The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.
Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N’s high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 µM as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-µ-molar range.
Tetherin-associated antiviral activity is induced by interferon-α, and it comprises protein-based tethers, that leads to the retention of fully formed virions on infected cell surfaces1. The necessity for tetherin glycosylation in the inhibition of virus release remains uncertain, implying the importance of glycosylation patterns on recombinantly expressed glycans for in vitro studies1,2, which depends on the conformation of (in the case of influenza virus) surface-expressed influenza hemagglutinin HA3,4. It has been noted that modification of oligosaccharide tethered to N-linked glycosylation is enough for tetherin-mediated restriction of HIV type-1 release2, while dimerization plays an essential role in preventing virus release, thereby involving the transmembrane domain or glycosyl-phosphatidyl-inositol (GPI)-anchor for tethering the budding virions5. Unique features are described for human and murine tetherin to block multiple enveloped viruses, retroviruses, and filoviruses. BST-2/tetherin is an interferon-inducible antiviral protein of the innate immunity1,6, acting with broad-spectrum antiviral activity and is antagonized by envelope glycoproteins5 to either translocate tetherin or disrupt the structure of tetherin6. For example, surface-expressed envelope glycoprotein HA and neuraminidase on influenza A virus are well known for tetherin antagonism in a strain-specific manner7, facilitating the recognition of host receptor binding sites8. Glycan-targeting antibodies are studied in the stoichiometry of their interactions with the rapidly customizing glycan shields on HA, resulting in binding affinity to influenza A H3N2 and H1N1 subtypes4.
To elucidate the binding mechanisms between antiviral agents and virus envelope spikes, i.e., carbohydrate ligands, and complementary immunological and spectroscopic methods, mono-, di- and tri-mannose moieties are chemically synthesized. The mannosylated peptides are created via azido glycosylation of glycosyl {beta}-peracetates to 1,2-trans glycosyl azides transformation9, mimicking the typically found N-acetyl glucosamine and high-mannose oligosaccharides on the surface of life-threatening viruses. Triazole bioisosteres are utilized to mimic linkages that form the mannosylated residue of HA peptide10 and facilitate site-specific interactions with antiviral CV-N derivatives around the second N-linked glycosylation spot on the HA head domain (HA top with 4 N-linked glycans N54, N97, N181, N301)8,11,12. Interactions between glutamic acid (Glu) and arginine (Arg) and the resulting helix dipole manifestated good stability of both model peptides and proteins but are visualized using SPR. If compared with recognizing a single chemically synthesized glycosylation site on HA10 by directly inhibiting receptor binding on the glycan moieties, a higher affinity of a four-site mutated Fc structure to its receptor is shown to elicit effector functions in vivo, revealing the unrelated composition of N-linked glycans attached to Fc mutant to be mechanistically determined13.
CV-N displays antiviral activity against HIV14,15, influenza16, and Ebola virus, which is mediated by nanomolar binding to high-mannose oligosaccharide modifications on envelope spike proteins12,17,18,19. Influenza HA binding to one high-affinity carbohydrate-binding site (H) in CV-N or two Hs in covalently linked dimeric CVN2 is determined to have equilibrium dissociation constants (KD) = 5.7 nM (Figure 1A) and KD = 2.7 nM, respectively. Both CV-N and CVN2 harbor another one or two low-affinity carbohydrate-binding sites (L)s12,17,20,21. Ebola GP1,2 binds to 2H of CVN2 with affinities in the lower nanomolar range (KD = 26 nM). CV-N WT binding to Ebola GP1,2 and HA exhibits affinities from KD = 34 nM to KD = 5.7 nM (A/New York/55/04)12. Lectins, such as CV-N, which specifically target high-mannose glycans on the viral envelopes, further inhibit replication of hepatitis C virus, SARS-CoV, herpesvirus, Marburg virus, and measles virus22.
The small CV-N molecule has been studied thoroughly for more than 20 years as it functionalizes to bind a wide range of viruses to inhibit viral entry16,18. Structural analyses and binding affinity assays indicate cross-linking of two Ls in a domain-swapped CVN2 dimer by bivalent binding in the micromolar range to enhance avidity to viral envelope glycoproteins10,19. Selective binding of Manα1-2Manα on Man(8) D1D3 arms and Man(9) comprises two binding sites of differing affinities located on opposite protein protomers20, thereby reaching nanomolar binding affinities (Figure 1B). Thus, CVN2 is considered a pseudo-antibody concerning its application to bind epitopes on HIV gp120, similar to virus-neutralizing antibodies17. Herein, the author is interested in investigating the potential binding of CVN2 to the SARS-CoV-2 spike via its receptor-binding domain (RBD). Binding curves of immobilized human angiotensin-converting enzyme (ACE)-2 with the SARS-CoV-2 RBD result in KD = 4.7 nM for this biologically relevant binding interaction23.
