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Biological and Environmental Phenomena at the Interface

Quantification of Multivalency in Protein Oligomer Coated Nanoparticles Targeting Dynamic Membrane Glycan Receptors Jiake Lin, Kang Wang, Xiaoyu Xia, and Lei Shen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01605 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

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Quantification of Multivalency in Protein Oligomer Coated Nanoparticles Targeting Dynamic Membrane Glycan Receptors Jiake Lin,‡§ Kang Wang,†§ Xiaoyu Xia,‡ Lei Shen,*† †

School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan 430070, China ‡ School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China

Abstract. Multivalent binding of proteins to glycan receptors on the host cell quantitatively controls the initial adhesion of most viruses. Yet quantifying such multivalency in terms of binding valency has always been a challenge due to the hierarchy of multivalency involving multiple protein oligomers on the virus, limiting our understanding of virus adhesion and virulence. To address this challenge, we mimicked virus adhesion to cell surfaces by attaching protein oligomer coated nanoparticles (NPs) to fluidic glycolipid membranes with surface glycan density varying over 4 orders of magnitude. Using total internal reflection fluorescence microscopy to track single attached NPs, we show that the binding isotherms exhibit two regions, attributed to monovalent and multivalent protein/glycan interactions at low and high glycan densities, respectively. The bimodal binding curve allows to quantify the different valency and binding constants of monovalent and multivalent interactions. In addition, the competitive inhibition of multivalency by the glycopolymer presenting multiple glycan moieties is quantitatively appreciated. This work is essential to mapping and understanding the complex binding specificities of glycan-binding proteins and inhibitory drug designs and applications.

§

These authors contribute equally to this work.

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Introduction Multivalent interactions between multiple receptor proteins and multiple glycan moieties provide the basis for mechanisms of a wide range of cell surface processes.1-4 This is exemplified by virus recognition and attachment onto host cells, where the binding affinity and selectivity are greatly altered and controlled by simultaneous interactions between multiple proteins on the virus and multiple glycans moieties on the host cell surfaces.5 Such protein/glycan multivalency can dramatically enhance binding affinity with a multivalent enhancement factor of up to 108.1 As such, synthetic molecules presenting multiple copies of glycan moieties have been developed to effectively inhibit virus adhesion as competitive inhibitors.6-11 Understanding the mechanistic insights into protein/glycan multivalency is critical not only for revealing the biological basis of virus infection; quantitative knowledge of such multivalency also provide insights into strategies for inhibitory drug designs and applications. To quantify a multivalent protein/glycan interaction, the association and dissociation of multiple protein/glycan pairs have been characterized to obtain the relative kinetic (kon and koff) and thermodynamic parameters (Kd, ∆G, ∆S, ∆H).12-18 In general, the equilibrium constant of multivalent interaction involving N protein/glycan pairs can be related to that of the monovalent interaction by Kmulti=(Kmono)αN. α represents the degree of cooperativity—the influence of one binding event on subsequent events—which can be evaluated by comparing the average free energy of interaction between a protein and a glycan moiety in a multivalent interaction with that of the first monovalent interaction. Among these parameters, quantification of the valency (N) of a multivalent interaction has always been a challenge. This is due to the complex hierarchy of the mutivalency. Take the influenza A virus as an example (Scheme 1), the glycan-binding hemagglutinin (HA) proteins on the surface of the virus are usually present in a trimeric state (t-


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HA) and there is also a high density of t-HA.19 This two-tier multivalency complicates the multivalent binding process, and

so far, little relative

quantitative information has been reported because of limitations of current studies. First, the hierarchy of the multivalency makes the binding probability a strong function of surface glycan densities.1,20 To

Scheme 1. Illustration of the hierarchy of multivalent adhesion of an influenza A virus to the host cell surface.

quantify the valency and binding constants involved, one must control and vary the surface glycan density over many orders of magnitude, which is neglected in current studies. In addition, the hierarchy of the mutivalency is strongly mediated by the mobility of glycan moieties on the cell surface because the mobility provides the dynamic clustering of glycan moieties for promoting statistical binding matching.21,22 The lack of mobility on glycan immobilized arrays demonstrated to date cannot quantitatively control the cooperativity and valency in the multivalent interaction. In this work, we mimicked virus adhesion to a cell surface by attaching protein oligomer coated nanoparticles (NPs) to a fluidic glycolipid membrane. The density of glycan on the membrane was quantitatively tuned by simply adjusting the concentration of glycolipids over 4 orders of magnitude (10-6–10-2 nm-2). Two protein model systems were chosen, t-HA trimers of H7N9 avian influenza A virus and pentameric cholera toxin B subunits (p-CTB) of bacterial pathogen Vibrio cholerae with strong affinity for α2-3 linked sialic acid (SA) containing GM3 and ganglioside GM1 glycolipids, respectively. Using total internal reflection fluorescence microscopy (TIRFM) to track single attached NPs, we show the quantitative determination of the valency and binding constants for multivalent protein/glycan interactions. Our method also helps


