CMP-sialic acid synthetase in Drosophila requires N-glycosylation of a noncanonical site
Novikov B. Boland D.J. Mertsalov I. Scott H. Dauletbayeva S. Monagas-Valentin P. Panin V.
June 2025American Society for Biochemistry and Molecular Biology Inc.
Journal of Biological Chemistry
2025#301Issue 6
Sialylation plays important roles in animals, affecting numerous molecular and cell interactions. In Drosophila, sialylation regulates neural transmission and mediates communication between neurons and glia. Drosophila CMP-sialic acid synthetase (CSAS), a key enzyme of the sialylation pathway, is localized to the Golgi and modified by N-glycosylation, suggesting that this modification can affect CSAS function. Here, we tested this hypothesis using in vitro and in vivo approaches. We found that CSAS proteins from divergent Drosophila species have two conserved N-glycosylation sites, including the rarely glycosylated noncanonical N-X-C sequon. We investigated CSAS glycosylation by generating CSAS “glycomutants” lacking glycosylation sites and analyzing them in vivo in transgenic rescue assays. The removal of noncanonical glycosylation significantly decreased CSAS activity, while the canonical site mutation did not affect CSAS function. Although all glycomutants were similarly localized to the Golgi, the non-canonical glycosylation, unlike the canonical one, affected CSAS stability in vivo and in vitro. Our results suggested that CSAS functions as a dimer, which was also supported by protein structure predictions that produced a dimer recapitulating the crystal structures of mammalian and bacterial counterparts, highlighting the evolutionary conservation of the CSAS structure–function relationship. This conclusion was supported by the rescue of CSAS mutants using the human ortholog. The noncanonical CSAS glycosylation was discussed in terms of a potential mechanism of temperature-dependent regulation of sialylation in poikilotherms that modulates neural activity in heat shock conditions. Taken together, we uncovered an important regulation of sialylation in Drosophila, highlighting a novel interplay between glycosylation pathways in neural regulation.
CMP-sialic acid synthetase , Drosophila , glycosylation , noncanonical N-glycosylation , sialylation
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Department of Biochemistry and Biophysics, AgriLife Research, Texas A&M University, College Station, TX, United States
Texas A&M Institute of Genome Sciences & Society, Texas A&M University, College Station, TX, United States
Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russian Federation
Department of Molecular Biology and Genetics, al-Farabi Kazakh National University, Almaty, Kazakhstan
Department of Biochemistry and Biophysics
Texas A&M Institute of Genome Sciences & Society
Koltzov Institute of Developmental Biology
Department of Molecular Biology and Genetics
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