Identification of cytoplasmic sialidase NEU2-associated proteins by LC-MS/MS

Abstract

Abstract Background Cytoplasmic sialidase (NEU2) plays an active role in removing sialic acids from oligosaccharides, glycopeptides, and gangliosides in mammalian cells. NEU2 is involved in various cellular events, including cancer metabolism, neuronal and myoblast differentiation, proliferation, and hypertrophy. However, NEU2-interacting protein(s) within the cell have not been identified yet. Objective The aim of this study is to investigate NEU2 interacting proteins using two-step affinity purification (TAP) strategy combined with mass spectrometry analysis. Methods In this study, NEU2 gene was cloned into the pCTAP expression vector and transiently transfected to COS-7 cells by using PEI. The most efficient expression time of NEU2- tag protein was determined by real-time PCR and Western blot analysis. NEU2-interacting protein(s) were investigated by using TAP strategy combined with two different mass spectrometry experiment; LC-MS/MS and MALDI TOF/TOF. Results Here, mass spectrometry analysis showed four proteins; α-actin, β-actin, calmodulin and histone H1.2 proteins are associated with NEU2. The interactions between NEU2 and actin filaments were verified by Western blot analysis and immunofluorescence analysis. Conclusions Our study suggests that association of NEU2 with actin filaments and other protein(s) could be important for understanding the biological role of NEU2 in mammalian cells.