Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen

Abstract

α2,8‐Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and ‐IV. An assay system that enables differential testing of ST8SiaII and ‐IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB‐DP3 and DMB‐DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP‐activated and N‐substituted sialic acid derivatives. Of these derivatives, the non‐transferable CMP‐Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

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

“…Indeed, in only a few hours, the specificity of any ST can potentially be assessed toward any glycoproteins including bearing several glycosylation motifs. In the past decade, in vitro promiscuity toward substrate modifications of N -acyl chain has been reported for ST6Gal, ST3Gal, ST6GalNAc, and ST8Sia families, with both mammalian and bacterial systems. ,− This promiscuity should allow extension of MPSA to other STs in the future.…”

An efficient assay for identification and quantitative evaluation of potential polysialyltransferase inhibitors

“…8,16 A number of in vitro assays have been developed and reported for the evaluation of polyST enzyme activity, utilising the enzyme, the substrate CMP-sialic acid, and the acceptor NCAM. [17][18][19] The Gerardy-Schahn group recently reported the utility of a non-ganglioside fluorescent acceptor, DMB-DP3, as a convenient substitute for NCAM, enabling analysis of polymerisation. DMB-DP3 is a trimer (degree of polymerisation, DP = 3) of α-2,8-linked sialic acid ('DP3') derivatised with a fluorescent 1,2-diamino-4,5-methylenedioxybenzene (DMB) label thereby forming 'DMB-DP3'.…”

Recent advances in the analysis of polysialic acid from complex biological systems

“…Alternatively, ultrafiltration-based membranes can also be used to de-salt and concentrate free polySia from biological matrices (Rode et al, 2008;Liu, Zhan, Wu, Lin, & Yu, 2010;Ehrit et al, 2017). As examples, Rode et al and Liu et al both employed 10 kDa ultrafiltration membranes to concentrate biosynthesised polySia from large amounts of E.coli broth culture before further purification of polySia (Rode et al, 2008;Liu et al, 2010).…”