Evidence for the association of synaptotagmin with glutathione S-transferases: implications for a novel function in human breast cancer.
OBJECTIVE
To analyze the pattern of changes in GSTs in cancerous and adjacent non-cancerous tissues obtained from breast cancer patients undergoing surgery.


METHODS
Cytosolic GST purification, assay of GST, protein expression levels, and GST-synaptotagmin association were analyzed using standard biochemical techniques like GSH-affinity purification, spectrophotometry, SDS-PAGE, Western blots, and matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF).


RESULTS
GST activity in cancerous tissues (0.26 U/mg protein) was significantly higher (P < 0.05) as compared to those from adjacent non-cancerous tissues (0.14 U/mg protein) of breast cancer patients. Further analysis of GST subunits on SDS-PAGE and Western blots using class-specific GST antibodies revealed significant elevation in GST-pi levels in cancer tissues with no appreciable changes in GST-alpha and GST-mu. Along with the elevation of GST-pi levels, high molecular weight proteins (approximately 70 kDa) cross reacting with GST antibodies were detected only in surgically resected tumor biopsies but not in the non-cancerous tissues adjacent to the tumor. Based on MALDI-TOF analysis, the high molecular weight band was identified as synaptotagmin V bound to GST-M1 with 47% sequence coverage after processing on an MS-FIT search engine.


CONCLUSIONS
Our results suggest a novel putative functional role for the GST-synaptotagmin complex in human breast cancers. As this association of GST M1-synaptotagmin was not seen in adjacent non-cancerous tissues, this can be used as a marker for breast cancers.
Sreenath
A S
AS
Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
Kumar
K Ravi
KR
Reddy
G V
GV
Sreedevi
B
B
Praveen
D
D
Monika
S
S
Sudha
S
S
Reddy
M Gopal
MG
Reddanna
P
P
D000328:Adult; D015153:Blotting, Western; D001940:Breast / Q000201:enzymology; D001943:Breast Neoplasms / Q000201:enzymology / Q000503:physiopathology*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D018270:Carcinoma, Ductal, Breast / Q000201:enzymology / Q000503:physiopathology; D002846:Chromatography, Affinity; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C494295:SYT5 protein, human; D050857:Synaptotagmins; D005982:Glutathione Transferase