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, 50 (44), 9475-87

Mammalian Sialyltransferase ST3Gal-II: Its Exchange Sialylation Catalytic Properties Allow Labeling of Sialyl Residues in Mucin-Type Sialylated Glycoproteins and Specific Gangliosides

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Mammalian Sialyltransferase ST3Gal-II: Its Exchange Sialylation Catalytic Properties Allow Labeling of Sialyl Residues in Mucin-Type Sialylated Glycoproteins and Specific Gangliosides

E V Chandrasekaran et al. Biochemistry.

Abstract

While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some can also catalyze readily reversible reactions. Recently, we found that mammalian sialyltransferase ST3Gal-II can catalyze the formation of CMP-NeuAc from 5'-CMP in the presence of a donor containing the NeuAcα2,3Galβ1,3GalNAc unit [Chandrasekaran, E. V., et al. (2008) Biochemistry 47, 320-330]. This study shows by using [9-(3)H]- or [(14)C]sialyl mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and the NeuAcα2,3Galβ1,3GalNAc unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease, which is evident from the following. (a) Radiolabeleling of fetuin was ~2-fold stronger than that of asialo fetuin when CMP- [9-(3)H]NeuAc was generated in situ from 5'-CMP and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by ST3Gal-II. (b) ST3Gal-II exchanged radiolabels between [(14)C]sialyl fetuin and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by generating CMP-[(14)C]- and -[9-(3)H]NeuAc through 5'-CMP; only 20.3% (14)C and 28.0% (3)H remained with the parent compounds after the sialyl exchange. The [9-(3)H]sialyl-tagged MN glycophorin A, human chorionic gonadotropin β subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins, and haptoglobin were analyzed by using Pronase digestion, mild alkaline borohydride treatment, Biogel P6, lectin agarose, and silica gel thin layer chromatography. Sulfated and sialylated O-glycans were found in GlyCAM-1 and human placental glycoproteins. This technique has the potential to serve as an important tool as it provides a natural tag for the chemical and functional characterization of O-glycan-bearing glycoproteins.

