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. 2012 Oct 30;23(3):276–285. doi: 10.1093/glycob/cws141

Affinities of recombinant norovirus P dimers for human blood group antigens

Ling Han 2, Pavel I Kitov 2, Elena N Kitova 2, Ming Tan 3,4, Leyi Wang 3, Ming Xia 3, Xi Jiang 3,4, John S Klassen 2,1
PMCID: PMC3555502  PMID: 23118206

Abstract

Noroviruses (NoVs), the major cause of viral acute gastroenteritis, recognize histo-blood group antigens (HBGAs) as receptors or attachment factors. To gain a deeper understanding of the interplay between NoVs and their hosts, the affinities of recombinant P dimers (P2's) of a GII.4 NoV (VA387) to a library of 41 soluble analogs of HBGAs were measured using the direct electrospray ionization mass spectrometry assay. The HBGAs contained the A, B, H and Lewis epitopes, with variable sizes (2–6 residues) and different types (1–6). The results reveal that the P2's exhibit a broad specificity for the HBGAs and bind to all of the oligosaccharides tested. Overall, the affinities are relatively low, ranging from 400 to 3000 M−1 and are influenced by the chain type: 3 > 1 ≈ 2 ≈ 4 ≈ 5 ≈ 6 for H antigens; 6 > 1 ≈ 3 ≈ 4 ≈ 5 > 2 for A antigens; 3 > 1 ≈ 4 ≈ 5 ≈ 6 > 2 for B antigens, but not by chain length. The highest-affinity ligands are B type 3 (3000 ± 300 M−1) and A type 6 (2350 ± 60 M−1). While the higher affinity to the type 3 H antigen was previously observed, preferential binding to the types 6 and 3 antigens with A and B epitopes, respectively, has not been previously reported. A truncated P domain dimer (lacking the C-terminal arginine cluster) exhibits similar binding. The central-binding motifs in the HBGAs were identified by molecular-docking simulations.

Keywords: affinity, electrospray ionization mass spectrometry, histo-blood group antigens, norovirus, receptor

Introduction

Noroviruses (NoVs), a group of single-stranded, positive-sense RNA viruses in the Calciviridae family, are the major viral pathogens responsible for epidemic acute gastroenteritis in both developed and developing countries. Each year, the viruses infect roughly 20 million people (Lindesmith et al. 2003; Tan and Jiang 2007), resulting in approximately 200,000 deaths (Patel et al. 2008). Currently, there is no effective vaccine or antiviral against NoV infections. Human NoVs can be divided into two major genogroups (GI and GII), which contain at least 25 different genotypes (GI.1–8 and GII.1–17; Tan and Jiang 2005a). The GII.4 is the predominant genotype worldwide causing ∼80% of NoV gastroenteritis outbreaks (Johnston et al. 2007; Tan and Jiang 2007; Cannon et al. 2009; Yang et al. 2010; Baert et al. 2011).

The absence of an in vitro cell culture system or a suitable animal model has hindered the characterization of NoVs. Consequently, efforts have focused on the recombinant virus-like particles (VLPs). In vitro expression of NoV VP1, the major capsid protein, through recombinant baculoviruses results in the spontaneous assembly of VLPs that are structurally and antigenically indistinguishable from the authentic viruses (Jiang et al. 1992). X-ray crystallography analysis of Norwalk virus VLPs revealed that each of VP1 contains two major domains, the N-terminal shell (S) domain and the C-terminal protrusion (P) domain, linked by a flexible hinge (Prasad et al. 1999). The S domain forms the interior shell of the capsid, while the P domain is responsible for the exterior P dimer (P2) formation. The P domain exhibits high sequence variability and is important for host–receptor interactions and for the host immune response (Cao et al. 2007; Tan, Fang, Chachiyo, et al. 2008). On its own, the P domain forms homodimers called the P2's (Tan et al. 2004). The P2's can further assemble into larger complexes, a 12-mer small P particle (Tan, Fang, Xia, et al. 2011) and a 24-mer P particle (Tan and Jiang 2005a; Tan, Xia, et al. 2008; Bereszczak et al. 2012). In addition, a soluble P protein in the stool of NoV-infected patients, referred as P polypeptide, has been reported (Greenberg et al. 1981; Hardy et al. 1995) and contains most of the P domain, but lacks the highly conserved arginine (Arg) cluster at the C-terminus and forms a homodimer (Tan et al. 2006; Bu et al. 2008).

