title: N-linked glycosylation of a beetle (Apriona germari) cellulase Ag-EGase II is necessary for enzymatic activity.
abstract: 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.
authors.affiliation: Department of Applied Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea.
authors.forename: Ya Dong
authors.initials: YD
authors.lastname: Wei
authors.forename: Kwang Sik
authors.initials: KS
authors.lastname: Lee
authors.forename: Zhong Zheng
authors.initials: ZZ
authors.lastname: Gui
authors.forename: Hyung Joo
authors.initials: HJ
authors.lastname: Yoon
authors.forename: Iksoo
authors.initials: I
authors.lastname: Kim
authors.forename: Yeon Ho
authors.initials: YH
authors.lastname: Je
authors.forename: Sang Mong
authors.initials: SM
authors.lastname: Lee
authors.forename: Guo Zheng
authors.initials: GZ
authors.lastname: Zhang
authors.forename: Xijie
authors.initials: X
authors.lastname: Guo
authors.forename: Hung Dae
authors.initials: HD
authors.lastname: Sohn
authors.forename: Byung Rae
authors.initials: BR
authors.lastname: Jin
mesh_terms: 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
chemical_list: D019476:Insect Proteins; D002480:Cellulase