Figures

Figure 1
Figure 1. Exchange of Sialyl residues between CMP-[9-3H]NeuAc and NeuAcα2,3Galβ1,3 (GlcNAcβ1,6)GalNAcα-O-Al [1] catalyzed by ST3Gal-II
(Scheme I symbols: ◆:sialic acid, ●:Gal, ◻:GalNAc, ∎:GlcNAc): a) 0.2μCi [9-3H]CMP-NeuAc was mixed with cold CMP-NeuAc to concentrations of 20μM (low CMP-NeuAc concentration, high specific activity) or 200μM (high concentration, low specific activity). This was added to 0–0.8mM [1] in the presence of 0.16mU enzyme at 37°C for 4h in 100mM NaCa codylate buffer at pH6.0. Reaction volume was 20μl. The product was diluted to 1.0ml using 10mM Hepes pH7.5 containing CaCl2 and MnCl2 and fractioned on a WGA agarose column. Radiolabeled product formed bound WGA agarose affinity column, matrix that binds [1]. b) 0.5mM [9-3H]CMP-NeuAc and 0.3mM [1] were incubated with ST3Gal-II under reaction conditions of panel a and radiolabeled product was isolated using Biogel P2 column. Radiolabeled [1] appeared as a peak between 40–45ml with unreacted [9-3H] CMP-NeuAc appearing at 60mL (not shown). V0 denotes void volume. c) [9-3H]NeuAc labeled product from Biogel P2 column in panel b was subjected to TLC plates in three different solvent systems: 1-propanol/NH4OH/H2O (12/2/5 v/v) developed once (lanes 1,2); CHCl3:CH3OH:H2O (5/4/1/v/v) developed twice (lane 3,4); and ethyl acetate: Pyridine:H2O:Acetic Acid Acetic Acid (5/5/3/1 v/v) developed once (lane 5, 6). In each case, the first lane is the unreacted acceptor while the second lane quantifies the radioactivity associated with product scraped from sections of the TLC plate. d) Reaction mixtures (RM) with the following compositions were incubated at 37°C for 2h in 100mM NaCa codylate buffer, pH6.0: i) RM containing 150μM (0.4μCi) [9-3H]NeuAcα2,3Galβ1,3(GlcNAcβ1,6)GalNAcα-OAl [9-3H[1]] (donor) along with 0.8mU ST3Gal-II but no CMP-NeuAc. ii) RM containing [9-3H[1]] and 1.0mM CMP-NeuAc, but lacking ST3Gal-II. iii) RM containing all components including donor, CMP-NeuAc and enzyme. Products formed were fractionated on a WGA agarose column. Only iii) contained components that did not bind WGA agarose e) WGA agarose affinity chromatography was performed similarly as panel d) with all components in RM except that the amount of CMP-NeuAc was varied. f) TLC of RMs ii) (lane 1) and ii) (lane 2) from panel d) using solvent CHCl3:CH3OH:H2O (5/4/1/v/v) developed twice. Position where radioactivity peak appears is marked by square bracket, along with migration distance for NeuAc, CMP-NeuAc and [9-3H[1]] which were determined in independent run. g. Separation of radiolabeled product from acceptor NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,6 (NeuAcα2,3Galβ1,3) GalNAcα-O-Me using Biogel P2 column. h. TLC runs of the radio labeled product and the acceptor using the solvent system 2-propanol/NH4OH/H2O (12/2/5/ v/v). i. CMP-NeuAc was incubated in cacodylade buffer for 4h at either −20°C or 37°C. Dowex-1-formate chromatography was then performed by elution with 3.0mL each of water and various NaCl concentrations as indicated. CMP-NeuAc eluted from the column at 0.3– 0.4M NaCl and free NeuAc at 0.10–0.15M NaCl.
Figure 2
Figure 2. Enzymatic exchange of sialyl residues between [14C]sialyl fetuin and [9-3H]NeuAcα2,3 Galβ1,3GalNAcβ1,3Galα-O-Me
In scheme II, 5′-CMP acts as the sialic acid acceptor to form radiolabeled CMP-NeuAc via the reverse sialylation reaction. The newly formed CMP-NeuAc participates in the transfer of sialic acid to fetuin and NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me via the exchange mechanism. [14C] Sialyl Fetuin (2mg in 100μl water) and 100μl of [93H] NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me (0.75mM) was diluted into 400μl volume using 0.15M Na cacodylate pH 6.0. 100mU cloned ST3Gal-II was added in the absence (panel a) or presence (panel b) of 10mM 5′-CMP. Reaction proceeded at 37°C for 20h. Reaction mixtures were diluted to 1.0ml with water and fractionated using the Biogel P2 column. The product identity was verified by mass spectrometry as indicated by the molecular weights in the figure.
Figure 3
Figure 3. Enzymatic exchange of sialyl residues between CMP-NeuAc and gangliosides