By contrast, selected immunoglobulin classes recognize specific and consistent structural protein patterns, which impart a substrate for affinity maturation in the membrane-anchored HA regions24. CV-N shows highly potent activity in almost all influenza A and B viruses16, and it is a broadly neutralizing antiviral agent. Our knowledge is incomplete on the location of targeted epitopes on the stem of HA1 and HA2 that possibly involve epitopic structures for glycan-targeting by highly neutralizing antibodies and as compared with lectin binding25.
Figure 1: Schematic representation of the SPR binding assay for CV-N to virus envelope spikes. (A) SPR Assay for CV-N binding to ligand: HA full-length protein (90 kDa). Kinetic data set (5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 0 nM) showing real-time double-referenced binding to influenza HA A/New-York/55/04 (H3N2). (B) CVN2L0 variant V2 binding to immobilized ligand DM within a concentration range of 500 nM to 16 µM. Sequence: L residues are highlighted in yellow. H residues are highlighted in gray. E58 and R73 are a replacement for cysteines in the wildtype protein and make V2 a stable protein fold with three instead of four disulfide bonds Please click here to view a larger version of this figure.
Whereas the glycan shield on the membrane-distal HA top part induces high-affinity binding to CV-N12, CVN2 binding to HA adjacent to a disulfide bridge of the HA top part has further been observed at its low-affinity sites10,12. Various polar interactions and interaction sites are identified in carbohydrate-binding by CV-N. These interactions are verified by generating knock-out variants in the binding site to correlate binding affinities to in silico predicted glycosylation12. Thus, the project aims to compare previously tested chemically mannosylated HA peptides in binding affinity and specificity with short peptide sequences from SARS-related 2019-nCoV spikes and SARS-CoV-2, naturally occurring modified by a small number of different N-linked glycosylation sites and O-linked glycosylation. Using cryo-electron microscopy and binding assays, Pinto and coworkers report a monoclonal antibody, S309, that potentially recognizes an epitope on SARS-CoV-2 spike protein containing a conserved glycan within the Sarbecovirus subgenus, without competing with receptor attachment26. The protocol of this study describes how designing, expressing, and characterizing CV-N variants are important to study how CV-N and CVN2 bind to glycosylated proteins and synthetic mannosylated peptides using the SPR technology10,12.
Tandem-linked dimer CVN2L027 and binding-site variants (V2-V5) are recombinantly expressed and variants are with disulfide bond replacements (C58E and C73R) (Figure 2A). Also, a mutant with a single-point mutation E41A is prepared because this position has been seen as an intermolecular cross-contacting residue. This mutant is another interesting molecule for SPR binding measurements between the lectin and high-mannose oligosaccharides deciphering binding domains and allows comparison with the dimeric form. The domain-swapped crystal structure of CVN2 shows a flexible linker, that extends between 49 and 54 residues. The two domains can continue moving around the hinge as rigid bodies, developing either a monomer through intramolecular domain interactions (domain A -residues 1-39;90-101- with domain B -residues 40-89) or a dimer by intermolecular domain swapping [domain A (of the first monomer) with domain B (of the second), and domain B (of the first monomer) with domain A (of the second copy)]. There are no close interactions between the two protomers' A and B domains, except for Glu4128. The gene for CV-N can be developed using a repetitive PCR method with 40-mer synthesized oligos29 and is then subcloned into the NdeI and BamHI sites of pET11a for transformation (electroporation) into electrocompetent cells as described by Keeffe, J.R.27. The protein, which is used for achieving the respective crystal structure (PDB ID 3S3Y), includes an N-terminal 6-histidine purification tag followed by a Factor Xa protease cleavage site. Site-directed mutagenesis is utilized to make point mutations, switch codons, and insert or delete single or multiple bases or codons for amino acid exchange. These transformations provide invaluable insight into protein function and structure. Recombinantly expressed and purified CV-N, CVN2, and CVN3 have been biophysically well studied20,21,27, are cheap to produce, and therefore used to characterize binding assays to glycans immobilized onto SPR sensor chips. Conventional enzyme-linked immunosorbent assay (ELISA) provides less reproducibility concerning the immobilization technique of glycan ligands and transforms real-time binding of various binding-site variants, which is shown for SPR, into endpoint assays.
