Global Mass Spectrometry and Transcriptomics Array Based Drug Profiling Provides Novel Insight into Glucosamine Induced Endoplasmic Reticulum Stress

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. 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. Glucosamine (GlcN) 1The abbreviations used are:GlcNGlucosamineAPI5apoptosis inhibitor 5CANXcalnexinCASP2caspase-2DEHPBis (2-Ethylhexyl)(Phthalate)FDRfalse discovery rateGEOGene Expression OmnibusGLU2Bglucosidase 2 subunit betaMlog ratiosMMP12matrix metalloproteinase 12mziXICmass, charge and isotope-dependent extracted ion chromatogramsO-GlcNAcO-linked β-N-acetylglucosamineOGAO-GlcNAcaseOGTO-GlcNAc transferasePCMspolycyclodi-methylsiloxanePIpropidium iodidePKCprotein kinase CPLBPhospholambanPSMspeptide spectral matchesqPCRquantitative polymerase chain reactionRIPAradioimmune precipitation assayRMArobust multichip averageSERCAsarco/endoplasmic reticulum calcium ATPaseUDP-GalNAzUridine 5′-diphospho-N-azidoacetylgalactosamine.1The abbreviations used are:GlcNGlucosamineAPI5apoptosis inhibitor 5CANXcalnexinCASP2caspase-2DEHPBis (2-Ethylhexyl)(Phthalate)FDRfalse discovery rateGEOGene Expression OmnibusGLU2Bglucosidase 2 subunit betaMlog ratiosMMP12matrix metalloproteinase 12mziXICmass, charge and isotope-dependent extracted ion chromatogramsO-GlcNAcO-linked β-N-acetylglucosamineOGAO-GlcNAcaseOGTO-GlcNAc transferasePCMspolycyclodi-methylsiloxanePIpropidium iodidePKCprotein kinase CPLBPhospholambanPSMspeptide spectral matchesqPCRquantitative polymerase chain reactionRIPAradioimmune precipitation assayRMArobust multichip averageSERCAsarco/endoplasmic reticulum calcium ATPaseUDP-GalNAzUridine 5′-diphospho-N-azidoacetylgalactosamine. induces the posttranslational modification O-linked β-N-acetylglucosamine (O-GlcNAc) on serines and threonines (2Hart G.W. Housley M.P. Slawson C. Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins.Nature. 2007; 446: 1017-1022Crossref PubMed Scopus (1077) Google Scholar, 3Jones S.P. Zachara N.E. Ngoh G.A. Hill B.G. Teshima Y. Bhatnagar A. Hart G.W. Marban E. Cardioprotection by N-acetylglucosamine linkage to cellular proteins.Circulation. 2008; 117: 1172-1182Crossref PubMed Scopus (187) Google Scholar). Studies have demonstrated that GlcN treatment can lead to glucose intolerance and the death of pancreatic β-cells. For example, O-GlcNAc plays a role in the chronic complications of diabetes mellitus and insulin resistance (4Vosseller K. Wells L. Lane M.D. Hart G.W. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5313-5318Crossref PubMed Scopus (393) Google Scholar, 5Akimoto Y. Hart G.W. Hirano H. Kawakami H. O-GlcNAc modification of nucleocytoplasmic proteins and diabetes.Med. Mol. Morphol. 2005; 38: 84-91Crossref PubMed Scopus (42) Google Scholar). The clinical relevance of GlcN, a dietary supplement used in osteoarthritis patients, is unclear (6McCarty M.F. The neglect of glucosamine as a treatment for osteoarthritis–a personal perspective.Med. Hypotheses. 1994; 42: 323-327Crossref PubMed Scopus (59) Google Scholar). Previous investigations of long-term GlcN administration did not reveal risks or concerns (7Reginster J.Y. Deroisy R. Rovati L.C. Lee R.L. Lejeune E. Bruyere O. Giacovelli G. Henrotin Y. Dacre J.E. Gossett C. Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial.Lancet. 2001; 357: 251-256Abstract Full Text Full Text PDF PubMed Scopus (1057) Google Scholar), which is also consistent with recent studies (8Silbert J.E. Dietary glucosamine under question.Glycobiology. 2009; 19: 564-567Crossref PubMed Scopus (15) Google Scholar). However, in recent clinical studies, GlcN oral administration (8Silbert J.E. Dietary glucosamine under question.Glycobiology. 