TY - JOUR
AU - Klassen, John S
AB - Abstract Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis. Many HuNoVs recognize histo-blood group antigens (HBGAs) as cellular receptors or attachment factors for infection. It was recently proposed that HuNoV recognition of HBGAs involves a cooperative, multistep binding mechanism that exploits both known and previously unknown glycan binding sites. In this study, binding measurements, implemented using electrospray ionization mass spectrometry (ESI-MS) were performed on homodimers of the protruding domain (P dimers) of the capsid protein of three HuNoV strains [Saga (GII.4), Vietnam 026 (GII.10) and VA387 (GII.4)] with the ethyl glycoside of the B trisaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-OC2H5) and free B type 1 tetrasaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-(1→3)-d-GlcNAc) in an effort to confirm the existence of new HBGA binding sites. After correcting the mass spectra for nonspecific interactions that form in ESI droplets as they evaporate to dryness, all three P dimers were found to bind a maximum of two B trisaccharides at the highest concentrations investigated. The apparent affinities measured for stepwise binding of B trisaccharide suggest positive cooperativity. Similar results were obtained for B type 1 tetrasaccharide binding to Saga P dimer. Based on these results, it is proposed that HuNoV P dimers possess only two HBGA binding sites. It is also shown that nonspecific binding corrections applied to mass spectra acquired using energetic ion source conditions that promote in-source dissociation can lead to apparent HuNoV–HBGA oligosaccharide binding stoichiometries and affinities that are artificially high. Finally, evidence that high concentrations of oligosaccharide can induce conformational changes in HuNoV P dimers is presented. affinity, binding stoichiometry, ESI-MS, HBGA, norovirus Introduction Human noroviruses (HuNoVs) are a genus of nonenveloped, single-stranded, positive sense RNA viruses in the Caliciviridae family that cause acute gastroenteritis (Donaldson et al. 2010). HuNoVs can be genetically classified into two major genogroups (GI and GII), which contain more than 30 different genotypes (e.g., GI.1–9 and GII.1–22) (Zheng et al. 2006). Among them, GII.4 is the predominant genotype and accounts for ~80% of HuNoV gastroenteritis outbreaks (Patel et al. 2008; Siebenga et al. 2009). The capsid of HuNoV consists of a single major viral protein, VP1, which encapsulates the RNA genome (Jiang et al. 1992). Structurally, VP1 consists of two major domains—the N-terminal shell (S) and the C-terminal protruding (P) domains. The S domain is involved in the formation of the interior icosahedral shell of the capsid, while the P domain forms the dimeric protrusions of the capsid that is responsible for host cell recognition (Prasad et al. 1999). It is known that most HuNoVs recognize histo-blood group antigens (HBGAs) as cellular receptors or attachment factors (Donaldson et al. 2010; Tan and Jiang 2010, 2005). HBGAs are fucosylated glycoproteins or glycolipids abundantly found on the mucosal epithelia of gastrointestinal track, as well as in biological fluids, including saliva, milk and blood (Oriol 1990; Ravn and Dabelsteen 2000). HuNoVs interact with HBGAs in a strain specific manner, with the l-fucose residue serving as the core recognition motif (Tan and Jiang 2011, 2014). Recently, it was shown that HuNoVs also bind to acidic glycosphingolipids (gangliosides), raising the possibility that gangliosides, along with HBGAs, are cellular receptors or attachment factors for HuNoV recognition and infection (Han et al. 2014; Wegener et al. 2017). X-ray crystallographic structures of the P domain dimers of many GI and