Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized due to challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, sulfotransferases, and other glycan modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one, the sialyltransferase ST6GALNAC2. Many enzymes were produced at high yields and similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

No abstract

No abstract

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N -linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch selective manner to give asymmetrical structures commonly found in Nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in 10 or fewer chemical and enzymatic steps into multi-antennary N -glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N -glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart allowing each arm to be elaborated into a unique appendage.

During circulation in humans and natural selection to escape antibody recognition for decades, A/H3N2 influenza viruses emerged with altered receptor specificities. These viruses lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. Examination of receptor specificities of A/H3N2 viruses reveals that recent viruses compensated for decreased binding of the prototypic human receptor by recognizing α2,6-sialosides on extended LacNAc moieties. Erythrocyte glycomics shows an absence of extended glycans providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installing functional receptors on erythrocytes, allows antigenic characterization of recent A/H3N2 viruses confirming the cocirculation of antigenically different viruses in humans. Computational analysis of HAs in complex with sialosides having extended LacNAc moieties reveals that mutations distal to the RBD reoriented the Y159 side chain resulting in an extended receptor binding site.

No abstract

No abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and the most abundant protein in coronaviruses and thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having post-translational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells, one was in-house expressed and purified without a signal peptide sequence and the other was commercially produced with a signal peptide channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation, as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for coronavirus disease and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development point-of-care diagnostics.