Changes in myosin structure and function in response to glycation

Nonenzymatic glycosylation (glycation) is recognized as an important post-t ranslational modification underlying alterations of structure and function of extracellular proteins. The effect of glycation on intracellular protein s is, on the other hand, less well known despite the vital importance of in tracellular proteins for cell, tissue, and organ function. The aim of this study was to explore the effects of glycation on the structure and function of skeletal muscle myosin. Myosin was incubated for up to 30 min with gluc ose and subsequently tested for structural and functional modifications by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and a single-fiber in vitro motility assay, respectively. MALDI spectra revealed glycation-related structural alterations as evidenced by the disappearance of specific Lys-C proteolysis products and the appearance of higher mass p eaks that are attributed to cross-linking by glucose. This change was paral leled by a significant reduction in the in vitro motility speed, suggesting a structure-related decline in myosin mechanics in response to glucose exp osure. Further evidence that early glycation products form in the regulator y regions of the myosin molecule is derived from the fact that there is com plete reversal of motility speed after reaction with the Schiff base-cleavi ng agent hydroxylamine hydrochloride. Thus, glycation of skeletal muscle my osin has a significant effect on both the structural and functional propert ies of the protein, a finding that is important in understanding the mechan isms underlying the impairment in muscle function associated with aging and diabetes.