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.

MATERIALS AND 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.

CONCLUSION:

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: Pukkavadee Netsirisawan, Daranee Chokchaichamnankit, Chantragan Srisomsap, Jisnuson Svasti, Voraratt Champattanachai
• Journal: Cancer genomics & proteomics (Cancer Genomics Proteomics) 2015 Jul-Aug Vol. 12 Issue 4 Pg. 201-9 ISSN: 1790-6245 [Electronic] Greece
• PMID: 26136220 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Chemical References

• Culture Media, Serum-Free
• Neoplasm Proteins
• N-Acetylglucosaminyltransferases
• hexosaminidase C
• beta-N-Acetylhexosaminidases

Topics

• Breast Neoplasms (enzymology, metabolism, pathology)
• Cell Compartmentation
• Cell Line, Tumor
• Culture Media, Serum-Free
• Electrophoresis, Gel, Two-Dimensional
• Extracellular Space (metabolism)
• Female
• Glycosylation
• Humans
• Intracellular Space (metabolism)
• Mass Spectrometry
• N-Acetylglucosaminyltransferases (metabolism)
• Neoplasm Proteins (metabolism)
• Proteomics (methods)
• Reproducibility of Results
• beta-N-Acetylhexosaminidases (metabolism)