N-linked glycosylation of a beetle (Apriona germari) cellulase Ag-EGase II is necessary for enzymatic activity.
We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family (GHF) 45 of the mulberry longicorn beetle, Apriona germari (Ag-EGase II), has three potential N-linked glycosylation sites; these sites are located at amino acid residues 56-59 (NKSG), 99-102 (NSTF), and 237-239 (NYSstop). In the present study, we analyze the functional role of these potential N-linked glycosylation sites. Tunicamycin treatment completely abolished the enzymatic activity of Ag-EGase II. To further elucidate the functional role of the N-linked glycosylation sites in Ag-EGase II, we have assayed the cellulase enzyme activity in Ser58Gln, Thr101Gln, or Ser239Gln mutants. Lack of N-linked glycosylation site at residues 99-102 (NSTF), the site of which is conserved in known beetle GHF 45 cellulases, showed loss of enzyme activity and reduced the molecular mass of the enzyme. In contrast, mutations in Ser58Gln or Ser239Gln affected neither the activity nor the apparent molecular mass of the enzyme, indicating that these sites did not lead to N-linked glycosylation. The present study demonstrates that N-linked glycosylation at residues 99-102 (NSTF), while not essential for secretion, is required for Ag-EGase II 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
Kwang Sik
KS
Gui
Zhong Zheng
ZZ
Yoon
Hyung Joo
HJ
Kim
Iksoo
I
Je
Yeon Ho
YH
Lee
Sang Mong
SM
Zhang
Guo Zheng
GZ
Guo
Xijie
X
Sohn
Hung Dae
HD
Jin
Byung Rae
BR
D019943:Amino Acid Substitution; D000818:Animals; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D046188:Protein Modification, Translational
D019476:Insect Proteins; D002480:Cellulase