The structure of human α-2,6-sialyltransferase reveals the binding mode of complex glycans

Abstract

Human β-galactoside α-2,6-sialyltransferase I (ST6Gal-I) establishes the final glycosylation pattern of many glycoproteins by transferring a sialyl moiety to a terminal galactose. Complete sialylation of therapeutic immunoglobulins is essential for their anti-inflammatory activity and protein stability, but is difficult to achieve in vitro owing to the limited activity of ST6Gal-I towards some galactose acceptors. No structural information on ST6Gal-I that could help to improve the enzymatic properties of ST6Gal-I for biotechnological purposes is currently available. Here, the crystal structures of human ST6Gal-I in complex with the product cytidine 5'-monophosphate and in complex with cytidine and phosphate are described. These complexes allow the rationalization of the inhibitory activity of cytosine-based nucleotides. ST6Gal-I adopts a variant of the canonical glycosyltransferase A fold and differs from related sialyltransferases by several large insertions and deletions that determine its regiospecificity and substrate specificity. A large glycan from a symmetry mate localizes to the active site of ST6Gal-I in an orientation compatible with catalysis. The glycan binding mode can be generalized to any glycoprotein that is a substrate of ST6Gal-I. Comparison with a bacterial sialyltransferase in complex with a modified sialyl donor lends insight into the Michaelis complex. The results support an SN2 mechanism with inversion of configuration at the sialyl residue and suggest substrate-assisted catalysis with a charge-relay mechanism that bears a conceptual similarity to serine proteases.

Enzymatic Basis for N-Glycan Sialylation

“…6). Similar interactions with CMP were observed in the structure of human ST6GAL1 in complex with CMP (29). The acceptor sites in the sialyltransferases are more diverse, yet the general positions and angles of approach for the distinct disaccharide acceptors to the sugar donor and catalytic His residues are remarkably similar between ST6GAL1, ST3GAL1, and CstII (Fig.…”

“…A single GlcNAc residue at each site is sufficient to maintain enzyme function, because EndoF1 cleavage does not compromise enzyme solubility or catalytic characteristics (Table 2). For human ST6GAL1, a partial PNGase digestion resulted in the removal of a single N-glycan equivalent to Asn-158 in the rat enzyme (29), but the remaining biantennary complex N-glycan at the equivalent of Asn-144 in the rat enzyme was retained and contributed to contacts in the active site in the adjoining unit in the crystal lattice and helped to stabilize the loop that was disordered in the rat ST6GAL1 structure.…”

“…The structure revealed several conserved features with ST3GAL1 (25), the bacterial GT42 sialyltransferase, CstII (30), and the recently published structure of human ST6GAL1 (29), including the sialylmotif region involved in binding the sugar-nucleotide donor. Modeling, molecular dynamics (MD) 5 simulations, and kinetic analysis of site-directed mutants provide evidence for a broadly similar positioning of sugar donor, glycan acceptor, and catalytic base for all four enzymes.…”

“…Insights into the domain structures and specificities of the mammalian sialyltransferases have come from extensive enzymatic studies on wild type and mutant enzymes (20,26,27), from the crystal structures of a pig ST3GAL1 involved in the synthesis of Neu5Ac␣2,3Gal linkages on O-linked glycans (25), and a structure of human ST6GAL1 that was published while this manuscript was being completed (29). The overall domain organization among the sialyltransferases is similar (28).…”

Arabinogalactan biosynthesis: Implication of AtGALT29A enzyme activity regulated by phosphorylation and co-localized enzymes for nucleotide sugar metabolism in the compartments outside of the Golgi apparatus

“…The results indicate that the charge status of Y144 likely affects the AtGALT29A GalT activity, which may be regulated by an unknown kinase/phosphatase system. The predicted 3D structure of AtGALT29A using rat a-2,6-sialyltransferase 6 as a template indicates that the Y144 site is located in the globular catalytic domain, far (35 A ) from the catalytic site, and on the surface of the globular domain (Fig. 1B).…”