:[9-3H] and [14C] sialic acid were incorporated into bovine brain ganglioside upon addition of either CMP-[14C]NeuAc or CMP-[9-3H]NeuAc to bovine brain gangliosides in the presence of ST3Gal-II. TLC separation used CHCl3:CH3OH:0.2% aqueous CaCl2 (v/v 60:40:9) as the mobile phase. TLC plate containing [14C] sialyl ganglioside mixture was developed by auto radiography (lane A1). TLC plate containing the same sample was also charred with H2S04 in ethanol (lane A2) to locate the gangliosides. Tritium was located on the TLC plates by scraping silica gel from 0.5cm width segments, soaking silica in 2.0ml water and then counting the radioactivity by scintillation counting (lane A3). Structure of gangliosides is shown in cartoon notation (▴:glucose, ◆:sialic acid, ●:Gal, ◻:GalNAc ). Radioactive exchange occurs at the NeuAcα2,3Galβ1,3GalNAc arm of gangliosides.
Figure 4
Figure 4. Characterization [9-3H] Sialyl tagged glycoproteins:
A. Comparing the fragments arising from pronase digestion of [9-3H] Sialyl tagged glycoproteins by Biogel P6 chromatography: a) MN Glycophorin A; b) GlyCAM-1; c) Human placental glycoproteins; d) bovine casein macroglycopeptide (MGP); e) Cowper's gland mucin (CGM) and f) human haptoglobin B. Biogel P6 chromatography of [9-3H]sialylated products arising from mild alkaline borohydride treatment of various glycoconjugates. a) HCGβ, b) MN Glycophorin A, c) GlyCAM-1, d) CD43, e) Human placental glycoproteins (HPG), f) bovine casein macroglycopeptide (MGP) g) Cowper's gland mucin (CGM) and h) human haptoglobin.
Figure 5
Figure 5. Analysis of HCGβ:
WGA-agarose affinity chromatography of the Biogel P6 fractions arising from pronase-digestion of [9-3H] sialyl HCGβ. a. Biogel P6 fractionation of Pronase digested [9-3H]sialyl HCGβ. WGA-agarose chromatography of : b. Pronase fraction A; c. Pronase fraction B; d. Pronase fraction C and e. Pronase fraction D. f. Biogel P6 chromatography of [9-3H]sialylated products arising from mild alkaline borohydride treatment of HCGβ g. TLC analysis of fractions A–C from panel f.
Figure 6
Figure 6. WGA-agarose chromatography of [9-3H] Sialyl Fetuin and the Biogel P6 fractions A, B and C arising from pronase-digested [9-3H] Sialyl Fetuin: a)
Biogel P6 fractionation of pronase digested [9-3H]sialyl Fetuin. WGA-agarose chromatography of b) [9-H3]sialyl Fetuin, c) Pronase fraction C, d) Pronase fraction B and e) Pronase fraction A. f) Biogel P6 elution of alkaline borohydride treated [14C] sialyl Fetuin, g) Thin layer chromatography of the Biogel P6 fractions isolated from alkaline borohydride treatment of [9-3H] Fetuin show the presence of one major fraction with radioactivity. h) TLC of dominant fraction identified in panel g (fraction B from panel f) was developed by autoradiography. i) SDS-PAGE of [14C]sialyl fetuin before and after PNGase F treatment. 10 μg (14C-sialylfetuin) was applied to lanes 1,2,3,4, and 20 μg to lanes 5,6,7,8. Lanes 1, 2, 5 and 6 contained PNGase F treated fetuin and 3, 4, 7 and 8 fetuin without PNGase F treatment.
Figure 7
Figure 7. Thin layer chromatography of the Biogel P6 fractions isolated from alkaline borohydride treatment of [9-3H] sialylated glycoproteins:
Lanes 1, 2: MNGlycophorin fractions A, B; Lanes 3, 4: GlyCAM-1 fractions A, B; Lanes 5–7 human placental glycoprotein fractions A, B, C; Lane 8: bovine casein MGP fraction; Lanes 9,10: CGM fractions A, B. Lane 11–13 Fractions A, B, C prepared from [14C] sialyl identical to human placental glycoprotein 5–7 only these were labeled using CMP-[14C] NeuAc and the TLC images were developed by autoradiography.
Figure 8
Figure 8. Autoradiography of apotransferrin and haptoglobin
Apotransferrin (panel a) and haptoglobin (panel b) were sialylated using the exchange reaction. These proteins were treated with PNGaseF to remove N-glycans as indicated. 10 μg of each protein was then analyzed using SDS-PAGE with Coomassie blue staining being used to visualize protein and phosphorimaging being applied to monitor 14C-NeuAc incorporation. All samples were analyzed in duplicate.

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