Synthesis of biologically active N- and O-linked glycans with multi-sialylated poly-N-acetyllactosamine extensions using P. damsela α2-6 sialyltransferase

Abstract

Sialosides on N- and O-linked glycoproteins play a fundamental role in many biological processes and synthetic glycan probes have proven to be valuable tools for elucidating these functions. Though sialic acids are typically found α2-3 or α2-6-linked to a terminal non-reducing end galactose, poly-LacNAc extended core-3 O-linked glycans isolated from rat salivary glands and human colonic mucins have been reported to contain multiple internal Neu5Acα2-6Gal epitopes. Here, we have developed an efficient approach for the synthesis of a library of N- and O-linked glycans with multi-sialylated poly-LacNAc extensions, including naturally occurring multi-sialylated core-3 O-linked glycans. We have found that a recombinant α2-6 sialyltransferase from Photobacterium damsela (Pd2,6ST) exhibits unique regioselectivity and is able to sialylate internal galactose residues in poly-LacNAc extended glycans which was confirmed by MS/MS analysis. Using a glycan microarray displaying this library, we found that Neu5Acα2-6Gal specific influenza virus hemagglutinins, siglecs and plant lectins are largely unaffected by adjacent internal sialylation, and in several cases the internal sialic acids are recognized as ligands. Polyclonal IgY antibodies specific for internal sialoside epitopes were elicited in inoculated chickens.

Sialosides on N- and O-linked glycoproteins play many important roles in biological recognition.¹ These glycans present sialic acids as terminal substituents that are ideally positioned to interact with glycan binding proteins,² including the Siglecs (sialic acid-binding immunoglobulin-type lectins),^3,4 and viral hemagglutinins.⁵ The sialic acids on N-acetyllactosamine (LacNAc, Galβ1-4GlcNAc) chains of glycoproteins are typically linked α2-3 or α2-6 to a terminal non-reducing end galactose.⁶ However, poly-LacNAc extended core-3 O-linked glycans isolated from rat salivary glands have been reported to contain multiple internal Neu5Acα2-6Gal groups (Figure 1A).⁷ Similar multi α2-6-sialylated core-3 O-linked glycans have been reported on human colonic mucins.^8,9 Although internally sialylated glycans are found in certain gangliosides¹⁰ and milk oligosaccharides,¹¹ these remain the only reports describing the Neu5Acα2-6Gal epitope on internal Gal residues of poly LacNAc chains. The extent of expression and biological function of this novel glycan sequence has not been explored due to the lack of specific chemical and biological probes. Here we report the use of Pd2,6ST to synthesize a library of structures including N- and O-linked glycans having poly-LacNAc chains with multiple Neu5Acα-2,6-Gal epitopes (1-26, Figure 1B). The binding specificity of a panel of glycan binding proteins (lectins) and polyclonal IgY antibodies for these multi-sialylated glycans has been evaluated using a custom glycan microarray.

Figure 1.

(A) Poly-LacNAc extended Core-3 O-linked glycans with multiple Neu5Acα2-6Gal groups isolated from rat salivary⁷ and human colonic^8,9 mucins. (B) Synthetic sialoside targets (1-26), including Core-3 O-linked structures (15-17) isolated from colonic mucins. Sp1=O-ethyl amine, Sp2=O-Threonine, and Sp3=N-(KVA)NKT.

A bacterial α2-6 sialyltransferase from Photobacterium damsela (Pd2,6ST)^12,13 has been used previously to synthesize sialosides on glycans with terminal Gal residues and was shown to react with fucosylated and α2-3-sialylated substrates.^14-16 In our recent studies, we attempted to use recombinant Pd2,6ST to sialylate poly-LacNAc extended glycans, and surprisingly both ¹H NMR and MS analysis suggested that multiple sialic acids were being transferred to the substrates instead of one on the terminal Gal as expected. Subsequent MS/MS fragmentation showed that multi-sialylated glycans were formed by sialylation of the terminal and internal galactose positions along the poly-LacNAc backbone. This unprecedented regioselectivity of Pd2,6ST suggested a simple enzymatic strategy for synthesis of multi-sialylated poly-LacNAc chains with sialic acid on both terminal and internal Gal units.