Binding-affinity variant CVN2L0-V2 (an intact fold of homodimeric CV-N with a disulfide bridge substitution10) is expressed with a His-tag in Escherichia coli (E. coli), purified over Ni-NTA column applying affinity chromatography and tested for binding to HA (H3N2), monomannosylated HA-peptide and dimannosylated HA-peptide using SPR. Chemically mannosylated peptides, or HA and S protein, all are ligands and amine coupled to the hydrophilic chip surface via reactive esters or biotin-streptavidin protein engineering. The same procedure of sequential runs is applied to those ligands, injecting various dilutions of CV-N and variants of CV-N (and CVN2) to obtain kinetic information for the molecular interaction analyses as described below30. RBD-immobilized SPR sensor chip is used for binding studies on CV-N to S peptides, and affinities are compared to SARS-CoV-2 binding with the human ACE2.
For the present study, a CVN-small ubiquitin-like modifier (SUMO) fusion protein has been used in enzyme-linked immunosorbent assays instead of CV-N and is suitable for cell-based assays. Recombinant full-length influenza A virus HA H3 protein is obtained commercially (see Table of Materials) or expressed in mammalian HEK293 cell lines and baculovirus-infected insect cells according to standard protocols12. Wuhan-1 spike protein is expressed in mammalian HEK293 cells. The synthesis of monomannosylated peptide (MM) and dimannosylated peptide (DM) allows the detection of homogeneous ligands to CVN2 and monomannosylated small molecule10.
1. Creating CV-N constructs
2. Preparation of LB-agar plates with plasmid DNA transformed cells
3. Cloning
4. Site-directed mutagenesis
5. Transformation of bacterial cells
6. Expression and protein purification
Figure 2: CV-N sequences and expression. (A) CVN2 without a linker between each CV-N repeat (101 amino acids each) and four disulfide bridges is expressed in pET11a vector in E. coli. (B) Expressions of two independent colonies for CV-N (monomer) and CVN2 (dimer). (C) Disulfide bond variants are purified and analyzed on SDS-PAGE. A low molecular weight marker (6 µL) is used as a reference. WT = CVN2L0 bearing four disulfide bridges as marked in (A). V2 is a variant with a disulfide bond replacement by polar residues at positions 58 and 73. V3-V5 are variants with two remaining S-S bonds and either polar (C58E-C73R) or non-polar (C58W-C73M) substituting amino acids or a combination of these residue pair substitutions. (D) HPLC chromatograms of purified CVN2L0 are elutated at a flow rate of 1 mL/min with a linear gradient from 5%-65% buffer B in buffer A over 30 min. Buffer A is: 0.1% (v/v) trifluoroacetic acid in ddH2O, buffer B is: 0.08% (v/v) trifluoroacetic acid in acetonitrile. Protein is analyzed on a high-performance silica gel 300-5-C4 (150 x 4.6 mm) column at 214 nm and 280 nm. Please click here to view a larger version of this figure.
7. SPR spectroscopy
8. SPR binding assay for CV-N binding to HA, S protein, and RBD
A dimeric domain-swapped CVN2L0 molecule is tested for binding to the HA top region in three separate SPR experiments and binding affinity is presented in KD values. Domain B is assumed to comprise H-binding sites, which are impacted by replacing a disulfide bond into ionic residues, and domain A forms L10,18. Single injections of CVN2L0 and variants V2 (three disulfide bridges) and V5 (two disulfide bridges) are first tested for binding to the HA-coupled sensor chip to estimate binding capacities before setting up the kinetics measurement using the autosampler and running table editor (Figure 3A and Supplementary Figure 2). Repeated injections are automated for a series of CVN2L0, V2, and V5 at various concentrations in the nanomolar and µ-molar range in the system over time (Figure 3B-D). Correlating the number of intact Cys-Cys bonds, CVN2L0 is binding immobilized HA at KD = 255 nM when reaching good saturation. In this case, both KD values, calculated either from kinetic data or by fitting the equilibrium data to the Langmuir binding isotherm, are in moderate agreement (Table 1 and Supplementary Figure 3, Supplementary Figure 4).
