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Thrombospondin type 1 repeat-derived C-mannosylated peptide attenuates synaptogenesis of cortical neurons induced by primary astrocytes via TGF-β

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Abstract

C-Mannosylation is a rare type of protein glycosylation and is reportedly critical for the proper folding and secretion of parental proteins. Still, the effects of C-mannosylation on the biological functions of these modified proteins remain to be elucidated. The Trp-x-x-Trp (WxxW) sequences, whose first tryptophan (Trp) can be C-mannosylated, constitute the consensus motifs for this glycosylation modification and are commonly found in thrombospondin type 1 repeats that regulate molecular functions of thrombospondin 1 in binding and activation of transforming growth factor β (TGF-β). TGF-β plays critical roles in the control of the central nervous system including synaptogenesis. Here, we investigated whether C-mannosylation of the synthetic Trp-Ser-Pro-Trp (WSPW) peptide may confer certain functions to this peptide in TGF-β-mediated synaptogenesis. By using primary cultured rat astrocytes and cortical neurons, we found that the C-mannosylated WSPW (C-Man-WSPW) peptide, but not non-mannosylated WSPW peptide, suppressed astrocyte-conditioned medium (ACM)-stimulated synaptogenesis. C-Man-WSPW peptide inhibited both ACM- and recombinant mature TGF-β1-induced activations of Smad 2, an important mediator in TGF-β signaling. Interactions of recombinant mature TGF-β with the C-Man-WSPW peptide were similar to those with non-C-mannosylated WSPW peptide. Taken together, our results reveal a novel function of C-mannosylation of the WxxW motif in signaling and synaptogenesis mediated by TGF-β. Molecular details of how C-mannosylation affects the biological functions of WxxW motifs deserve future study for clarification.

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Data availability

All data generated or analyzed during this study are included in this published article.

Abbreviations

ACM:

Astrocyte-conditioned medium

C-Man-WSPW peptide :

C-Mannosylated WSPW peptide

FBS:

Fetal bovine serum

PBS:

Phosphate-buffered saline

PSD-95:

Postsynaptic density protein 95

TGF-β:

Transforming growth factor β

TSP:

Thrombospondin

TSR:

Thrombospondin type 1 repeat

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Funding

This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan JP16H06290 (to Y.Ito); a Wakayama Medical Award for Young Researchers 2013 and 2015 (to M.I.); and the Japan Society for the Promotion of Science Grant-in-Aid 19K09784 to M.I. and 20K09605 to K.N.

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Author notes

  1. Kazuchika Nishitsuji and Midori Ikezaki contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Wakayama Medical University, Wakayama, 641-8509, Japan

    Kazuchika Nishitsuji, Midori Ikezaki & Yoshito Ihara

  2. Laboratory of Functional Molecule Chemistry, Pharmaceutical Department and Institute of Medicinal Chemistry, Hoshi University, Tokyo, 142-8501, Japan

    Shino Manabe

  3. Research Center for Pharmaceutical Development, Graduate School of Pharmaceutical Sciences & Faculty of Pharmaceutical Sciences, Tohoku University, Miyagi, 980-8578, Japan

    Shino Manabe

  4. Unité de Glycobiologie Structurale Et Fonctionnelle, UMR 8576, CNRS, Université de Lille, 59655, Villeneuve d’Ascq, France

    Kenji Uchimura

  5. RIKEN Cluster for Pioneering Research, Saitama, 351-0198, Japan

    Yukishige Ito

  6. Graduate School of Science, Osaka University, Osaka, 560-0043, Japan

    Yukishige Ito

Authors
  1. Kazuchika Nishitsuji
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  2. Midori Ikezaki
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  3. Shino Manabe
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  4. Kenji Uchimura
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  5. Yukishige Ito
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  6. Yoshito Ihara
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Contributions

M.I. performed the experiments. S.M. and Y. Ito prepared the peptides and biotinylated peptides. Y. Ihara contributed experimental materials and tools. K.U. interpreted the data, wrote the paper, and supervised the entire project. K.N. designed the research, performed the experiments, interpreted the data, and wrote the paper. K.N. and Y. Ihara take full responsibility for the entire manuscript. All authors reviewed the results and approved the final version of the manuscript.

Corresponding authors

Correspondence to Kazuchika Nishitsuji or Yoshito Ihara.

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Nishitsuji, K., Ikezaki, M., Manabe, S. et al. Thrombospondin type 1 repeat-derived C-mannosylated peptide attenuates synaptogenesis of cortical neurons induced by primary astrocytes via TGF-β. Glycoconj J 39, 701–710 (2022). https://doi.org/10.1007/s10719-021-10030-y

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