Cell entry of severe acute respiratory coronavirus-2 (SARS-CoV-2) and other CoVs can occur via two distinct routes. Following receptor binding by the spike glycoprotein, membrane fusion can be triggered by spike cleavage either at the cell surface in a transmembrane serine protease 2 (TMPRSS2)-dependent manner or within endosomes in a cathepsin-dependent manner. Cellular sialoglycans have been proposed to aid in CoV attachment and entry, although their functional contributions to each entry pathway are unknown.
View Article and Find Full Text PDFSmall-molecule sensors that are selective for particular sugars are rare. The synthesis of BODIPYs appended with two boronic acid units is reported, alongside cellular staining/labelling and turn-on fluorescence binding data for carbohydrates. The structural frameworks were designed using computational methods, leaning on the chelation characteristics of bis(boronic acids) and the photophysical properties of BODIPYs.
View Article and Find Full Text PDFGlycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan-protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands.
View Article and Find Full Text PDFExo-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.
View Article and Find Full Text PDFAll 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.
View Article and Find Full Text PDFExo-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 identifysialyltransferase 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.
View Article and Find Full Text PDFα-Dystroglycan (α-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xylα1,3-GlcAβ1,3-) termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrate length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein.
View Article and Find Full Text PDFTools to interrogate glycoconjugate-protein interactions in the context of living cells are highly attractive for the identification of critically important functional binding partners of glycan-binding proteins. These interactions are challenging to study due to the low affinity and rapid dissociation rates of glycan-protein binding events. The use of photo-cross-linkers to capture glycan-protein interaction complexes has shown great promise for identifying binding partners involved in these interactions.
View Article and Find Full Text PDFMass spectrometry-based shotgun glycomics (MS-SG) is a rapid, sensitive, label-, and immobilization-free approach for the discovery of natural ligands of glycan-binding proteins (GBPs). To perform MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissues are screened against a target GBP using catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS). Because glycan concentrations are challenging to determine, ligand affinities cannot be directly measured.
View Article and Find Full Text PDFChronic hepatitis C virus (HCV) infections continue to be a major contributor to liver disease worldwide. HCV treatment has become highly effective, yet there are still no vaccines or prophylactic strategies available to prevent infection and allow effective management of the global HCV burden. Glycan-dependent interactions are crucial to many aspects of the highly complex HCV entry process, and also modulate immune evasion.
View Article and Find Full Text PDFContemporary 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.
View Article and Find Full Text PDFComplex N-glycans of glycoproteins of the zona pellucida (ZP) of human oocytes have been implicated in the binding of spermatozoa. The termini of these unusual bi-, tri-, and tetra-antennary N-glycans consist of the tetrasaccharide sialyl-Lewis (SLe ), which was previously identified as the minimal epitope for sperm binding. We describe here the chemoenzymatic synthesis of highly complex triantennary N-glycans derived from ZP carrying SLe moieties at the C-2 and C-2' arm and a sialyl-Lewis -Lewis (SLe -Le ) residue at the C-6 antenna and two closely related analogues.
View Article and Find Full Text PDFA fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes.
View Article and Find Full Text PDFCell-surface engineering strategies that permit long-lived display of well-defined, functionally active molecules are highly attractive for eliciting desired cellular responses and for understanding biological processes. Current methodologies for the exogenous introduction of synthetic biomolecules often result in short-lived presentations, or require genetic manipulation to facilitate membrane attachment. Herein, we report a cell-surface engineering strategy that is based on the use of a CMP-Neu5Ac derivative that is modified at C-5 by a bifunctional entity composed of a complex synthetic heparan sulfate (HS) oligosaccharide and biotin.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
July 2017
Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray.
View Article and Find Full Text PDFLow-molecular-weight ice recrystallization inhibitors (IRIs) are ideal cryoprotectants that control the growth of ice and mitigate cell damage during freezing. Herein, we describe a detailed study correlating the ice recrystallization inhibition activity and the cryopreservation ability with the structure of -aryl-glycosides. Many effective IRIs are efficient cryoadditives for the freezing of red blood cells (RBCs).
View Article and Find Full Text PDFDuring cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol.
View Article and Find Full Text PDFIn North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at -80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment.
View Article and Find Full Text PDFMost antifreeze proteins (AFPs) exhibit two types of "antifreeze activity" - thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity. The mechanism of TH activity has been studied in depth and is the result of an adsorption of AFPs to the surface of ice with an ice-binding face (IBF). In contrast, the mechanism of ice recrystallization and its inhibition is considerably less understood.
View Article and Find Full Text PDFThe inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization.
View Article and Find Full Text PDFA library of peptides and glycopeptides containing (4R)-hydroxy-L-proline (Hyp) residues were designed with a view to providing stable polyproline II (PPII) helical molecules with antifreeze activity. A library of dodecapeptides containing contiguous Hyp residues or an Ala-Hyp-Ala tripeptide repeat sequence were synthesized with and without α-O-linked N-acetylgalactosamine and α-O-linked galactose-β-(1→3)-N-acetylgalactosamine appended to the peptide backbone. All (glyco)peptides possessed PPII helical secondary structure with some showing significant thermal stability.
View Article and Find Full Text PDFThe ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized.
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