NoVs recognize the human histo-blood group antigens (HBGAs; Huang et al. 2003, 2005; Tan and Jiang 2005b), which play an important role in host susceptibility of NoV. The HBGAs are complex carbohydrates that consist of oligosaccharides covalently linked to proteins or lipids. They are generally present on red blood cells, mucosal epithelia or as free antigens in body fluids, such as blood, saliva, milk and the intestinal contents (Oriol 1990). Although the HBGA phenotype is determined by the terminal part of the oligosaccharide chain linked to protein or lipid, the antigen determinants can be associated with different carbohydrate structures, i.e. precursor chain types. There are six possible types of precursor chains (Oriol 1990). Of these, types 1–4 are widely distributed in red blood cells, mucosal epithelia and different organs (Ravn and Dabelsteen 2000), whereas type 6 chain mainly exists in milk and urine (Oriol 1990). The type 5 structure has not been detected in human tissue or secretions. At present, the biological significance of the different HBGA chain types is not fully understood. The carbohydrate moieties of the HBGAs represent the minimum epitope for NoV recognition (Huang et al. 2005; Tan, Xia, et al. 2008; de Rougemont et al. 2011; Fiege et al. 2012). NoVs recognize the human HBGAs in a strain-specific manner and distinct NoV–HBGA-binding patterns have been described (Huang et al. 2005). The HBGA-binding sites of NoVs are located at the P2 interface, and the recombinant P2's are shown to be structurally the same as those of VLP (Cao et al. 2007; Choi et al. 2008; Chen et al. 2011). Thus, the recombinant P2 and its complex form, the P particle, have been used as models for NoV–HBGA interactions extensively (Tan et al. 2004, 2006, 2009; Tan and Jiang 2005b; Tan, Fang, Chachiyo, et al. 2008; Tan, Xia, et al. 2008; Tan, Fang, Xia, et al. 2011; Tan, Huang, et al. 2011; Tan and Jiang 2012).

At present, there are few quantitative data available for the interactions between the NoV VLPs and HBGAs. Peters and coworkers recently investigated such interactions using saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy (Fiege et al. 2012). l-fucose (l-fuc) was identified as the minimal structure recognized by a GII.4 VLP (Ast6139), and the association constants (Ka) of VLP and the HBGA fragments containing α-l-Fuc are, at best, ∼104 M−1. In another study, affinities of 2.6 × 103 and 2.2 × 103 M−1 were measured for a GII.10 P2 binding to H type 2 trisaccharide and l-fuc, respectively (Hansman et al. 2012).

Here, we describe the first quantitative study of the interactions between GII.4 P2's, in both their full-length and truncated forms, with HBGA oligosaccharides using the direct electrospray ionization mass spectrometry (ESI-MS) assay (Wang et al. 2003). The affinities of both P2's for a library of 41 HBGA oligosaccharides, comprising A, B, H and Lewis antigens, were measured at 25°C and pH 7. In addition, molecular-docking simulations were performed to elucidate the structural basis for the trends in the measured affinities.

Results and discussion

HBGA affinities

The direct ESI-MS assay was performed to test for specific binding between the P2 and the tr-P2 and each of the 41 HBGA oligosaccharides and to quantify their affinities at pH 7 and 25°C. Shown in Figure 1 are ESI mass spectra acquired for an aqueous ammonium acetate (10 mM) solution of the P2 (12 μM) and of the tr-P2 (12 μM). From the mass spectra, it can be seen that the recombinant NoV VA387 P domain exists predominantly as a dimer (i.e. P2) under these solution conditions, with only protonated P2n+ ions detected. The measured MW of 69 312 ± 2 Da of the P2 is in good agreement with the theoretical value of 69 311 Da. The truncated P domain also forms a dimer (i.e. tr-P2) under these conditions. However, the mass spectrum reveals evidence of three different protein species. In addition to signal for the protonated ions (tr-P2n+) of the expected tr-P2 (measured MW 69 004 ± 2 Da, theoretical MW 69 006 Da), ions corresponding to protonated ions of proteins with MW of 68 763 ± 8 Da and 69 160 ± 10 Da were detected. As illustrated in Supplementary data, Figure S3, the three isoforms of tr-P2 exhibit similar affinities for the HBGA.

Fig. 1.

Fig. 1.

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Direct ESI-MS analysis of NoV VA387 P2 and tr-P2 at pH 7 and 25°C. Representative mass spectra acquired for 10 mM ammonium acetate solution with (A) P2 (12 μM) and (B) tr-P2 (12 μM).

Shown in Figure 2 are typical ESI mass spectra measured for aqueous ammonium acetate solutions (10 mM) with P2 (12 μM) and 20, 70 and 100 μM A type 6 tetrasaccharide (L17), respectively. The scFv (10 μM), which served as Pref, was present in all of the ESI solutions used for affinity measurements. According to the ESI-MS data, the P2 binds up to two molecules of L17, i.e. (P2 + qL17)n+, where q = 0–2 and n = 15–18. Signals corresponding to unbound and bound Pref ions were also detected, i.e. (Pref + qL17)n+, where q = 0–2 and n = 9–11, which indicates that nonspecific binding of P2 to L17 occurred during the ESI process. As seen in Figure 2D–F, after correction for nonspecific binding, no ions corresponding to the specific (P2 + 2L17) complex were identified (El-Hawiet et al. 2011). Therefore, the P2 binds to a single molecule of L17 under these solution conditions, with a Ka of 2400 ± 150 M−1. A summary of the Ka values obtained for the P2 binding to each of the 41 HBGA oligosaccharides is listed in Table I.

Fig. 2.

Fig. 2.

