Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution

　

Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution | PNAS

Qinghong Zeng * , † , Martijn A. Langereis † , ‡ , Arno L. W. van Vliet ‡ , Eric G. Huizinga * , § , and Raoul J. de Groot ‡ , §
*Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Sciences, and
‡Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands
　

"Both, the influenza C (INF-C) virus haemagglutinin esterase fusion and bovine coronavirus (BCoV) haemagglutinin esterase surface glycoproteins exhibit a lectin binding capability and a receptor-destroying 9-O-acetyl esterase activity that recognise 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2)-containing glycans. "

ASTRACT
The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer–monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design.

冠状病毒血凝素酯酶的结构为研究冠状病毒和流感病毒的进化提供了思路

ASTRACT

血凝素-酯酶(hemagglutinin-esterases HEs)是一类病毒包膜糖蛋白，它通过扮演凝集素和受体破坏酶(RDEs)的角色，介导与o -乙酰化唾液酸的可逆连接。相关的HEs发生在C型流感病毒、toro-病毒和冠状病毒中，这显然是最近的基因横向转移事件的结果。在这里，我们报告了冠状病毒(CoV) HE及其受体的晶体结构。我们证明CoV HE起源于一种类似于流感c的HE融合蛋白(HEF)。在这个过程中，他从一个三聚体转变为一个二聚体，而融合域的残余被用来建立新的单体-单体接触。虽然RNA -乙酰甾醇酶域的结构设计没有改变，但是HE受体结合域经历了重构，以至于配体现在以相反的方向结合。这是令人惊讶的，因为HEF位点的结构在甲型流感HA中被保存了更大的进化距离，尽管受体特异性和广泛的抗原变异发生了转换。显然，HA和HEF受到比HE更严格的选择约束，限制了它们对其他绑定点拓扑的探索。我们属性浸他受体结合部位的可塑性进化灵活性赋予的功能冗余他和同伴之间突起蛋白S. 我们的发现提供了独特的见解独立蛋白质的结构和功能影响进化后interviral基因交换和开放的潜在途径广谱抗病毒药物设计。

sialic acidx-ray crystallographyglycoproteinlectinnidovirus

Many different respiratory and enteric viruses initiate infection by binding to sialic acids. These nine-carbon amino sugars are common terminal residues of glycoconjugates and occur in a wide variety, which arises from differential modification of the parental molecule, 5-N-acetyl neuraminic acid (Neu5Ac), and from differences in glycosidic linkage (1). Although viruses may attain host and cell specificity by being selective for particular sialic acid subtypes (1–4), even these receptors may still occur abundantly on off-target host cells (including the infected cell), on viral progeny, and on soluble and insoluble molecules in the extracellular environment. To prevent irreversible binding to “decoy receptors” and consequent loss of infectivity, orthomyxo-, paramyxo-, and coronaviruses encode virion-associated receptor-destroying enzymes (RDEs) (5–7). In turn, RDEs make prime targets for antiviral intervention, as exemplified by the influenza A virus neuraminidase (8–10).

The coronaviruses are a group of enveloped (+)strand RNA viruses that comprises several important pathogens of human clinical and veterinary importance, including severe acute respiratory syndrome (SARS)-coronavirus, human respiratory coronavirus (HCoV) OC43 and its proposed zoonotic ancestor (11), bovine coronavirus (BCoV). HCoV-OC43 and BCoV use 9-O-acetylated sialic acids as receptor and correspondingly possess sialate-9-O-acetylesterases as RDE (12, 13). Enzymatic activity is associated with the hemagglutinin-esterase (HE), a homodimeric class I membrane protein (for a review, see ref. 7). Although among coronaviruses, HE is exclusive to members of phylogenetic subgroup 2a (7), closely related proteins do occur in toroviruses (14–16) and in orthomyxoviruses of mammals (17) and fish (18). Best studied is the influenza C virus HE fusion protein (HEF), a homotrimeric multifunctional spike protein that mediates virion attachment, receptor destruction, and membrane fusion (19–22).

The presence of homologous HE genes in the genomes of viruses from three distinct taxa is generally ascribed to RNA recombination and would have required two lateral gene transfer events at the minimum (7, 14, 23). The source of the HE module and the direction of gene flow remain to be established, however (7). Moreover, the influenza, toro-, and coronavirus HEs followed independent evolutionary trajectories, the functional and structural consequences of which are poorly understood. Unlike HEF, CoV HEs lack membrane fusion activity and, in the virion, they are accessory to the spike protein S, a receptor-binding fusion protein. Furthermore, although CoV HEs do bind to sialic acid, their role in virion attachment is subject to debate (7). Reportedly, CoV HEs serve primarily as RDE (24), but this view is at variance with observations that esterase-deficient HE promotes viral spread in vivo (25). Intriguingly, there is no obvious sequence homology between the HEF receptor-binding site and corresponding regions in HE, raising the question of whether CoV HEs possess a separate receptor-binding site or mediate sialic acid-binding and catalysis through a single site in the esterase pocket (7).