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to quantitatively appreciate the competitive inhibition of protein/glycan multivalency by the glycopolymer presenting multiple glycan moieties. Experimental Section Materials. The recombinant cholera toxin (CT) and polyhistidine-tagged HA proteins, expressed from the pathogenic Vibrio 5 holera and H7N9 avian influenza A virus (A/Shanghai/1/2013), were purchased from Sigma-Aldrich (USA) and Sino Biological Ltd. (China), respectively. SEC characterization provided by the sponsor demonstrates the trimeric state of HA oligomers in buffer solutions. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Texas Red-dihexadecanoyl-phosphatidylethanolamine

(TR-DHPE),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide-

(polyethylene glycol)) (maleimide-PEG-DSPE), nickel-1,2-dioleoyl-sn-glycero-3-([N-(5-amino1-carboxypentyl)iminodiacetic acid]succinyl)-nickel salts (Ni2+-NTA-DOGS) lipids, GM1 and GM3 glycolipids were purchased from Avanti Polar Lipids (USA). Trisaccharide Neu5Ac-α2-3Gal-β1-4-Glc containing glycopolymer was purchased from GlycoTech Inc. (USA). All chemicals were used as received without further purification. Tris(hydroxymethyl)aminomethane (Tris) buffer was freshly prepared from Tris salt to give a pH 7.4 at 25 oC. Synthesis of SiO2 nanoparticles (NPs). SiO2 NPs were synthesized through the Stöber method.23 First, 5 mL of ammonia solution (~37%) in 22 mL of ethanol was added into a conical flask (250 mL) and kept at 30 oC for 10 mins by stirring at 350 rpm. Then, 0.4 mL of tetraethyl orthosilicate (TEOS) in 22 mL of ethanol was slowly added. After 30 mins, 4 mL of ammonia solution (~37%) and 1mL of TEOS in 44 mL of ethanol were successively added into the flask. Finally, the mixture was stirred at 30 oC for 6 hrs. The obtained SiO2 NPs were purified by


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centrifugation and re-dispersed in water three times with the assistant of sonication. The diameter of SiO2 NPs is ~150 nm as determined by TEM. Preparation of spherically supported lipid bilayer membranes (SSLBMs) coated fluorescent SiO2 NPs. We chose SSLBMs composed of DSPC and 0.2 mol% of TR-DHPE on SiO2 NPs to obtain fluorescent SiO2 NPs with mechanical stability. With high phase transition temperature (Tc~55 oC), the DSPC membrane behaves in a solid lamellar gel phase with good mechanical stability at room temperature.24 Typically, the DSPC and 0.2 mol% TR-DHPE lipids were dispersed in Tris buffer (0.5 mg/mL of DSPC) and incubated at 70 oC above the Tc of the DSPC for a minimum of 2 hrs with periodic shaking to form multilamellar vesicles (MLVs). The MLVs suspension was then extruded through a polycarbonate filter with a pore size of 100 nm 11 times to yield a small unilamellar vesicles (SUVs) clear solution. After that, 100 µL of the SiO2 NPs (2 mg/mL in water) dispersion was mixed with 500 µL of SUVs solution, incubated at 70 oC for a minimum of 2 hrs with successive shaking, and then allowed to cool to room temperature. Finally, the fluorescent SSLBMs-SiO2 NPs were centrifuged at 3000 rpm and redispersed in Tris buffer. The centrifuge/washing steps were repeated 3 times. Preparation of maleimide labelled SiO2 NPs. The preparation of maleimide labelled SiO2 NPs is similar as that of fluorescent SSLBMs-SiO2 NPs (Scheme S1 in Supporting Information (SI)). First, the DSPC, 0.2 mol% of TR-DHPE and 0.3 mol% of maleimide-PEG-DSPE lipids were dispersed in Tris buffer (0.5 mg/mL of DSPC) and incubated at 70 oC for 2 hrs with periodic shaking to form MLVs. The MLVs suspension was then extruded through a polycarbonate filter with a pore size of 100 nm 11 times to yield a SUVs clear solution. After that, 100 µL of the SiO2 NPs (2 mg/mL in water) dispersion was mixed with 500 µL of the maleimide SUVs solution, incubated at 70 oC for 2 hrs with successive shaking, and then


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allowed to cool to room temperature. Finally, the maleimide labelled SSLBMs-SiO2 NPs were centrifuged at 3000 rpm and re-dispersed in Tris buffer. The centrifuge/washing steps were repeated 3 times. 0.3 mol% of maleimide-PEG-DSPE in DSPC membrane yields that the interdistance of maleimide groups on SiO2 NPs is about ~12 nm. Preparation of Ni2+ labelled SiO2 NPs. The preparation of Ni2+ labelled SiO2 NPs is similar as that of maleimide-labeled SiO2 NPs (Scheme S2 in SI). First, the DSPC, 0.2 mol% of TRDHPE and 0.3 mol% of Ni2+-NTA-DOGS lipids were dispersed in Tris buffer (0.5 mg/mL of DSPC) and incubated at 70 oC for 2 hrs with periodic shaking to form MLVs. The MLVs suspension was then extruded through a polycarbonate filter with a pore size of 100 nm 11 times to yield a SUVs clear solution. After that, 100 µL of the SiO2 NPs (2 mg/mL in water) dispersion was mixed with 500 µL of the Ni2+ containing SUVs solution, incubated at 70 oC for 2 hrs with successive shaking, and then allowed to cool to room temperature. Finally, the Ni2+ labelled SSLBMs-SiO2 NPs were centrifuged at 3000 rpm and re-dispersed in Tris buffer. The centrifuge/washing steps were repeated 3 times. Synthesis of thiol-functional pentameric cholera toxin B subunits (p-CTB). Cholera toxin (CT) consists of five B subunits (molecular weight of 10600) and two peptides linked by a single disulfide bond (A1-S-S-A2). The molecular weights of A1 and A2 are 23000 and 5500, respectively. Each B subunit also contains one internal disulfide bond which is stable unless it is reduced at high temperature in urea. We selectively reduced the A1-S-S-A2 disulfide bond at room temperature to obtain thiol-functional p-CTB following the previously reported method.25 Typically, 12 µL of CT water solution (1 mg/mL) and 20 µL of dithiothreitol (DTT, 10 mM) were added into Tris Buffer (pH=9) containing 1mM of ethylene diaminete traacetic acid (EDTA). This mixture was then incubated for one hour at room temperature. Finally, the mixture