2009; 19: 564-567Crossref PubMed Scopus (15) Google Scholar) did not and the used in was not with the observed in the in after the oral administration of GlcN, which (8Silbert J.E. Dietary glucosamine under question.Glycobiology. 2009; 19: 564-567Crossref PubMed Scopus (15) Google Scholar, R. Glucosamine and to PubMed Scopus Google Scholar). studies have for the use of GlcN in the treatment of osteoarthritis and studies that in to for osteoarthritis patients, GlcN is in For example, GlcN observed in Glucosamine in associated with O-linked N-acetylglucosamine protein modification and 2007; PubMed Scopus Google Scholar), the of an in to Glucosamine inhibitor calnexin discovery Expression 2 subunit metalloproteinase charge and isotope-dependent extracted ion O-linked β-N-acetylglucosamine O-GlcNAc protein kinase spectral polymerase chain precipitation multichip reticulum calcium 5′-diphospho-N-azidoacetylgalactosamine. Glucosamine inhibitor calnexin discovery Expression 2 subunit metalloproteinase charge and isotope-dependent extracted ion O-linked β-N-acetylglucosamine O-GlcNAc protein kinase spectral polymerase chain precipitation multichip reticulum calcium 5′-diphospho-N-azidoacetylgalactosamine. GlcN in C. K. Glucosamine of of 2009; PubMed Scopus Google Scholar, C. 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The GlcN 2 and and the of O-HexNAcylation of a known of is consistent with the “yin-yang” hypothesis for phosphorylation and by Hart and G.W. O-linked the “yin-yang” of and PubMed Scopus Google Scholar). Previous studies have demonstrated for O-GlcNAc in the and The of two regulated O-HexNAc sites on proteins known to be ER that O-GlcNAc a role in the ER in to Our data that the induced of of to GlcN in cell to of in the We that and of lead to ER which is by the proteins to the the proteome of GlcN treatment by MS-based this in the We this is is that on the cell can lead to and of the C. H. of in the endoplasmic reticulum cell Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). address this to in of is the that resistance to treatment combined with GlcN treatment. We that GlcN an in of cell death However, higher GlcN the that a clinical on the combined of treatment and GlcN or is the global of and proteins in and cell lines a that and proteins are in a to glucose as a to the The ER stress that to cell as by the in and protein and of and activation can be by the from GlcN The is to be by from by for O-GlcNAc for for and for O-GlcNAc were We that the “yin-yang” between phosphorylation and O-HexNAcylation that we not by by of and the hypothesis of a “yin-yang” between phosphorylation and G.W. O-linked the “yin-yang” of and PubMed Scopus Google Scholar, L. Hart G.W. O-GlcNAc is in a with protein Full Text Full Text PDF PubMed Scopus Google Scholar, C. Hart G.W. between O-GlcNAc and the of protein PubMed Scopus Google Scholar). examples from a “yin-yang” between and to a “yin-yang” on specific protein this we two additional examples of a “yin-yang” between O-HexNAcylation and phosphorylation. For example, the between O-HexNAcylation and phosphorylation on observed in our in which the modification sites were in two different our in with O-HexNAcylation the phosphorylation of nuclear proteins the hypothesis of global “yin-yang” between and phosphorylation. The “yin-yang” between and phosphorylation observed GlcN treatment an alternative hypothesis to GlcN cell and GlcN an from to and and the of proteins the for studies in The data in this that two protein calnexin and which in the protein folding and quality in are after GlcN treatment. Furthermore, different of the O-HexNAc and from GLU2B and calnexin were observed in different cellular GLU2B and calnexin are involved in ER stress this a for a between O-HexNAc and ER stress as either a or a and that O-HexNAc in the O-GlcNAc plays a role in the ER in of