title: Proteomic Analysis Reveals Aberrant O-GlcNAcylation of Extracellular Proteins from Breast Cancer Cell Secretion.
abstract: BACKGROUND
  O-GlcNAcylation is a unique intracellular protein modification; however, few extracellular O-GlcNAc-modified proteins have been discovered. We have previously demonstrated that many cellular proteins were aberrant in O-GlcNAcylation in breast cancer tissues. In the present study, therefore, we investigated whether O-GlcNAc-modified proteins were abnormally secreted from breast cancer cells.
  
  
  METHODS
  Intracellular and extracellular proteins were prepared from cell lysates of breast cancer cells (MCF-7 and MDA-MB-231) and normal breast cells (HMEC) and from their serum-free media (SFM), respectively. O-GlcNAcylation level was examined by immunoblotting. O-GlcNAc-Modified proteins were identified using two-dimensional gel electrophoresis and Liquid Chromatography-tandem Mass Spectrometry.
  
  
  RESULTS
  O-GlcNAcylation level was significantly increased in the extracellular compartment of both types of cancer cells compared to normal cells. Interestingly, O-GlcNAc patterns differed between intracellular and extracellular proteins. Proteomic analysis revealed that many O-GlcNAc spots in MCF-7 secretions were abnormally increased in comparison to those in HMEC secretions. Among these, transitional endoplasmic reticulum ATPase (TER ATPase) and heat-shock 70 kDa (HSP70) were confirmed to be O-GlcNAc-modified. The levels of O-GlcNAc-HSP70 and O-GlcNAc-TER ATPase were higher in SFM from MCF-7 cells than in that from HMEC.
  
  
  CONCLUSIONS
  O-GlcNAcomic study of the extracellular compartments reveals aberrant O-GlcNAc-secreted proteins, which may be of interest as potential biomarkers in breast cancer.
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Laksi, Bangkok, Thailand.
authors.forename: Pukkavadee
authors.initials: P
authors.lastname: Netsirisawan
authors.affiliation: Laboratory of Biochemistry, Chulabhorn Research Institute, Laksi, Bangkok, Thailand.
authors.forename: Daranee
authors.initials: D
authors.lastname: Chokchaichamnankit
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Laksi, Bangkok, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Laksi, Bangkok, Thailand.
authors.forename: Chantragan
authors.initials: C
authors.lastname: Srisomsap
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Laksi, Bangkok, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Laksi, Bangkok, Thailand.
authors.forename: Jisnuson
authors.initials: J
authors.lastname: Svasti
authors.affiliation: Applied Biological Sciences Program, Chulabhorn Graduate Institute, Laksi, Bangkok, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Laksi, Bangkok, Thailand voraratt@cri.or.th.
authors.forename: Voraratt
authors.initials: V
authors.lastname: Champattanachai
mesh_terms: D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D002451:Cell Compartmentation; D045744:Cell Line, Tumor; D016895:Culture Media, Serum-Free; D015180:Electrophoresis, Gel, Two-Dimensional; D005110:Extracellular Space / Q000378:metabolism*; D005260:Female; D006031:Glycosylation; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D013058:Mass Spectrometry; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D016895:Culture Media, Serum-Free; D009363:Neoplasm Proteins; D017351:N-Acetylglucosaminyltransferases; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases
keywords: Breast cancer; O-GlcNAcylation; extracellular proteins; heat-shock 70 kDa; transitional endoplasmic reticulum ATPase