GII strains complexed with HBGA oligosaccharides have been solved (Cao et al. 2007; Choi et al. 2008; Chen et al. 2011; Hansman et al. 2011; Shanker et al. 2011, 2014; Kubota et al. 2012; Hao et al. 2015; Liu et al. 2015,; Singh et al. 2015; Koromyslova et al. 2017; Weichert et al. 2016). The P dimers are found to be highly symmetric complexes, possessing two identical HBGA binding sites (Cao et al. 2007; Choi et al. 2008; Chen et al. 2011; Hansman et al. 2011; Shanker et al. 2011, 2014; Kubota et al. 2012; Hao et al. 2015; Liu et al. 2015; Singh et al. 2015; Weichert et al. 2016; Koromyslova et al. 2017). According to solution binding measurements performed on the Vietnam 026 P dimer (GII.10), using saturation-transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, and the VA387 P dimer (GII.4), using native mass spectrometry (electrospray ionization mass spectrometry (ESI-MS) performed under native conditions), HBGA oligosaccharide affinities for HuNoV P dimers are low, with apparent (macroscopic) association constants (Ka,app)<3,000 M−1 (Hansman et al. 2012; Han et al. 2013). Recently reported structural and in vitro binding data, however, suggest that HuNoV–HBGA binding may involve a cooperative, multistep mechanism that exploits both known and, previously, unknown glycan binding sites. Crystallographic data obtained for the Vietnam 026 P dimer complexed with l-fucose revealed four bound monosaccharides, two at the known HBGA binding sites and two at new binding sites located at the dimer interface (Koromyslova et al. 2015). Interestingly though, when complexed with B trisaccharide, only two bound ligands (at the known binding sites) were identified in the crystal structure (Koromyslova et al. 2015). The binding isotherms for methyl-α-l-fucose, citrate and several HBGA oligosaccharides with the P dimer of the Saga strain (GII.4), measured by STD-NMR spectroscopy, revealed successive binding steps with surprisingly large positive cooperativity factors (i.e., large Hill coefficients) (Mallagaray et al. 2015). Based on these data, it was concluded that the Saga P dimer binds up to two molecules of fucose and H disaccharide, three molecules of citrate and four molecules of the B trisaccharide (methyl glycoside) and B type 1 tetrasaccharide (free oligosaccharide). To further support the existence of additional HBGA binding sites, ESI-MS measurements were performed to quantify the stoichiometry of the interactions between the B type 1 tetrasaccharide and the Saga P dimer. Although, the ESI mass spectra appear to show the presence of up to four bound tetrasaccharides, the distributions (following correction for nonspecific binding during the ESI process, vide infra) are inconsistent with the affinities measured by STD-NMR. Moreover, the apparent affinities, calculated from the reported distributions decrease with increasing ligand concentration, suggesting the possibility of systematic error in the ESI-MS measurements. Given the critical role that HBGA recognition plays in HuNoV infection (Tan and Jiang 2005; Tan and Jiang 2010), an accurate description of their interactions with HuNoVs is of fundamental importance and can guide the development of therapeutics to treat and prevent HuNoV infections. With the goal of establishing the existence of the additional HBGA binding sites, a quantitative ESI-MS study of the binding of Saga, Vietnam 026 and VA387 P dimers to the ethyl glycoside of the B trisaccharide and free B type 1 tetrasaccharide was carried out. In addition to providing new insights into the nature of HuNoV–HBGA interactions, the results of this study highlight practical challenges in applying ESI-MS to measure the binding stoichiometries and affinities of low affinity protein–glycan complexes