Traditional lectins exhibit broad binding specificity for cell-surface carbohydrates, and generating anti-glycan antibodies is challenging due to low immunogenicity. Nevertheless, it is necessary to develop glycan binding proteins for single-cell glycosylation pathway analysis. Here, we test the hypothesis that protein engineering of mammalian glycosyltransferases can yield glycan-binding proteins with defined specificity. Introducing an H302A mutation, based on rational design, into porcine ST3Gal1 abolishes its enzymatic activity, but results in a lectin that specifically binds sialylated core-2 O-linked glycans (Neu5Acα2-3Galβ1-3[GlcNAc(β1-6)]GalNAcα). To improve binding, we develop a mammalian cell-surface display platform to screen variants. One ST3Gal1 mutant (sCore2) with three mutations, H302A/A312I/F313S exhibits enhanced binding specificity. Spectral flow cytometry and tissue microarray analysis using sCore2 reveal distinct cell- and tissue-specific sialyl core-2 staining patterns in human blood cells and paraffin-embedded tissue sections. Overall, glycosyltransferases can be engineered to generate specific glycan binding proteins, suggesting that a similar approach may be extended to other glycoenzymes.
© 2025. The Author(s).

Mucin-type O-glycans on glycoproteins are pivotal for biology and impact the quality of biotherapeutics. Furthermore, glycans on host cells serve as ligands for lectins/adhesins on bacteria for bacterium-host interactions in the colonization or attachment/invasion of bacteria. Defining the structure-function relationship of O-glycans is hindered by a lack of enzyme(s) to release sialylated O-glycans from glycoproteins. Here we show identification of endo-α-N-acetylgalactosaminidases (O-glycanases, GH101) with broad substrate specificities, termed Peptide:O-Glycosidase (POGase). In 5 POGase orthologs identified, we characterize one that releases sialylated O-glycans from glycopeptides, glycoproteins and biotherapeutics. Three peptide motifs differentiate the POGase existing in phylum Actinomycetota from known O-glycanases in other bacteria. While the GH101 domain classifies POGases, other domains confer the efficient enzyme activity and binding to major glycans decorating epithelial cells. The dual functional POGases encompassing broader O-glycanase and adhesin activities will facilitate the study of O-glycomics, quality assessment of biotherapeutics, and development of microbiology and medicine.
© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.

O-GalNAc glycans, also known as mucin-type O-glycans, are primary constituents of mucins on various mucosal sites of the body and also ubiquitously expressed on cell surface and secreted proteins. They have crucial roles in a wide range of physiological and pathological processes, including tumor growth and progression. In addition, altered expression of O-GalNAc glycans is frequently observed during different disease states. Research dedicated to unraveling the structure-function relationships of O-GalNAc glycans has led to the discovery of disease biomarkers and diagnostic tools and the development of O-glycopeptide-based cancer vaccines. Many of these efforts require amino acid-linked O-GalNAc core structures as building blocks to assemble complex O-glycans and glycopeptides. There are eight core structures (cores one to eight), from which all mucin-type O-glycans are derived. In this protocol, we describe the first divergent synthesis of all eight cores from a versatile precursor in practical scales. The protocol involves (i) chemical synthesis of the orthogonally protected precursor (3 days) from commercially available materials, (ii) chemical synthesis of five unique glycosyl donors (1-2 days for each donor) and (iii) selective deprotection of the precursor and assembly of the eight cores (2-4 days for each core). The procedure can be adopted to prepare O-GalNAc cores linked to serine, threonine and tyrosine, which can then be utilized directly for solid-phase glycopeptide synthesis or chemoenzymatic synthesis of complex O-glycans. The procedure empowers researchers with fundamental organic chemistry skills to prepare gram scales of any desired O-GalNAc core(s) or all eight cores concurrently.
© 2024. Springer Nature Limited.

Sialic acid residues found as terminal monosaccharides in various types of glycan chains in cell surface glycoproteins and glycolipids have been identified as important contributors of cell-cell interactions in normal vs. abnormal cellular behavior and are pivotal in diseases such as cancers. In vertebrates, sialic acids are attached to glycan chains by a conserved subset of sialyltransferases with different enzymatic and substrate specificities. ST6Gal I is a sialyltransferase using activated CMP-sialic acids as donor substrates to catalyze the formation of a α2,6-glycosidic bond between the sialic acid residue and the acceptor disaccharide LacNAc. Understanding sialyltransferases at the molecular and structural level shed light into their function. We present here two human ST6Gal I structures, which show for the first time the enzyme in the unliganded state and with the full donor substrate CMP-Neu5Ac bound. Comparison of these structures reveal flexibility of the catalytic loop, since in the unliganded structure Tyr354 adopts a conformation seen also as an alternate conformation in the substrate bound structure. CMP-Neu5Ac is bound with the side chain at C5 of the sugar residue directed outwards at the surface of the protein. Furthermore, the exact binding mode of the sialic acid moiety of the substrate directly involves sialylmotifs L, S and III and positions the sialylmotif VS in the immediate vicinity. We also present a model for the ternary complex of ST6Gal I with both the donor and the acceptor substrates.Copyright © 2020. Published by Elsevier Inc.

Nucleotide-sugar transporters (NSTs) facilitate eukaryotic cellular glycosylation by transporting nucleotide-sugar conjugates into the Golgi lumen and endoplasmic reticulum for use by glycosyltransferases, while also transferring nucleotide monophosphate byproducts to the cytoplasm. Mutations in this family of proteins can cause a number of significant cellular pathologies, and wild type members can act as virulence factors for many parasites and fungi. Here, we describe an in vitro assay to measure the transport activity of the CMP-sialic acid transporter (CST), one of seven NSTs found in mammals. While in vitro transport assays have been previously described for CST, these studies failed to account for the fact that 1) commercially available stocks of CMP-sialic acid (CMP-Sia) are composed of ~10% of the higher-affinity CMP and 2) CMP-Sia is hydrolyzed into CMP and sialic acid in aqueous solutions. Herein we describe a method for treating CMP-Sia with a nonselective phosphatase, Antarctic phosphatase, to convert all free CMP to cytidine. This allows us to accurately measure substrate affinities and transport kinetics for purified CST reconstituted into proteoliposomes.Copyright © The Authors; exclusive licensee Bio-protocol LLC.