TY - JOUR
T1 - Specificity of enzymatic in vitro glycosylation by PNGase F
T2 - A comparison of enzymatic and non-enzymatic glycosylation
AU - Jeong, Hee Yong
AU - Lee, Ji Youn
AU - Park, Tai Hyun
N1 - Funding Information:
The authors wish to acknowledge the financial support of the Korea Science & Engineering Foundation through the Nano Bio-Electronic & System Center, Seoul National University, Seoul, Korea.
PY - 2004/12/1
Y1 - 2004/12/1
N2 - Enzymatic in vitro glycosylation is possible using a reverse reaction of peptide-N-glycosidase F (PNGase F), and non-enzymatic in vitro glycosylation occurs when the sugar residue is one or two units long. To identify the differences between enzymatic and non-enzymatic glycosylation, glycosylation sites were analyzed by the acid hydrolysis of glycopeptides followed by MALDI-TOF mass spectrometric analysis. Pentapeptide (Arg-Lys-Asp-Val-Tyr) and octapeptide (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr) were used in this study, and the sequence of the octapeptide was appropriately chosen to investigate the specificity of enzymatic glycosylation by considering the characteristics of PNGase F and non-enzymatic glycosylation. N,N′-Diacetylchitobiose was aminated prior to the glycosylation reaction at an amination extent of 60%. The glycosylation site was very specific to the aspartate residue in the enzymatic reaction, while non-enzymatic glycosylation occurred at arginine or lysine residues. PNGases F can be effectively used for the glycosylation of the non-glycosylated recombinant proteins produced in prokaryotic cells.
AB - Enzymatic in vitro glycosylation is possible using a reverse reaction of peptide-N-glycosidase F (PNGase F), and non-enzymatic in vitro glycosylation occurs when the sugar residue is one or two units long. To identify the differences between enzymatic and non-enzymatic glycosylation, glycosylation sites were analyzed by the acid hydrolysis of glycopeptides followed by MALDI-TOF mass spectrometric analysis. Pentapeptide (Arg-Lys-Asp-Val-Tyr) and octapeptide (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr) were used in this study, and the sequence of the octapeptide was appropriately chosen to investigate the specificity of enzymatic glycosylation by considering the characteristics of PNGase F and non-enzymatic glycosylation. N,N′-Diacetylchitobiose was aminated prior to the glycosylation reaction at an amination extent of 60%. The glycosylation site was very specific to the aspartate residue in the enzymatic reaction, while non-enzymatic glycosylation occurred at arginine or lysine residues. PNGases F can be effectively used for the glycosylation of the non-glycosylated recombinant proteins produced in prokaryotic cells.
KW - Acid hydrolysis
KW - In vitro glycosylation
KW - MALDI-TOF mass spectrometry
KW - N,N′-Diacetylchitobiose
KW - Peptide-N-glycosidase F (PNGase F)
UR - http://www.scopus.com/inward/record.url?scp=7544229461&partnerID=8YFLogxK
U2 - 10.1016/j.enzmictec.2004.08.010
DO - 10.1016/j.enzmictec.2004.08.010
M3 - Article
AN - SCOPUS:7544229461
SN - 0141-0229
VL - 35
SP - 587
EP - 591
JO - Enzyme and Microbial Technology
JF - Enzyme and Microbial Technology
IS - 6-7
ER -