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was dialyzed against Tris buffer for 1 week. Polyacrylamide gel experiments (Fig. S1 in SI) show that the reduced CT exhibit two main peaks attributed to componets A1 and p-CTB with A2, which is consistent with the previous results. Preparation of Protein oligomer coated NPs. In the text, p-CTB and t-HA coated SiO2 NPs are called p-CTB@SiO2 and t-HA@SiO2 NPs for short, respectively. P-CTB@SiO2 NPs were generated by attaching thiol-functional p-CTB onto maleimide-labeled SiO2 NPs via maleimidemediated conjugation under near neutral conditions (Scheme S1). Typically, 100 µL of maleimide-labeled SiO2 NPs suspension (0.2 mg/mL in water) was mixed with excess (1 mL) of thiol-functional p-CTB in Tris buffer (10 µg/mL) and incubated at 4 oC overnight. The suspension was then centrifuged at 3000 rpm and re-suspended in Tris buffer. The centrifuge/washing steps were repeated 3 times to obtain purified p-CTB@SiO2 NPs. THA@SiO2 NPs were generated by mixing 1 mL of polyhistidine-tagged t-HA in Tris buffer (10 µg/mL) with 100 µL of Ni2+-labeled SiO2 NPs suspension (0.2 mg/mL in water) (Scheme S2). This conjugation is highly efficient via the chelated effect between polyhistidine groups of t-HA and Ni2+ ions on NPs surface. The t-HA@SiO2 NPs were also purified through 3 times centrifuge/washing steps. 0.3 mol% of maleimide or Ni2+ functionality in SLBs yields that the inter-distance of functional groups for anchoring protein oligomers on SiO2 NPs is ~12 nm. Since the maleimide- and Ni2+-mediated conjugation is highly efficient, the inter-distance of immobilized protein oligomers on SiO2 NPs is estimated to be ~12 nm. Fluidic glycolipid membranes. The glycolipid SLBs platforms were generated on freshly cleaned SiO2 substrates mounted with a home-made holder apparatus. Briefly, the mixture of DOPC and glycolipid (GM1 or GM3) were prepared in CHCl3, dried, and re-suspended in Tris buffer to a concentration of 0.5 mg/mL. The content of glycolipid was quantitatively adjusted


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over 4 orders of magnitude (10-5–10-1 mol%). Then, the suspension of the lipids was filtered through a polycarbonate filter with 100 nm pores 11 times to yield a small unilamellar vesicles (SUVs) clear solution. Finally, the SUVs solution was deposited onto the freshly cleaned SiO2 substrates and incubated for one hour at room temperature under controlled humidity. The excess vesicle above the SLBs were removed from the SiO2 substrates by extraction with fresh Tris buffer 10 times from the holder apparatus. Total internal reflection fluorescence microscopy (TIRFM). TIRFM experiments were carried out at room temperature on a commercial instrument based on an inverted microscope (Nikon, Ti-S). A green He-Ne laser (λexc=532 nm) was directed into a 100X objective lens (Nikon, PlanApo TIRF, NA=1.45) with a higher angle than the critical angle and fluorescence images were acquired on an EM-CCD camera (Andor, iXon+). The penetration depth of an evanescent wave was estimated to be ~100 nm and it allowed us to selectively probe fluorescent NPs located near the glass-water interface. A series of fluorescence images were acquired. We obtained single NPs trajectories (positions vs. time) from the time-dependent images using the NIS-elements software (Nikon). The time-dependent fluorescence images (80×80 µm2) are shown in movie formats, with time steps per frame of 100 ms on each surface. Results and Discussion Generation of protein oligomer coated NPs. To generate protein oligomer coated NPs, we hybridized protein oligomers onto SSLBMs formed on SiO2 NPs. We chose SSLBMs coated SiO2 NPs for three reasons. First, SiO2 NPs are ideal for interfacing with biological systems due to their chemical inertness and biocompatibility. Second, SiO2 NPs can be easily and completely coated with a single, fluid SSLBM as a model for virus interfaces. Third, a surface modification on SSLBMs coated SiO2 NPs allows a direct protein immobilization without changing the


