1. Regulation of N-glycosylation and secretion of Isthmin-1 by its C-mannosylation.
- Author
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Yoshimoto S, Katayama K, Suzuki T, Dohmae N, and Simizu S
- Subjects
- Amino Acid Motifs, Amino Acid Substitution, Cell Line, Tumor, Culture Media, Conditioned chemistry, Fibroblasts cytology, Fibroblasts metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Glycosylation, Humans, Mannose chemistry, Mutation, Recombinant Proteins genetics, Recombinant Proteins metabolism, Thrombospondins genetics, Mannose metabolism, Protein Processing, Post-Translational, Thrombospondins metabolism
- 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 Trp
223 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)- Published
- 2021
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