Satoshi Yoshimoto1, Kazuhiro Katayama1, Takehiro Suzuki2, Naoshi Dohmae2, Siro Simizu3. 1. Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. 2. Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako 351-0198, Japan. 3. Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. Electronic address: simizu@applc.keio.ac.jp.
Abstract
BACKGROUND: C-mannosylation is a type of protein glycosylation. Human Isthmin-1 (ISM1) is a 52-kDa secreted protein with a thrombospondin type 1 repeat (TSR) domain, containing two consensus C-mannosylation sequences at Trp223 and Trp226. In this study, we sought to examine the role of C-mannosylation in the secretion of ISM1. METHODS: We established and cultured an ISM1-overexpressing HT1080 cell line and purified recombinant ISM1 for analysis from the conditioned medium by LC-MS/MS. Subcellular localization of ISM1 was observed by confocal fluorescence microscopy. RESULTS: We found that ISM1 is C-mannosylated at Trp223 and Trp226 in the TSR domain. To determine the functions of the C-mannosylation of ISM1, we established a C-mannosylation-defective mutant ISM1-overexpressing HT1080 cell line and measured its secretion of ISM1. The secretion of ISM1 decreased significantly in this mutant ISM1-overexpressing line compared with wild-type cells. Furthermore, ISM1 was N-glycosylated only in these C-mannosylation-defective cells. CONCLUSIONS: ISM1 is C-mannosylated in its TSR domain, and the status of the C-mannosylation of ISM1 affects its N-glycosylation. GENERAL SIGNIFICANCE: The C-mannosylation of ISM1 regulates its N-glycosylation status.
BACKGROUND: C-mannosylation is a type of protein glycosylation. HumanIsthmin-1 (ISM1) is a 52-kDa secreted protein with a thrombospondin type 1 repeat (TSR) domain, containing two consensus C-mannosylation sequences at Trp223 and Trp226. In this study, we sought to examine the role of C-mannosylation in the secretion of ISM1. METHODS: We established and cultured an ISM1-overexpressing HT1080 cell line and purified recombinant ISM1 for analysis from the conditioned medium by LC-MS/MS. Subcellular localization of ISM1 was observed by confocal fluorescence microscopy. RESULTS: We found that ISM1 is C-mannosylated at Trp223 and Trp226 in the TSR domain. To determine the functions of the C-mannosylation of ISM1, we established a C-mannosylation-defective mutant ISM1-overexpressing HT1080 cell line and measured its secretion of ISM1. The secretion of ISM1 decreased significantly in this mutant ISM1-overexpressing line compared with wild-type cells. Furthermore, ISM1 was N-glycosylated only in these C-mannosylation-defective cells. CONCLUSIONS:ISM1 is C-mannosylated in its TSR domain, and the status of the C-mannosylation of ISM1 affects its N-glycosylation. GENERAL SIGNIFICANCE: The C-mannosylation of ISM1 regulates its N-glycosylation status.