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ESI mass spectra acquired in positive-ion mode for aqueous ammonium acetate solutions (10 mM) at pH 7 and 25°C containing NoV VA387 P2 (12 μM), (A) 20 μM L17 (A type 6 tetrasaccharide, MW 691 Da), (B) 70 μM L17 and (C) 100 μM L17. A Pref (10 μM) was added to each solution to correct the mass spectra for the occurrence of nonspecific carbohydrate–protein binding during ESI process. Normalized distribution of L17 [at concentrations of (D) 20 μM, (E) 70 μM and (F) 100 μM] bound to the P2 before and after correcting the ESI mass spectra [shown in (A), (B) and (C)] for the nonspecific ligand binding.

Table I.

Apparent association constants, Ka (M−1) for the binding of the HBGA oligosaccharides (L1–L41) and HMOs (L42–L43) with the NoV VA387 P2 and tr-P2, measured at 25°C and pH 7 by the direct ESI-MS assaya

HBGA P2 tr-P2
L1 H disaccharide α-l-Fuc-(1 → 2)-d-Gal 470 ± 70 650 ± 60
L2 H trisaccharide type 1 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)-β-d-GlcNAc-OC8H15 500 ± 90 460 ± 70
L3 H trisaccharide type 2 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 4)-d-GlcNAc 400 ± 50 300 ± 60
L4 H trisaccharide type 3 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)-α-d-GalNAc-OC8H15 1300 ± 130 1100 ± 300
L5 H trisaccharide type 5 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)-β-d-Gal-OC8H15 700 ± 100 790 ± 90
L6 H trisaccharide type 6 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 4)-d-Glc 680 ± 70 [650 ± 50]b 500 ± 110
L7 H tetrasaccharide type 1 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)-β-d-GlcNAc-(1 → 3)-d-Gal 630 ± 30 850 ± 50
L8 H tetrasaccharide type 2 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 4)-β-d-GlcNAc-(1 → 4)-d-Gal 420 ± 80 490 ± 60
L9 H tetrasaccharide type 4 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)-β-d-GalNAc-(1 → 3)-d-Gal 570 ± 40 600 ± 100
L10 H pentasaccharide type 1 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)-β-d-GlcNAc-(1 → 3)-β-d-Gal-(1 → 4)-d-Glc 610 ± 70 710 ± 80
L11 H pentasaccharide type 2 α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 4)-β-d-GlcNAc-(1 → 3)-β-d-Gal-(1 → 4)-d-Glc 500 ± 40 540 ± 80
L12 A trisaccharide α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-d-Gal 850 ± 90 690 ± 30
L13 A tetrasaccharide type 1 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-GlcNAc-OC8H15 1200 ± 130 1200 ± 200
L14 A tetrasaccharide type 2 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 4)-β-d-GlcNAc-OC8H15 570 ± 60 680 ± 80
L15 A tetrasaccharide type 3 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-α-d-GalNAc-OC8H15 1600 ± 100 1100 ± 130
L16 A tetrasaccharide type 5 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-Gal-OC8H15 1120 ± 80 1020 ± 50
L17 A tetrasaccharide type 6 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 4)-d-Glc 2400 ± 150 [2350 ± 60]b 1900 ± 160
L18 A pentasaccharide type 1 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-GlcNAc-(1 → 3)-d-Gal 1300 ± 100 830 ± 70
L19 A pentasaccharide type 2 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 4)-β-d-GlcNAc-(1 → 3)-d-Gal 560 ± 30 430 ± 90
L20 A pentasaccharide type 4 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-GalNAc-(1 → 3)-d-Gal 1040 ± 60 1000 ± 110
L21 A hexasaccharide type 1 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-GlcNAc-(1 → 3)-β-d-Gal-(1 → 4)-d-Glc 1200 ± 120 1200 ± 150
L22 A hexasaccharide type 2 α-d-GalNAc-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 4)-β-d-GlcNAc-(1 → 3)-β-d-Gal-(1 → 4)-d-Glc 620 ± 70 530 ± 90
L23 B trisaccharide α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-OC2H5 1230 ± 90 1200 ± 200
L24 B tetrasaccharide type 1 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-GlcNAc-OC8H15 1300 ± 200 1100 ± 200
L25 B tetrasaccharide type 2 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 4)-β-d-GlcNAc-OC8H15 820 ± 90 800 ± 110
L26 B tetrasaccharide type 3 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-α-d-GalNAc-OC8H15 3000 ± 300 2400 ± 200
L27 B tetrasaccharide type 5 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-Gal-OC8H15 1400 ± 260 1090 ± 90
L28 B tetrasaccharide type 6 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 4)-d-Glc 1200 ± 90 [1200 ± 120]b 1100 ± 100
L29 B pentasaccharide type 1 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-GlcNAc-(1 → 3)-d-Gal 1060 ± 70 1220 ± 60
L30 B pentasaccharide type 2 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 4)-β-d-GlcNAc-(1 → 3)-d-Gal 780 ± 30 730 ± 90
L31 B pentasaccharide type 4 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-GalNAc-(1 → 3)-d-Gal 1320 ± 60 1200 ± 100
L32 B hexasaccharide type 1 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 3)-β-d-GlcNAc-(1 → 3)-β-d-Gal-(1 → 4)-d-Glc 1400 ± 100 1300 ± 190
L33 B hexasaccharide type 2 α-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 2)]-β-d-Gal-(1 → 4)-β-d-GlcNAc-(1 → 3)-β-d-Gal-(1 → 4)-d-Glc 860 ± 60 820 ± 80
L34 Lea trisaccharide β-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 4)]-d-GlcNAc 360 ± 50 480 ± 70
L35 Lea tetrasaccharide β-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 4)]-β-d-GlcNAc-(1 → 3)-d-Gal 480 ± 40 590 ± 90
L36 LeX trisaccharide β-d-Gal-(1 → 4)-[α-l-Fuc-(1 → 3)]-d-GlcNAc 550 ± 70 690 ± 80
L37 LeX tetrasaccharide β-d-Gal-(1 → 4)-[α-l-Fuc-(1 → 3)]-β-d-GlcNAc-(1 → 3)-d-Gal 610 ± 50 670 ± 70
L38 Leb tetrasaccharide α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 4)]-d-GlcNAc 630 ± 70 590 ± 90
L39 Leb pentasaccharide α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)-[α-l-Fuc-(1 → 4)]-β-d-GlcNAc-(1 → 3)-d-Gal 500 ± 60 490 ± 60
L40 LeY tetrasaccharide α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 4)-[α-l-Fuc-(1 → 3)]-d-GlcNAc 680 ± 40 690 ± 90
L41 LeY pentasaccharide α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 4)-[α-l-Fuc-(1 → 3)]-β-d-GlcNAc-(1 → 3)-d-Gal 490 ± 80 400 ± 70
L42 3′-siallyllactose α-d-Neu5Ac-(2 → 3)-β-d-Gal-(1 → 4)-d-Glc NBc n.d.d
L43 6′-siallyllactose α-d-Neu5Ac-(2 → 6)-β-d-Gal-(1 → 4)-d-Glc NBc n.d.d
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aUncertainties correspond to one standard deviation.

