title: Introduction of bisecting GlcNAc in N-glycans of adenylyl cyclase III enhances its activity.
abstract: Adenylyl cyclases (ACs) catalyze the synthesis of cAMP in response to extracellular and intracellular signals and are responsible for a wide variety of biological activities including cell growth, differentiation, and metabolism. There are nine, currently known, isoforms of transmembrane ACs, and the primary structure of the catalytic unit and the potential N-glycosylation sites are highly conserved among them. The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) to N-glycans. We have been studying the function of GnT-III on signaling molecules. In this study, we report on the effects of a bisecting GlcNAc on AC signaling. We established GnT-III stable expressing cell lines of Neuro-2a mouse neuroblastoma cells and B16 mouse melanoma cells. Forskolin-induced AC activation and downstream signaling, such as the synthesis of cAMP and the phosphorylation of transcriptional factor CRE-binding protein were upregulated in the GnT-III transfectants compared with mock transfectants or a dominant negative mutant of GnT-III-transfected cells. Since endogenous AC expression levels in Neuro-2a and B16 cells were too low to permit the glycosylation status to be examined, AC type III (ACIII) was overexpressed in a stable expression system using Flp-In-293 cells. The N-glycans of ACIII in the GnT-III transfectants were confirmed to be modified by the introduction of a bisecting GlcNAc, and AC activity was found to be significantly up-regulated in the GnT-III transfectants. Thus, the structure of N-glycans of ACIII regulates its enzymatic activity and downstream signaling.
authors.affiliation: Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Motoko
authors.initials: M
authors.lastname: Takahashi
authors.forename: Yukinao
authors.initials: Y
authors.lastname: Shibukawa
authors.forename: Shunichi
authors.initials: S
authors.lastname: Yokoe
authors.forename: Jianguo
authors.initials: J
authors.lastname: Gu
authors.forename: Eiji
authors.initials: E
authors.lastname: Miyoshi
authors.forename: Koichi
authors.initials: K
authors.lastname: Honke
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry*; D000262:Adenylyl Cyclases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
chemical_list: D007527:Isoenzymes; D011134:Polysaccharides; D005576:Colforsin; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D000262:Adenylyl Cyclases; C421784:adenylate cyclase 3; D000117:Acetylglucosamine