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affinity characteristics of the protein.26-28 In this work, we chose SSLBMs composed of DSPC and 0.2 mol% of TR-DHPE on ~150 nm (in diameter) SiO2 NPs to prepare fluorescent SiO2 NPs with mechanical stability.24 By introducing maleimide-PEG-DSPE or Ni2+-NTA-DOGS lipids into the DSPC membrane, the fluorescent SiO2 NPs were labelled with maleimide group and Ni2+ ion to anchor thiol-tagged p-CTB and polyhistidine-tagged t-HA protein oligomers, respectively.29,30 These readily adaptable procedures make the anchored p-CTB and t-HA present their glycan binding sites protruding to the solution without changing the affinity characteristics of the protein. Construction of fluidic glycolipid membranes. To achieve a fluidic glycolipid membrane, we incorporated glycolipids (GM1 or GM3) into a small unilamellar vesicles (SUVs) solution of dioleoyl-phosphatidylcholine (DOPC) and formed supported lipid bilayers (SLBs) on planar glass cover slips, with precisely known glycan densities on SLBs.31 The density of glycans on SLBs surface was precisely tuned by varying the concentration of glycolipids in the SUVs over at least 4 orders of magnitude. The fluidity of the SLBs was verified by tracking the twodimensional (2D) diffusivity of lipid molecules through TIRFM measurements (Fig. S2 in SI). p-CTB/GM1 multivalency. As a representative of multivalent protein/glycan interactions, bacterial pathogen Vibrio cholerae utilizes the well-known cell receptor cholera toxin (CT) to bind the GM1 glycan present on mammalian cells.32 High-resolution structures of CT indicate that one CT consists of five CTB subunits (p-CTB) and can bind up to five GM1 glycan moieties. We first investigated the adsorption of p-CTB@SiO2 NPs on GM1 containing DOPC SLBs by carrying out TIRFM experiments for each SLBs surface in contact with a 3.6 pM solution of pCTB@SiO2 NPs. This experiment allows us to study the dynamics of adsorbed NPs by distinctly tracking the trajectory of single NPs on the SLBs (Fig. 1a). From the time-dependent fluorescent


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Figure 1. (a) The single molecule trajectories (40 ms per step) and (b) mean squared displacement (MSD) as a function of time (t) for p-CTB@SiO2 NPs on neat DOPC and 0.1 mol% GM1 containing DOPC SLBs. (b) French grey lines represent for each of 100 trajectories. Symbols are the average MSD over all trajectories. Solid lines are linear fits to MSD=4Dt, where D is the two-dimensional diffusion coefficient.

images, we obtained the trajectories of positions of single adsorbed NPs as a function of time. The 2D diffusion coefficients (D) of NPs can be obtained from the trajectories by fitting the mean square displacement (MSD) against the elapsed time (t), i.e., MSD=4Dt (Fig. 1b). To provide more quantitative results on the diffusion measurements, a large number of more than ~103 single NPs trajectories were tracked to calculate the D distribution of adsorbed NPs. Our TIRFM results show that p-CTB@SiO2 NPs are trapped on GM1 containing DOPC SLBs within small local areas with long surface residence time (>60 s). The apparent D is estimated to be ~0.05±0.02 µm2/s. In contrast, p-CTB@SiO2 NPs on neat DOPC SLBs exhibit noticeable translational movement (D~1.6±0.6 µm2/s) with very short surface residence time (~1 s). This D value is closed to the theoretical value of 0.4 µm2/s on SLBs determined by the classical hydrodynamic interaction regime. 33 These results indicate that the translational motion of pCTB@SiO2 NPs dragged by the hydrodynamic interaction with DOPC lipids is frozen by the


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multivalent p-CTB/GM1 interaction. Counting the number of trapped p-CTB@SiO2 NPs on SLBs enables quantitative analysis of p-CTB@SiO2 NPs binding isotherms caused by pCTB/GM1 multivalency.


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Figure 2. (a–d) Total internal reflection fluorescence microscope images of p-CTB@SiO2 NPs trapped on DOPC SLBs containing 0.001, 0.01, 0.1 and 0.5 mol% GM1 glycolipid after a fixed time of 300, 300, 180 and 100s, respectively. The trapped NPs appears as spherical spots, as illustrated by yellow arrows. Scale bar: 20 µm. (e) Surface densities of adsorbed p-CTB@SiO2 NPs on GM1 containing DOPC SLBs as a function of time. Black solid curves present for Langmuir isotherm based fits.

Fig. 2a–2d show four representative images of p-CTB@SiO2 NPs adsorbed on 0.001, 0.01, 0.1 and 0.5 mol% GM1 containing DOPC SLBs, respectively, with each binding kinetics presented in Fig. 2e. The binding processes were stopped after a fixed time (300, 300, 180, and 100 s for cases in Fig. 2a–2d, respectively) until the binding curve plateaus as a steady state were reached. The kinetics are in agreement with the first-order exponential model, suggesting that pCTB@SiO2 NPs binding proceeds via the Langmuir mechanism. Fig. 3 summarizes quantitatively the steady density of adsorbed p-CTB@SiO2 NPs as a function of GM1 density on DOPC SLBs. As can be seen, the binding curve is characterized by two distinct regions. At low GM1 density (≤6×10-4 nm-2), the NPs density increases with the increase of GM1 density and saturates at ~0.004 NPs/µm2. At high GM1 density (>6×10-4 nm-2), the NPs density rises again


