Reversible Sialylation:  Synthesis of Cytidine 5‘-Monophospho-N-acetylneuraminic Acid from Cytidine 5‘-Monophosphate with α2,3-Sialyl O-Glycan-, Glycolipid-, and Macromolecule-Based Donors Yields Diverse Sialylated Products^†

Abstract

Sialyltransferases transfer sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer alpha2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcalpha2,3Galbeta1,3GalNAcalpha unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5'-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming alpha2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5'-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5'-CMP had a wide optimum range (pH 5.2-7.2 and 4.8-6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5'-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5'-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5'-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.

Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures

“…In cases where radiolabeled donor compounds were prepared by use of this column, the peak fractions containing radioactivity were pooled, lyophilized to dryness, dissolved in a small volume of water, and stored frozen at −20°C for further experimentation [35].…”

“…The bound material was then eluted with 0.5 M GlcNAc (WGA) or 0.2 M Gal (PNA or RCA-I) in the same buffer. SNA-I-agarose (Vector Lab) affinity chromatography was also carried out as above, except that fractions of 2.0 mL were collected and the bound material was eluted with 0.5 M lactose [35]. The supplier Vector Lab indicates that this SNA binds preferentially to sialic acid attached to terminal Gal in α2-6 linkage.…”

“…Hydrophobic chromatography This was done with a Sep.-Pak C18 cartridge (Waters, Milford, MA) and the product, which was bound, was eluted with 3.0 mL of methanol, flash evaporated to dryness, dissolved in small volume of water and stored at −20°C for further experimentation [27,35,37].…”

“…When an aliquot of this [ 14 C] fucosylated glycopeptide was tested on AAL-agarose, it showed 100 % binding [43]. C 44, C 45 : Fetuin asialo O-glycosidic glycopeptide (400 μg) was [9-3 H] sialylated using ST3Gal II (20 mU) or ST6GalNAc I (10 mU) [35] as above and isolated by Biogel P2 column chromatography. C 46 : Asialo BSM (800 μg) was [9-3 H] sialylated using ST3Gal II (20 mU) [35] and isolated by Biogel P2 column chromatography.…”

“…C 44, C 45 : Fetuin asialo O-glycosidic glycopeptide (400 μg) was [9-3 H] sialylated using ST3Gal II (20 mU) or ST6GalNAc I (10 mU) [35] as above and isolated by Biogel P2 column chromatography. C 46 : Asialo BSM (800 μg) was [9-3 H] sialylated using ST3Gal II (20 mU) [35] and isolated by Biogel P2 column chromatography. C 47 : Asialo BSM (2 mg) was [9-3 H] sialylated with ST6GalNAc I (10 mU) by using ST3Gal II (20 mU) and [9-3 H]NeuAcα2-3Galβ1-3(4-FGlcNAcβ1-6)GalNAcα-O-Al as the sialyl donor in reverse sialylation [35] and isolated by Biogel P2 chromatography.…”