Srikanth B, Ravindra G, Sachin ME, Prajna H, Lu GY, Bale MS and Shashikala RI
Background: TF antigen specific Sclerotium rolfsii lectin (SRL) inhibits human colon epithelial cancer HT29 cell growth by induction of apoptosis through cell surface binding and has tumor suppressing effect in vivo as reported earlier. Here we report the purification, identification and characterization of SRL binding membrane proteins from HT29 cells.
Methods and Findings: Membrane proteins from HT29 cells were isolated by phase separation and purified by affinity chromatography using SRL-Sepharose- 4B matrix. Affinity purified proteins were subjected to in-gel and in-solution trypsin digestion, analysed by ESI-Q-TOF LC-MS and spectrum mill software. Considering the specificity of SRL towards O-glycans, the presence of O-GalNAc sites in SRL interacting proteins were tested using NetOGlyc software. Western blotting was performed to validate the MS identified proteins. A major protein band around 25kDa following in-gel trypsin digestion was identified as Keratin 1 by MS. In-solution trypsin digestion followed by MS identified 25 SRL interacting proteins namely, keratins, heat shock proteins, tubulins, pyruvate kinase M1/M2, peroxiredoxin-1, ATP synthase subunit alpha, mitochondrial, retinal dehydrogenase 1, actin, annexin-A2, prohibitin, ADP/ATP translocase-2 and alpha enolase. NetOGlyc software analysis revealed 21 proteins positive for O-glycosylation sites including keratins alone containing 27 to 50 O-GalNAc sites. Keratin 1 identified and validated by western blotting as major SRL interacting protein showed 49 O-GalNAc sites.
Conclusion: SRL binding membrane proteins from human colon epithelial cancer HT29 cells have been identified and characterized. Identified proteins contain O-GalNAc sites and are known to be involved in cell survival, apoptosis and tumorigenesis. The present study provides insights in studying the mechanism of SRL induced apoptosis and to explore lectin for its clinical implications.