Chemical and Enzymatic Synthesis of DisialylGb5 and Other Sialosides for Glycan Array Assembly and Evaluation of Siglec-Mediated Immune Checkpoint Inhibition.

Aberrant glycosylation, especially sialylation, on cell surface is often associated with cancer progression and immunosuppression. Over-sialylation of stage-specific embryonic antigen-4 (SSEA-4) to generate disialylGb5 (DSGb5) was reported to trigger Siglec-7 recognition and suppress NK-mediated target killing. In this study, efficient chemo-enzymatic and programmable one-pot methods were explored for the synthesis of DSGb5 and related sialosides for assembly of glycan microarrays and evaluation of binding specificity toward Siglecs-7, 9, 10, and 15 associated with immune checkpoint inhibition.

Structure-Based Mechanism and Specificity of Human Galactosyltransferase β3GalT5.

Human β1,3-galactosyltransferase 5 (β3GalT5) is a key enzyme involved in the synthesis of glycans on glycoproteins and glycolipids that are associated with various important biological functions, especially tumor malignancy and cancer progression, and has been considered as a promising target for development of anticancer agents. In this study, we determined the X-ray structures of β3GalT5 in complex with the stable donor analogue UDP-2-fluorogalactose or the native donor substrate UDP-galactose (UDP-Gal) and several glycan acceptors at different reaction steps. Based on the structures obtained from our experiments, β3GalT5 catalyzes the transfer of galactose from UDP-Gal to a broad spectrum of glycan acceptors with an S2-like mechanism; however, in the absence of a glycan acceptor, UDP-Gal is slowly converted to UDP and two other products, one is galactose through an S2-like mechanism with water as an acceptor and the other is an oxocarbenium-like product, presumably through an S1-like mechanisms.

Expression of Human β3GalT5-1 in Insect Cells as Active Glycoforms for the Efficient Synthesis of Cancer-Associated Globo-Series Glycans.

The globo-series glycosphingolipids (GSLs) are unique glycolipids exclusively expressed on the cell surface of various types of cancer and have been used as targets for the development of cancer vaccines and therapeutics. A practical enzymatic method has been developed for the synthesis of globo-series glycans, where the conversion of Gb4 to Gb5 (SSEA-3) glycan based on the microbial galactosyltransferase LgtD is relatively inefficient compared to other steps. To improve the efficiency, we explored the two human galactosyltransferase (β3GalT5) isozymes in cancer cells for this reaction and found that isozyme 1 (β3GalT5-1) is more active than isozyme 2 (β3GalT5-2).

Glycosite-deleted mRNA of SARS-CoV-2 spike protein as a broad-spectrum vaccine.

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response.