• BMB Reports
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• 제51권12호
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• pp.609-610
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• 2018

Glycosylation is one form of protein modification and plays a key role in protein stability, function, signaling regulation and even cancer. NleB and SseK are bacterial effector proteins and possess glycosyltransferase activity, even though they have different substrate preferences. NleB/SseKs transfer the GlcNAc sugar to an arginine residue of host proteins, leading to reduced $NF-{\kappa}B-dependent$ responses. By combining X-ray crystallography, NMR, molecular dynamics, enzyme kinetic assays and in vivo experiments, we demonstrated that a conserved HEN (His-Glu-Asn) motif in the active site plays a key role in enzyme catalysis and virulence. The lid-domain regulates the opening and closing of the active site and the HLH domain determines the substrate specificity. Our findings provide evidence for the enzymatic mechanism by which arginine can be glycosylated by SseK/NleB enzymes.

• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.191-195
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• 2003

The in vitro glycosylation of pentapeptide (Arg-Lys-Asp-Val-Tyr; RKDVY) and RNase A was carried out using PNGase F (peptide-N-glycosidase F), and the results were analyzed using MALDI-TOF-MS. Aminated N,N-diretyl chitobiose was used as the sugar in the glycosylation reaction, and the amination yield of N,N'-diacetyl chitobiose was about $60\%$. To reduce the water activity and shift the reaction equilibrium to a reverse reaction, 1,4-dioxane or ethylene glycol was used as the organic solvent in the enzymatic glycosylation. A certain extent of nonenzymatic glycosylaton, known as the Maillard reaction, was also observed, which occurs on an arginine or lysine residue when the length of tie sugar residue is one or two. However, the extent of glycosylation was much higher in the enzymatic reaction, indicating that PNGase F can be effectively used to produce glycopeptides and glycoproteins in vitro.

• Bulletin of the Korean Chemical Society
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• 제25권12호
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• pp.1791-1800
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• 2004

Angiotensin I, a model decapeptide, was glycosylated and partially hydrolyzed with HCl (6 N, 80 $^{\circ}C$, 4 h), aminopeptidase, and carboxypeptidase Y. A single peptide mass map obtained from truncated peptides in the partial acid hydrolysate of angiotensin and its glycosylation product mixture by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry enabled sequencing of angiotensin by a combinatorial procedure. MALDI-TOF and electrospray ionization (ESI) tandem mass spectrometric results indicate that both the N-terminal amino group of aspartic acid and the guanidinium group of the second residue arginine are glycosylated.