Biochimica et biophysica acta. General subjects · 2020
The fibrinogen C-terminal domain is seldom C-mannosylated but its C-mannosylation is important for the secretion of microfibril-associated glycoprotein 4.
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Osada Yoshiyuki, Suzuki Takehiro, Mizuta Hayato, Mori Kento, Miura Kazuki, Dohmae Naoshi, Simizu Siro
Аннотация
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.
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.