Specificity And Universality Of The Sugar Accepor Of OGT

O-GlcNAcylation is widely located on nucleocytoplasmic proteinsand participates in various physiological processes. But O-GlcNAc status on numerous proteins remains unknown. To better understand the sequence specificity in O-GlcNAcylation, computational analysis combined with experimental study were performed in this work. Structural analysis of38O-GlcNAcylated proteins indicated that the modification occurred predominantly at random coil region or hinge region, suggesting that primary amino acids sequence should play decisive roles in protein O-GlcNAcylation. In addition, frequency analysis of317O-GlcNAcylated peptides from human, rat and mouse revealed a signature sequence around the modification site (Position0):PPVS/TSATT. Based on the sequence, a peptide array was designed to investigate amino acids preference. At three positions (-2,-1and+2), the presence of uncharged amino acids with small side chains could confer high reactivity in O-GlcNAcylation. The observation was further convinced on an O-GlcNAcylated protein, Bovine Crystalline-a, by site-directed mutagenesis and western blot.Combining all these findings, it can be concluded that a substrate (peptide or protein) with Pro, Ala at position-2, and/or Val, Ala, Thr, Ser at position-1, and/or Ala, Ser, Pro, Thr, Gly at position+2would exhibit high reactivity. The result will contribute to predicting O-GlcNAc status of a protein and further functional studies.In addition, phage display and ELSA were performed to investigate universality of the sugar acceptor of OGT.4peptide sequences were screened from30phage clones. Detailed work should be further performed to convince these peptides.