Proteomic Identification of Glycosylphosphatidylinositol Anchor-dependent Membrane Proteins Elevated in Breast Carcinoma

Abstract

Background: GPI-anchored proteins are elevated in breast carcinoma. Results: We utilized mass spectrometry and molecular biology techniques to capture and identify GPI-anchored proteins from breast carcinoma. Conclusion: Increased levels of GPI anchor addition contributes to the dedifferentiation of malignant breast epithelial cells. Significance: We have identified new potential diagnostic and therapeutic targets for breast carcinoma.

Elevated levels of glycosylphosphatidylinositol (GPI) anchored proteins in plasma from human cancers detected by C. septicum alpha toxin

“…The membrane was blocked in 5% milk/1X TBST (Blotto Solution) overnight at 4°C. The blot was incubated with biotin labeled alpha toxin (2 μ g/ml) expressed, purified, and labeled as described previously [4]. Bound toxin was detected using a 1:5,000 dilution of streptavidin-HRP (Vector Labs, Burlingame, CA) before washing and detection using Western Lightening Plus (Perkin Elmer).…”

“…For example, breast carcinoma has significant elevations in the GPAA1 and PIGT subunits of the GPIT due to chromosomal amplifications. The increased expression of these non-catalytic subunits was found to significantly increase both the tumorigenicity and the overall levels of GPI anchored proteins in breast carcinoma [1,4]. …”

“…Their abundance in specific tissues may be lower due to the regulation of specifically expressed GPI acceptor proteins. We have been utilizing alpha toxin to capture and identify GPI anchored proteins by mass spectrometry [4]. Our studies have revealed that alpha toxin binds with the GPI glycan region as shown by retained binding of the toxin after removal of the lipid portion of the GPI anchor with GPI-specific phospholipase C. Furthermore, the diversity of GPI anchored proteins that bind the toxin indicates that the binding occurs via the GPI glycan without peptide requirements.…”

“…Our studies have revealed that alpha toxin binds with the GPI glycan region as shown by retained binding of the toxin after removal of the lipid portion of the GPI anchor with GPI-specific phospholipase C. Furthermore, the diversity of GPI anchored proteins that bind the toxin indicates that the binding occurs via the GPI glycan without peptide requirements. Therefore, alpha toxin can be used as a lectin specific for the GPI anchor [4]. …”

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines

“…The technology now exists to associate altered glycomes directly with genetic disease and epigenetic changes, as well as with transcriptomes, metabolomes, proteomes and other –omics [5–9], providing further insights into the integrated biology of tumour cells and the mechanisms of disease. The potential of glycosylated epitopes in clinical medicine is well recognised; however until recently, translation into practice has been hampered by the lack of rapid, reliable, reproducible technologies for glycan analysis.…”