and suggest that high concentrations of oligosaccharide can affect HuNoV P dimer conformation. Results and Discussion B trisaccharide–HuNoV P dimer interactions Direct ESI-MS binding measurements were carried out on the B trisaccharide with the P dimers of Saga, Vietnam 026 and VA387 to establish the number of carbohydrate binding sites and, from the measured apparent affinities for stepwise ligand binding, whether the binding sites are equivalent and independent. Representative ESI mass spectra acquired for aqueous (300 mM ammonium acetate, pH 7.0, 25°C) solutions of the three P dimers are shown in Supplementary data, Figure S2. The mass spectra revealed that all three P domain proteins exist predominantly as homodimers under the solution conditions investigated. The measured MW of the Saga P dimer (P2Saga), 69,751 ± 5 Da (Supplementary data, Figure S2A and Table SI) is in reasonable agreement with the theoretical MW (69,734 Da). The MW of the VA387 P dimer (P2VA387), 69,317 ± 3 Da (Supplementary data, Figure S2B and Table SII), also agrees with the theoretical MW (69,312 Da). In the case of Vietnam 026 P dimer, three different dimers (P2Viet-I, MW 70,141 ± 11 Da, P2Viet-II, MW 70,495 ± 10 Da, and P2Viet-III, MW 70,806 ± 11 Da) were identified from the mass spectrum (Supplementary data, Figure S2C and Table SIII). ESI-MS measurements performed under denaturing conditions (data not shown) revealed the presence of three different PViet monomer isoforms, with MWs of 34,883 ± 7 Da (PViet-I), 35,247 ± 4 Da (PViet-II), and 35,549 ± 5 Da (PViet-III). The MW of PViet-III is in reasonable agreement with the theoretical MW (35,511 Da) calculated from the P domain sequence of the HuNoV Vietnam 026 strain, while PViet-II and PViet-I appear to correspond to truncated (by two (35,224 Da) and five (34,854 Da) residues, respectively, at the C-terminus). Based on these results, it is proposed that association of PViet-II with PViet-I leads to P2Viet-I, homodimerization of PViet-II produces P2Viet-II, and association of PViet-II with PViet-III produces P2Viet-III. To simplify data analysis, only the ions corresponding to P2Viet-II were taken into account for the affinity measurements. Binding of the B trisaccharide, at concentrations ranging from 100 μM to 800 μM, to the P dimers of the three HuNoV was monitored using direct ESI-MS measurements. Representative ESI mass spectra acquired for aqueous solutions (300 mM ammonium acetate, pH 7.0, 25°C) of Saga P dimer (3 μM), B trisaccharide (100 μM or 800 μM) and Pref are shown in Figure 1. As described above, two different Pref—scFv (Figure 1A and B; Supplementary data, Table SIV) and cytoc (Figure 1C and D; Supplementary data, Table SV)—were used to correct the mass spectra for the nonspecific ligand binding during the ESI process. At 100 μM B trisaccharide, ions corresponding to free P dimer, and P dimer bound up to two B trisaccharides were detected. At 800 μM, P dimer ions bound to up to five B trisaccharides were observed. Fig. 1. View largeDownload slide (A)–(B) ESI mass spectra acquired in positive ion mode at Cone voltage 50 V for aqueous ammonium acetate (300 mM, pH 7.0 and 25°C) solution of HuNoV Saga P dimer (P2Saga, 3 μM), scFv (Pref, 1 μM) with (A) 100 μM and (B) 800 μM B trisaccharide (L). (C)–(D) ESI mass spectra acquired in positive ion mode at Cone voltage 50 V for aqueous ammonium acetate solution (300 mM, pH 7.0 and 25°C) of P2Saga (3 μM), cytoc (Pref, 1 μM) with (C) 100 μM and (D) 800 μM B trisaccharide. Insets, normalized distributions of L bound to P2Saga before (apparent) and after correction for nonspecific ligand binding using the reference protein method (with scFv or cytoc as Pref) or the Kns method. Fig. 1. View largeDownload slide (A)–(B) ESI