To further our understanding of the function and evolution of coronavirus HEs, we expressed BCoV HE and solved its structure, free and in complex with its receptor.

Results and Discussion
Expression, Purification, and Biochemical Analysis of CoV HE.
We produced the ectodomain of BCoV HE (residues 19–388) in enzymatically active (HE) and inactive form (HE0, containing an active site Ser40-to-Ala substitution) as Fc-fusion proteins by transiently transfecting N-acetylglucosaminyltransferase-deficient HEK293S cells with expression plasmids encoding the respective molecules. Soluble CoV HE was purified by protein A-affinity chromatography of the Fc chimeras followed by on-the-bead thrombin digestion. The proteins were tested in a series of assays to confirm that biological functions had been retained also as an indication for proper folding. Indeed, in cleavage assays with a synthetic di-O-acetylated Sia (5-N-acetyl-4,9-di-O-acetylneuraminic acid α-methylglycoside, αNeu4,5,9Ac32Me) as substrate, HE-Fc displayed sialate-9-O-acetylesterase activity (Fig. 1 A). In addition, HE-Fc, but not its esterase-deficient derivative HE0-Fc, specifically destroyed BCoV receptors on rat erythrocytes and on Madin–Darby bovine kidney (MDBK) cells (Fig. 1 B and C). Moreover, HE-Fc and HE0-Fc retained lectin activity as demonstrated by hemagglutination assay (Figs. 1 C and 4 E) and by Sia-specific immunofluorescence staining of tissue culture cells (Fig. 1 D).

Fig. 1.
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Fig. 1.
BCoV HE-Fc chimeras retain esterase and lectin activity. (A) HE-Fc displays sialate-9-O-acetylesterase activity. Purified HE-Fc was assayed for substrate specificity as described (16) with the synthetic di-O-acetylated sialic acid analogue αNeu4,5,9Ac32Me as substrate. Graphs show total ion current gas chromatograms. Sialic acids were identified by mass spectrometry: αNeu4,5,9Ac32Me (peak 1), αNeu4,5Ac22Me (peak 2); peaks 1′ and 3 represent nonsialic acid compounds. (B) HE-Fc, but not esterase-deficient HE0-Fc, destroys BCoV receptors on the surface of tissue culture cells. MDBK cell monolayers were mock-treated, treated with neuraminidase (NA), or with the HE-Fc chimeras and subsequently infected with BCoV. Infected cells were visualized by an in situ acetylesterase staining. (C) HE-Fc destroys BCoV receptors on rat erythrocytes. Erythrocytes were mock-treated or treated with HE-Fc and subsequently subjected to hemagglutination assay with two-fold dilutions of BCoV (+) or with PBS (−). (D) HE0-Fc lectin activity allows immunohistochemical detection of 9-O-acetylated sialic acid on monolayers of polarized MDBK cells (green). Cell contacts were visualized by staining for PAN-Cadherin (red) and nuclei by staining with HOECHST 33258 (blue).

Structure Determination and Overall Structure.
Crystals of free HE and of a complex of HE0 with αNeu4,5,9Ac32Me diffracted to 2.1 and 1.8 Å resolution, respectively. The structures were solved by molecular replacement by using influenza C HEF residues 35–410 as search model (for detailed crystallographic statistics, see Table 1).

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Table 1.
Data collection and refinement statistics

The data reveal that the CoV HE monomer is composed of three modules, each with an equivalent in influenza C HEF subunit 1 (HEF1): a receptor-binding domain (R), an acetylesterase domain (E), and a membrane-proximal domain (MP; Fig. 2 A–C). There is no equivalent of HEF subunit 2 (HEF2), which in HEF comprises the bulk of the fusion domain (F) (Fig. 2 B and C). Bound to the R domain is a well defined ligand molecule [supporting information (SI) Fig. S1] providing first and direct evidence for the presence of a genuine receptor-binding site. This observation unambiguously defines BCoV HE as a receptor-binding protein per se.

Fig. 2.
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Fig. 2.
Structure of HE and comparison to HEF. (A) Ribbon representation of the BCoV HE dimer. One HE monomer is colored gray, the other by domain: lectin domain (R, blue) with bound αNeu4,5,9Ac32Me (cyan sticks) and potassium ion (magenta sphere); esterase domain (E, green) with Ser-His-Asp active site triad (magenta sticks); membrane-proximal domain (MP, red). (B) Structure of the influenza C HEF trimer. One monomer is colored by domain with domains R and E color-coded as in a; the fusion domain F is shown in red. Remaining monomers are shown in pink or gray. (C) Linear order of the sequence segments in HE and HEF color-coded by domains as in a and b. Gray segments indicate the transmembrane domain in HE and the fusion peptide in HEF. The arrowhead indicates the HEF cleavage site. (D) Top views showing the arrangement of the lectin domains in the BCoV HE homodimer (Left) and the influenza C HEF homotrimer (Right). The four-stranded β-sheets forming a continuous eight-stranded β-sheet in the HE dimer are emphasized by darker coloring.

