Dr. Feng Shao's laboratory and Dr. Lei Liu's group together developed synthesis method for a novel type of glycopeptides and a specific RabMab antibody recognizing GlcNAc-modified arginine.

    On Oct. 29, 2014, Dr. Feng Shao's laboratory at National Institute of Biological Sciences (NIBS), Beijing, in collaboration with Dr. Lei Liu's group at Tsinghua University, chemically synthesized a novel glycopeptides and therefore generated highly specific and sensitive antibodies for GlcNAc-modified arginine, a novel type of posttranslational modification. The work entitled "Synthesis of and Specific Antibody Generation for Glycopeptides with Arginine N-GlcNAcylation" is published online in the journal Angew. Chem. Int. Ed.

    Protein glycosylation is a general posttranslational modification and plays important roles in biology. In a recent study from Dr. Shao's laboratory (Li et al., Nature, 2013), they discover that the NleB family of virulence factors from Enteropathogenic E. coli (EPEC) employs a novel N-acetylglucosamine (GlcNAc) transferase activity to modify an arginine residue in host death-domain proteins, block death  receptors-mediated inflammation and host cell death, and therefore promote bacterial colonization and induce pathological effects in infected animals. The arginine GlcNAcylation modification, catalyzed by NleB, differs from the canonical O-GlcNAcylation in eukaryotes in the modification site and the glycol-linkage, but both modifications use UDP-GlcNAc as the sugar donor, involve a similar enzymatic mechanism, and occur in the cytoplasm or nuclei. UDP-GlcNAc is the end product of the hexosamine biosynthetic pathway. The functionality of this pathway as well as the metabolic efficiency of UDP-GlcNAc is influenced by the level of cellular glucose, amino acid, and fatty acid. UDP-GlcNAc connects the dynamic and interconnected metabolism of three major nutrients in cells. Thus, it is possible that arginine GlcNAcylation may play a similar and universal role as the endogenous O-GlcNAcylation in regulating cellular processes such as gene transcription, signal transduction, cell growth and differentiation, circadian rhythm, and immunity in eukaryotes.

    To further study the biological function of this newly discovered protein posttranslational  modification, Dr. Shao's laboratory collaborated with Dr. Lei Liu's laboratory at Tsinghua University, designed a silver-assisted solid phase synthesis method, and established the first general strategy for a rapid and cost-effective synthesis of glycopeptides carrying single or multiple arginine N-GlcNAcyl  groups. Using this peptide as the immunogen, the researchers successfully generated antibodies that could specifically recognize arginine N-GlcNAcylated peptides or proteins. The antibody only recognizes GlcNAcylated arginine in dependently of the peptide backbone, and shows a high sensitivity and can detect NleB modification of host death-domain targets during EPEC infection. More importantly, the antibody does not cross-react with the endogenous O-GlcNAcylation modification in eukaryotes. The study is the first to synthesize GlcNAcylated arginine-containing peptides and generate a high-specific antibody for arginine  GlcNAcylation modification. The desired property of the antibody shall allow for development of new diagnostic tools for enterobacterial infections as well as probing the existence and functionality of endogenous arginine GlcNAcylation in eukaryotes.

    Dr. Shan Li, a visiting scholar in the Shao laboratory, and graduate student Man Pan in the Liu laboratory are co-first authors of the paper. Xiang Li in the Liu group has also contributed to this work. Drs. Lei Liu, Hong-Gang Hu and Feng Shao are co-corresponding authors. The research was supported by the 973 National High-Tech. Projects, China National Science Foundation, and the Beijing Municipal Government.

http://dx.doi.org/doi:10.1002/anie.201407824