Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4)

Kazuki Miura, Takehiro Suzuki, Hongkai Sun, Haruka Takada, Yudai Ishizawa, Hayato Mizuta, Naoshi Dohmae, Siro Simizu

研究成果: Article査読

5 被引用数 (Scopus)

抄録

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.

本文言語English
論文番号129833
ジャーナルBiochimica et Biophysica Acta - General Subjects
1865
3
DOI
出版ステータスPublished - 2021 3月

ASJC Scopus subject areas

  • 生物理学
  • 生化学
  • 分子生物学

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