In the virion, CoV HE forms dimers that are disulphide-linked through Cys385 near the C-terminal membrane anchor (7). This bond, although present in HE crystals (Fig. S2), was not visible in electron density maps because of disorder of residues 377–388. The biological dimer could, however, be identified uniquely from the proximity of C termini and size of interaction surface. It consists of identical molecules, related by twofold crystallographic symmetry, with an interface comprised of two major protein contact regions (CRs). CR1, associated with 952 Å2 buried solvent-accessible surface, involves a continuous eight-stranded β-sheet bridging the two R domains (Fig. 2 D). CR2 buries 1,182 Å2 solvent-accessible surface and involves MP.

Origin and Evolution of CoV HE.
The structure of MP provides a decisive clue to the origin of CoV HE. As shown in Fig. 3, MP is strikingly similar to the F1/F2 globular region of HEF1 (22) and the F′ subdomain in the influenza A virus HA (26). In the native metastable forms of HEF and HA, these subdomains pack against a heptad-repeat loop that, as a prelude to membrane fusion, refolds into an α-helix at endosomal pH (26). HEF and HA are evolutionarily related but diverged long ago; their fusion domains are similar in structure, as are their R domains, but overall sequence identity has dwindled to only 12% (22). In contrast, HEF1 and CoV HE share 30% identity, indicative of a more recent split-off (7, 23). The presence in HE of an element that already must have been part of a fusion protein ancestral to HA and HEF (22) thus indicates that CoV HE originates from an HEF-like fusion protein and strongly argues against evolutionary models in which orthomyxo- and coronaviruses independently acquired an HE module solely comprised of R and E (7). In the simplest scenario (23), a group 2a CoV progenitor obtained through recombination the 5′-end of an HEF gene, including the coding sequence for the fusion peptide that came to serve as transmembrane domain (Fig. 2 C). Without the HEF2 helical subdomain to direct trimerization, fortuitous interactions between transmembrane and/or F2 segments might have caused the HE protein to assemble into dimers instead; of note, the face of MP involved in dimer formation is equivalent to that of HEF segment F2 contacting the heptad repeat loop (Fig. 3). A four-stranded β-sheet in the R domain that remains unpaired in HEF and HA trimers (22) may have been remodeled subsequently to create a second, membrane-distal intermonomer contact to increase dimer stability (Fig. 2 D).

Fig. 3.
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Fig. 3.
MP is a remnant of a fusion domain. (A) Ribbon representations of MP in the HE dimer and of the fusion domains of the HEF and HA monomers. MP and corresponding subdomains in HEF and HA are shown in red; the remainder of the HEF and HA fusion domains are shown in gray. A conserved disulfide bond is shown in yellow stick representation. (B) Superposition of HE MP (red) and corresponding HEF (magenta) and HA subdomains (cyan). Sequence identity and rms deviations on Cα positions for HE-HEF and HE-HA are 33% and 1.9 Å, and 22% and 2.5 Å, respectively.

The Esterase Domain.
The E domain of CoV HE, harboring the RDE activity, has an SGNH-hydrolase fold (27) highly similar to that of HEF (1.4-Å rms deviation on Cα positions at 34% sequence identity). In fact, the active-site architecture of the HEF sialate-9-O-acetylesterase is fully conserved in BCoV HE with Ser40, His329, and Asp326 as catalytic triad and with the side chain of Asn104 and the NH groups of Ser40 and Gly75 forming an oxyanion hole (Fig. 4 A). HE crystals soaked with 9-O-acetylated sialic acid did not contain this substrate in the enzymatic pocket. Instead, the oxyanion hole was occupied by an acetate ion, i.e., one of the catalytic products. The HEF and HE E domains do differ in size and structure of an extended surface loop (residues 47–54) that shields the catalytic pocket and that may prevent antibodies from accessing the functionally important esterase site (Fig. 4 A). This loop is highly variable even among coronavirus HEs (7), tentatively identifying it as a site of antigenic variation.

