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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 Trp 223 and Trp 226 . 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 Trp 223 and Trp 226 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)