mass spectra acquired in positive ion mode at Cone voltage 50 V for aqueous ammonium acetate (300 mM, pH 7.0 and 25°C) solution of HuNoV Saga P dimer (P2Saga, 3 μM), scFv (Pref, 1 μM) with (A) 100 μM and (B) 800 μM B trisaccharide (L). (C)–(D) ESI mass spectra acquired in positive ion mode at Cone voltage 50 V for aqueous ammonium acetate solution (300 mM, pH 7.0 and 25°C) of P2Saga (3 μM), cytoc (Pref, 1 μM) with (C) 100 μM and (D) 800 μM B trisaccharide. Insets, normalized distributions of L bound to P2Saga before (apparent) and after correction for nonspecific ligand binding using the reference protein method (with scFv or cytoc as Pref) or the Kns method. At all B trisaccharide concentrations investigated, the ESI mass spectra showed evidence of nonspecific binding. For example, at 100 μM, signal corresponding to Pref bound to one B trisaccharide was detected (Figure 1A and C); and up to four bound B trisaccharides were observed at 800 μM (Figure 1B and D). These results indicate that nonspecific binding of B trisaccharides to the P dimer occurs during ESI and needs to be corrected for. The normalized distributions of Saga P dimer species before and after correction (using the Pref distributions) at 100 μM and 800 μM are shown in Figure 1A–D. Also shown are the corrected distributions calculated using the Kns method assuming two binding sites. It can be seen that the corrected distributions obtained for the two Pref are indistinguishable within error. The similarity of the results obtained with two structurally different Pref supports the reliability of the correction method. The corrected distributions calculated with the Kns method also show a maximum of two bound B trisaccharides, although slightly more ligand-bound P dimer is predicted compared to the distributions calculated with the Pref method. Summarized in Figure 2A–C are the corrected distributions (based on the Pref and Kns methods) of bound B trisaccharide measured at each of the ligand concentrations investigated. It can be seen that, in all cases, following correction for nonspecific binding, the Saga P dimer binds a maximum of two B trisaccharides. Shown in Figure 2D are plots of f (the fraction of bound P dimer, calculated from Eq. (2) following correction for nonspecific binding using the two different Pref and the Kns method) versus ligand concentration. Also shown is the corresponding plot calculated from the binding data reported by Peters and coworkers (Mallagaray et al. 2015). According to the STD-NMR data, the first ‘step’ in the isotherm should be visible at B trisaccharide concentrations >600 μM. However, the ESI-MS data show no evidence of ‘step-like’ character at these concentrations. Fig. 2. View largeDownload slide (A)–(C) Normalized distributions of B trisaccharide bound to Saga P dimer (P2Saga) determined by ESI-MS after correction for nonspecific ligand binding using the reference protein method: (A) Pref = scFv and (B) Pref = cytoc, or (C) the Kns method. (D) Plots of the fraction of B trisaccharide-bound P2Saga versus ligand concentration. Each curve represents the best fit of the binding model to the experimental data. Also shown is the binding isotherm calculated using the stepwise binding model reported by Mallagaray et al. (2015). The error bars represent one standard deviation and were determined from at least three replicate measurements. Fig. 2. View largeDownload slide (A)–(C) Normalized distributions of B trisaccharide bound to Saga P dimer (P2Saga) determined by ESI-MS after correction for nonspecific ligand binding using the reference protein method: (A) Pref = scFv and (B) Pref = cytoc, or (C) the Kns method. (D) Plots of the fraction of B trisaccharide-bound P2Saga versus ligand concentration. Each