Fig. 4.
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Fig. 4.
HE and HEF display conserved enzymatic but divergent receptor-binding sites. (A) Superposition of active site residues of HE (green) and HEF (gray). An acetate ion occupying the oxyanion hole in HE is shown in stick representation (carbon, cyan; oxygen, red). Catalytic triad and oxyanion hole hydrogen bonds are indicated by dashed black lines. Also shown is an adjacent loop that is highly variable also among CoVs. (B) Comparison of the receptor-binding regions of HE, HEF, and HA. Bound ligands, αNeu4,5,9Ac32Me in HE, αNeu5,9Ac22Me in HEF, and αNeu5Ac2Me in HA are shown in stick representation (carbon, cyan; nitrogen, blue; oxygen, red). Proteins are depicted in similar orientations obtained by superposition of their conserved central cores. (C) Ligand bound to the HE receptor-binding site in stick representation. Water molecules are shown as red spheres, a potassium ion as magenta sphere. Hydrogen bonds with receptor are shown as dashed black lines. (D) Surface representation of the HE receptor-binding site revealing two pockets accommodating the 9-O- and 5-N-acetyl groups of the receptor. (E) The effect of Ala substitutions on receptor binding. Relative binding affinity of wild-type HE and its derivatives was assessed by hemagglutination assay with rat erythrocytes and twofold serial dilutions of each of the HE-Fc chimeras (1,000 to 0.5 ng of per well, arrow).

A Receptor-Binding Site in CoV HE.
The R domain mediates receptor recognition (Fig. 1 D). Although its core, an eight-stranded “Swiss roll,” is structurally well conserved and closely resembles that of HEF and HA (Fig. S3), its receptor-binding region is unique. This region is shaped by five variable loops, four grafted on the conserved core and one extending from the E domain. Differences between HE and HEF in loop size and conformation result in dramatically different binding sites that engage 9-O-acetylated sialic acid in nearly opposite orientations. Compared with the HEF–receptor complex, the receptor analogue bound to HE is rotated by 147° and shifted by 4 Å (Figs. 2 D and 4 B). Conversely, HEF and HA are similar with respect to receptor orientation and binding site topology (22) (Figs. 4 B and Fig. S3). In HE, a β-hairpin, replacing an α-helix in HEF and HA interacts with the receptor through hydrogen bonds from main chain atoms of Leu212 and Asn214 to the 5-N-acetyl- and carboxylate groups, respectively, and from the side chain of Ser213 to O-8 (Fig. 4 C). The α-2-O-methyl group of the receptor analogue points into the solvent, of relevance, because at this position, the natural ligand would be linked to the penultimate residue of the sugar chain. The 5-N-acetyl group fits into a pocket, its methyl interacting with a hydrophobic patch formed mainly by Phe211 (Fig. 4 D). The 9-O-acetyl methyl of the receptor analogue docks into an adjacent hydrophobic pocket, formed by Tyr184, Phe211, Leu266, and Leu267 (Fig. 4 C and D), with the latter two residues in a loop stabilized by a potassium ion coordinated by Asp220, Ser221, Gln222, Ser263, Glu265, Leu267, and one water (Fig. S4). Given that the 9-O-acetyl moiety is a prerequisite for receptor binding and serves as a switch for virion attachment and release, this hydrophobic pocket must be key to ligand recognition. Indeed, Ala substitution of Phe211 or of Leu266 and Leu267 in tandem abrogated receptor binding, whereas substitution of Tyr184 decreased affinity significantly (Fig. 4 E).

Plasticity of the CoV HE Receptor-Binding Site.
HEF also binds 9-O-acetylated sialic acid by contacting the 5-N-acetyl group through a hydrophobic patch and by burying the 9-O-acetyl in a nonpolar pocket (22). Therefore, despite the considerable differences in receptor-binding site architecture, HEF and HE use similar strategies to discriminate functional receptors from non-O-acetylated sialic acids. It is, however, surprising that the HE receptor-binding site has changed to this extent at all. Over a much larger evolutionary distance, the architecture of the HEF receptor-binding site has been preserved in HA, despite differences in receptor specificity (Neu5,9Ac2 vs. α2,3- or α2,6-linked Neu5Ac, respectively) and throughout antigenic variation (22, 26, 28) (Fig. 4 B). Unlike HA and HEF, however, CoV HE acts in concert with another receptor-binding protein, S, a fusion protein essential for entry, and whereas HE-mediated virion attachment may be advantageous (25), it is not vital (16, 25, 29). We propose that redundancy in function between S and HE (24) afforded CoVs leeway to explore HE-binding site topologies and even to shift in coreceptor usage from 9-O- to 4-O-acetylated sialic acids in one branch of murine coronaviruses (16, 30–32).

The plasticity of the CoV HE receptor-binding site contrasts with the strict conservation of the E domain and the esterase catalytic site. From a pragmatic point of view, this may potentially allow the development of broad-spectrum antivirals targeting both orthomyxo- and coronavirus sialate-O-acetylesterases.

Materials and Methods
Construction of Expression Plasmids.
A synthetic codon-optimized sequence for the HE ectodomain of BCoV strain Mebus (amino acid residues 19–388) was cloned in a derivative of expression plasmid S1-Ig (33). The resulting construct, pCD5-BCoVHE-T-Fc, encodes a chimeric HE protein provided with an N-terminal CD5 signal peptide and at its C terminus, separated by a thrombin cleavage site, the Fc domain of human IgG1. To allow expression of an enzymatically inactive HE-Fc chimera (HE0-Fc), we generated a derivative of pCD5-BCoVHE-T-Fc in which the codon for the esterase catalytic residue Ser40 was substituted by Ala by site directed mutagenesis using the QuikChange XL II kit (Stratagene). Ala substitutions of key residues in the HE receptor-binding site were made likewise.

