Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition

The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the trimannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.     

Loading of the antigen-presenting protein CD1d with synthetic glycolipids

CD1 proteins present mammalian and microbial lipid and glycolipid antigens to different subsets of T cells. Few such antigens have been identified and the binding of these to CD1 molecules has mainly been studied by using responding T cells in cellular assays or recombinant solid-phase CD1 proteins. In the present study we use four different glycolipids, some of which contain tumor-associated carbohydrate antigens, to develop a procedure to easily detect binding of glycolipids to CD1 proteins on viable cells. Two of these glycolipids are novel glycoconjugates containing alpha-D-N-acetylgalactosamine (alpha-GalNAc) that were prepared by a combined solution and solid-phase approach. The key step, a Fischer glycosylation of 9-fluorenylmethoxycarbonylaminoethanol with GalNAc, furnished the alpha-glycoside 4 in 34% yield. Cells were incubated with glycolipids and stained with monoclonal antibodies specific for the carbohydrate part. The level of glycolipid bound to cells was then determined by flow cytometry with a secondary antibody labeled with fluorescein isothiocyanate. All four glycolipids were found to bind to CD1d but with different selectivity. The loading was dose dependent and could be inhibited by an established CD1d ligand, alpha-galactosylceramide. Through use of this procedure, glycolipids were selectively loaded onto CD1d expressed on professional antigen-presenting cells for future use as cellular vaccines. Moreover, the glycolipids described in this study represent novel CD1d-binding ligands that will be useful derivatives in the study of CD1d-dependent immune responses, for example, against tumors.     

The Uptake of Trehalose Glycolipids by Macrophages Is Independent of Mincle

Trehalose glycolipids play an important role in the pathogenesis of Mycobacterium tuberculosis and are used as adjuvants for vaccines; however, much still remains unanswered about the mechanisms through which these glycolipids exert their immunomodulatory potential. Recently, the macrophage‐inducible C‐type lectin Mincle was determined to be the receptor for trehalose glycolipids, yet the role played by Mincle in glycolipid uptake is unknown. Accordingly, we developed several fluorescent trehalose glycolipid reporter systems that can be used to study the uptake of soluble trehalose glycolipids and glycolipid‐coated particles by macrophages. Our studies revealed that, although Mincle is essential for the activation of macrophages by trehalose glycolipids, the receptor does not play a role in the uptake of these glycolipids or of glycolipid‐coated particles.     

Identification of Ustilago cynodontis as a new producer of glycolipid biosurfactants, mannosylerythritol lipids, based on ribosomal DNA sequences

Mannosylerythritol lipids (MELs) are one of the most promising glycolipid biosurfactants known because of their multifunctionality and biocompatibility. The search for novel producers of MELs was undertaken based on the analysis of ribosomal DNA sequences on basidiomycetous yeasts. The bermuda grass smut fungus Ustilago cynodontis NBRC 7530, which taxonomically relates to Pseudozyma shanxiensis known as a MEL-C producer, was found to accumulate glycolipids in the cultured medium. Under a shake flask culture with soybean oil, the amount of the glycolipids was 1.4 g/L for 7 days at 25 degrees C. As a result of the structural characterization, the main glycolipids was identified as 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol, and the major fatty acids were C(14) and C(16) ones. The glycolipid was highly hydrophilic MEL-C, and very similar to those produced by P. shanxiensis. The fungi of the genus Ustilago are thus likely to be potential producers of MELs as well as the yeasts of the genus Pseudozyma.     

Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M. smegmatis

Lipomannan and lipoarabinomannan are integral components of the mycobacterial cell wall. Earlier studies demonstrated that synthetic arabinan and arabinomannan glycolipids acted as inhibitors of mycobacterial growth, in addition to exhibiting inhibitory activities of mycobacterial biofilm. Herein, it is demonstrated that synthetic mannan glycolipids are better inhibitors of mycobacterial growth, whereas lipoarabinomannan has a higher inhibition efficiency to biofilm. Syntheses of mannan glycolipids with a graded number of mannan moieties and an arabinomannan glycolipid are conducted by chemical methods and subsequent mycobacterial growth and biofilm inhibition studies are conducted on Mycobacterium smegmatis. Growth inhibition of (73±3) % is observed with a mannose trisaccharide containing a glycolipid, whereas this glycolipid did not promote biofilm inhibition activity better than that of arabinomannan glycolipid. The antibiotic supplementation activities of glycolipids on growth and biofilm inhibitions are evaluated. Increases in growth and biofilm inhibitions are observed if the antibiotic is supplemented with glycolipids, which leads to a significant reduction of inhibition concentrations of the antibiotic.     

