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N -Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sd a Synthase B4GALNT2.

Virginie CogezDorothée VicogneCéline SchulzLucie PortierGiulia VenturiJerome de RuyckMathieu DecloquementMarc F LensinkGuillaume BrysbaertFabio Dall'OlioSophie Groux-DegrooteAnne Harduin-Lepers
Published in: International journal of molecular sciences (2023)
The Sd a carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 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 a and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N -glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N -glycan. We explored the influence of this N -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 N -glycan on each monomer corroborated these findings and suggested that N -glycosylation of each B4GALNT2 isoform controlled their biological activity.
Keyphrases
  • endoplasmic reticulum
  • poor prognosis
  • endothelial cells
  • binding protein
  • transcription factor
  • gene expression
  • high resolution
  • mass spectrometry
  • amino acid
  • monoclonal antibody