Protein Expression and Purification.
HEK293S cells lacking N-acetylglucosaminyl-transferase I (GnTI) activity (34) were transiently transfected with the expression plasmids using polyethyleneimine. At 18 h after transfection, the transfection mixture was replaced by 293 SFM II expression medium (Invitrogen), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter), penicillin (100 units/ml), streptomycin (100 μg/ml), glutaMAX (Gibco), and 1.5% DMSO. Tissue culture supernatants were harvested 5–6 days after transfection, and HE-Fc and HE0-Fc were purified by protein A-affinity chromatography. For crystallography, the HE and HE0 ectodomains were cleaved off by on-the-bead thrombin digestion, concentrated to 10–15 mg/ml, and dialyzed against crystallization buffer (Tris·HCl, pH 8.2; 50 mM NaCl).

Biological and Biochemical Characterization of HE-Fc Chimeras.
Molecular weights of the proteins were determined by SDS/PAGE (under reducing and nonreducing conditions) and by gel filtration chromatography. O-acetylesterase activity was routinely measured by pNPA assay (19). Enzymatic de-O-acetylation of αNeu4,5,9Ac32Me was analyzed by GC-MS (16, 35). To assess esterase activity of the expression products toward cell-bound 9-O-acetylated sialic acids, MDBK cell monolayers in 16-mm diameter wells were treated with 20 μg/ml HE-Fc or HE0-Fc in PBS for 2 h at 37°C and subsequently infected with BCoV strain Mebus. Monolayers, mock-treated or treated with Vibrio cholerea neuraminidase (NA; 0.6 units/ml), were included as controls. Infected cells were visualized by staining using an in situ acetylesterase assay (36). Receptor-destroying activity toward 9-O-acetylated sialic acids on rat erythrocytes was assessed by incubating the cells with 20 μg/ml HE-Fc in PBS for 2.5 h at 37°C, before hemagglutination assay with twofold serial dilutions of BCoV. In situ detection of 9-O-acetylated sialic acids in cultured cells was demonstrated by performing a standard indirect immunofluorescence assay. Briefly, monolayers of polarized MDBK cells, grown on glass coverslips, were fixed with 2% paraformaldehyde and successively incubated with ≈25 μg/ml HE0-Fc in PBS, 0.05% Tween20 and with FITC-conjugated anti-human IgG1 (Nordic, 1:100) as secondary antibody. The samples were mounted on glass slides in FluorSave (Calbiochem) and examined by confocal fluorescence microscopy (Leica TCS SP2).

Crystallization.
Crystallization conditions were screened by the sitting-drop vapor diffusion method using a Honeybee 961 (Genomic Solutions). Drops were set up with 0.1 μl of HE protein solution in 10 mM Tris·HCl, pH 8.0, and 0 l reservoir solution. Diffracting crystals were obtained from JCSG solution D12 [0.04 M KH2PO4, 16% (wt/vol) PEG 8000, 20% (wt/vol) glycerol] (JCSG Technologies) and PEGIon-lite solution 89 [0.1 M KH2PO4, 10% (wt/vol) PEG 3350] (Hampton Research) at 18°C. Crystals for diffraction experiments were grown with the hanging-drop vapor diffusion method set up by hand with 0.8 μl of reservoir solution and 0.8 μl of protein solution at 18°C. Under both crystallization conditions, crystals of wild-type HE and HE0 grew within 2 weeks with the shape of hexagonal bipyramids to a size of ≈0.25 × 0.16 × 0.16 mm. Their space group is P6522. Crystals of wild-type HE grown at 0.1 M KH2PO4, 10% (wt/vol) PEG 3350 were flash-frozen in liquid nitrogen using reservoir solution with 22.5% (wt/vol) glycerol as the cryoprotectant.

Data Collection and Structure Solution.
To determine the HE structure in complex with its receptor analog Neu4,5,9Ac32Me (16), HE0 was used to prevent substrate degradation during soaking. Crystals obtained from 0.1 M KH2PO4, 10% (wt/vol) PEG 3350 cracked during ligand soaking and the flash-freezing procedure. Therefore, we used for substrate-soaking crystals of the mutant grown from 0.04 M KH2PO4, 16% (wt/vol) PEG 8000, 20% (wt/vol) glycerol, which did not require addition of cryoprotectant. Soaking was performed by adding 2 μl of reservoir solution with 10 mM Neu4,5,9Ac32Me directly into the margin of the drop with crystals, resulting in a final substrate concentration of ≈7 mM. Crystals were flash-frozen after 5–10 min.

