Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation

Abstract

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior.

Keywords: extrinsic glycosylation; glycosyltransferases; platelets; serum.

Fig. 1.

Schematic diagram of galactosyl modification of GlcNAc and sialyl- and fucosyl- modifications of LacNAc and mucin-type Core 1 disaccharides studied in this paper. Except for those reactions marked by asterisks, extracellular glycosyltransferases that are freely circulating or increased upon platelet activation can construct the glycan structures indicated. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 2.

Activity toward GlcNAc and mucin-type Core 1 acceptor in WT murine serum and plasma. Panels A–C: Glycosyltransferase activity toward LacNAc, mucin-type Core 1 and GlcNAc acceptors in WT murine plasma and serum. A comparison of glycosyltransferase activity, namely SiaT (A), FucT (B) and GalT (C), in WT murine plasma (filled circle) and serum (unfilled squares) was assessed by measuring the amount of radiolabeled nucleotide-sugar donor transferred various acceptors (n = 5). SiaT activity toward Type-II LacNAc, whether displaying α2,3- or α2,6- linkage, was further deconvoluted using agarose- bound SNA lectin. GalT activity toward GlcNAc and SiaT activity toward mucin-type Core 1 acceptor is increased in WT murine serum compared to plasma by 1.3- and 2.2-fold, respectively. SiaT activity toward type-II LacNAc is predominantly α2,3-linked, shown by lectin chromatography. Using MSⁿ analysis, it was determined that FucT activity in murine plasma and serum toward Type-II LacNAc produces predominantly Lewis^x (Le^x) structures (Figure 3).

Fig. 3.

MSⁿ determined linkage of glycosylation on fucosylated LacNAc acceptors. Panels A and B show the structures and representative fragment masses for the fucosylated Type-I LacNAc, H1 and Lewis a, respectively whilst panels C and D show the structures and representative fragment masses for the fucosylated Type-II acceptor, H2 and Lewis x, respectively. The m/z 660 fragment of the latter set is emphasized to indicate that this is the ion fragment analyzed for linkage identification (Panel F). Panel E shows the fucosylated trisaccharide isolated from the product formed by incubation of a Type-I LacNAc with mouse serum and GDP-Fuc. Panel F shows a zoomed in view of the m/z 660 fragment of the product formed by incubation of a Type-II LacNAc with mouse serum and GDP-fucose. Both spectra seem to indicate mixtures of the relevant Lewis and H antigen structures. Diagnostic ions are indicated with the arrows.

Fig. 4.

MSⁿ for spectral identification of galactosylated GlcNAc products. Panels A and B show the Type-I (Gal(β3)GlcNAc-O-Bn) and Type-II (Gal(β4)GlcNAc-O-Bn) LacNAc, respectively, with fragment masses identified. Panels C and D show the MS2 spectra of the Type-I and Type-II standard materials, respectively. Panel E shows the MS2 spectrum of the galactosylated product isolated from the reaction mixture. The pattern of fragments matches the spectrum of the Type-II standard. Panel F shows the MS3 spectrum of the m/z 486 fragment, empirically showing the β4-linkage of the product. Mass spectral identification of the Type-I and Type-II LacNAc relies on the differing bond labilities of the Gal(β3)- and Gal(β4)GlcNAc linkages relative to the O-benzyl bond. For Type-I acceptor, the Gal-β1,3 linkage is relatively more labile and thus its MS² spectrum is dominated by products of that cleavage. For Type-II LacNAc, the O-benzyl bond is weaker than the Gal-β1,4 linkage and the B-type Gal-GlcNAc fragment dominates the spectrum, though Gal-GlcNAc cleavage products are detectable at low abundance. Identification of the unknown is based on a simple comparison of the obtained spectrum with those of the standards. Further clarification of the linkage can be made by the presence of the cross ring cleavage (m/z 329) found in the Type-II LacNAc spectra as well as those of the GalT reaction product.

Fig. 5.