bValues obtained from the ESI-MS titration experiments.

cNB = no binding detected.

dn.d. = not determined.

Glc, glucose; Neu5Ac, N-acetyl neuraminic acid.

Because the interactions between the P2 and the HBGA oligosaccharides are quite weak, it was desirable to establish the reliability of the ESI-MS-binding protocol used for the measurements. To this end, ESI-MS titration experiments were carried out on a small number of HBGA oligosaccharides (L6, L17 and L28, which represent H, A and B type 6 antigens) using a fixed P2 concentration (12 μM) and seven or more different HBGA concentrations (10–120 μM). Figure 3 shows the plot of the fraction of L17-bound P2 [R/(R + 1)] versus L17 concentrations and the curve of fitting Eq. (4) to the experimental data (after correction for nonspecific binding). Nonlinear fitting yields a Ka value of 2350 ± 60 M−1. In a similar way, Ka values of 650 ± 50 and 1200 ± 120 M−1 were determined for ligands L6 and L28, respectively. Notably, the affinities obtained from the titration experiments are in excellent agreement with the values obtained using a limited number of HBGA oligosaccharide concentrations.

Fig. 3.

Fig. 3.

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Fraction of the ligand-bound P2 [i.e. R/(R + 1)] versus ligand concentration measured for L6, L17 and L28, which represent H, A and B type 6 antigens. The titration experiments were carried out on aqueous ammonium acetate solutions (10 mM) at pH 7 and 25°C containing P2 (12 μM), Pref (10 μM) and ligand concentrations of between 10 and 120 μM. The solid curves correspond to the best fit of Eq. (4) to the experimental data for each ligand.

As a further test the reliability of the ESI-MS assay, measurements were performed on two human milk oligosaccharides (HMO), 3′-siallyllactose and 6′-siallyllactose, which were reported not to bind to the P2 (Fiege et al. 2012). Supplementary data, Figure S4 shows representative ESI mass spectra acquired for aqueous ammonium acetate solutions (10 mM) with P2 (12 μM) and 60 μM of 3′-siallyllactose (L42) or 6′-siallyllactose (L43) and scFv (10 μM). Although there were signals corresponding to the binding of each of the oligosaccharides to the P2, these were found to be due entirely to nonspecific binding.

The ESI-MS binding measurements revealed that the P2 recognizes all HBGA oligosaccharides investigated. However, the binding is uniformly weak, ranging from ∼400 to ∼3000 M−1. The highest-affinity ligands for the P2 are B tetrasaccharide type 3 (L26) and A tetrasaccharide type 6 (L17), with Ka values of 3000 ± 300 M−1 and 2350 ± 60 M−1, respectively. Interestingly, the affinities measured for the P2 are similar in magnitude to that estimated for the VLP of another GII.4 NoV (Ast6139; Fiege et al. 2012). Given that the P2 only possesses two binding sites, while the VLP has 180 sites and assuming that the binding sites of the P2 resembles those of the VLP, the apparent affinities of the VA387 VLP for these soluble oligosaccharides are expected to range from 104 to 106 M−1.