We first examined the reactivity of Pd2,6ST on LacNAc oligomers of varying length and Gal composition (Scheme 1). Substrates 27-30 were prepared as previously described.¹⁷ Reaction of 27, which contains a single internal Gal residue, with Pd2,6ST readily gave mono-sialylated 31 in 87% yield. Treatment of 28 and 29 with Pd2,6ST gave glycans 32 and 33, respectively. NMR and MS analysis confirmed the addition of two Neu5Ac residues in both. Finally, sialylation of tri-LacNAc 30 using Pd2,6ST and excess CMP-Neu5Ac remarkably gave 34 which contains three Neu5Ac residues. Sialosides 31-34 were subjected to hydrogenation conditions to give 1, 3-5 respectively. In contrast, recombinant human α2-6-sialyltransferase (hST6Gal-I),¹⁸ an enzyme that catalyzes the α2-6-sialylation of terminal Gal residues, gave no reaction on the internal Gal of 27. However, we found that disialo 32 could also be prepared by galactosylation of internal sialylated 31 using β1-4-galactosyltransferase/UDP-4′-Gal-epimerase fusion protein (β4GalT-GalE),¹⁸ followed by sialylation of the terminal Gal using hST6Gal-I (Scheme S1). Thus, the ability of hST6Gal-I to sialylate the glycans with internal sialic acid suggests a plausible biosynthetic mechanism where the multi-sialylated poly-LacNAc extensions are formed by iterative terminal sialylation by ST6-Gal I followed by LacNAc chain extension. We next explored Pd2,6ST catalyzed sialylations of poly-LacNAc on various N- and O-linked core structures (Scheme 1). Core-3 O-linked glycans 12-14 with sialic acid on the GalNAc residue were prepared from 36 using recombinant chicken ST6GalNAc-I (Scheme S2).¹⁹ Treatment of 12-14 with Pd2,6ST using excess CMP-Neu5Ac readily gave the multisialylated glycans 15-17. The addition of the Neu5Ac residues was confirmed by integration of the Neu5Ac H-3ax and H-3eq methylene protons in the ¹H-NMR and was supported by MS analysis. This report represents the first synthesis of the naturally occurring glycans 15-17.

Scheme 1.

Enzymatic transformations.^a Pd2,6ST catalyzed sialylation of poly-LacNAc extended glycans.

^aReagents and Conditions: (a) Pd2,6ST, CMP-Neu5Ac (CMP-Neu5Gc for preparing 26); (b) H₂, Pd/C, H₂O. CMP = cytidine monophosphate, Neu5Ac = N-acetyl neuraminic acid; Neu5Gc = N-glycolyl neuraminic acid.

Also, we sialylated a panel of poly-LacNAc extended biantennary core-4 O-linked (37-40) and N-linked glycans (41-44) (Scheme 1). The substrates 37-44 were prepared as previously described.²⁰ Remarkably, reaction using Pd2,6ST and excess CMP-Neu5Ac (or CMP-Neu5Gc for 26) gave the multi-sialylated products 18-26. MS analysis of the per-methylated 19, 20, and 25 showed the multisialylation products with minor amounts of under-sialylated products (Figure 2 and S3-S5). MS/MS fragmentation revealed that in addition to the terminal Gal residue of poly-LacNAc, including extended N- and O-linked glycans, Pd2,6ST can also sialylate internal Gal in α2,6 linkage, and that adjacent LacNAc units are concurrently modified. Typical yields for the Pd2,6ST catalyzed sialylations ranged between 80-95%.

Figure 2.

Mass spectrometric analysis of N-linked glycan 25. (top) MS analysis of per-methylated product mixture; (bottom) MS/MS fragmentation analysis of [M+Na]⁺ peak m/z 6034.

Exploiting the unique regioselectivity of Pd2,6ST, we were also able to synthesize di-LacNAc structures with varied sialylation patterns between the terminal and internal Gal positions (Scheme 2). The terminal Gal residue of 28 was first capped with either an α296 (45, 46) or α2-3-linked (47, 48) sialic acid by reaction using hST6Gal-I or Pasteurella multocida α2-3-sialyltransferase 1 (PmST1)²¹ respectively. Next, the internal Gal positions of each were α2-6-sialylated by treatment with Pd2,6ST to give 49-53, following the hydrogenation conditions to give 6-10 with ethyl amine linkers. MS analysis of the products confirmed that a second sialic acid was added which matched the integrations in the ¹HNMR.

Scheme 2.

Enzymatic transformations.^a Pd2,6ST catalyzed internal sialylation of mono-sialylated diLacNAc fragments.

^aReagents and Conditions: (a) hST6Gal-I, CMP-Neu5Gc (for 45); hST6Gal-I, CMP-Neu5Ac (for 46); PmST1, CMP-Neu5Gc (for 47); PmST1, CMP-Neu5Ac (for 48); (b) Pd2,6ST, CMP-Neu5Ac (for 49, 51, and 53) or CMP-Neu5Gc (50 and 52); (c) H₂, Pd(OH)₂, H₂O.

