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A Rapid and Sensitive MicroPlate Assay (MPSA) Using an Alkyne-Modified CMP-Sialic Acid Donor to Evaluate Human
Kiamungongo Clairene Filipe1, Sushmaa Dangudubiyyam1, Cédric Lion1
1Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, Faculté des sciences et Technologies, F-59000, Lille, France.
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Human sialyltransferases primarily utilize CMP-Sias, especially transferring Neu5Ac from CMP-Neu5Ac to various acceptors. Advances in chemical biology have led to the synthesis of novel CMP-Sia donors suitable for bioorthogonal reactions in cell-based assays. However, the compatibility of these donors with all human enzymes remains uncertain. We synthesized a non-natural CMP-Sia donor with an alkyne modification on the N-acyl group of Neu5Ac, which was effectively used by human ST6Gal I and ST3Gal I. A sensitive MicroPlate Sialyltransferase Assay (MPSA) was developed and expanded to a panel of 13 human STs acting on glycoproteins. All assayed enzymes tolerated CMP-SiaNAl, allowing for the determination of kinetic parameters and turnover numbers. This study enhances the biochemical characterization of human sialyltransferases and opens new avenues for developing sialyltransferase inhibitors.