The binding data in Table I reveal that, overall, the A and B antigens bind more strongly to the P2 than do the H and Lewis antigens. This finding appears to be consistent with the previously proposed NoV binding model, in which VA387 can recognize the HBGAs through either a α-d-N-acetyl galactosamine (GalNAc) or a α-d-galactose (α-d-Gal) epitope, and a α-l-Fuc epitope, using two different binding pockets (Figure 4; Tan and Jiang 2005a). The A and B antigens possess both epitopes, which could be the reason for the stronger binding than those of the H and Lewis antigens, which lack the α-d-GalNAc/α-d-Gal epitope. The binding data also indicate that both 1,2-linked α-l-Fuc (H epitope) and 1,3/4-linked α-l-Fuc (Lea and LeX epitope) are recognized by the P2 with comparable affinities.

Fig. 4.

Fig. 4.

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An interaction model for the NoV VA387 P2 binding to HBGA oligosaccharides adapted from the proposed model for the NoV–HBGA binding (Tan and Jiang 2005a). (A) The binding model proposed for A/B/H oligosaccharides; illustrated by an A/B/H type 1 antigen. (B) The binding model proposed for Lewis oligosaccharides and illustrated by a Leb antigen. This model assumes that VA387 P2 can accommodate both the H epitope and A/B epitope independently in two nearby binding pockets. In addition, the VA387 P2 can also accommodate Lewis epitope in the H epitope-binding pocket.

The present data also indicate that the precursor chain type influences the strength of HBGA binding. For the H antigens, type 3 displays higher affinity over the remaining types 1, 2, 4, 5 and 6, which exhibit similar affinities ranging from 400 to 700 M−1. The finding that type 2 binds with comparable affinity as 1, 4, 5 and 6 is in agreement with results determined by the STD-NMR spectroscopy reported by Peters and coworkers (Fiege et al. 2012). However, this is inconsistent with those measured by the enzyme-linked immunosorbent assay, which suggested that VA387 VLP does not bind to H type 2 trisaccharide (Huang et al. 2005). For the A antigens, type 6 exhibits the strongest, type 2 the weakest; while types 1, 3, 4 and 5 exhibit similar affinities ranging from 1200 to 1600 M−1. For the B antigens, type 3 displays the highest affinity, and similar to the A antigens, type 2 the weakest; while types 1, 4, 5 and 6 exhibit similar affinities (in the range of 1100–1400 M−1).

The affinities of the tr-P2 for the 41 listed HBGAs were also measured (Table I). Overall, they are similar in magnitude to those measured for the P2. These results indicate that the elimination of the C-terminal Arg tail, which is remote from the binding pocket, does not influence substantially the binding of the P domain with the HBGAs. This finding contrasts the results of a previous study, which suggested that the removal of Arg tail eliminated binding to the HBGA (Tan et al. 2006).

Docking analysis

To further elucidate the influence of structure of the HBGA on the strength of their interactions with the P2, molecular-docking analysis was performed on each of the HBGA oligosaccharides investigated. The results of this analysis are summarized below.

H antigens

Docking analysis revealed that all of the H antigens (L1–L11) share a common binding motif comprising the α-l-Fuc-(1 → 2)-β-d-Gal disaccharide. In each case, the α-l-Fuc residue engages in hydrogen bonds (H-bonds) with T344, R345, D374 of chain A and G442′ of chain B, while the β-d-Gal residue forms H-bonds with residues D391′ and S441′ in chain B (Supplementary data, Figure S5). Atomic coordinates derived from the energy optimized structures of type H oligosaccharides docked into the binding site of the NoV VA387 P2 are given in the Supplementary data type H oligosaccharides. With the exception of the type 3, additional H-bonds were identified for the H antigen trisaccharides. However, as noted above, the P2 exhibits the highest affinity for the type 3 H antigen. Therefore, it would seem that these putative stabilizing interactions do not significantly enhance binding. Docking of the larger oligosaccharides leads to a similar conclusion—there is no obvious correlation between the number of putative H-bonds identified in the docking results and the measured affinities.

A and B antigens

As described above, the P2 exhibits, with a few exceptions, higher affinities for the A and B antigens compared with the H antigen. These findings can be reasonably explained by the participation of the α-d-GalNAc (A antigens) or α-d-Gal (B antigen) residues in binding interactions, in addition to those involving the α-l-Fuc-(1 → 2)-β-d-Gal moiety. According to the docking results for the A antigens (Supplementary data, Figure S5 and Supplementary data type A oligosaccharides), the α-d-GalNAc residue of the A antigens forms H-bonds with residues K348′ and S441′ (chain B). Moreover, residue I389′ (chain B) may engage in van der Waals interactions with the N-acetyl group of terminal α-d-GalNAc. The docking results for the B antigens (Supplementary data, Figure S5 and Supplementary data type B oligosaccharides) reveal that the α-d-Gal residue forms H-bonds with residues A346 (chain A) and Q331′, K348′ and S441′ (chain B). As was the case for the H antigens, additional H-bonds were identified for some of the larger A and B oligosaccharides. However, these putative interactions do not translate to significant changes in affinity.