Conformationally constrained CCK8 analogues obtained from a rationally designed peptide library as ligands for cholecystokinin type B receptor

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.     

Pineapple Lectin AcmJRL Binds SARS-CoV-2 Spike Protein in a Carbohydrate-Dependent Fashion**

The highly glycosylated spike protein of SARS-CoV-2 is essential for infection and constitutes a prime target for antiviral agents and vaccines. The pineapple-derived jacalin-related lectin AcmJRL is present in the medication bromelain in significant quantities and has previously been described to bind mannosides. Here, we performed a large ligand screening of AcmJRL by glycan array analysis, quantified the interaction with carbohydrates and validated high-mannose glycans as preferred ligands. Because the SARS-CoV-2 spike protein was previously reported to carry a high proportion of high-mannose N-glycans, we tested the binding of AcmJRL to the recombinantly produced extraviral domain of spike protein. We could demonstrate that AcmJRL binds the spike protein with a low-micromolar K_D in a carbohydrate-dependent fashion.     

Unravelling the Carbohydrate‐Binding Preferences of the Carbohydrate‐Binding Modules of AMP‐Activated Protein Kinase

The β subunit of adenosine monophosphate (AMP)‐activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a carbohydrate‐binding module (CBM) that interacts with glycogen. Although the β1‐ and β2‐CBMs are structurally similar, with strictly conserved ligand‐contact residues, they show different carbohydrate affinities. β2‐CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into β2‐CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why β2‐CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for β2 and Thr for β1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant β2‐Thr101Δ/Val134Thr differs from that of β1‐CBM, and mutant β1‐Thr101ins/Thr134Val differs from that of β2‐CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild‐type β‐CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant β1‐CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of β2‐CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.     

Chemical Tools for Studying the Biological Function of Glycolipids

Glycolipids play an important role in many biological processes and to this end, synthetic chemists have developed a variety of new techniques and “chemical tools” that allow for the study of glycolipids in vitro and in vivo. The types of probes prepared include fluorescent, radio‐labelled, biotinylated and photoreactive ones, as well as others based on liposomes, microarrays and other supramolecular constructs—each of which offers its own advantages, as is discussed. A number of more specialised probes, such as metabolically engineered glycolipids and photopolymerisable glycolipids, have also been prepared in order to investigate various processes including substrate specificities and binding interactions. The purpose of this review is to present the key approaches that can be used for the development of glycolipid probes, organised according to application, and also to discuss the limitations of such strategies, which include the nontrivial task of ensuring that the probe does not adversely influence the biological activity of the parent compound. On the whole, it is exciting to see what can be achieved through the development of chemical probes as tools to study biological processes, and it is envisioned that the reader will be inspired by the large number of superb studies highlighted here and will be encouraged to undertake further work in this research area.     

Monolayers from synthetic glycolipids

Summary Synthetic glycolipids have been prepared by coupling of (a) aliphatic amines with lactone derivatives, or (b) fatty acids with amino derivatives of mono- and disaccharides via amide linkage. The aliphatic chain was varied with respect to the chain length and to its chemical composition (fluorination, functional end group). The influence of the glycolipid structure on the monolayer properties was investigated. Stable films are obtained with most of the products mainly due to strong interactions by hydrogen bonds in the subphase. Polymeric films may be produced by polycondensation in the subphase using particular crosslinking agents for the carbohydrate head group.     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

Role of glycolipids in breadmaking

In wheat flour used for breadmaking, glycolipids are essential “endogenous surfactants” (1). Bread volume and crumb structure of wheat flour‐based bakery products are influenced by glycolipids, without the decisive mode of action being conclusively known. Little information on how glycolipids from other plant sources perform as wheat flour improvers is available. In this paper we review our work done on revealing the baking performance of the four major glycolipid classes present in commercial lecithins (sterol glucosides (SG), acylated sterol glucosides (ASG), cerebrosides and digalactosyl diacylglycerides (DGDG). Our purpose is to better understand the role of these substances in the baking process. Our results suggest that there are two groups of glycolipids with excellent baking performance. Both groups were found to have an impact on the dough liquor rather than on the gluten‐starch matrix. The cerebrosides and DGDG most likely influenced the overall baking result by directly stabilizing the dough liquor/gas cell interface, while ASG and SG did this indirectly through a synergistic effect with a dough liquor constituent, most probably the endogenous flour lipids.     

