Identificaton of novel Cystoisospora suis enolase 2 interaction proteins in IPEC-J2 cells using Co-IP/LC-MS.

Enolase 2 (ENO2) is a crucial an important glycolytic enzyme involved in glycolysis and is also recognised as a multifunctional protein. However, its interacting proteins in intestinal porcine epithelial cells (IPEC-J2 cells) remain unclear. This study investigates the interacting proteins of Cystoisospora suis (Biester et Murray, 1934) ENO2 (CsENO2) in IPEC-J2 cells. According to our previous research, CsENO2 is highly expressed in sporulated oocysts. Co-immunoprecipitation/liquid chromatography-mass spectrometry (Co-IP/LC-MS) was conducted using the pcDNA3.1-Flag vector carrying the enolase 2 gene, which was transfected into IPEC-J2 cells to co-immunoprecipitate with cellular proteins. The Co-IP/LC-MS-based protein sequencing identified three cellular proteins: NME2, RPS3, and Vimentin. Subsequently, cDNAs encoding the complete coding sequences of these proteins were cloned into the pGEX-4T-1 expression vector. Immunofluorescence assays revealed the localisation of CsENO2 in sporulated oocysts and sporozoites. Most CsENO2 protein was found in the cytoplasm of sporulated oocysts. In sporozoites, CsENO2 protein was primarily distributed around and outside the nucleus. This study aims to identify CsENO2-interacting proteins in IPEC-J2 cells and determine the localisation of CsENO2 within sporulated oocysts and sporozoites.