Lewis antigens

The Lewis structures are characterized by the Lewis epitope (i.e. 1,3-linked α-l-Fuc in LeX and LeY antigens or 1,4-linked α-l-Fuc in Lea and Leb antigens). Although lacking the central-binding motif, the α-l-Fuc-(1 → 2)-β-d-Gal disaccharide, the LeX and Lea antigens nevertheless bind to the P2 with affinities similar to those of the H antigens. From the docking analysis (Supplementary data, Figure S5 and Supplementary data Lewis type oligosaccharides), the 1,3/4-linked α-l-Fuc residue in LeX and Lea antigens is able to form H-bonds with residues T344, R345, D374 (chain A) and G442′ (chain B). In addition, the β-d-N-acetyl glucosamine (GlcNAc) residue forms H-bonds to residues S441′ and D391′ (chain B). The LeY and Leb antigens possess both the α-l-Fuc-(1 → 2)-β-d-Gal and the 1,3/4-linked α-l-Fuc motifs, both of which are recognized by the protein. However, the docking results indicate that the P2 binds preferentially to the 1,3/4-linked α-l-Fuc motif (Supplementary data, Figure S5 and Supplementary data Lewis type oligosaccharides). The 1,3/4-linked α-l-Fuc residue in LeY and Leb antigens forms H-bonds with residues T344, R345, D374 (chain A) and G442′ (chain B). For the LeY antigens, the β-d-GlcNAc residue also interacts with the residue D391′ (chain B). This interaction is not present for the Leb antigens; instead, the 1,2-linked α-l-Fuc residue interacts with the residue G392′ (chain B).

Conclusion

In summary, the interactions between the NoV VA387 P2 and the tr-P2 with a library of 41 HBGA oligosaccharides were quantified for the first time. The results of the binding measurements performed at 25°C and pH 7 indicate that the P2 binds to all of the HBGA oligosaccharides with affinities ranging from 400 to 3000 M−1. The affinities of the tr-P2, which lacks the Arg cluster at the C-terminus, for the HBGAs are similar in magnitude to those measured for the P2. Based on the affinities measured for the P2, the apparent affinities of NoV VA387 for the HBGA are estimated between 104 and 106 M−1. Efforts are now underway in our laboratory to quantify directly the affinities of the VA387 VLP, as well as the corresponding P particle, for the HBGA oligosaccharides.

Overall, the P2 exhibits higher affinities for the A and B antigens compared with those of the H and Lewis antigens. This finding is consistent with a proposed NoV binding model, in which VA387 can recognize the HBGAs through both a α-d-GalNAc-(1 → 3)/α-d-Gal-(1 → 3) epitope, and a α-l-Fuc(1 → 2) epitope, using two different binding sites. From the binding measurements, the influence of the chain type on the affinities was found to be: 3 > 1 ≈ 2 ≈ 4 ≈ 5 ≈ 6 for H antigens; 6 > 1 ≈ 3 ≈ 4 ≈ 5 > 2 for A antigens; 3 > 1 ≈ 4 ≈ 5 ≈ 6 > 2 for B antigens. The central-binding motifs in the HBGAs were identified from molecular-docking simulations. However, the modeling did not reveal the structural basis for the influence of chain type on the measured affinities.

Materials and methods

Proteins

Two forms of P2's of NoV strain VA387 (GII.4) were studied. The first one, referred as P2 [molecular weight (MW) 69 311 Da], was formed from the full-length P domain with an amino acid sequence spanning residues 222–539 of VA387 VP1 (AAK84679.2). The second one, referred as the truncated P dimer (tr-P2, MW 69 006 Da), was formed by a truncated P domain lacking the C-terminal Arg cluster with the sequence spanning residues 222–535 of VA387 VP1 (Tan et al. 2006). Both P2's were expressed in bacteria through the Glutathione S-transferase Gene Fusion System (GE Healthcare Life Sciences, Piscataway, NJ) and purified as described previously (Tan et al. 2004; Bu et al. 2008). A single-chain fragment (scFv, MW 26 539 Da) of the monoclonal antibody Se155-4, which served as a reference protein (Pref) to correct ESI mass spectra for the occurrence of nonspecific ligand binding, was produced using recombinant technology as described elsewhere (Zdanov et al. 1994). Each protein was concentrated and dialyzed against aqueous 50-mM ammonium acetate (pH 7) using Amicon Ultra 0.5 mL centrifugal filters (Millipore Corp., Billerica, MA) with a MW cutoff of 10 kDa and stored at −20°C until needed. The concentrations of protein stock solutions were measured by ultraviolet absorption.

Carbohydrates

A complete list of the HBGA oligosaccharides, which range in size from di- to hexasaccharide, is given in Table I; their structures are shown in Supplementary data, Figure S1. Compounds L2, L4–L6, L13–L16 and L24–L27 (Meloncelli and Lowary 2009; Meloncelli and Lowary 2010; Meloncelli et al. 2011) were donated by Prof. Todd Lowary (University of Alberta); compound L23 was donated by Alberta Innovates Technology Futures (Alberta, Canada); compounds L3, L34, L36, L38 and L40 were purchased from Dextra (Reading, UK); compounds L1, L6–L12, L17–L22, L28–L33, L35, L37, L39 and L41 were purchased from Elicityl SA (Crolles, France). Two compounds, 3′-siallylactose (L42) and 6′-siallylactose (L43), which served as negative controls, were purchased from IsoSep AB (Sweden). Stock solutions of each oligosaccharide were prepared by dissolving a known amount of the solid sample in ultrafiltered water (Milli-Q, Millipore) to yield a concentration of 1 mM. The solutions were stored at −20°C until needed.