Figure 3: SPR binding assay for ligand binding via multivalent interactions. CVN2 (WT) and binding site-variants V2 and V5 with disulfide replacements in the high-affinity carbohydrate-binding pocket are tested for binding covalently immobilized HA. (A) Left and right flow cell binding curves of CVN2, V2, and V5 can be extracted from the SPR run protocol, and sensorgrams are summarized in an overlay. (B) Sensorgram from conventional SPR experiment shown as kinetic analyses for binding of CVN2L0 to HA, injecting analyte concentrations from 10-4 to 10-8 M. CVN2L0 is measured up to the highest concentration at 1.5 µM (top line) and up to 500 nM (bottom line), for which no saturation on the chip surface nor equilibrium binding is achieved. RU = Response units. A CMD500D sensor chip is used. (C) SPR sensorgram for V2 binding to HA. (D) V5 binding to HA. The figure (B-D) is reproduced with permission from reference10. Please click here to view a larger version of this figure.
ka (M-1s-1) | kd (s-1) | KD [HA] Kinetic |
KD [HA] Equilibrium |
|
CVN2 | 5.1 e3 | 1.3 x10-3 | 255 nM | 246 nM |
V2 | 4.0 e3 | 1.1 x10-3 | 275 nM | 255 nM |
V5 | 1.2 e3 | 5.8 x10-2 | 5 µM | 4.9 µM |
CVN2L0 | 2.07 e4 | 3.0 x10-3 | 147 nM | 600 nM |
2.27 e4 | 3.0 x10-3 | 133 nM | 490 nM |
Table 1: Binding affinities of CVN2 and variants to HA measured via SPR. Scrubber 2.0 (a BioLogic software) is utilized to fit real-time SPR association and dissociation curves and to calculate the equilibrium dissociation constants (KD) from kinetic and equilibrium data. Ka = association rate constant. Kd = dissociation rate constant.
Whereas KD values for binding of CVN2L0 and V2 to HA are both in the nanomolar range, V5 is diminished in binding (Table 1). In V5, two disulfide bonds are replaced by ion-pairing residues that retrain potential ionic interactions between mutated Glu and Arg residues (Figure 2A,3D). Primary data is analyzed following the integrated rate of association and rate of dissociation equations, which describe the binding kinetics of the interaction of soluble CV-N with immobilized ligand and the dissociation of the formed complex from the chip surface. Two independent measurements are performed, and sensorgrams, equivalent to those obtained from replicates are analyzed. KD is calculated as the quotient of koff/kon (Supplementary Table 1)10,35. However, KD is related to the equilibrium data that fit the Langmuir binding isotherm36, where the equilibrium binding response is plotted as a function of the analyte concentration (Supplementary Figure 3A,4A,3B,4B). In a concentration-dependent manner, dissociation rate constant kd is determined post analyses and incorporated into association phase fitting (kon) to estimate the association rate constant ka. (Table 1, Supplementary Figure 3C, Supplementary Figure 4C).
Next, the binding of CV-N to HA is compared to its interaction with RBD. CV-N WT binding to SARS-CoV-2 RBD is measured at weaker affinity (KD = 260 µM, Figure 4A) as compared with binding to HA (KD = 5.7 nM)8,10,12 and binding to S protein (KD = 18.6 µM, Figure 5). Concentration versus response is plotted for CV-N WT binding to RBD, assuming specific targeting of possibly conserved carbohydrates on the RBD S1 subunit (Figure 4A). The same affinity plot is shown for CVN2L0-V4 binding to DM that is no longer analyzable due to the replacement of disulfide bonds which interfere with high-affinity binding to small glycosylated peptides (Figure 4B).
CVN2L0-V4 (2 disulfide bonds opposite to unsymmetric replacement of cysteine residues by either Glu-Arg residues or amphipathic amino acids Trp and Met) can form non-polar hydrogen bond networks around the pseudo-domain B carbohydrate-binding site10. Higher density of glycans on HA protein reaches binding with polar Glu-Arg residues instead of cystines (Supplementary Table 1), and an association with mannosylated peptides (KD = 10 µM for DM) between CVN2L0 and modifications into Glu-Arg, but shows discontinuous dissociation of variants from this monoglycosylated peptide, in particular when H is modified on both monomers. For the interaction between CVN2L0-V4 with DM, the response of 56 µM CVN2L0-V4 injection yields a higher response than Rmax. (Figure 4B). V5 (2x Glu-Arg) fails in dissociation from surface-bound DM (data not shown). In sum, response curves of lower concentrations decline before ending the injection for all sensorgrams on CVN2 binding to peptide without native H and monomer binding to RBD.
