Synthesis of Biologically Active N- and O-Linked Glycans with Multisialylated 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.

CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells

“…Analogues 3k and 3l are multisialylated di-LacNAc products, which were prepared with bacterial sialyl-transferase. 33–34 Notably, the N-glycans are derived from a natural glycan isolated from eggs, ( SGP , Figure 3A) which is attached to asparagine, facilitating subsequent conjugation strategies. 35 …”

Efficient chemoenzymatic synthesis of an N-glycan isomer library

“…For example, by Danishefsky 7a, 15 constructed a core pentasaccharide by glycosylating a trisaccharide with a monosaccharide thioether donor; Unverzagt 16 synthesized multi-antennary complex type N-glycans with acetated Schmidt’s trichloroacetimidate donor; Wong 8a used oligosaccharyl fluorides as donors to produce bi-, tri- and tetra-antennary complex type N-glycans; Boons 9 developed a strategy by sequential removal of the protecting groups, and then by chemical glycosylation using a diverse set of trifluoroacetimidate donors. In these cases, to prepare various glycosyl donors, a temporary anomeric protecting group was used and then transformed into trichloroacetimidates, fluorides or trifluoroacetimidates, depending on the choice of glycosylation reaction.…”

“…6 Among chemically synthesized N-glycans, only few contains terminal sialic acid (Sia) due to difficulties in sialic acid chemistry, 7 which was later overcome by enzymatic glycosylation using sialyltransferases. 8 Most recently, Boons developed a strategy for chemoenzymatic synthesis of asymmetrical N-glycans, and 14 tri-antennary complex N-glycans were obtained. 9 Nevertheless, only a few N-glycan structures were prepared in each report, mainly due to their complexity and diversity.…”

“…8,9 However, even though gel filtration has been applied for decades in purifying glycans, it is time-consuming, less efficient, and may waste significant portion of products in preparing small quantities of precious N-glycans. Therefore, a more reliable and rapid N-glycan purification approach is yet to be developed.…”

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

“…In many studies, glycosylation has been shown to affect the viral attachment or penetration steps via the modification of cellular receptors or viral envelope glycoproteins. For instance, the O-linked glycoproteins of ␣2,3-or ␣2,6-linked sialic acids were shown to play important roles during infection by IAV and other viruses, including the coxsackievirus A24 variant and enterovirus 70 (42)(43)(44)(45). Furthermore, the envelope proteins of many viruses, such as human immunodeficiency virus type 1, HCV, Ebola virus, and herpes simplex virus 1, have been shown to be modified by O-linked glycosylation, which influences the attachment of viral virions to the cell surface (42,(46)(47)(48).…”