The fibrinogen C-terminal domain is seldom C-mannosylated but its C-mannosylation is important for the secretion of microfibril-associated glycoprotein 4

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 9; p. 129637
• Main Authors: Osada, Yoshiyuki, Suzuki, Takehiro, Mizuta, Hayato, Mori, Kento, Miura, Kazuki, Dohmae, Naoshi, Simizu, Siro
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2020
• Subjects: amino acid sequences; C-mannosylation; Carrier Proteins - metabolism; cell adhesion; Cell Line, Tumor; cell lines; consensus sequence; Extracellular Matrix Proteins - metabolism; fibrinogen; Fibrinogen - chemistry; Fibrinogen - metabolism; Fibrinogen C-terminal domain; fluorescence; fluorescence microscopy; glycoproteins; Glycoproteins - metabolism; Glycosylation; homeostasis; Humans; liquid chromatography; Mannose - metabolism; Microfibril-associated glycoprotein 4; mutants; Protein Domains; protein secretion; Protein Transport; tandem mass spectrometry; Glycosylation; C-mannosylation; Microfibril-associated glycoprotein 4; Fibrinogen C-terminal domain
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129637

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. 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. 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. 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. These results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion. •The fibrinogen C-terminal domain of MFAP4 is C-mannosylated at Trp235.•C-mannosylation of MFAP4 regulates its secretion and subcellular localization.•The fibrinogen C-terminal domain is a novel C-mannosylation domain with important functions.