Figure 4: SPR analyses of (A) CV-N WT monomer binding to RBD. KD is calculated by fitting kinetic data (left side, KD = 260 µM) and compared with affinity data (right side) using a simple biomolecular model for 1:1 binding between ligand and analyte (KD equil = 274 µM). Equilibrium binding response or binding capacity is plotted as a function of concentration in comparison with the SPR binding curves (0-605 µM CVN). H = High-affinity binding site. L = Low-affinity binding site. Both are found on CV-N and CVN2L0. (B) Dimeric CVN2L0-V4 binding to DM, assuming 2L without analyzable KD. CVN2L0-V4 concentrations are 56 µM, 28 µM, 14 µM, 7 µM, 3.5 µM. Please click here to view a larger version of this figure.
Figure 5: Mutant CVN-E41A and CV-N WT binding to (A) S glycoprotein. Arrows indicate the beginning of the association and dissociation phase. (B) CVN-E41A binding to RBD on SARS-CoV-2. The ligands are pre-immobilized onto HC polycarboxylate and CMD500D sensor chips. Covid-19 S1 subunit [Pinto, D. et al.26; and Barnes, C. et al.37] is used for RBD immobilization. Flow rate: 30 µL/min, 25 °C; plotted raw data. In comparison, the binding of human blood serum antibodies to RBD with a dilution factor of 1:200 is depicted. (C) SPR assay of CV-N WT binding to S glycoprotein that is immobilized to a response level of 400 µRIU to capture CV-N. Please click here to view a larger version of this figure.
It has been shown before that monomeric CV-N with a single L cannot sufficiently bind gp120, but that CV-N's neutralization ability requires cross-linking of two carbohydrate-binding sites and is predominantly restored by dimerization to functionalize with 2H12,19. Hence, a monomeric mutant CVN-E41A is expressed, which is suspected to destabilize pseudo-domain B or can interrupt connectivity with the second domain A, such as found in CV-N WT. CVN-E41A monomer reveals stability upon binding to S protein similar to CV-N monomer. Although SPR response for 605-680 µM analyte concentrations results in high response units and typical SPR sensorgrams for CV-N WT and E41A binding, respectively, the mutant is unstable when diluted (Figure 5).
Supplementary Figure 1: SPR sensorgram for capturing DM mimicry as part of influenza HA top. Screenshot: SPR data plot showing the SPR run protocol for the immobilization of DM onto SPR sensor chip CMD500D, which is coated with 500 nm carboxymethyl dextran hydrogel and suitable for kinetics analyses of low molecular weight analytes on high ligand density. Sensorchips are directly mounted onto the detector with emersion oil and fixed below a three-port flow cell. After quenching the activated chip surface with 1 M ethanolamine HCl pH 8.5, CVN2 is injected twice (concentration =1 µM and 2 µM, respectively). Purple: Difference between flow cell 1 and flow cell 2; response is recorded at an interval of 0.2 s. Blue: Flow cell 1: 3000-4000 micro-Refractive Index Units (µRIU) coated from a 400 nM solution of the ligand DM to an activated carboxylated surface. Red: Reference flow cell 2. Please click here to download this File.
Supplementary Figure 2: Manual run on HA-functionalized CMD500D sensor chip showing binding of dimeric CVN2L0-V5, V2, and CVN2L0. At infuse flow rates from 30-50 µL/min, CVN2L0 and disulfide bond variants expressed with a His-tag and purified over Ni-NTA are injected in the mid-µM-range and tested for binding to HA with an association phase of 3 min and dissociation phase of 2 min. Injections are V5 (15 µM), V2 (2.4 µM), 0 µM, CVN2L0 subcloned from a SUMO-fusion protein (1 µM), and CVN2L0 (2 µM). Running buffer at physiological pH is used for all experiments at 25 °C. All solutions are degassed and filtered (0.2 µm) before injection into the system. Purple: Difference between flow cell 1 and flow cell 2; response is recorded at an interval of 0.2 sec. Blue: Flow cell 1: 2540 µRIU HA. Red: Reference flow cell 2. Please click here to download this File.
Supplementary Figure 3: CVN2 binding to HA. Analyses of sensorgrams when curves are auto-aligned. (A) Sensorgrams zeroed and aligned using Scrubber software (left) and binding isotherm showing concentration-dependent response curve to bound analytes (right). (B) Binding isotherm expressed in percent capacity. (C) Computationally fitted theoretical association and dissociation curves. (D) Kinetics data on SPR sensorgrams without fitted curves. Please click here to download this File.