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Figure 3. Surface densities of adsorbed p-CTB@SiO2 NPs on GM1 containing DOPC SLBs as a function of GM1 density on the SLBs. Black solid curve presents fit to Eq. (3). Inset: schematic illustration of the hierarchy of multivalent adhesion of p-CTB@SiO2 NPs to the SLBs.

and saturates at a higher value of ~0.02 NPs/µm2. We attribute this bimodal binding curve to two types of NPs adsorption with different p-CTB/GM1 valency. The quantitative analysis of the binding isotherms is carried out using a simple Langmuir adsorption model as: (1)

(2) where [C]=2.16×109 NPs/mL =3.6 pM (1pM=10-12 M) is the concentration of p-CTB@SiO2 NPs in the solution; θg is the surface density of glycan; θ1 and θ2 are the surface densities of pCTB@SiO2 NPs with p-CTB/GM1 valency of n1 and n2, respectively. θ10 and θ20 are the saturation densities of the two valency types of adsorbed p-CTB@SiO2 NPs. Each term in parentheses represents the available surface sites for NPs binding. K1 and K2 are the equilibrium


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constants. Based on Eqs. (1) and (2), the total density (θ=θ1+θ2) of adsorbed p-CTB@SiO2 NPs is given by: (3)

Fitting (solid curve in Fig. 3) to Eq. (3) yields n1=1.2±0.2 and n2=5.4±1.0, with θ10=0.004±0.002 µm-2 and θ20=0.015±0.005 µm-2, respectively. This quantitative analysis distinguishes two types of p-CTB@SiO2 NPs binding processes: one NP binding to one GM1 moiety via a monovalent pCTB/GM1 interaction and one NP binding to ~5 GM1 moieties via a multivalent p-CTB/GM1 interaction. To study the interaction strength of monovalent and multivalent p-CTB/GM1 interactions, we further evaluated the dissociation constants (

) of each binding process. For

-1

Kdi =(Kiθng i)

example, at θg=6×10-4 nm-2 (low GM1 density region), Kd1=5.5×10-13 M and Kd2=6.6×10-10 M. Here, the monovalent p-CTB/GM1 interaction is 103 times more effective than multivalent pCTB/GM1 interaction. At high GM1 density region (θg=7×10-3 nm-2), Kd1=4.3×10-14 M and Kd2=1.7×10-16 M, where the multivalent p-CTB/GM1 interaction dominates. t-HA/GM3 multivalency. As another representative of protein/glycan multivalency, the viral t-HA protein oligomers of the influenza A virus bind specifically to the terminal Nacetylneuraminic acid (=SA) on glycan receptors of host epithelial cells. The recent outbreaks in human populations of the H1N1 swine virus and the H7N9 avian virus underscore the critical need for quantitative analysis of t-HA/glycan multivalency.17,18 The model system we chose is the recombinant t-HA from pathogenic H7N9 influenza A virus (A/Shanghai/1/2013) that binds preferentially to α2-3 linked SA, e.g., trisaccharide Neu5Ac-α2-3-Gal-β1-4-Glc like GM3.34 In solution, this recombinant HA proteins are present mostly in the trimeric form (t-HA) as on the virus surface.


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Figure 4. (a) Surface densities of adsorbed t-HA@SiO2 NPs on GM3 containing DOPC SLBs as a function of GM3 density on the SLBs. Black solid curve presents fit to Eq. (3). (b) Surface densities of adsorbed t-HA@SiO2 NPs on DOPC SLBs containing 0.1 mol% GM3 as a function of solution phase glycopolymer (inhibitor) concentrations.

The binding curve for t-HA@SiO2 NPs on GM3 SLBs in Fig. 4a is also characterized by two distinct Langmuir adsorption channels, which are attributed to monovalent and multivalent tHA/GM3 interactions. Fitting (solid curve in Fig. 4a) to Eq. (3) yields n1=1.3±0.3 and n2=9.0±1.0, with θ10=0.012±0.005 µm-2 and θ20=0.035±0.003 µm-2, respectively. This suggests a monovalent channel involving binding to one GM3 moiety per t-HA@SiO2 NPs and a multivalent channel corresponding to binding to ~9 GM3 moieties per t-HA@SiO2 NPs. At low GM3 density region (θg=6×10-4 nm-2), the monovalent binding channel is more effective than the multivalent binding channel with the familiar Kd1(=1.7×10-12 M) < Kd2(=3.0×10-11 M). When the GM3 density is at the high region (θg=9×10-4 nm-2), Kd1(=1.1×10-12 M) > Kd2(=5.0×10-13 M) are obtained. Here, the multivalent t-HA/GM3 interaction dominates. The t-HA/GM3 multivalency can be effectively inhibited by a glycopolymer presenting multiple copies of Neu5Ac-α2-3-Gal-β1-4-Glc groups. Here, each polymer chain contains 4 α2-3 linked SA moieties provided by the manufacturer. The glycopolymer was added to the tHA@SiO2 NPs solution as the competitive inhibitor for their binding to GM3 SLBs. Fig. 4b