Diffraction data of native and ligand-soaked crystals of BCoV HE were collected at European Synchrotron Radiation Facility station ID14-3 and ID 23-1, respectively. Diffraction data were processed by using XDS (37) and scaled using SCALA from the CCP4 suite (38). Molecular replacement was performed by using PHASER with Influenza C HEF residues 35–410 as template [Protein Data Bank (PDB) ID code 1FLC]. Density modification was carried out by using RESOLVE (PHENIX package) (39) and CNS (40). Approximately 280 residues were built automatically by ARP/WARP (41); the remaining residues were built manually with Coot (42). Refinement was carried out by using REFMAC (43); water molecules were added by using ARP/WARP. Molecular graphics were generated with PYMOL (http://pymol.sourceforge.net).

The structure of HE0 in complex with Neu4,5,9Ac32Me was solved by molecular replacement by using the wild-type HE structure as template. The structure was refined as described for wild-type HE. The library file for refinement of the ligand in REFMAC was made by adding chemical and geometric descriptions of the 4- and 9-O-acetyl groups to that of sialic acid (SIA) in the monomer library of the CCP4 suite by using the program SKETCHER (CCP4 Suite).

Acknowledgments
We thank Peter Rosenthal (MRC, Cambridge, UK) for providing coordinates of the HEF-inhibitor complex. We also thank P. Gros, J. de Groot-Mijnes, B. Bosch, P. Rottier, and P. Rosenthal for advice and for reading of this manuscript; G. Gerwig and J. Kamerling for help in preparing the sialic acid analogue; and P. Reeves (University of Essex, Colchester, UK) and M. Farzan (Harvard Medical School, MA) for sharing HEK293S GnTI(−) cells and expression plasmids, respectively. We acknowledge the European Synchrotron Radiation Facility for providing beamline facilities and the beamline scientists at ID-14-EH3 and ID23-1 for help with data collection. This work was supported by “Pionier” program and ECHO grants of the Council for Chemical Sciences of The Netherlands Organization for Scientific Research (NWO-CW) and by a grant of the Mizutani Foundation for Glycoscience.

Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution | PNAS
https://www.pnas.org/content/105/26/9065

　

J Virol. 1999 May; 73(5): 3737–3743.

Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus

Alfred Klausegger,1 Birgit Strobl,1 Gerhard Regl,1 Alexandra Kaser,1 Willem Luytjes,2 and Reinhard Vlasak1,*
Institute of Molecular Biology, Austrian Academy of Sciences, A-5020 Salzburg, Austria,1 and University of Leiden, Institute of Microbiology, Department of Virology, 2300 AH Leiden, The Netherlands2

ABSTRACT
We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse α1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC104150/

　

　

Proteolytic digestion of erythrocytes with ficin, bromelain or V-8 protease inhibited virus binding almost completely, suggesting that the erythrocyte receptor for influenza C virus is a glycoprotein.
Attachment of influenza C virus to human erythrocytes.
www.ncbi.nlm.nih.gov/pubmed/3049938
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Attachment of influenza C virus to human erythrocytes.
https://www.ncbi.nlm.nih.gov/pubmed/3049938

It was also found that treatment of erythrocytes with monoclonal antibody to the M or N blood group antigen greatly inhibited virus binding to the cells. These results, taken together, suggest that most influenza C virus receptors on human erythrocytes, if not all, reside on glycophorin A which is known to possess the M or N blood group activity.

Cited by: 20
Publish Year: 1988

　

PHYTONUTRIENTS IN LICORICE ROOT
Triterpenoid saponins (particularly glycyrrhizin or glycyrrhizic acid) are the major active ingredients of glabra. The concentration of glycyrrhizin in the root is about 2-6% depending on the method of assay. This phytonutrient shows significant expectorant, demulcent, anti-inflammatory and anti-allergic properties, making licorice root a popular herbal treatment of asthma. Glycyrrhizin is also what gives licorice its sweetness, being a substance roughly 50 times sweeter than usual sugar. Glycyrrhizin has recently been proved to reduce dental plaque formation, thus preventing caries.

Glycyrrhetinic acid, a pentacyclic triterpenoid derivative, is another compound with mild expectorant properties. Its main feature, however, is its anti-ulcer action.
Flavonoids (flavanones, chalcones, and isoflavonoids), present in a concentration of about 1-1.5%. Flavonoids are often reported to have antioxidant, anti-inflammatory, and antibacterial properties.

Phytosterols have a long-time proven cholesterol-lowering (anti-atherogenic) effect. Some studies also indicate these compounds may increase estrogen levels to a certain extent.

Helicobacter pylori: Helicobacter pylori is a germ believed to be one of the major causes of peptic ulcer. Glabridin, one of the main flavonoids of licorice, is able to effectively inhibit this bacterium, thus making licorice root a powerful remedy for bacterial gastritis and peptic ulcer.

