Identification and function of beta-1,4-galactosyltransferase associated proteins.

摘要

Studies in several laboratories demonstrated that sperm surface beta 1,4-galactosyltransferase (GalTase-I) binds to ZP3 and stimulates the acrosome reaction. To study the signaling events activated by GalTase-I, the cytoplasmic domain of GalTase-I was expressed as fusion proteins with maltose binding protein. These expressed fusion proteins and synthetic GalTase-I peptide were used to screen a pig testis phage display cDNA library. Several phage clones that possess high affinity with GalTase-I fusion proteins and synthetic peptide were identified. The identified proteins include a kinase anchor protein 9; rho-associated, coiled-coil containing protein kinase I; actin; sarcolemmal associated protein-3 and a protein has high homology to rhodopsin receptor. The interaction between phage clone and GalTase-I cytoplasmic domain was further tested and confirmed with GalTase-I binding assay and Far-Western assay.; Dot-blot and GalTase-I binding assay confirmed that actin is associated with GalTase-I in vitro in mouse sperm lysate; synthetic GalTase-I cytoplasmic peptide affinity precipitated actin in mouse sperm; furthermore, the affinity precipitation results demonstrated that actin form dimmers after acrosomal reaction; cytochalasin B inhibits actin polymerization also prevent ionphore A 23187 induced mouse sperm acrosomal reaction.; As an alternative method, synthetic GalTase-I peptide was immobilized to affinity isolate GalTase-I associated proteins in pig sperm lysate. Several proteins were found specifically associated with GalTase-I cytoplasmic domain as demonstrated in SDS-PAGE. Mass spectrometric sequencing determined that one of the proteins identified is homologous to human squamous cell carcinoma antigen.; In summary, a pig testis phage display cDNA library screening and protein affinity chromatography were applied to identify GalTase-I associated proteins. Several proteins were identified as candidates that interact with GalTase-I and potentially play important roles during fertilization. Deciphering proteins that interact with GalTase-I cytoplasmic domain will greatly enhance our ability to understand the signaling events activated by GalTase-I during the process of fertilization.