Abstract
We investigated the molecular effects of glucosamine supplements, a popular and safe alternative to nonsteroidal anti-inflammatory drugs, for decreasing pain, inflammation, and maintaining healthy joints. Numerous studies have reported an array of molecular effects after glucosamine treatment. We questioned whether the differences in the effects observed in previous studies were associated with the focus on a specific subproteome or with the use of specific cell lines or tissues. To address this question, global mass spectrometry- and transcription array-based glucosamine drug profiling was performed on malignant cell lines from different stages of lymphocyte development. We combined global label-free MS-based protein quantitation with an open search for modifications to obtain the best possible proteome coverage. Our data were largely consistent with previous studies in a variety of cellular models. We mainly observed glucosamine induced O-GlcNAcylation/O-GalNAcylation (O-HexNAcylation); however, we also observed global and local changes in acetylation, methylation, and phosphorylation. For example, our data provides two additional examples of "yin-yang" between phosphorylation and O-HexNAcylation. Furthermore, we mapped novel O-HexNAc sites on GLU2B and calnexin. GLU2B and calnexin are known to be located in the endoplasmic reticulum (ER) and involved in protein folding and quality control. The O-HexNAc sites were regulated by glucosamine treatment and correlated with the up-regulation of the ER stress marker GRP78. The occupancy of O-HexNAc on GLU2B and calnexin sites differed between the cytosolic and nuclear fractions with a higher occupancy in the cytosolic fraction. Based on our data we propose the hypothesis that O-HexNAc either inactivates calnexin and/or targets it to the cytosolic fraction. Further, we hypothesize that O-HexNAcylation induced by glucosamine treatment enhances protein trafficking.
Highlights
IntroductionStudies have demonstrated that GlcN treatment can lead to glucose intolerance and the death of pancreatic -cells
From the ‡Proteolysis in Diseases, IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal; §Human Genetics Department, National Institute of Health Dr Ricardo Jorge, Av
Lymphocyte-derived Cell Lines Respond Differently to GlcN—To define the effects of GlcN at the molecular level, we reasoned that a strong increase in the O-GlcNAcylation of proteins would be needed
Summary
Studies have demonstrated that GlcN treatment can lead to glucose intolerance and the death of pancreatic -cells. In recent clinical studies, GlcN oral administration [8] did not demonstrate any benefits, and the concentration used in vitro was not comparable with the levels observed in the plasma in vivo after the oral administration of GlcN, which raised skepticism [8, 9]. Global Glucosamine Profiling several studies have suggested caution for the use of GlcN in the treatment of osteoarthritis and other autoimmune diseases. GlcN exhibits anticancer properties in vitro [11, 12], and a recent review by Slawson et al [13] suggested several molecular mechanisms through which O-GlcNAcylation can play a regulatory role in cancer. The negative side effect from high drug dosages is more acceptable in the case of terminal cancers
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