Supplementary Figure 4: CVN2 binding to HA. Analyses of sensorgrams when curves are manually aligned. (A) Sensorgrams zeroed and aligned using Scrubber software (left) and binding isotherm showing concentration-dependent response curve to bound analytes (right). (B) Binding isotherm expressed in percent capacity. (C) Computationally fitted theoretical association and dissociation curves. (D) Kinetics data on SPR sensorgrams without fitted curves. Please click here to download this File.
Supplementary Table 1: Kinetic data obtained from SPR sensorgrams for the binding of CVN2L0 and Cys-Cys bond variants V2, V4, and V5 to HA using a Langmuir 1:1 binding model. KD [M] = koff/kon or kd/ka. All data is generated at 25 °C in HBS-EP(+) buffer.: Please click here to download this Table.
CV-N's binding affinity is correlated with the number of functional binding sites [2H on domains B, and 2L on domain(s) A when engineered as domain-swapped dimer]. A variant with an altered binding affinity (CVN2L0-V2, a homodimeric stable fold of CV-N comprising a disulfide bridge knock-out) is expressed in E. coli, purified, and positively tested for binding to HA-protein (H3N2) using SPR10, and shows a conformational change upon binding HA with either H or L carbohydrate-binding sites and KD1 = 49 nM and KD2 = 8 µM38. As the number of disulfide bridges near the glycan-targeting pocket in CVN2 decreased from 4 to 2, binding affinity to HA protein decreased (Figures 3B–D and Supplementary Table 1). Disulfide bond variants are created by substituting Cys and inserting polar residue pairs Glu – Arg with slightly decreasing stability. The variants otherwise represent the same molecule, and the four binding sites are functional, although multisite binding on the chip is affected for all variants based on two disulfide substitutions. A non-polar substitution of both disulfide bonds in the two high-affinity binding pockets makes variant CVN2L0-V3, which actively binds the HA-peptide in both its monomannosylated and dimannosylated form. The interactions with CVN2L0-V3 are confirmed for micromolar concentrations and binding with the synthetic peptides (MM and DM) in solution via STD-NMR. Notably, CVN2 SPR binding experiments to MM are not analyzable due to insufficient calculation of Rmax, a general drawback of the SPR technique, and weak binding affinities to the immobilized MM10. Increased ligand density of this peptide and other peptides reduces binding selectivity. Using SPR, dimeric CVN2L0 (2H+2L) with high stability shows multivalent binding to HA, which is expressed in insect cells, and specificity for dimannose on a single chemically engineered biomimicry for high-mannose oligosaccharides, assuming a binding model 1:1. Binding to oligo-mannosides is usually measured by isothermal titration calorimetry, which requires a higher concentration of ligand to be tittered against the lectin17.
Competitive binding to monomannose or any glycan smaller than Man(7) has not been found before either15, indicating the involvement of the chemical linkage with the peptide backbone in recognition of these glycan moieties. The number of mannose-mannose linkages in the target was varied, deciphering tryptophan interactions in the high-affinity glycan pocket. The type of modification of peptides with triazole-linked monomannose or triazole-linked dimannose may determine KD values, in particular to H. Concerning binding affinities of CVN2L0 (2H+2L) and CVN2L0-V2 (1H+2L) to DM, koff is to the maximum 10-fold higher concerning CVN2L0-V2 dissociation from DM versus HA10. Besides measuring kinetic rate constants, SPR allows to estimate the equilibrium constants for very slowly equilibrating systems without actually reaching equilibrium (Table 1). By contrast, kon expresses good relative response values for all binding curves of domain-swapped CVN2L0 and its disulfide bond variants (V2, V4, and V5) to HA (Supplementary Table 1), or DM, while the dissociation rate constant koff shows faster off-rates for V4 and V5, two variants bearing two functional L, and V4 with non-analyzable KD concerning DM (Figures 4B). Assigning domain B to H and domain A the respective L is a previous finding that basis for the disulfide bond variants to reveal different binding affinities in SPR, specified, to HA18,28.