Mass spectrometry

All of the binding measurements were carried out in positive-ion mode using a 9.4-T ApexQe Fourier-transform ion cyclotron resonance (FT ICR) mass spectrometer (Bruker-Daltonics, Billerica, MA) equipped with a modified nanoflow ESI (nanoESI) source. NanoESI tips were produced from borosilicate capillaries (1.0 mm outer diameter, 0.68 mm inner diameter) pulled to ∼5 μm using a P-97 micropipette puller (Sutter Instruments, Novato, CA). A platinum wire was inserted into the nanoESI tip, and a capillary voltage of ∼1.0 kV was applied to carry out ESI. Each ESI solution was prepared from stock solutions of P2 (or tr-P2), one of HBGA oligosaccharides and Se155-4 scFv, which served as Pref. Aqueous ammonium acetate (10 mM) was added to each solution. In all cases, the P2 (or tr-P2) was incubated with the HBGA oligosaccharide for ∼20 min at 25°C before ESI-MS analysis. For a limited number of HBGA, imidazole (10 mM), which is known to stabilize labile protein–ligand complexes during ESI-MS analysis (Sun et al. 2007; Bagal et al. 2009), was added to the solution to test for the occurrence of in-source (gas-phase) dissociation. Notably, the addition of imidazole did not result in a measurable increase (after correction for nonspecific binding) in the relative abundance of ligand-bound P2 (Supplementary data, Figure S2). These results established that the affinity measurements were not adversely affected by in-source dissociation. Ions/droplets produced by ESI were introduced into the mass spectrometer through a stainless steel capillary (0.43 mm inner diameter). The capillary voltage was 280 V. The ions were steered by a deflector (250 V) into the first funnel (150 V) and skimmer (20 V) and transmitted through the second funnel (7.5 V) and skimmer (5.0 V), and then accumulated in the hexapole (h1) for 0.6 s. The ions were then transferred through the quadrupole (using a low m/z cutoff of 1500) followed by further accumulation in a hexapole collision cell (h2) for 0.5 s. Ions were then transferred to the ion cyclotron resonance cell for detection. The pressure in the ICR cell region was ∼10−10 mbar. Data acquisition was performed using the ApexControl software (version 4.0, Bruker-Daltonics). The time-domain signal, consisting of the sum of 30 transients containing 32K data points per transient, was subjected to one zero-fill prior to Fourier transformation.

Determination of Ka values

A detailed description of the direct ESI-MS assay can be found elsewhere (Wang et al. 2003; Kitova et al. 2012) and only a brief overview is given here. The assay is based on the detection and quantification of free and ligand-bound protein ions by ESI-MS. The NoV VA387 P2 possesses two independent and equivalent HBGA-binding sites (Cao et al. 2007; Bu et al. 2008). However, as discussed in more detail below, the binding of the HBGA oligosaccharides is very weak (Ka < 104 M−1) and only a single bound ligand was observed (after correction for nonspecific binding) under the experimental conditions used. As a result, the reported Ka values correspond to the apparent association constant, which is a factor of two larger than the microscopic Ka.

For a given P2–HBGA ligand complex, referred to generally as PL (Eq. 1), the magnitude of Ka was determined from the ratio (R) of total abundance (Ab) of ligand-bound (PL) and free protein (P) ions, as measured by ESI-MS for solutions of known initial concentrations of protein ([P]0) and ligand ([L]0), Eq. (2):

graphic file with name cws141eq1.jpg (1)
graphic file with name cws141eq2.jpg (2)

where R is calculated using Eq. (3):

graphic file with name cws141eq3.jpg (3)

The Ka values reported in Table I correspond to the average value established from replicate (≥3) measurements performed on at least three different HBGA concentrations. In all cases, each ESI mass spectrum was corrected for the occurrence of nonspecific HBGA–protein binding during the ESI process using the Pref method (Sun et al. 2006). As described elsewhere, this technique involves the addition of a Pref, which does not bind specifically to the protein and ligand of interest, to the solution. The “true” abundance (in the absence of nonspecific binding) of the ligand-bound and unbound P2 is calculated from the measured abundances of ligand-bound and unbound P-dimer. The underlying assumption with the method, that nonspecific ligand binding is a random process and affects equally all proteins in solution regardless of their size or structure, has been rigorously tested and shown to be generally valid (Sun et al. 2006, 2010; Deng et al. 2010).

