Characterization of Human Sialyltransferase ST6GalNAc5 and Its Use in the Synthesis of Internally α2,6-Sialylated Glycans

In this study, we successfully expressed recombinant human sialyltransferase α-N-acetylgalactosaminide α-2,6-sialyltransferase 5 (hST6GalNAc5) in Expi293F cells (with 32.9 mg/L). Comprehensive kinetic and substrate scope analyses revealed that α-2,3-sialylation is critical for efficient catalysis, as evidenced by the acceptor reactivity trend: sialyl lacto-N-tetraose (SLNT) > GM1b ≫ lacto-N-tetraose (LNT) > GA1 glycan. Notably, SSEA-4, which shares a nonreducing glycan sequence (Sia-α-2,3-Gal-β-1,3-GalNAc-β-) similar to GM1b, served as the acceptor for disialosyl globopentaosylceramide (DSGb5) synthesis. To enhance enzymatic efficiency, we integrated a sugar nucleotide regeneration system (SNRS), enabling the preparative synthesis of disialyllacto-N-tetraose (DSLNT) with minimal enzyme consumption. This strategy facilitated a multicycle one-pot enzymatic approach for DSLNT synthesis, achieving a hundred-milligram-scale production with high efficiency. Overall, hST6GalNAc5 demonstrated broad applicability in synthesizing DSLNT derivatives, α-series gangliosides, and DSGb5, including previously inaccessible structures such as disialyl Lewis a and GH1cα. Glycan microarray analyses revealed distinct Siglec-binding profiles for these synthetic glycoconjugates. GH1cα and GQ1bα showed strong affinity toward Siglec-7, while RM2 was identified as a potent ligand for both Siglec-3 and Siglec-8. DSLNT and DSGb5 also exhibited selective interactions with Siglec-8. These results underscore the utility of hST6GalNAc5 as a powerful tool for glycoengineering and highlight the potential of the synthesized glycans for probing Siglec-mediated immunological functions.