curve represents the best fit of the binding model to the experimental data. Also shown is the binding isotherm calculated using the stepwise binding model reported by Mallagaray et al. (2015). The error bars represent one standard deviation and were determined from at least three replicate measurements. Based on the distributions (corrected) of ligand-bound Saga P dimer measured by ESI-MS, the apparent affinities, Ka,1 and Ka,2, were calculated (Table I): 540 ± 70 M−1 and 160 ± 30 M−1 (Pref = scFv), respectively; 670 ± 60 M−1 and 200 ± 60 M−1 (Pref = cytoc), respectively; and 970 ± 70 M−1 and 380 ± 80 M−1 (Kns method), respectively. The values obtained with the Kns correction method (Shimon et al. 2010) agree, within a factor of two, with the values obtained using the Pref method (Sun et al. 2006; Kitova et al. 2012). To our knowledge, this is the first direct comparison of the Kns method with the Pref method for correcting ESI mass spectra for nonspecific ligand binding. It is notable that, for all three data sets, the ratio Ka,1/Ka,2 is < 4. This finding suggests that B trisaccharide binding exhibits positive cooperativity (Lin et al. 2014). However, given the uncertainty in the affinities, the magnitude of the cooperativity factor could not be precisely determined. Table I. Summary of apparent association constants (Ka,q, M−1) and association constant ratio (Ka,q/Ka,q+1) for B trisaccharide binding to P dimers of HuNoV Saga, Vietnam 026, and VA387 strains measured in 300 mM aqueous ammonium acetate solutions (pH 7.0 and 25°C) using ESI-MSa Correction method Ka,q or Ka,q/Ka,q+1 Saga P dimer Vietnam 026 P dimer VA387 P dimer Pref = scFv Ka,1 540 ± 70 410 ± 80 970 ± 30 Pref = scFv Ka,2 160 ± 30 220 ± 80 380 ± 20 Pref = scFv Ka,1/Ka,2 3.4 ± 0.8 1.9 ± 0.6 2.6 ± 0.1 Pref = cytoc Ka,1 670 ± 60 620 ± 70 1210 ± 130 Pref = cytoc Ka,2 200 ± 60 220 ± 50 360 ± 90 Pref = cytoc Ka,1/Ka,2 3.4 ± 0.9 2.8 ± 1.0 3.4 ± 0.4 Kns method Ka,1 970 ± 70 800 ± 50 1420 ± 80 Kns method Ka,2 380 ± 80 370 ± 90 510 ± 70 Kns method Ka,1/Ka,2 2.6 ± 0.4 2.2 ± 0.6 2.8 ± 0.4 Correction method Ka,q or Ka,q/Ka,q+1 Saga P dimer Vietnam 026 P dimer VA387 P dimer Pref = scFv Ka,1 540 ± 70 410 ± 80 970 ± 30 Pref = scFv Ka,2 160 ± 30 220 ± 80 380 ± 20 Pref = scFv Ka,1/Ka,2 3.4 ± 0.8 1.9 ± 0.6 2.6 ± 0.1 Pref = cytoc Ka,1 670 ± 60 620 ± 70 1210 ± 130 Pref = cytoc Ka,2 200 ± 60 220 ± 50 360 ± 90 Pref = cytoc Ka,1/Ka,2 3.4 ± 0.9 2.8 ± 1.0 3.4 ± 0.4 Kns method Ka,1 970 ± 70 800 ± 50 1420 ± 80 Kns method Ka,2 380 ± 80 370 ± 90 510 ± 70 Kns method Ka,1/Ka,2 2.6 ± 0.4 2.2 ± 0.6 2.8 ± 0.4 aUncertainties correspond to one standard deviation. Table I. Summary of apparent association constants (Ka,q, M−1) and association constant ratio (Ka,q/Ka,q+1) for B trisaccharide binding to P dimers of HuNoV Saga, Vietnam 026, and VA387 strains measured in 300 mM aqueous ammonium acetate solutions (pH 7.0 and 25°C) using ESI-MSa Correction method Ka,q or Ka,q/Ka,q+1 Saga P dimer Vietnam 026 P dimer VA387 P dimer Pref = scFv Ka,1 540 ± 70 410 ± 80 970 ± 30 Pref = scFv Ka,2 160 ± 30 220 ± 80 380 ± 20 Pref = scFv Ka,1/Ka,2 3.4 ± 0.8 1.9 ± 0.6 2.6 ± 0.1 Pref = cytoc Ka,1 670 ± 60 620 ± 70 1210 ± 130 Pref = cytoc Ka,2 200 ± 60 220 ± 50 360 ± 90 Pref = cytoc Ka,1/Ka,2 3.4 ± 0.9 2.8 ± 1.0 3.4 ± 0.4 Kns method Ka,1 970 ± 70 800 ± 50 1420 ± 80 Kns method Ka,2 380 ± 80 370 ± 90 510 ± 70 Kns method Ka,1/Ka,2 2.6 ± 0.4 2.2 ± 0.6 2.8 ± 0.4 Correction method Ka,q or Ka,q/Ka,q+1 Saga P dimer Vietnam 026 P dimer VA387 P dimer Pref = scFv Ka,1 540 ± 70 410 ± 80 970 ± 30 Pref = scFv Ka,2 160 ± 30 220 ± 80 380 ± 20 Pref = scFv Ka,1/Ka,2 3.4 ± 0.8 1.9 ± 0.6 2.6 ± 0.1 Pref = cytoc Ka,1 670 ± 60 620 ± 70 1210 ± 130 Pref = cytoc Ka,2 200 ± 60 220 ± 50 360 ± 90 Pref = cytoc Ka,1/Ka,2 3.4 ± 