Glycobiology Advance Access originally published online on February 26, 2007
Glycobiology 2007 17(6):655-662; doi:10.1093/glycob/cwm022
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Introduction of bisecting GlcNAc in N-glycans of adenylyl cyclase III enhances its activity
2 Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
3 Division of Molecular Cell Biology, Department of Biomolecular Sciences, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan
4 Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan
5 Department of Biochemistry, Kochi Medical School, Nangoku, Kochi 783-8505, Japan
6 Department of Disease Glycomics, Research Institute for Microbial Diseases, 4th Floor, Center for Advanced Science & Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
1 To whom correspondence should be addressed; Tel: +81-6-6879-4137; Fax: +81-6-6879-4137; e-mail address: tani52{at}wd5.so-net.ne.jp
Received on January 9, 2007; revised on February 16, 2007; accepted on February 19, 2007
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 ß1,4-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.
Key words: adenylyl cyclase / bisecting GlcNAc / glycosylation / GnT-III / N-glycan