三萜皂苷(尤其是甘草酸或甘草酸)是山楂的主要活性成分。根据测定方法的不同，根中甘草酸的浓度约为2-6%。这种植物营养素具有显著的祛痰、镇痛、抗炎和抗过敏的特性，使甘草根成为治疗哮喘的常用草药。甘草酸也是甘草的甜味来源，这种物质的甜度大约是普通糖的50倍。甘草酸最近被证明可以减少牙菌斑的形成，从而预防龋齿。

甘草次酸是一种五环三萜类衍生物，是另一种具有温和祛痰作用的化合物。然而，它的主要特点是抗溃疡作用。

类黄酮(黄酮、查耳酮和异黄酮类)，浓度约为1-1.5%。黄酮类化合物常被报道具有抗氧化、抗炎和抗菌的特性。

幽门螺杆菌:幽门螺杆菌是一种细菌，被认为是造成消化性溃疡的主要原因之一。甘草素是甘草中主要的黄酮类化合物之一，能有效地抑制这种细菌，使甘草根成为治疗细菌性胃炎和消化性溃疡的有力药物。

　

Licorice Root Benefits Are Due To Its Potent Triple-Effect Expectorant
https://juicing-for-health.com/licorice-root

　

glycyrrhiza glabra
Glycyrrhiza glabra Common name: Licorice, Liquorice

Family: Fabaceae

Part used: Dried root & rhizome

Constituents: Triterpene saponins (glycyrrhizin, glycyrrhizic & glycyrrhetinic acid), flavonoids, iso-flavonoids, phytosterols, polysaccharides (glucans), coumarins, volatile oils, amino acids, gum, lignans, starch, sugars

Actions: General tonic, anti-depressant, adrenal restorative, adrenal tonic, anti-tumor, anti-viral, anti-inflammatory, anti-tussive, mucoprotector, expectorant, detoxifier, demulcent, hepatoprotector, anti-ulcer, mild laxative, aldosterone-like action, anti-spasmodic

Medical uses: Is specific for the treatment of peptic ulcers and Addison’s disease. Also used for adrenal insufficiency, corticosteroid withdrawal, hypoglycemia and protection against cataracts in diabetes. Inflammatory conditions also benefit from it’s use such as allergies and autoimmune disease, lyme disease, and protects against carcinogens. It will soothe gastrointestinal inflammation and irritations such as GERD, gastritis, IBD, and ulcers. For rheumatism, muscle spasm & cramps, and joint inflammation it can be used both internally and topically.

Pharmacology:

Triterpene saponins (glycyrrhizin, glycyrrhizic acid) antagonize effect of estrogen, inhibit growth of HIV & DNA/RNA viruses, and are anti-inflammatory.
Glycyrrhetinic acid is an aglycone similar to natural corticosteroids which have an adrenocorticomimetic action. Is also anti-viral, hepatoprotective, inhibits EBV activation and is anti-inflammatory and anti-tumor.
Flavonoids and isoflavonoids are anti-microbial and estrogenic.
Phytosterols have an estrogenic action.
Polysaccharides (glucans) are demulcent.

DGL (deglycyrrhinized licorice) is a preparation where most of the GL has been removed. It inhibits the formation of ulcers, reduces gastric secretions, and protects gastric mucosa against damage.
Pharmacy: Powdered root: 1-4g TID. Decoction: 1 tsp/cup, simmer 15 min, TID. Tincture: (1:1, 20%) 2-6ml QD.

Contraindications: Use with caution in anemia, hypertension, cardiovascular disorders, edema associated with heart failure, liver problems, kidney insufficiency, hypokalemia, hypothyroidism, fibrocystic breasts, male infertility or erectile dysfunction.

Toxicity: Minimal adverse effects if intake is less than 10mg/day. Long term use may reduce thyroid function and basal metabolic rate. High doses of long term use may cause hypokalemia, symptoms of hyperaldosteronism and amenorrhea.

Interactions: With diuretics, cardiac glycosides, corticosteroids, MAOIs, blood pressure medications, spironolactone, hormonal therapies, laxatives and K+ depleting medications.

甘草glabra

甘草俗称:甘草、甘草

家庭:蝶形花科

部分使用:干燥的根和根茎

主要成份:三萜皂苷(甘草酸、甘草酸、甘草次酸)、黄酮类、异黄酮类、植物甾醇、多糖(葡聚糖)、香豆素、挥发油、氨基酸、树胶、木脂素、淀粉、糖类

作用:一般滋补、抗抑郁、肾上腺恢复、肾上腺强化剂、抗肿瘤、抗病毒、抗炎、止咳、粘膜保护剂、祛痰、解毒、镇痛、保肝、抗溃疡、轻泻、醛固酮类作用、抗痉挛

医疗用途:特别用于治疗消化性溃疡和艾迪生氏病。也用于肾上腺机能不全，皮质类固醇停药，低血糖和预防糖尿病白内障。炎症条件也受益于它的使用，如过敏和自身免疫性疾病，莱姆病，并防止致癌物。它可以缓解胃肠道炎症和刺激，如GERD，胃炎，IBD，溃疡。对于风湿病，肌肉痉挛和抽筋，和关节炎症，它可以用于内部和局部。

