MicroPlate Sialyltransferase Assay: A Rapid and Sensitive Assay Based on an Unnatural Sialic Acid Donor and Bioorthogonal Chemistry

Abstract

Mammalian sialyltransferases transfer sialic acids onto glycoproteins and glycolipids within the Golgi apparatus. Despite their key role in glycosylation, the study of their enzymatic activities is limited by the lack of appropriate tools. Herein, we developed a quick and sensitive sialyltransferase microplate assay based on the use of the unnatural CMP-SiaNAl donor substrate. In this assay, an appropriate acceptor glycoprotein is coated on the bottom of 96-well plate and the sialyltransferase activity is assessed using CMP-SiaNAl. The alkyne tag of SiaNAl enables subsequent covalent ligation of an azido-biotin probe via CuAAC and an antibiotin-HRP conjugated antibody is then used to quantify the amount of transferred SiaNAl by a colorimetric titration. With this test, we evaluated the kinetic characteristics and substrate preferences of two human sialyltransferases, ST6Gal I and ST3Gal I toward a panel of asialoglycoprotein acceptors, and identified cations that display a sialyltransferase inhibitory effect.

Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation

“…Microsomal fractions containing the full length fish enzyme were used in enzymatic assays, but again, no significant enzymatic activity could be detected (data not shown). We therefore chose to apply the quick and sensitive MPSA developed recently to assess the sialyltransferase activity of human recombinant ST3Gal I and ST6Gal I onto glycoprotein acceptors [45]. In this assay, the acceptor glycoprotein is coated on the bottom of 96-well plate and the crude sialyltransferase activity is assessed using CMP-Sia N Al, a high-energy donor form of the unprotected alkyne-tagged sialic acid reporter Sia N Al that is readily used by these two human sialyltransferases [43,44] followed by covalent ligation of an azido-probe via a Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC) [61,62].…”

“…After transfer onto a nitrocellulose membrane (Biotrace, Pall corporation, Ann Arbor, MI, USA), the radioactive products were detected and quantified by radio-imaging using a Personal Molecular Imager FX (Bio-Rad, France). Sialyltransferase assays were also performed using the MPSA described recently [45]. Briefly, 400 ng of glycoprotein acceptor in 100 µL of sodium bicarbonate buffer (20 mM pH 9,6) or glycolipids mixture were coated in 96-well plate wells (F8 MaxiSorp Loose Nunc-Immuno Module ThermoScientific) overnight at 4 °C.…”

“…As a first step towards bringing a functional basis to the distribution of the various diSia motifs described in the zebrafish tissues, we analyzed the spatio-temporal profile of the zebrafish st8sia6 -like gene during the zebrafish embryogenesis. In an attempt to define its biochemical activity, we expressed a recombinant enzyme and took advantage of chemoenzymatic glycan labeling strategies using unnatural CMP-activated sialic acid reporters [43,44] and of the newly developed MicroPlate Sialyltransferase Assay (MPSA) [45] to show that the enzyme was not active on sialylated fetuin. Towards understanding this unexpected data, we assessed the evolutionary relationships of fish mono-α2,8-sialyltransferases (i.e., ST8Sia I, ST8Sia V, ST8Sia VI and ST8Sia VII).…”

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

“…[36,38,42] HPLC analysiso ff luorescently labeled substrates [33] is indirect, and surrogate substrates may show ak inetic behavior different from the native substrates. [43] The only continuous assay for SiaT activity is based on the indirect measurement of CMP release by an enzymatic link to NADPH consumption, which is inappropriate for discrimination of sialidase activity. [44] In contrast to HPLC, radiolabeling, capillary electrophoresis or ESI-MS assays, which requiree xpensive dedicated instrumentation,a ssays based on monitoring ap Hs hifta saconsequenceo fr eaction progress are simple to operate in continuous mode, [45] anda lso universal in that they are independent of substrate variations.…”

Unliganded and CMP-Neu5Ac bound structures of human α-2,6-sialyltransferase ST6Gal I at high resolution

“…Finally, CMP-Neu5Ac is bound with the side chain at C-5 of the sugar residue directed towards empty space at the surface of the protein. It is known that CMP-Neu5Ac sialic acid modified at the N-acyl position with a bulkier alkynyl group (Noel et al, 2018) can serve as the donor substrate both for 2,6-sialylation of N-glycans and 2,3-sialylation of O-glycans (Noel et al, 2017), and Sun et al (2016) showed that even biotin at the N-acyl position can be tolerated and used to . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.…”