Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs.

Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups, which can be used to study glycans and their interactions with glycan-binding proteins. In recent years, strategies to edit cellular glycans by installing monosaccharides and their derivatives using glycosyltransferase enzymes have rapidly expanded. However, analogous methods to introduce chemical reporter-functionalized type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars bearing azide, alkyne, and diazirine functionalities on the C2-acetamido group using the mutant uridylyltransferase AGX1F383A. The unnatural UDP-GlcNAc derivatives were examined as substrates for the human GlcNAc-transferase B3GNT2, where it was found that modified donors were tolerated for transfer, albeit to a lesser extent than the natural UDP-GlcNAc substrate. When the GlcNAc derivatives were examined as acceptor substrates for the human Gal-transferase B4GalT1, all derivatives were well tolerated and the enzyme could successfully form derivatized LacNAcs. B3GNT2 was also used to exo-enzymatically install GlcNAc and unnatural GlcNAc derivatives on cell-surface glycans. GlcNAc- or GlcNAz-engineered cells were further extended by B4GalT1 and UDP-Gal, producing LacNAc- or LacNAz-engineered cells. Our proof-of-concept glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural type 2 LacNAc motifs.

One-Step Selective Labeling of Native Cell Surface Sialoglycans by Exogenous α2,8-Sialylation.

Exo-enzymatic glycan labeling strategies have emerged as versatile tools for efficient and selective installation of terminal glyco-motifs onto live cell surfaces. Through employing specific enzymes and nucleotide-sugar probes, cells can be equipped with defined glyco-epitopes for modulating cell function or selective visualization and enrichment of glycoconjugates. Here, we identifyCampylobacter jejunisialyltransferase Cst-II I53S as a tool for cell surface glycan modification, expanding the exo-enzymatic labeling toolkit to include installation of α2,8-disialyl epitopes. Labeling with Cst-II was achieved with biotin- and azide-tagged CMP-Neu5Ac derivatives on a model glycoprotein and native sialylated cell surface glycans across a panel of cell lines. The introduction of modified Neu5Ac derivatives onto cells by Cst-II was also retained on the surface for 6 h. By examining the specificity of Cst-II on cell surfaces, it was revealed that the α2,8-sialyltransferase primarily labeled N-glycans, with O-glycans labeled to a lesser extent, and there was an apparent preference for α2,3-linked sialosides on cells. This approach thus broadens the scope of tools for selective exo-enzymatic labeling of native sialylated glycans and is highly amenable for the construction of cell-based arrays.

Glycosyltransferases as versatile tools to study the biology of glycans.

All cells are decorated with complex carbohydrate structures called glycans that serve as ligands for glycan-binding proteins (GBPs) to mediate a wide range of biological processes. Understanding the specific functions of glycans is key to advancing an understanding of human health and disease. However, the lack of convenient and accessible tools to study glycan-based interactions has been a defining challenge in glycobiology. Thus, the development of chemical and biochemical strategies to address these limitations has been a rapidly growing area of research. In this review, we describe the use of glycosyltransferases (GTs) as versatile tools to facilitate a greater understanding of the biological roles of glycans. We highlight key examples of how GTs have streamlined the preparation of well-defined complex glycan structures through chemoenzymatic synthesis, with an emphasis on synthetic strategies allowing for site- and branch-specific display of glyco-epitopes. We also describe how GTs have facilitated expansion of glyco-engineering strategies, on both glycoproteins and cell surfaces. Coupled with advancements in bioorthogonal chemistry, GTs have enabled selective glyco-epitope editing of glycoproteins and cells, selective glycan subclass labeling, and the introduction of novel biomolecule functionalities onto cells, including defined oligosaccharides, antibodies, and other proteins. Collectively, these approaches have contributed great insight into the fundamental biological roles of glycans and are enabling their application in drug development and cellular therapies, leaving the field poised for rapid expansion.

Single-cell RNA-sequencing reveals transcriptional dynamics of estrogen-induced dysplasia in the ovarian surface epithelium.

Estrogen therapy increases the risk of ovarian cancer and exogenous estradiol accelerates the onset of ovarian cancer in mouse models. Both in vivo and in vitro, ovarian surface epithelial (OSE) cells exposed to estradiol develop a subpopulation that loses cell polarity, contact inhibition, and forms multi-layered foci of dysplastic cells with increased susceptibility to transformation. Here, we use single-cell RNA-sequencing to characterize this dysplastic subpopulation and identify the transcriptional dynamics involved in its emergence. Estradiol-treated cells were characterized by up-regulation of genes associated with proliferation, metabolism, and survival pathways. Pseudotemporal ordering revealed that OSE cells occupy a largely linear phenotypic spectrum that, in estradiol-treated cells, diverges towards cell state consistent with the dysplastic population. This divergence is characterized by the activation of various cancer-associated pathways including an increase in Greb1 which was validated in fallopian tube epithelium and human ovarian cancers. Taken together, this work reveals possible mechanisms by which estradiol increases epithelial cell susceptibility to tumour initiation.