A nonradioactive glycosyltransferase assay is described here. This method takes advantage of specific phosphatases that can be added into glycosyltransferase reactions to quantitatively release inorganic phosphate from the leaving groups of glycosyltransferase reactions. The released phosphate group is then detected using colorimetric malachite-based reagents. Because the amount of phosphate released is directly proportional to the sugar molecule transferred in a glycosyltransferase reaction, this method can be used to obtain accurate kinetic parameters of the glycosyltransferase. The assay can be performed in multiwell plates and quantitated by a plate reader, thus making it amenable to high-throughput screening. It has been successfully applied to all glycosyltransferases available to us, including glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosyltransferases, galactosyltransferases, fucosyltransferases and sialyltransferases. As examples, we first assayed Clostridium difficile toxin B, a protein O-glucosyltransferase that specifically monoglucosylates and inactivates Rho family small GTPases; we then showed that human KTELC1, a homolog of Rumi from Drosophila, was able to hydrolyze UDP-Glc; and finally, we measured the kinetic parameters of human sialyltransferase ST6GAL1.
This study provides the first direct evidence of AMD stage- and region-specific changes in retinal protein levels and highlights potential novel, disease-related proteins and biochemical pathways for future studies of AMD.
The proteasome mediates pathways associated with oxidative stress and inflammation, two pathogenic events correlated with age-related macular degeneration (AMD). In human donor eyes corresponding to four stages of AMD, we found the proteasomal chymotrypsin-like activity increased in neurosensory retina with disease progression. Increased activity correlated with a dramatic increase in the inducible subunits of the immunoproteasome, which was not due to an increase in CD45 positive immune cells in the retina. The novel observation of proteasome transformation may reflect retinal response to local inflammation or oxidative stress with AMD.
Consistently modified proteins indicate preferred protein targets for oxidation by HNE. HNE-modified proteins were not different between regions or stages, suggesting that pathways for detoxification of HNE or removal of damaged proteins are adequate. Consistent levels of HNE-modified proteins suggest that HNE is not a sensitive retinal biomarker for AMD.
Crystallins are small heat shock proteins with chaperone function that prevent heat- and oxidative stress-induced aggregation of proteins. This is the first report describing modifications of alphaA crystallin in the sensory retina, including altered content and truncation with aging. Proteins from adult, middle age, and old Fischer 344 Brown Norway rats were compared. Western immunoblotting was used to evaluate alphaA crystallin content and identify protein spots on two-dimensional gels containing alphaA crystallin. The type and site of multiple post-translational modifications were identified by mass spectrometry. We found the content of alphaA crystallin was significantly decreased in the oldest rats. On two-dimensional gels, retinal crystallins resolved into multiple spots with altered migration, indicative of changes in intrinsic charge and/or truncation. Post-translational modifications that were identified included oxidation, phosphorylation, deamidation, acetylation, and truncation. In samples from rats of all ages, a highly modified N-terminus containing these modifications was found. We also observed an age-dependent difference in the extent of N- and C-terminal truncation. These results suggest that protection against stress-induced protein aggregation is compromised in the aged retina.
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