0.9 2.8 ± 1.0 3.4 ± 0.4 Kns method Ka,1 970 ± 70 800 ± 50 1420 ± 80 Kns method Ka,2 380 ± 80 370 ± 90 510 ± 70 Kns method Ka,1/Ka,2 2.6 ± 0.4 2.2 ± 0.6 2.8 ± 0.4 aUncertainties correspond to one standard deviation. To provide additional support for the reliability of the ESI-MS derived affinities, the thermodynamic parameters for B trisaccharide binding to Saga P dimer were measured by ITC. Notably, the ITC data exhibited a thermodynamic profile (Supplementary data, Figure S3) typical of weak binding, i.e., [P] × Ka « 1 (Freyer and Lewis 2008; Dutta et al. 2015), which suggest that the B trisaccharide–Saga P dimer interactions are low affinity, with apparent association constants <1000 M−1. This finding is consistent with the ESI-MS affinity data. The Ka,1 and Ka,2 values measured for B trisaccharide binding to the Saga P dimer are similar in magnitude to the values reported for other HBGA oligosaccharides and HuNoV P dimers (Hansman et al. 2012; Han et al. 2013). However, they are ~1/30th of the values determined by STD-NMR spectroscopy for B trisaccharide binding to the Saga P dimer (Mallagaray et al. 2015). Moreover, the (corrected) distributions measured in the present study differ significantly from the ESI-MS derived distributions reported by Peters and coworkers (Mallagaray et al. 2015). The reported distributions, which were measured for 300 mM ammonium acetate solutions (pH 7.4) containing Saga P dimer (2.25 μM), cytoc (19 μM) and 200 μM or 500 μM B type 1 tetrasaccharide, suggest the presence of Saga P dimer bound to as many as four tetrasaccharide ligands (after correction of the nonspecific ligand binding). However, there are two notable aspects to the reported ESI-MS distributions. First, they are inconsistent with the proposed (based on STD-NMR data) binding model, which suggests that the Saga P dimer would exist predominantly bound to one ligand under the solution conditions used for ESI-MS. Secondly, the Ka,q extracted from the two (corrected) distributions suggest a decrease in affinity with increasing ligand concentration. To test whether the findings for Saga P dimer are general, binding measurements were carried out on the B trisaccharide with the P dimers of HuNoV Vietnam 026 and VA387. Measurements were performed using identical solution and instrumental conditions as those used for the Saga P dimer (Supplementary data, Figures S4 and S5). The corrected (for nonspecific binding) distributions and resulting binding isotherms are shown in Figure 3. As was found with the Saga P dimer, the Vietnam 026 and VA387 P dimers bind to a maximum of two B trisaccharides at the highest ligand concentration investigated (Figure 3A and B). The former result is consistent with the crystallographic data, wherein the Vietnam 026 P dimer is found to bind to two molecules of B trisaccharide (Koromyslova et al. 2015). According to the ESI-MS data (Figure 3C and D), the Ka,1 and Ka,2 values for B trisaccharide binding to the Vietnam 026 P dimer are (Table I): 410 ± 80 M−1 and 220 ± 80 M−1 (Pref = scFv); 620 ± 70 M−1 and 220 ± 50 M−1 (Pref = cytoc); and 800 ± 50 M−1 and 370 ± 90 M−1 (Kns method). For binding the VA387 P dimer the Ka,1 and Ka,2 values are: 970 ± 30 M−1 and 380 ± 20 M−1 (Pref = scFv); 1210 ± 130 M−1 and 360 ± 90 M−1 (Pref = cytoc); and 1420 ± 80 M−1 and 510 ± 70 M−1 (Kns method). Importantly, the B trisaccharide affinity for VA387 P dimer is in reasonable agreement with the Ka,1 determined previously using lower ligand concentrations (Han et al. 2013). Taken together, the results of the present binding measurements strongly suggest that, generally, HuNoV P dimers possess only two B trisaccharide binding sites. Fig. 3. View largeDownload slide (A)–(B) Normalized distributions of B trisaccharide