<i>N</i>-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd<sup>a</sup> Synthase B4GALNT2
The Sd<sup>a</sup> carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human <i>B4GALNT2</i> gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and...
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| Format: | Article |
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MDPI AG
2023-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/4/4139 |
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| author | Virginie Cogez Dorothée Vicogne Céline Schulz Lucie Portier Giulia Venturi Jérôme de Ruyck Mathieu Decloquement Marc F. Lensink Guillaume Brysbaert Fabio Dall’Olio Sophie Groux-Degroote Anne Harduin-Lepers |
| author_facet | Virginie Cogez Dorothée Vicogne Céline Schulz Lucie Portier Giulia Venturi Jérôme de Ruyck Mathieu Decloquement Marc F. Lensink Guillaume Brysbaert Fabio Dall’Olio Sophie Groux-Degroote Anne Harduin-Lepers |
| author_sort | Virginie Cogez |
| collection | DOAJ |
| description | The Sd<sup>a</sup> carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human <i>B4GALNT2</i> gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sd<sup>a</sup> and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual <i>N</i>-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type <i>N</i>-glycan. We explored the influence of this <i>N</i>-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an <i>N</i>-glycan on each monomer corroborated these findings and suggested that <i>N</i>-glycosylation of each B4GALNT2 isoform controlled their biological activity. |
| first_indexed | 2024-03-11T08:40:53Z |
| format | Article |
| id | doaj.art-718199075d484811b20971a1a49b3239 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T08:40:53Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-718199075d484811b20971a1a49b32392023-11-16T21:10:03ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-02-01244413910.3390/ijms24044139<i>N</i>-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd<sup>a</sup> Synthase B4GALNT2Virginie Cogez0Dorothée Vicogne1Céline Schulz2Lucie Portier3Giulia Venturi4Jérôme de Ruyck5Mathieu Decloquement6Marc F. Lensink7Guillaume Brysbaert8Fabio Dall’Olio9Sophie Groux-Degroote10Anne Harduin-Lepers11CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceDepartment of Medical and Surgical Sciences (DIMEC), University of Bologna, General Pathology Building, Via San Giacomo 14, 40126 Bologna, ItalyCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceDepartment of Medical and Surgical Sciences (DIMEC), University of Bologna, General Pathology Building, Via San Giacomo 14, 40126 Bologna, ItalyCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceCNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F-59000 Lille, FranceThe Sd<sup>a</sup> carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human <i>B4GALNT2</i> gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sd<sup>a</sup> and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual <i>N</i>-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type <i>N</i>-glycan. We explored the influence of this <i>N</i>-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an <i>N</i>-glycan on each monomer corroborated these findings and suggested that <i>N</i>-glycosylation of each B4GALNT2 isoform controlled their biological activity.https://www.mdpi.com/1422-0067/24/4/4139B4GALNT2dimerglycosyltransferase<i>N</i>-glycanunusual N-X-C glycosylation site |
| spellingShingle | Virginie Cogez Dorothée Vicogne Céline Schulz Lucie Portier Giulia Venturi Jérôme de Ruyck Mathieu Decloquement Marc F. Lensink Guillaume Brysbaert Fabio Dall’Olio Sophie Groux-Degroote Anne Harduin-Lepers <i>N</i>-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd<sup>a</sup> Synthase B4GALNT2 International Journal of Molecular Sciences B4GALNT2 dimer glycosyltransferase <i>N</i>-glycan unusual N-X-C glycosylation site |
| title | <i>N</i>-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd<sup>a</sup> Synthase B4GALNT2 |
| title_full | <i>N</i>-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd<sup>a</sup> Synthase B4GALNT2 |
| title_fullStr | <i>N</i>-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd<sup>a</sup> Synthase B4GALNT2 |
| title_full_unstemmed | <i>N</i>-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd<sup>a</sup> Synthase B4GALNT2 |
| title_short | <i>N</i>-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd<sup>a</sup> Synthase B4GALNT2 |
| title_sort | i n i glycan on the non consensus n x c glycosylation site impacts activity stability and localization of the sd sup a sup synthase b4galnt2 |
| topic | B4GALNT2 dimer glycosyltransferase <i>N</i>-glycan unusual N-X-C glycosylation site |
| url | https://www.mdpi.com/1422-0067/24/4/4139 |
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