Synthetic Arabinomannan Heptasaccharide Glycolipids Inhibit Biofilm Growth and Augment Isoniazid Effects in Mycobacterium smegmatis

Biofilm formation, involving attachment to an adherent surface, is a critical survival strategy of mycobacterial colonies in hostile environmental conditions. Here we report the synthesis of heptasaccharide glycolipids based on mannopyranoside units anchored on to a branched arabinofuranoside core. Two types of glycolipids—2,3‐branched and 2,5‐branched—were synthesized and evaluated for their efficacies in inhibiting biofilm growth by the non‐pathogenic mycobacterium variant Mycobacterium smegmatis. Biofilm formation was inhibited at a minimum biofilm growth inhibition concentration (MBIC) of 100 μg mL^?1 in the case of the 2,5‐branched heptasaccharide glycolipid. Further, we were able to ascertain that a combination of the drug isoniazid with the branched heptasaccharide glycolipid (50 μg mL^?1) potentiates the drug, making it three times more effective, with an improved MBIC of 30 μg mL^?1. These studies establish that synthetic glycolipids not only act as inhibitors of biofilm growth, but also provide a synergistic effect when combined with significantly lowered concentrations of isoniazid to disrupt the biofilm structures of the mycobacteria.     

Probe Molecules for Pulsed-Field-Gradient Diffusion Nuclear Magnetic Resonance Experiments on Micelles

Nuclear magnetic resonance (NMR) diffusion measurements of surfactants suffer from fast exchange of the surfactant between the micellar aggregate and bulk solution. Therefore, hydrophobic probe molecules are commonly used to directly measure aggregate diffusion under the assumption that the probe molecules remain solubilized inside the micelles. Aggregate size is then determined or estimated from the aggregate diffusion value. Unfortunately, the probe molecule also experiences rapid exchange between the aggregate and bulk, leading to inaccurate or unreasonable micelle diffusion values, a fact that has often been ignored in the literature. In this article, we present a systematic evaluation of probe molecules in cationic and anionic surfactants, obtained by measuring the diffusion of probe molecules with varying hydrophobicity. We find that an octanol–water partition coefficient of at least ~5 is required for correct measurements of aggregate diffusion in the systems studied. Notably, some commonly used probes have a partition coefficient much lower than five and are therefore not suitable for aggregate diffusion measurements. Consideration of these results will help researchers obtain accurate results for micelle sizing or partitioning studies with pulsed-field-gradient NMR.     

Comparison of the Reactivity of Carbohydrate Photoaffinity Probes with Different Photoreactive Groups

A judicious choice of photoreactive group is critical in successful photoaffinity labeling studies of small molecule–protein interactions. A set of carbohydrate‐based photoaffinity probes was prepared to compare the effects of three major photoreactive groups on the efficiency and selectivity of crosslinking a binding protein with low affinity. We showed that, despite the low crosslinking yield, the diazirine probe displayed the high ligand‐dependent reactivity consistent with the ideal mechanism of photoaffinity labeling. Moreover, we demonstrated that, among the three photoreactive groups, only the diazirine probe achieved highly selective crosslinking of a low‐affinity binding protein in cell lysate.     