bound to (A) Vietnam 026 P dimer (P2Viet) and (B) VA387 P dimer (P2VA387) by ESI-MS after correction for nonspecific ligand binding using the reference protein method (Pref = scFv). (C)–(D) Plots of the fraction of B trisaccharide-bound (C) P2Viet and (D) P2VA387 versus ligand concentration. Nonspecific ligand binding was corrected using the reference protein method (Pref = scFv or cytoc) and the Kns method. Each curve represents the best fit of the binding model. The error bars represent one standard deviation and were determined from at least three replicate measurements. Fig. 3. View largeDownload slide (A)–(B) Normalized distributions of B trisaccharide bound to (A) Vietnam 026 P dimer (P2Viet) and (B) VA387 P dimer (P2VA387) by ESI-MS after correction for nonspecific ligand binding using the reference protein method (Pref = scFv). (C)–(D) Plots of the fraction of B trisaccharide-bound (C) P2Viet and (D) P2VA387 versus ligand concentration. Nonspecific ligand binding was corrected using the reference protein method (Pref = scFv or cytoc) and the Kns method. Each curve represents the best fit of the binding model. The error bars represent one standard deviation and were determined from at least three replicate measurements. B type 1 tetrasaccharide-Saga P dimer interactions Although the binding properties of the B trisaccharide and B type 1 tetrasaccharide are not expected to be significantly different for the P dimers investigated, ESI-MS binding measurements were performed on the B type 1 tetrasaccharide (200–600 μM) with Saga P dimer (Figure 4). Notably, under the same conditions as used for the B trisaccharide, a maximum (after correction for nonspecific binding) of two bound ligands was detected and the apparent affinities (840 ± 90 M−1 (Ka,1) and 320 ± 80 M−1 (Ka,2)) are similar in magnitude to those of the B trisaccharide for the Saga P dimer. However, these values are 23–31% of the recently reported Ka,1 (2700 ± 1200 M−1) and Ka,2 (1400 ± 710 M−1) values derived from ESI-MS measurements (Mallagaray et al. 2015). Additionally, in this earlier study, the presence of a third (1200 ± 750 M−1 (Ka,3)) and fourth (2500 ± 2100 M−1 (Ka,4)) bound B type 1 tetrasaccharide was observed after correction for nonspecific binding (Mallagaray et al. 2015 and Table II). Fig. 4. View largeDownload slide (A)–(B) ESI mass spectra acquired in positive ion mode for aqueous ammonium acetate (300 mM, pH 7.0 and 25°C) solutions containing Saga P dimer (P2Saga, 2.25 μM), cytoc (Pref, 1 μM), with (A) 200 μM and (B) 500 μM B type 1 tetrasaccharide (L) at Cone voltage 50 V, 130 V and 150 V. Insets: normalized distributions of (P2Saga + qL) species before (apparent) and after correction for the nonspecific ligand binding using the reference protein method (Pref = cytoc). (C) Plots of the fraction of B type 1 tetrasaccharide-bound P2Saga (after correction for nonspecific ligand binding) versus ligand concentration measured at different Cone voltages. Each curve represents the best fit of the binding model to the experimental data. (D) Apparent Ka,q (q = 1–4) values measured at different Cone voltages after correction for nonspecific ligand binding. The error bars represent one standard deviation. Fig. 4. View largeDownload slide (A)–(B) ESI mass spectra acquired in positive ion mode for aqueous ammonium acetate (300 mM, pH 7.0 and 25°C) solutions containing Saga P dimer (P2Saga, 2.25 μM), cytoc (Pref, 1 μM), with (A) 200 μM and (B) 500 μM B type 1 tetrasaccharide (L) at Cone voltage 50 V, 130 V and 150 V. Insets: normalized distributions of (P2Saga + qL) species before (apparent) and after correction for the nonspecific ligand binding using the reference protein method (Pref = cytoc). (C) Plots of the fraction of B type 