Increased activity toward mucin-type Core 1 is attributed to ST3Gal1 released by activated platelets. Panel A: Mouse models displaying low-platelet count did not show observed increase in serum SiaT activity toward Type-III acceptor. The difference plasma (filled circles) and serum (unfilled squares) SiaT activity in WT mouse is compared to two thrombocytopenic mouse models (ST3GalIV^{− /−} and WT^GP1b-alpha). SiaT activity toward mucin-type Core 1 was not differentiated in plasma (filled circle) and serum (unfilled square) in the two thrombocytopenic mouse models. Additionally, levels of SiaT active toward mucin-type Core 1 in plasma were lowered in thrombocytopenic mouse models compared to WT mice. Panel B: Increased activity toward mucin-type Core 1 is contributed directly by platelets. The supernatant of activated isolated platelets were examined for SiaT activity (pooled platelets, n = 4). Each μL in the assay contained supernatant from approximately 5 × 10⁶ of platelets, activated with thrombin. Both activated WT and ST3GalIV^{− /−} platelet supernatant contains equally active SiaT active toward mucin-type Core 1, showing that the platelets directly supply SiaT active toward mucin-type Core 1 and that lowered serum activity observed in ST3GalIV^−/− is due to low-platelet count in the system, and not a SiaT deficiency within the platelets. No observable transfer of [H3]-NeuNAc toward Type-I and Type-II LacNAc by activated WT platelet supernatant was observed. Panel C: Lowered activity of plasma, serum and platelet supernatant from ST3Gal1^loxP Pf4-Cre mice show that the increased SiaT activity toward mucin-type Core 1 is attributed to ST3Gal1. ST3Gal1^loxP Pf4-Cre plasma and serum show no difference in SiaT activity toward mucin-type Core 1 (n = 3). Supernatant of activated isolated ST3Gal1^loxP Pf4-Cre platelets (unfilled diamonds) show no activity toward mucin-type Core 1 (unpooled platelets, n = 4).

Fig. 6.

Comparing glycosyltransferase activity in healthy human plasma and serum samples to murine samples. Panels A–C: No significant difference between plasma and serum glycosyltransferase activities were observed in human samples. A comparison of the glycosyltransferase activity, namely GalT (A), SiaT (B) and FucT (C), in healthy human plasma (filled circle) and serum (unfilled squares) was assessed by measuring the amount of radiolabeled nucleotide-sugar donor transferred to GlcNAc and LacNAc acceptors (n = 3). In human compared to mouse, increased SiaT activity toward Type-II, particularly α2,6-sialylation, and mucin-type Core 1 acceptors are observed. Additionally, SiaT activity toward mucin-type Core 1 is also increased in human plasma compared to murine plasma; however, serum levels of the same SiaT activity are comparable between human and murine systems. FucT activity toward LacNAc acceptors is ~2-fold higher in human circulation than murine whereas GalT activity is ~5-fold lower in human circulation compared to murine systems. Additionally, fucosylation of Type-II acceptor, though higher in mice, seems to contain equal mixtures of both Le^x and H2 structures, unlike the murine blood, which had dominantly Le^x structures (MSⁿ analysis, Supplementary data, Figure S1).

Fig. 7.

Comparing glycosyltransferase activity between human and murine platelets. Panels A: SiaT activity. Panels B: GalT activity. Panels (A and B)1: Activated platelet supernatant. Panels (A and B)2: Activated platelets. Human (filled diamond, n = 3, unpooled), Mouse (unfilled diamond, n = 4 or 5, pooled). Human platelets contain more glycosyltransferase activity than mouse platelets. When activated, mouse platelets contain GalT and only the SiaT active toward mucin-type core 1 structure. Human platelets also release the same glycosyltransferases, and additionally have higher SiaT activity toward Type-II acceptor, particularly ST6Gal-1, which creates an α-2,6 linkage to the acceptor. Panel C1: FucT activities in human and mouse platelets toward LacNAc disaccharides. Mouse and human platelets fucosylate Type-II LacNAc with different ratios of Lewis x and H2 structures. Type-I LacNAc fucosylation by mouse platelets is not detected whether by radiolabel- or mass spectrometry-based assays and is barely discernible for fucosylation by human platelets.