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shows quantitatively the surface density of adsorbed t-HA@SiO2 NPs on SLBs containing 0.1 mol% GM3 as a function of glycopolymer concentrations in the solution. The surface density of adsorbed t-HA@SiO2 NPs decreases from ~0.032 µm-2 to ~0.015 µm-2 with increasing glycopolymer concentration to ~1.5 nM. Here, the multivalent t-HA/GM3 interaction is inhibited, but the monovalent channel is not. As the polymer concentration is further increased to ~4.5 nM, the surface density of adsorbed t-HA@SiO2 NPs decreases to ~0.005 µm-2, indicating that the monovalent t-HA/GM3 interaction is also inhibited within experimental uncertainty. These results show that our method can quantitatively screen for monovalent and multivalent inhibitors by precisely varying the density of surface glycan moieties on fluidic glycan SLBs. A detailed study of the inhibitory efficacy on the binding between virus and cell membranes by a series of anti-viral agents is of definite interest and deserves future study. The dynamic glycan SLBs with precisely varying density of surface glycan moieties over more than 4 orders of magnitude allow us to quantify the different valency of multivalent protein/glycan interactions. On fluidic SLBs at low glycan density, weak and monovalent binding occurs. Only one protein oligomer on NPs binds to one glycan moiety (n1=1 in Figs. 3 and 4) at every instant of experimental time. As the surface glycan density reaches above a critical value, the multivalent protein/glycan interaction is turned on, and the relative valency n2 is ~5 and ~9 for p-CTB/GM1 and t-HA/GM3 multivalency, respectively. As for p-CTB@SiO2 NPs, one p-CTB@SiO2 NP binds to ~5 GM1 moieties. Previous studies indicate that the binding constant of p-CTB with the first GM1 ligand is lower by a factor of 4 than that with the second GM1 ligand. 35 That is to say p-CTB/GM1 interaction is a positively cooperative multivalent interaction where the binding of p-CTB with GM1 ligand is enthalpically enhanced by that of subsequent events. With this in mind, we can deduce that each p-CTB preferentially binds to a


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cluster of five GM1 moieties on the fluidic SLBs surface. That is to say the adsorption of pCTB@SiO2 NPs is determined by the hierarchy of multivalency due to the possibility of one pCTB oligomer per NPs (see the inset of Fig. 3). Note that the inter-GM1 distance at the critical density (6×10-4 nm-2) is ~40 nm, 4 times longer than the inter-p-CTB distance (~12 nm) on NPs. This indicates that it is the mobility of the glycan moiety that allows the occurrence of multivalent p-CTB/GM1 interaction. As for t-HA@SiO2 NPs, our results show that one NP binds ~9 GM3 moieties. In principle, one t-HA has three binding domains with specificity for GM3 and can bind 3 GM3 moieties. However, this may be not true in reality. High-resolution structures of t-HA indicate that the inter-binding site distance is ~2 nm at the tip of the trimer.19 To allow for multivalent interaction with 3 GM3 moieties, the GM3 must also cluster to a similar short dimension, which will likely introduce the steric hindrance that reduces the binding valency (from trivalent to divalent or monovalent) and cooperativity, thus decreasing the multivalent affinity. Because of no statement regarding the cooperativity for t-HA/GM3 multivalency, it is reasonable to conclude that t-HA@SiO2 NPs binding is determined by the hierarchy of multivalency due to the number of ~3–9 t-HA oligomers per NPs. As a future research direction, we will address this hierarchy and cooperativity of multivalency by using inactive influenza virus nanoparticles as model systems. Conclusions In conclusion, we apply dynamic self-assembly of glycolipids in fluidic SLBs to quantitatively profile and characterize the binding processes of protein oligomer coated NPs using two model systems, the adhesions of p-CTB to GM1 and t-HA to GM3 glycans. This method allows us to mimic the virion adhesion on cell membranes and determine the monovalent and multivalent protein/glycan binding channels. Our results show that protein oligomers on NPs surface


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Langmuir

switches glycan affinity from monovalent to multivalent as the density of mobile glycan moieties increases over 4 orders of magnitude range. The different valency and binding constants for monovalent and multivalent protein/glycan interactions were quantitatively determined by fitting the binding isotherms via a bimodal Langmuir adsorption model. This method was also applied to quantitatively appreciate the inhibition of protein/glycan multivalent interactions by the glycopolymer presenting multiple glycan moieties. This work is essential to mapping and understanding the complex binding specificities of glycan-binding proteins, especially the complex hierarchy of the multivalency, that is one of the greatest challenges in glycomics. Supporting information The Supporting Information is available free of charge on the ACS Publications website. Illustrations of the materials preparation, generation of fluidic glycolipid membranes and the TIRFM characterizations. Author information (L. S.) [email protected] Competing financial interests The authors declare no competing financial interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21574051 and 21404046), the Fundamental Research Funds for the Central Universities (WUT: 2018IVA021) and the Excellent Dissertation Cultivation Project of Wuhan University of Technology (No. 2017-YS-080). References

1

Mammen, M.; Choi, S. K.; Whitesides, G. M. Polyvalent Interactions in Biological Systems:


19

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Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew. Chem. Int. Ed. 1998, 37, 2754-2794. 2

Varki, A. Glycan-based Interactions Involving Vertebrate Sialic-Acid-Recognizing Proteins. Nature 2007, 446, 1023-1029.

3

Van Kooyk, Y.; Rabinovich, G. A. Protein-glucan Interactions in the Control of Innate and Adaptive Immune Responses. Nat. Immunol. 2008, 9, 593-601.

