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Glycosylphosphatidylinositol-anchoring is required for the proper transport and glycosylation of classical arabinogalactan protein precursor in tobacco BY-2 cells

Daiki Nagasato, Yuto Sugita, Yuhei Tsuno, Rutsuko Tanaka, Maki Fukuda, View ORCID ProfileKen Matsuoka
doi: https://doi.org/10.1101/2020.10.19.346049
Daiki Nagasato
1Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka, Japan
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Yuto Sugita
1Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka, Japan
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Yuhei Tsuno
1Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka, Japan
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Rutsuko Tanaka
2Chikushigaoka High School, Fukuoka, Japan
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Maki Fukuda
3School of Agriculture, Kyushu University, Fukuoka, Japan
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Ken Matsuoka
1Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka, Japan
3School of Agriculture, Kyushu University, Fukuoka, Japan
4Faculty of Agriculture, Kyushu University, Fukuoka, Japan
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  • ORCID record for Ken Matsuoka
  • For correspondence: kenmat@agr.kyushu-u.ac.jp
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Abstract

Arabinogalactan proteins (AGPs) are extracellular proteoglycans with many O-linked glycan chains. Precursors to many AGPs contain a C-terminal signal for the addition of a glycosylphosphatidylinositol (GPI)-anchor, but the role of this modification has not been elucidated. NtAGP1, a tobacco precursor to AGP, comprises a signal peptide, an AGP-coding region, and a GPI-anchoring signal, and it is classified as a member of the classical AGP family. Using green fluorescent protein (GFP) and sweet potato sporamin (SPO) as tags and tobacco BY-2 cells as the host, we analysed the transport and modification of NtAGP1. The fusion protein of GFP or SPO and NtAGP1 expressed in BY-2 cells migrated as a large smear on SDS-polyacrylamide gel. A confocal microscopic analysis indicated that the GFP and NtAGP1 fusion protein localized to the plasma membrane (PM) and intracellular structures. Fractionation studies of microsomes indicated that most of the fusion protein of SPO and NtAGP1 (SPO-AGP) localized to the PM. In contrast, the expression of mutants without a GPI-anchoring signal yielded several forms. The largest forms migrating as large smears on the gel were secreted into the culture medium, whereas other forms were recovered in the endomembrane organelles. A comparison of the glycan structures of the microsomal SPO-AGP and the secreted mutant SPO-AGP without a GPI-anchoring signal using antibodies against AGP glycan epitopes indicated that the glycan structures of these proteins differ. These observations indicate that a GPI-anchoring signal is required for both the proper transport and glycosylation of the AGP precursor.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Several new data are included and some other results are improved from the previous version. Supplemental data are also updated.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted February 01, 2023.
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Glycosylphosphatidylinositol-anchoring is required for the proper transport and glycosylation of classical arabinogalactan protein precursor in tobacco BY-2 cells
Daiki Nagasato, Yuto Sugita, Yuhei Tsuno, Rutsuko Tanaka, Maki Fukuda, Ken Matsuoka
bioRxiv 2020.10.19.346049; doi: https://doi.org/10.1101/2020.10.19.346049
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Glycosylphosphatidylinositol-anchoring is required for the proper transport and glycosylation of classical arabinogalactan protein precursor in tobacco BY-2 cells
Daiki Nagasato, Yuto Sugita, Yuhei Tsuno, Rutsuko Tanaka, Maki Fukuda, Ken Matsuoka
bioRxiv 2020.10.19.346049; doi: https://doi.org/10.1101/2020.10.19.346049

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