To assess the potential of these novel compounds (1-26) to serve as ligands for glycan binding proteins known to mediate cell surface biology, a custom glycan microarray was constructed from this sialoside library and screened against several sialoside binding proteins (Figure 3A-E). Glycans with a terminal α2-3 or α2-6-linked sialic acid or no sialic acid (asialo) were included as controls (A-M, Figure 3A-E). Controls L and M were only included in the screen against Siglec-F and polyclonal IgY (Figure 3C and 3E). The array was constructed by direct printing on glass slides activated with N-hydroxy succinimide, reacting with the free amino group on the aglycone of each glycan to form a covalent amide bond.

Figure 3.

Glycan microarray binding analyses as measured by fluorescence intensity for (A) S. nigra agglutinin (SNA), (B) recombinant human Siglec-2 Fc chimera, (C) murine Siglec-F Fc chimera, (D) influenza A viral hemagglutinin H1 (A/Kentucky/07), and (E) total chicken polyclonal IgY (elicited with glycan 4). (yellow) Sialosides 1-26; (green/grey) controls A-M. Controls L and M were only included in the screen against Siglec-F and polyclonal IgY. See supporting information for details.

Sambucus nigra agglutinin (SNA),²² which is known to bind terminal α2-6-linked sialosides (e.g. control glycans B-H), bound all synthetic glycans with terminal α2-6-linked sialic acid (3, 5-7, 15, 17, 19, 21, 23, 25, and 26), and surprisingly also bound two glycans, 16 and 18, which contained only internal sialic acid (Figure 3A). Several members of the sialic acid binding siglec receptor family were also examined.³ The B cell siglec CD22 (Siglec-2) is known to be specific for glycans with a terminal Siaα2-6Galβ1-4GlcNAc sequence, exhibiting equal affinity for glycans with the sialic acids Neu5Ac or Neu5Gc.²³ Multi-sialylated glycans that bound strongly included the linear fragments (3, 5-7), biantennary N- and O-linked glycan structures (19, 21, 23, 25, and 26) and two glycans with internal sialic acid only (20 and 24) (Figure 3B). The eosinophil siglec, murine Siglec-F, as expected showed strong preference for the control glycans L and M, in keeping with its known specificity for these sulfated α2-3-sialylated structures (Figure 3C).²⁴ However, Siglec-F remarkably also bound strongly to many of the novel multi-sialylated glycans (4, 5, 10, 18-25), including several with internal sialic acids only (4, 18, 20, 22, and 24). Notably, the switch from Neu5Ac to Neu5Gc largely abrogates binding to Siglec-F. A recombinant hemagglutinin (HA) from a seasonal human influenza virus (H1 A/KY/07)²⁵ was also examined (Figure 3D). The HA which is known to bind glycans with the terminal sequence Neu5Acα2-6Gal (C, D and F) bound exclusively to glycans with this terminal sequence (3, 5, 7, 17, 19, 21, 23, and 25), and did not bind to glycans with only internal sialic acids. Lack of binding to glycans with no terminal sialic acids help document the fidelity of the enzymatic synthesis during repetitive synthesis of LacNAc repeats.

Lastly, we used the glycan array to screen polyclonal IgY antibodies isolated from eggs of chickens inoculated with glycan 4 displayed on a viral scaffold as described in the supporting information (Figure 3E). The chicken polyclonal IgY strongly recognized glycans with internal sialic acid only (1, 4, 16, 18, 20, 22, and 24) and remarkably showed no binding to the α2-6-sialylated control glycans with terminal α2-6 sialic acids only (B-H). This demonstrates that the chicken immune system can recognize the internal sialic acids distinct from the terminal sialic acids, suggesting that such antibodies could be used as immune-probes to aid in the elucidation of the functions of this novel class of glycans.

In summary, we report that recombinant P. damsela α2-6-sialyltransferase is able to transfer sialic acid to internal galactose residues in poly-LacNAc backbones forming multi-sialylated glycans. This unique specificity has allowed us to synthesize a library of N- and O-linked glycans with multisialylated poly-LacNAc extensions, including poly-LacNAc extended core-3 O-linked glycans previously identified in rat salivary and human colonic mucins. Screening a glycan microarray of this library has demonstrated that sialic acid specific binding proteins, including a plant lectin (SNA) and members of the siglec family of immune cell receptors can recognize the internal sialic acids as ligands. Based on our ability to generate IgY antibodies specific for glycans with internal sialic acids, we anticipate that these novel glycans will facilitate investigations into the biological roles of this unique class of multi-sialylated poly-N-acetyl-lactosamine glycans.