Species-specific activity of glycolipid ligands for invariant NKT cells

The immunomodulatory glycolipid α-galactosylceramide (α-GalCer) binds to CD1d and exhibits potent activity as a ligand for invariant CD1d-restricted natural killer-like T cells (iNKT cells). Structural analogues of α-GalCer have been synthesised to determine which components are required for CD1d presentation and iNKT cell activation, however, to date the importance of the phytosphingosine 4-hydroxyl for iNKT cell activation has been disputed. To clarify this, we synthesised two 4-deoxy α-GalCer analogues (sphinganine and sphingosine) and investigated their ability to activate murine and human iNKT cells. Analysis revealed that the analogues possessed comparable activity to α-GalCer in stimulating murine iNKT cells, but were severely compromised in their ability to stimulate human iNKT cells. Here we determined that species-specific glycolipid activity was due to a lack of recognition of the analogues by the T-cell receptors on human iNKT cells rather than insufficient presentation of the analogues on human CD1d molecules. From these results we suggest that glycolipids developed for potent iNKT cell activity in humans should contain a phytosphingosine base.     

Reactivity Analysis of New Multivalent Electrophilic Probes for Affinity Labeling of Carbohydrate Binding Proteins

We have designed and synthesized six different multivalent electrophiles as carbohydrate affinity labeling probes. Evaluation of the reactivity of the electrophiles against peanut agglutinin (PNA) and Ricinus communis agglutinin (RCA) showed that p- and m-aryl sulfonyl fluoride are effective protein reactive groups that label carbohydrate binding lectins in a ligand-dependent fashion at a nanomolar probe concentration. Analysis of the selectivity of affinity labeling in the presence of excess BSA as a nonspecific protein indicated that m-arylsulfonyl fluoride is a more selective protein-reactive group, albeit with attenuated reactivity. Further analysis showed that the labeling efficiency of the multivalent electrophilic probes can be improved by employing reaction conditions involving 25 °C instead of typically employed 4 °C. Both isomers of arylsulfonyl fluoride groups together represent promising affinity labels for target identification studies that could serve as more efficient alternatives to photoreactive groups.     

Determination of Carbohydrate‐Binding Preferences of Human Galectins with Carbohydrate Microarrays

Galectins are a class of carbohydrate‐binding proteins named for their galactose‐binding preference and are involved in a host of processes ranging from homeostasis of organisms to immune responses. As a first step towards correlating the carbohydrate‐binding preferences of the different galectins with their biological functions, we determined carbohydrate recognition fine‐specificities of galectins with the aid of carbohydrate microarrays. A focused set of oligosaccharides considered relevant to galectins was prepared by chemical synthesis. Structure–activity relationships for galectin–sugar interactions were determined, and these helped in the establishment of redundant and specific galectin actions by comparison of binding preferences. Distinct glycosylations on the basic lactosyl motifs proved to be key to galectin binding regulation—and therefore galectin action—as either high‐affinity ligands are produced or binding is blocked. High‐affinity ligands such as the blood group antigens that presumably mediate particular functions were identified.     

Anti-Tumor Effect of Orally Administered Spinach Glycolipid Fraction on Implanted Cancer Cells, Colon-26, in Mice

We succeeded in purifying a major glycolipid fraction from a green vegetable, spinach. This fraction consists mainly of three glycolipids: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG). In a previous study, we found that the glycolipid fraction inhibited DNA polymerase activity, cancer cell growth and tumor growth with subcutaneous injection. We aimed to clarify oral administration of the glycolipid fraction, suppressing colon adenocarcinoma (colon-26) tumor growth in mice. A tumor graft study showed that oral administration of 20 mg/kg glycolipid fraction for 2 weeks induced a 56.1% decrease in the solid tumor volume (P < 0.05) without any side-effects, such as loss of body weight or major organ failure, in mice. The glycolipid fraction induced the suppression of colon-26 tumor growth with inhibition of angiogenesis and the expression of cell proliferation marker proteins such as Ki-67, proliferating cell nuclear antigen (PCNA), and Cyclin E in the tumor tissue. These results suggest that the orally administered glycolipid fraction from spinach could suppress colon tumor growth in mice by inhibiting the activities of neovascularization and cancer cellular proliferation in tumor tissue.     

High Resolution Crystal Structures of the Cerebratulus lacteus Mini-Hb in the Unligated and Carbomonoxy States

The nerve tissue mini-hemoglobin from Cerebratulus lacteus (CerHb) displays an essential globin fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O(2) dissociation and ultra-high O(2) affinity. However, CerHb affinity for O(2) is similar to that of mammalian myoglobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O(2) ligand, the overall binding schemes for CO and O(2) are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.