For a limited number of oligosaccharides, Ka was determined using a titration approach (Daniel et al. 2002), where the initial concentration of the P2 was fixed and the concentration of the HBGA ligand was varied. The value of Ka was established from nonlinear regression analysis of the experimentally determined concentration dependence of the fraction of ligand-bound protein, [R/(R + 1)], using Eq. (4):

graphic file with name cws141eq4.jpg (4)

Docking simulations

Molecular models of the interactions between the HBGA oligosaccharides and the P2 were obtained through a combination of molecular-docking and molecular dynamics (MD) simulations. Autodock Vina (Trott and Olson 2010) was used to dock the disaccharide and trisaccharide ligands. Binding modes for the larger HBGA were reconstructed from poses found for smaller, related, HBGA. The crystal structure of the P domain of VA387 bound to the B trisaccharide ligand (L23) was used as a model of the P2-binding site (pdb entry: 2obt; Cao et al. 2007). Protons were added to all heteroatoms using MGL Tools (Scripps Research Institute, La Jolla, CA). The structures of the HBGA ligands were generated using the online Glycam molecular builder at http://glycam.ccrc.uga.edu/ccrc. The grid box parameters were assigned using the following procedure. Open Babel obfit command was used to align the Fuc moiety in the ligand with that of L23 in the crystal structure. The parameters of a minimal rectangular box, which accommodates the ligand, were expanded 2 Å in each direction. For each ligand, poses were generated using Autodock Vina with an exhaustiveness set at 64 and arbitrary random seed values. Only poses for which the position of the l-fuc moiety matched (with root-mean-square deviation <1 Å) the position of that residue in the crystal structure and all interglycosidic dihedral angles were in agreement with an exoanomeric effect were considered (El-Hawiet et al. 2011). The resulting poses were then refined using MD simulations. The protein was protonated with “reduce” and its parameters and partial charges were assigned according to the Amber2003 force field (ff03.r1) using antechamber and sleap. The assigning of AM1 bond charge correction model atomic charges for the ligands was conducted using the sqm module in Amber 11. The general Amber force field was used for the HBGA ligands. The ligands were placed in the binding site according to poses generated by docking. The resulting complex was neutralized by the addition of Na+ counter ions and placed in a rectangular box of TIP3P water that extended 8 Å in all dimensions from the outer limits of the complex.

The MD simulations were performed using Amber 11 on a CentOS 6.0 computer. After minimization [first 1000 steps using steepest descent, then 1000 steps with the conjugate gradients with restrained solute (500 kcal mol−1 Å−2)], the system was heated to 300 K with weak restraints to the solute (10 kcal mol−1 Å−2) and equilibrated for 30 ps with controlled pressure and temperature. Each 200-ps production run was performed on partially constrained complex (amino acid residues that did not contact the ligand were restrained at 1 kcal mol−1 Å−2) at 1 atm, 300 K using the Langevin heat bath with 1 ps−1 collision frequency. Nonbonded interactions were evaluated with 8 Å cutoff, and the SHAKE constraint system for bonds to hydrogen was enabled. Long-range electrostatic interactions were treated with particle-mesh Ewald periodic boundary conditions.

The larger HBGA ligands (L2–L11, L13–L22, L24–L33, L35 and L37–L41) were modeled based on the structure of the smallest member of the congeneric series, the corresponding disaccharide for the H antigens and trisaccharide for the A, B and Lewis antigens. Putative bound poses of each of larger congeners were prepared by superimposing their atomic coordinates with the atomic coordinates of the corresponding di- or trisaccharide template in the complex. To alleviate possible clashes with the protein, the ligand position was adjusted using a local search method in Autodock Vina. The resulting structure was then subjected to MD simulations.

Supplementary data

Supplementary data for this article are available online at http://glycob.oxfordjounals.org/.

Conflict of interest

None declared.

Abbreviations

ESI-MS, electrospray ionization mass spectrometry; FT, Fourier-transform; Fuc, fucose; Gal, galactose; GalNAc, N-acetyl galactosamine; GlcNAc, N-acetyl glucosamine; HBGA, histo-blood group antigens; HMO, human milk oligosaccharides; ICR, ion cyclotron resonance; MD, molecular dynamics; nanoESI, nanoflow ESI; NoV, norovirus; P2, P dimer; Pref, reference protein; scFv, single-chain variable fragment; STD-NMR, saturation transfer difference nuclear magnetic resonance; tr-P2, truncated P dimer; VLP, virus-like particle.

Supplementary Material

Supplementary Data
supp_23_3_276__index.html (1.1KB, html)

Acknowledgements

The authors thank the Alberta Glycomics Centre (AGC) and the Natural Sciences and Engineering Research Council of Canada (NSERC) for supporting this research and Prof. Todd Lowary (University of Alberta) and Alberta Innovates Technology Futures for generously providing carbohydrate ligands used in this work. L.H. also acknowledges an Alberta Innovates Graduate Student Scholarship, and X.J. and M. T. acknowledge support from the National Institutes of Health of the United States of America.

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Associated Data

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Supplementary Materials

Supplementary Data
supp_23_3_276__index.html (1.1KB, html)
supp_cws141_cws141supp.doc (9.9MB, doc)
supp_cws141_cws141supp_docking_A_pdb.rar (1.5MB, rar)
supp_cws141_cws141supp_docking_B_pdb.rar (1.5MB, rar)
supp_cws141_cws141supp_docking_H_pdb.rar (1.5MB, rar)
supp_cws141_cws141supp_docking_Lewis_pdb.rar (825.9KB, rar)

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