4

Imberty, A.; Varrot, A. Microbial Recognition of Human Cell Surface Glycoconjugates. Curr. Opin. Struc. Biol. 2008, 18, 567-576.

5

Smith, A. E.; Helenius, A. How Viruses Enter Animal Cells. Science 2004, 304, 237-242.

6

Sigal, G. B.; Mammen, M.; Dahmann, G.; Whitesides, G. M. Polyacrylamides Bearing Pendant Alpha-sialoside Groups Strongly Inhibit Agglutination of Erythrocytes by Influenza Virus: The Strong Inhibition Reflects Enhanced Binding Through Cooperative Polyvalent Interactions. J. Am. Chem. Soc. 1996, 118, 3789-3800.

7

Papp, I.; Sieben, C.; Ludwig, K.; Roskamp, M.; Bottcher, C.; Schlecht, S.; Herrmann, A.; Haag, R. Inhibition of Influenza Virus Infection by Multivalent Sialic-Acid-Functionalized Gold Nanoparticles. Small 2010, 6, 2900-2906.

8

Ribeiro-Viana, R.; Sánchez-Navarro, M.; Luczkowiak, J.; Koeppe, J. R.; Delgado, R.; Rojo, J.; Davis, B. G. Virus-like Glycodendrinanoparticles Displaying Quasi-equivalent Nested Polyvalency Upon Glycoprotein Platforms Potently Block Viral Infection. Nat. Commun. 2012, 3, 1303-1311.

9

Zanini, D.; Roy, R. Synthesis of New Alpha-thiosialodendrimers and Their Binding Properties to The Sialic Acid Specific Lectin From Limax Flavus. J. Am. Chem. Soc. 1997, 119, 20882095.

10

Muñoz, A.; Sigwalt, D.; Illescas, B. M.; Luczkowiak, J.; Rodríguez-Pérez, L.; Nierengarten, I.;

Holler, M.; Remy, J. S.; Buffet, K.; Vincent, S. P.; Rojo, J.; Delgado, R.; Nierengarten, J. F.; Martín, N. Synthesis of Giant Globular Multivalent Glycofullerenes as Potent Inhibitors in A Model of Ebola Virus Infection. Nat. Chem. 2016, 8, 50-57. 11

Joshi, A.; Vance, D.; Rai, P.; Thiyagarajan, A.; Kane, R. S. The Design of Polyvalent

Therapeutics. Chem. Eur. J. 2008, 14, 7738-7747.


20

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

12

Shi, J.; Yang, T.; Kataoka, S.; Zhang, Y.; Diaz, A. J.; Cremer, P. S. GM1 Clustering Inhibits

Cholera Toxin Binding in Supported Phospholipid Membranes. J. Am. Chem. Soc. 2007, 129, 5954-5961. 13

Robinson, A.; Fang, J. M.; Chou, P. T.; Liao, K. W.; Chu, R. M.; Lee, S. J. Probing Lectin and

Sperm With Carbohydrate-Modified Quantum Dots. ChemBioChem 2005, 6, 1899-1905. 14

Yu, L.; Huang, M.; Wang, P. G.; Zeng, X. Cross-linked Surface-grafted Glycopolymer for

Multivalent Recognition of Lectin. Anal. Chem. 2007, 79, 8979-8986. 15

Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. Surface Plasmon Resonance

Imaging Studies of Protein-Carbohydrate Interactions. J. Am. Chem. Soc. 2003, 125, 6140-6148. 16

Liang, P. H.; Wang, S. K.; Wong, C. H. Quatitative Analysis of Carbohydrate-Protein

Interactions Using Glycan Microarrays: Determination of Surface and Solution Dissociation Constants. J. Am. Chem. Soc. 2007, 129, 11177-11184. 17

Shi, Y.; Zhang, W,; Wang, F.; Qi, J. X.; Wu, Y.; Song, H.; Gao, F.; Bi, Y.; Zhang, Y. F.; Fan, Z.;

Qin, C. F.; Sun, H. L.; Liu, J. H.; Haywood, J.; Liu, W. J.; Gong, W. M.; Wang, D. Y.; Shu, Y. L.; Wang, Y.; Yan, J. H.; Gao, G. F. Structures and Receptor Binding of Hemagglutinins from Human-Infecting H7N9 Influenza Viruses. Science 2013, 342, 243-247. 18

Srinivasan, A.; Viswanathan, K.; Raman, R.; Chandrasekaran, A.; Raguram, S.; Tumpey, T. M.;

Sasisekharan, V.; Sasisekharan, R. Quantitative Biochemical Rationale for Differences in Transmissibility of 1918 Pndemic Influenza A Viruses. Proc. Natl. Acad. Sci. USA 2008, 105, 2800-2805. 19

Skehel, J. J.; Wiley, D. C. Receptor Binding and Membrane Fusion in Virus Entry: The

Influenza Hemagglutinin. Annu. Rev. Biochem. 2000, 69, 531-569. 20

Cairo, C. W.; Gestwicki, J. E.; Kanai, M.; Kiessling, L. L. Control of Multivalent Interactions

by Binding Epitope Density. J. Am. Chem. Soc. 2002, 124, 1615-1619. 21

Rai, P.; Padala, C.; Poon, V.; Saraph, A.; Basha, S.; Kate, S.; Tao, K.; Mogridge, J.; Kane, R. S.

