Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase curtails its activity

Mikolajczyk, K.; Bereznicka, A.; Szymczak-Kulus, K.; Haczkiewicz-Lesniak, K.; Szulc, B.; Olczak, M.; Rossowska, J.; Majorczyk, E.; Kapczynska, K.; Bovin, N. V.; Lisowska, M.; Kaczmarek, R.; Miazek, A.; Czerwinski, M.

doi:10.1101/2021.02.04.429793

bioRxiv · 10.1101/2021.02.04.429793

Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase curtails its activity

Mikolajczyk, K.·Bereznicka, A.·Szymczak-Kulus, K.·Haczkiewicz-Lesniak, K.·Szulc, B.·Olczak, M.·Rossowska, J.·Majorczyk, E.·Kapczynska, K.·Bovin, N. V.·Lisowska, M.·Kaczmarek, R.·Miazek, A.·Czerwinski, M.

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Abstract

N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Gal1[->]4Gal (Gb3 and the P1 antigen) and Gal1[->]4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

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Mikolajczyk, K., Bereznicka, A., Szymczak-Kulus, K., Haczkiewicz-Lesniak, K., Szulc, B., Olczak, M., Rossowska, J., Majorczyk, E., Kapczynska, K., Bovin, N. V., Lisowska, M., Kaczmarek, R., Miazek, A., Czerwinski, M.. 2021-02-04. Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase curtails its activity. https://doi.org/10.1101/2021.02.04.429793

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