N-glycosylation is necessary for enzymatic activity of a beetle (Apriona germari) cellulase.
We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family 45 cloned from the mulberry longicorn beetle, Apriona germari (Ag-EGase I), is composed of 237 amino acid residues and has a potential N-glycosylation site at 97-100 amino acid residues (NSTF). We here describe the N-glycosylation and its role for enzymatic activity of the Ag-EGase I. The N-glycosylation of Ag-EGase I was revealed by the treatment of tunicamycin to the recombinant virus-infected insect Sf9 cells and by endoglycosidase F to the purified recombinant Ag-EGase I, demonstrating that the carbohydrate moieties are not necessary for secretion but essential for Ag-EGase I enzyme activity. To further elucidate the functional role of the N-glycosylation in Ag-EGase I, we have assayed the cellulase enzyme activity in Thr99Gln mutant. Lack of N-glycosylation in Ag-EGase I showed no substantial enzyme activity. This result demonstrates that N-glycosylation at site 97-100 amino acid residues (NSTF) is essential for enzyme activity.
Wei
Ya Dong
YD
Department of Applied Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea.
Lee
Seong Jin
SJ
Lee
Kwang Sik
KS
Gui
Zhong Zheng
ZZ
Yoon
Hyung Joo
HJ
Kim
Iksoo
I
Je
Yeon Ho
YH
Guo
Xijie
X
Sohn
Hung Dae
HD
Jin
Byung Rae
BR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002241:Carbohydrates / Q000737:chemistry; D002460:Cell Line; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D001517:Coleoptera; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007313:Insecta; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D002241:Carbohydrates; D011994:Recombinant Proteins; D002480:Cellulase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase