vanderbilt University, for expert technical help, and Dr. Vadim Pedchenko, Vanderbilt University, for helpful discussions. The authors also thank the Vanderbilt Center for Structural Biology for the generous use of their computational facilities. Funding. This work was supported by grants DK-65138 and DK-18381 from the National Institutes of Health (NIH). K.L.B. was supported by NIH grant 5T-32HL-94296-06. C.D. was supported by NIH research fellowship award T32-DK-007569-24S. H.M. was supported by NIH research fellowship grant DK-65123. J.A. and N.B. were supported by the Vanderbilt Aspirnaut program. Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. K.L.B., C.D., K.L.R., M.-Z.Z., and P.V. researched the data and wrote the manuscript. O.A.S., H.M., J.A., and N.B. researched the data and contributed to the discussion. A.F., R.H., and B.G.H. contributed to the discussion and reviewed and edited the manuscript. P.V. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
This work was supported by grants DK-65138 and DK-18381 from the National Institutes of Health (NIH). K.L.B. was supported by NIH grant 5T- 32HL-94296-06. C.D. was supported by NIH research fellowship award T32-DK- 007569-24S. H.M. was supported by NIH research fellowship grant DK-65123. J.A. and N.B. were supported by the Vanderbilt Aspirnaut program.