Yoshiyuki Osada1, Takehiro Suzuki2, Hayato Mizuta1, Kento Mori1, Kazuki Miura1, Naoshi Dohmae2, Siro Simizu3. 1. Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Japan. 2. Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science Wako, 351-0198, Japan. 3. Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Japan. Electronic address: simizu@applc.keio.ac.jp.
Abstract
BACKGROUND: C-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation. METHODS: We established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope. RESULTS: We found that MFAP4 is C-mannosylated at Trp235 in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion. CONCLUSIONS: Our results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion. GENERAL SIGNIFICANCE: These results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.
BACKGROUND: C-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation. METHODS: We established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope. RESULTS: We found that MFAP4 is C-mannosylated at Trp235 in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion. CONCLUSIONS: Our results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion. GENERAL SIGNIFICANCE: These results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.