Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments, using an extensive heparan sulfate (HS) oligosaccharide library, showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. A hexasaccharide composed of IdoA2S-GlcNS6S repeating units was identified as the minimal binding epitope. Surface plasmon resonance showed the SARS-CoV-2 spike protein binds with a much higher affinity to heparin ( K D = 55 nM) compared to the RBD ( K D = 1 μM) alone. It was also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. However, exogenous administered heparin or a highly sulfated HS oligosaccharide inhibited RBD binding to cells. Furthermore, an enzymatic removal of HS proteoglycan from physiological relevant tissue resulted in a loss of RBD binding. The data support a model in which HS functions as the point of initial attachment allowing the virus to travel through the glycocalyx by low-affinity high-avidity interactions to reach the cell membrane, where it can engage with ACE2 for cell entry. Microarray binding experiments showed that ACE2 and HS can simultaneously engage with the RBD, and it is likely no dissociation between HS and RBD is required for binding to ACE2. The results highlight the potential of using HS oligosaccharides as a starting material for therapeutic agent development.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments using an extensive heparan sulfate (HS) oligosaccharide library showed the spike of SARS-CoV-2 can bind HS in a length-and sequence-dependent manner. Hexa-and octasaccharides composed of IdoA2S-GlcNS6S repeating units were identified as optimal ligands. Surface plasma resonance (SPR) showed the SARS-CoV-2 spike protein binds with higher affinity to heparin (KD 55 nM) compared to the receptor binding domain (RBD, KD 1 µM) alone. An octasaccharide composed of IdoA2S-GlcNS6S could inhibit spike-heparin interaction with an IC50 of 38 nM. Our data supports a model in which the RBD of the spike of SARS-CoV-2 confers sequence specificity for HS expressed by target cells whereas an additional HS binding site in the S1/S2 proteolytic cleavage site enhances the avidity of binding. Collectively, our results highlight the potential of using HS oligosaccharides as a therapeutic agent by inhibiting SARS-CoV-2 binding to target cells. KEYWORDSSARS-CoV-2, coronavirus, heparan sulfate, heparin, spike glycoprotein, microarray, surface plasma resonances was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs, and thus, the reported synthetic principles will find broad applicability.

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.

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Sialylglycopeptide (SGP) is a complex bi-antennary N-glycan bearing a short peptide fragment that can be isolated from the yolk of hen eggs. This natural product has gained popularity as a starting material for the semi-synthesis of N-glycans. We have found that current isolation methods provide a glycopeptide contaminated with several related structures, one being a glycopeptide having a hexose directly attached to peptide backbone, most like through the hydroxyl containing side chain of the threonine moiety. Furthermore, current methods employ fresh egg yolks that need to be lyophilized and involve several tedious purification steps. Herein, we report a convenient method for the isolation of gram quantities of homogeneous SGP from commercially available egg yolk powder using solid/liquid extraction and HILIC-HPLC purification.

As a traditional soybean product with good quality and a healthy food with many functional components, tofu is increasingly consumed in people's daily life. Traditional tofu processing consists of numerous steps, including the soaking and grinding of soybean seeds, heating of the soybean slurry, filtering, and addition of coagulants, and others. The properties of soybean seeds, processing scale, soaking and heating conditions, type and concentration of coagulant, and other factors collectively impact the processing steps and the final tofu quality. The generation of whole soybean tofu with more nutritive value comparing with traditional tofu has been successfully reported by several studies. As one of the most important functional component, isoflavones and their presence in tofu are also influenced by the above-mentioned factors, which influence the nutritive value of tofu. Research investigating the influence of tofu processing conditions on the quality and isoflavone profiles of tofu are the subject of this review. Issues that should be further studied to investigate the influence of processing conditions on the quality and nutritive value of tofu are also introduced.

Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell lines, further confirming the in silico predictions.