药理学:

三萜皂苷(甘草酸、甘草酸)具有拮抗雌激素作用，抑制HIV病毒和DNA/RNA病毒的生长，具有抗炎作用。

甘草次酸是一种与天然糖皮质激素相似的苷元，具有促肾上腺皮质激素的作用。同时具有抗病毒、保肝、抑制EBV活化、抗炎、抗肿瘤的作用。

黄酮类化合物和异黄酮类化合物具有抗微生物和雌激素作用。

植物甾醇具有雌激素作用。

多糖(葡聚糖)是破乳的。

DGL(脱糖甘草糖)是一种大部分GL被去除的制剂。它能抑制溃疡的形成，减少胃分泌物，保护胃粘膜免受损害。

粉末根:1-4g TID。煎煮:1茶匙/杯，小火煮15分钟，TID。酊剂:(1:1,20%)每日2-6ml。

禁忌症:贫血、高血压、心血管疾病、心力衰竭引起的水肿、肝脏问题、肾功能不全、低钾血症、甲状腺功能减退、纤维囊性乳房、男性不育症或勃起功能障碍者慎用。

毒性:每日摄取量少于10毫克，不良反应最少。长期使用可能降低甲状腺功能和基础代谢率。长期高剂量使用可能引起低钾血症、高醛固酮增多症和闭经症状。

相互作用:与利尿剂，心糖，皮质类固醇，MAOIs，降压药，螺内酯，激素治疗，泻药和K+消耗药物。

　

Medical Applications of Liposomes | ScienceDirect
https://www.sciencedirect.com/book/9780444829177

The mechanics of the lung depend upon two, unrelated, properties of phospholipids. At birth, they reduce the burden of expanding an air/water interface from 1 cm 2 to 2 m 2 by lowering surface tension. Subsequently, plaques of condensing dipalmitoylphosphatidylcholine help ratchet the alveolar spaces open and keep them dry for the rest of life.

The role of surfactants in the formulation of elastic ...
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833371

Apr 08, 2016 · An edge activator is usually a single chain surfactant that causes the destabilization of the lipid bilayer of the vesicle and increases the vesicle-elasticity or fluidity by lowering its interfacial tension.4. Flexibility of an elastic liposome membrane is achieved by mixing suitable surface-active agents in the proper ratios.

Cited by: 9
Publish Year: 2016
Author: Sima Singh, Harsh Vardhan, Niranjan G Kotla, Balaji

　

Int J Nanomedicine. 2016; 11: 1475–1482.

The role of surfactants in the formulation of elastic liposomal gels containing a synthetic opioid analgesic

Sima Singh,1,* Harsh Vardhan,1,* Niranjan G Kotla,2 Balaji Maddiboyina,3 Dinesh Sharma,4 and Thomas J Webster5,6

Abstract
Transdermal drug delivery systems have made significant contributions to the medical community, but have yet to completely substitute oral or parenteral delivery. Recently, various strategies have been used to augment the transdermal delivery of therapeutics. Primarily, they include iontophoresis, electrophoresis, sonophoresis, chemical permeation enhancers, microneedles, and vesicular systems. Among these strategies, elastic liposomes appear promising. Elastic vesicle scaffolds have been developed and evaluated as novel topical and transdermal delivery systems, with an infrastructure consisting of hydrophobic and hydrophilic moieties together, and as a result, such scaffolds can accommodate drug molecules with a wide range of solubility. High deformability of these vesicles provides for better penetration of intact vesicles. This system is much more efficient at delivering low- and high-molecular-weight drugs to the skin in terms of quantity and depth. In this work, elastic liposomes of Tramadol HCl were prepared using a solvent evaporation method with different surfactants and were characterized using microscopy, and particle size, shape, drug content, ex vivo release, and zeta potential were also calculated. The prepared elastic liposomes were found to be in the range of 152.4 nm with a zeta potential of −22.4 mV; the entrapment efficiencies of the selected formulation was found to be 79.71%±0.27%. All formulations in the form of a gel were evaluated for physicochemical properties and were found to be homogeneous with no grittiness, and the pH of all formulations was found to be neutral. The optimized selected elastic liposomal formulation followed the Higuchi equation and Fickian diffusion and released the drug for a period of 24 hours. The overall results provide much promise for the continued investigation of deformable vesicles as transdermal drug carriers.

Keywords: elastic liposome, skin delivery, occlusion, hydration-gradient, hydrogel

The role of surfactants in the formulation of elastic liposomal gels containing a synthetic opioid analgesic
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833371/

　

　