Statistical Pattern Matching Facilitates The Design of Polyvalent Inhibitors of Anthrax and Cholera Toxins. Nat. Biotech. 2006, 24, 582-586. 22

Zhu, X. Y.; Holtz, B.; Wang, Y.; Wang, L. X.; Orndorff, P. E.; Guo, A. Quantitative Glycomics

From Fluidic Glycan Microarrays. J. Am. Chem. Soc. 2009, 131, 13646-13650.


21

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

23

Page 22 of 23

Green, D. L.; Lin, J. S.; Lam, Y. F.; Hu, M. Z.; Schaefer, D. W.; Harris, M. T. Size, Volume

Fraction, and Nucleation of Stober Silica Nanoparticles. J. Colloid Interface Sci. 2003, 266, 346-358. 24

Ahmed, S.; Wunder, S. L. Effect of High Surface Curvature on The Main Phase Transition of

Supported Phospholipid Bilayers on SiO2 Nanoparticles. Langmuir 2009, 25, 3682-3691. 25

Gill, D. M. Arrangement of Subunits in Cholera Toxin. Biochemistry 1976, 15, 1242-1248.

26

Loidl-Stahlhofen, A.; Schmitt, J.; Nöller, J.; Hartmann, T.; Brodowsky, H.; Schmitt, W.;

Keldenich, J. Solid-Supported Biomolecules on Modified Silica Surfaces – A Tool For Fast Physicochemical Characterization and High-Throughput Screening. Adv. Mater. 2001, 13, 1829-1834. 27

Conway, J. W.; Madwar, C.; Edwardson, T. G.; McLaughlin, C. K.; Fahkoury, J.; Lennox, R.

B.; Sleiman, H. F. Design Behavior of DNA Cages Anchored on Spherically Supported Lipid Bilayers. J. Am. Chem. Soc. 2014, 136, 12987-12997. 28

Fu, R.; Gill Jr., R. L.; Kim, E. Y.; Briley, N. E.; Tyndall, E. R.; Xu, J.; Li, C.; Ramamurthi, K.

S.; Flanagan, J. M.; Tian, F. Spherical Nanopaticle Supported Lipid Bilayers For The Structural Study of Membrane Geometry-Sensitive Molecules. J. Am. Chem. Soc. 2015, 137, 1403114034. 29

Merrill, S. A.; Hanson, P. I. Activation of Human VPS4A by ESCRT-III Protein Reveals

Ability of Substrates to Relieve Enzyme Autoinhibition. J. Biol. Chem. 2010, 285, 3542835438. 30

Mantovani, G.; Lecolley, F.; Tao, L.; Haddleton, D. M.; Clerx, J.; Cornelissen, J. J. L. M.

Design and Synthesis of N-maleimido-functionalized Hydrophilic Polymers via CopperMediated Living Radical Polymerization: A Suitable Alternative to PEGylation Chemistry. J. Am. Chem. Soc. 2005, 127, 2966-2973. 31

Shen, L.; Wang, Y.; Lin, C. I.; Liu, H. W.; Guo, A.; Zhu, X. Y. Membrane Enviroment Can

Enhance The Interaction of Glycan Binding Protein to Cell Surface Glycan Receptors. ACS Biol. Chem. 2014, 9, 1877-1884. 32

Fan, E.; Merritt, E. A.; Verlinde, C. L. M.; Hol, W. G. J. AB5 Toxins: Structures and Inhibitors

Design. Curr. Opin. Struc. Biol. 2000, 10, 680-686.


22

Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

33

Saffman, P. G.; Delbrück, M. Brownian-Motion in Biological-Membranes. Proc. Natl. Acad.

Sci. U.S.A. 1975, 72, 3111-3113. 34

Watanabe, T.; Kiso, M.; Fukuyama, S.; Nakajima, N.; Imai, M.; Yamada, S.; Murakami, S.;

Yamayoshi, S.; Iwatsuki-Horimoto, K.; Sakoda, Y.; Takashita, E.; McBrige, R.; Noda, T.; Hatta, M.; Imai, H.; Zhao, D. M.; Kishida, N.; Shirakura, M.; de Vries, R. P.; Shichinohe, E.; Ishikawa, I.; Watanabe, S.; Ito, M.; Sakai-Tagawa, Y.; Sugita, Y.; Uraki, R.; Yamaji, R.; Eisfeld, A.; Zhong, G. X.; Fan, S. F.; Ping, J. H.; Maher, E. A.; Hanson, A.; Uchida, Y.; Saito, T.; Ozawa, M.; Neumann, G.; Kida, H.; Odagiri, T.; Paulson, J. C.; Hasegawa, H.; Tashiro, M.; Kawaoka, Y. Characterization of H7N9 Influenza A Viruses Isolated From Humans. Nature 2013, 501, 551555. 35

Schoen, A.; Freire, E. Thermodynamics of Intersubunit Interactions in Cholero-Toxin Upon

Binding to The Oligosaccharide Protion of Its Cell-Surface Receptor, Ganglioside-GM1. Biochemitry 1989, 28, 5019-5024.


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Quantification of Multivalency in Protein Oligomer ... - ACS Publications

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