Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4)
| Autor: | Siro Simizu, Kazuki Miura, Takehiro Suzuki, Hongkai Sun, Naoshi Dohmae, Haruka Takada, Yudai Ishizawa, Hayato Mizuta |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Amino Acid Motifs Genetic Vectors Biophysics Gene Expression Biochemistry Substrate Specificity 03 medical and health sciences 0302 clinical medicine Cell Line Tumor Disintegrin Humans Protein Interaction Domains and Motifs Secretion Aggrecans Molecular Biology Metalloproteinase Thrombospondin Binding Sites biology Chemistry ADAMTS Fibroblasts Recombinant Proteins Cell biology carbohydrates (lipids) Kinetics 030104 developmental biology ADAMTS4 Amino Acid Substitution 030220 oncology & carcinogenesis Mannosylation Mutation Proteolysis ADAMTS4 Protein biology.protein Thermodynamics Mannose Protein Processing Post-Translational Intracellular Protein Binding |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - General Subjects. 1865:129833 |
| ISSN: | 0304-4165 |
| DOI: | 10.1016/j.bbagen.2020.129833 |
| Popis: | Background C-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear. Methods We established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan. Results We identified that ADAMTS4 is C-mannosylated at Trp404 in the metalloprotease domain and at Trp523, Trp526, and Trp529 in the thrombospondin type 1 repeat (TSR). The replacement of Trp404 with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp523, Trp526, and Trp529 with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity. Conclusions Our results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity. General significance Because C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect