Biochemical and Biophysical Research Communications, Vol.329, No.1, 331-336, 2005
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 arnino acid residues (NSTF). We here describe the N-glycosylation and its role for enzymatic activity of the Ag-EGase I. The N-glycosvlation 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. (C) 2005 Elscvier Inc. All rialits reserved.
Keywords:Apriona germari;baculovirus;beetle;cellulase;N-glycosylation;endoglucanase;enzyme;insect cells