The current emergence of the novel coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high-resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor-binding domain of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N-and O-glycosylation are strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research toward the development of a suitable vaccine candidate.
Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate-based inhibitors inside the cell. Due to the existence of metabolic feedback loops, they also act to prevent the de novo synthesis of the natural substrates, resulting in a global, family-wide shutdown of sialyl- and/or fucosyltransferases and remodeling of cell surface glycans. As an example of the functional consequences, the inhibitors drastically reduce expression of the sialylated and fucosylated ligand Sialyl Lewis X on myeloid cells, resulting in loss of binding to selectins and impaired leukocyte rolling.
Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.
The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilmmode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.
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