SPR is one of the leading tools for biomolecular interaction analysis in biomedical research35. If a specific, timed control of the bulk analyte concentration is feasible in the SPR instrument, the quantitative evaluation of the kinetics of binding progress between macromolecules is possible, with increasing interest in analyzing multi-protein complexes39. The challenge in its use is the controlled positioning of one of the interaction components on the widely used carboxymethyl dextran, or HC polycarboxylate hydrogel surface, which captures small and large biomolecules. To optimize immobilization and binding capacity of the chip surface, streptavidin-biotin sandwich immobilization and immobilization via protein His-tag to an anti-his antibody are available40. The biophysical characteristics of proteins or functional groups of small molecules might interfere with immobilization results, but any difficulties are experienced with the herein described glycoproteins and glycopeptides that are covalently bound via EDC/NHS chemistry. The immobilized glycans, either glycoprotein or the synthetic homogenously mono- or dimannosylated glycopeptides, are used on re-usable chips to screen various engineered CV-N variants, and binding assays are applied to fully purified and recombinantly expressed lectins. Impurities in injected protein solution are damaging the SPR channel system. Although experimental procedures are described on an in vitro system in this study, the glycosylation pattern on viral spikes may be recognized selectively by each analyte concentration of this small model protein as injected over time with optimized mass transfer limitation36. The size of CV-N is ~11 kDa and supports reproducible SPR data for its binding to viral spike proteins.
CV-N's binding specificity to high-mannose glycans is confirmed by the bivalent binding interaction with the dimannosylated peptide mimetic rather than binding to structurally functional glycans and the mono-mannosylated peptide using isothermal titration calorimetry (data not shown). A variant with the point mutation Glu41Ala expresses the monomeric 101-residue protein with two intertwined carbohydrate-binding sites on each protomer. Therefore, this mutation site abolishes a contacting residue between the high- and low-affinity carbohydrate sites and reduces molecular binding strength to N-acetyl-D-glucosamine. Binding of CVN-E41A to SARS-CoV-2 spike protein, bearing complex-type N-linked glycosylation and O-glycosylation, is achieved in the SPR, but CV-N binding to this spike is for both the spike protein and RBD without physiological relevance, as measured at micromolar concentrations (Figure 4,5).
Mannosylated peptides developed and generated in this study are used as protein scaffolds to screen the antiviral agents' binding characteristics by SPR and NMR, as they represent invariable ligands for carbohydrate-binding assays attached to a defined amino acid sequence10. Moreover, STD-NMR spectroscopy is a label-free technique, such as SPR, which allows the characterization of carbohydrate-binding by conformational selection10,41. In such settings, STD-NMR allows for the development of rapid screening methods for a large library of compounds to identify bioactive ligands via binding, epitope mapping, and direct determination of KD under different experimental conditions. Accurate KD values can be obtained by analyzing the protein-ligand association curve utilizing STD values at the limit of zero saturation time41,42. Therefore, the interrogation of protein-ligand interactions by the STD-NMR method was accomplished, but KDs are calculated on the SPR system.
Human 2019-nCoV (Wuhan-Hu-1-2019 novel coronavirus) is estimated to differ among 11 (out of 18) predicted N-linked glycosylation sites to SARS-CoV43. Epitope/paratope mapping reveals a few interplays with host-derived N-glycans and negligible contributions of antibody somatic hypermutations to epitope contacts37. The ability of antiviral lectins and broadly neutralizing antibodies to bind highly conserved epitopes on the influenza HA glycoprotein is most important to the rational design of vaccines for preventive and therapeutic use. Further investigation, however, is needed to evaluate the effect of immune-suppressive N-linked glycosylation around the host receptor binding site. The data may provide insight into the potential of virus spike proteins to escape from antibodies elicited during infection or to bind non-immunogenic CV-N and thus guide structure-based computational protein design for new optimized antiviral CVN2 variants. By contrast, the affinity of glycosylated Fc amino acid mutant to its receptor to elicit effector functions in vivo, uses a composition of glycosylation sites, and the number of glycans involved, may therefore be important to binding affinity but may not be determined for neutralization ability.
Taken together, fewer rotamers are predicted to bind carbohydrate ligands to proteins by computational protein design than protein-protein interactions alone. Domain-swapped CVN2, however, bearing various numbers of carbohydrate-binding sites, provides the necessary stability and flexibility to reveal different binding-affinities attributed to H and to form the multivalent interactions between oligomannose clusters on viral envelope spikes and neutralizing antibodies (NAb), thus allowing for inhibition assays to be experimentally validated.
The authors have nothing to disclose.
The author acknowledges Dr. Christian Derntl from the Department for Biotechnology and Microbiology at the TU Wien and the Department of Medicine III, Division of Nephrology and Dialysis at the Medical University of Vienna, especially Dr. Markus Wahrmann for technical and scientific support. Protein expression in mammalian cells was supported by the Department of Biotechnology at the University of Natural Resources and Life Sciences (BOKU) Vienna. The author wants to express her deep acknowledgement to Dr. Nico Dankbar from XanTec bioanalytics in Duesseldorf, Germany, for helpful scientific discussions on performing the SPR binding assays.
Äkta primeplus | Cytiva | ||
Amicon tubes | Merck | C7715 | |
Ampillicin | Sigma-Aldrich | A5354 | |
Beckmann Coulter Cooler Allegra X-30R centrifuge | Beckman Coulter | B06320 | |
Cell spreader | Sigma-Aldrich | HS86655 | silver stainless steel, bar L 33 mm |
Custom DNA Oligos | Sigma-Aldrich | OLIGO | |
Custom Gensynthesis | GenScript | #1390661 | cloning vector: pET27b(+) |
Cytiva HBS-EP+ Buffer 10, 4x50mL | Thermo Scientific | 50-105-5354 | |
Dionex UlitMate 3000 | Thermo Scientific | IQLAAAGABHFAPBMBFB | |
Dpn I restriction enzyme (10 U/μL) | Fisher Scientific | ER1701 | |
DTT | Merck | DTT-RO | |
EDC | Merck | 39391 | |
EDTA | Merck | E9884 | |
Eppendorf Safe-Lock Tubes | Eppendorf | 30120086 | |
Eppendorf Safe-Lock Tubes | Eppendorf | 30120094 | |
Eppendorf Minispin and MiniSpin Plus personal microcentrifuge | Sigma-Aldrich | Z606235 | |
Ethanol | Merck | 51976 | |
Ethanolamine HCl | Merck | E6133 | |
Falcon 50mL Conical Centrifuge Tubes | Fisher Scientific | 14-432-22 | |
Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap, Sterile, 25/Pack | Corning | 352057 | |
Glucose | Merck | G8270 | |
Glycine HCl | Merck | 55097 | |
HA H3 protein | Abcam | ab69751 | |
HEPES | Merck | H3375 | |
His-select Ni2+ | Merck | H0537 | |
Imidazole | Merck | I2399 | |
IPTG | Merck | I6758 | |
Kanamycin A | Sigma-Aldrich | K1377 | |
Kromasil 300-5-C4 | Nouryon | ||
LB agar | Merck | 52062 | |
LB agar | Merck | 19344 | |
LB Lennox | Merck | L3022 | |
Lysozyme | Merck | 10837059001 | |
Magnesium chloride | Merck | M8266 | |
Magnesium sulfate | Merck | M7506 | |
NaH2P04 | Merck | S0751 | |
NanoDrop UV-Vis2000c spectrophotometer | Thermo Scientific | ND2000CLAPTOP | |
NaOH | Merck | S5881 | |
NHS | Merck | 130672 | |
NZ amine (casein hydrolysate) | Merck | C0626 | |
PBS | Merck | 806552 | |
PD MidiTrap G-10 | Sigma-Aldrich | GE28-9180-11 | |
Peptone | Merck | 70171 | |
pET11a | Merck Millipore (Novagen) | 69436 | |
PMSF | Merck | PMSF-RO | |
QIAprep Spin Miniprep Kit (1000) | Qiagen | 27106X4 | |
Reichert Software Package Autolink1-1-9 | Reichert | ||
Reichert SPR SR7500DC Dual Channel System | Reichert | ||
Scrubber2-2012-09-04 for data analysis | Reichert | ||
SDS | Merck | 11667289001 | |
Site-directed mutagenesis kit incl pUC18 control plasmid | Stratagene | #200518 | |
Sodim chloride | Merck | S9888 | |
Sodium acetate.Trihydrate | Merck | 236500 | |
SPR sensor chip C19RBDHC30M | XanTec bioanalytics | SCR C19RBDHC30M | |
SPR sensor chip CMD500D | XanTec bioanalytics | SCR CMD500D | |
Sterilin Standard 90mm Petri Dishes | Thermo Scientific | 101R20 | |
TBS | Merck | T5912 | 10x, solution |
Triton-X100 | Merck | T8787 | |
Tryptone | Merck | 93657 | |
Tween20 | Merck | P1379 | |
Vortex-Genie 2 Mixer | Merck | Z258423 | |
X-gal | Merck | XGAL-RO | |
XL1-Blue Supercompetent Cells | Stratagene | #200236 | |
Yeast extract | Merck | Y1625 |