1 tetrasaccharide-bound P2Saga (after correction for nonspecific ligand binding) versus ligand concentration measured at different Cone voltages. Each curve represents the best fit of the binding model to the experimental data. (D) Apparent Ka,q (q = 1–4) values measured at different Cone voltages after correction for nonspecific ligand binding. The error bars represent one standard deviation. Table II. Summary of apparent association constants (Ka,q, M−1) for B type 1 tetrasaccharide binding to HuNoV Saga P dimer measured in 300 mM aqueous ammonium acetate solution (pH 7.0 and 25°C) using ESI-MS at different Cone voltagesa q 50 V 130 V 150 V Literatureb 1 840 ± 90 1750 ± 240 1910 ± 260 2700 ± 1200 2 320 ± 80 800 ± 40 1020 ± 150 1400 ± 710 3 Not observed 470 ± 30 760 ± 130 1200 ± 750 4 Not observed 350 ± 190 760 ± 280 2500 ± 2100 q 50 V 130 V 150 V Literatureb 1 840 ± 90 1750 ± 240 1910 ± 260 2700 ± 1200 2 320 ± 80 800 ± 40 1020 ± 150 1400 ± 710 3 Not observed 470 ± 30 760 ± 130 1200 ± 750 4 Not observed 350 ± 190 760 ± 280 2500 ± 2100 aUncertainties correspond to one standard deviation. bValues calculated from the distributions reported by Mallagaray et al. (2015). View Large Table II. Summary of apparent association constants (Ka,q, M−1) for B type 1 tetrasaccharide binding to HuNoV Saga P dimer measured in 300 mM aqueous ammonium acetate solution (pH 7.0 and 25°C) using ESI-MS at different Cone voltagesa q 50 V 130 V 150 V Literatureb 1 840 ± 90 1750 ± 240 1910 ± 260 2700 ± 1200 2 320 ± 80 800 ± 40 1020 ± 150 1400 ± 710 3 Not observed 470 ± 30 760 ± 130 1200 ± 750 4 Not observed 350 ± 190 760 ± 280 2500 ± 2100 q 50 V 130 V 150 V Literatureb 1 840 ± 90 1750 ± 240 1910 ± 260 2700 ± 1200 2 320 ± 80 800 ± 40 1020 ± 150 1400 ± 710 3 Not observed 470 ± 30 760 ± 130 1200 ± 750 4 Not observed 350 ± 190 760 ± 280 2500 ± 2100 aUncertainties correspond to one standard deviation. bValues calculated from the distributions reported by Mallagaray et al. (2015). View Large As highlighted above and discussed in detail elsewhere (Sun et al. 2006, 2010; Kitova et al. 2012), nonspecific ligand binding represents a significant source of error in the measurement of low affinity protein–oligosaccharide interactions by ESI-MS. While it is possible to correct for nonspecific ligand binding using the Pref method, successful implementation of the method requires negligible in-source (gas-phase) dissociation of both the specific and nonspecific protein–oligosaccharide complexes (Sun et al. 2010). If this condition is not met, the correction method may introduce artefacts into the measured binding stoichiometries and affinities. It is possible that in-source dissociation is responsible for the differences in binding stoichiometries and affinities of the B type 1 tetrasaccharide for Saga P dimer measured in the present and previous studies. As described below, support for this hypothesis is found in the fact that it was possible to achieve distributions (corrected) similar to those reported previously (Mallagaray et al. 2015) by using ion source conditions that promote in-source dissociation. Shown in Figure 4A and B is a comparison of ESI mass spectra and corresponding distributions of bound B type 1 tetrasaccharide measured for aqueous ammonium acetate solution (300 mM, pH 7.0 and 25°C) containing Saga P dimer (2.25 μM), B type 1 tetrasaccharide (200 μM or 500 μM) and Pref at Cone voltages of 50 V, 130 V and 150 V. It can be seen that up to four bound B type 1 tetrasaccharides were detected (after correction) at the higher Cone voltages (130 V and 150 V). This result is consistent with that from the previous ESI-MS binding study carried out using energetic ion source conditions (Mallagaray et al. 2015). The apparent affinities (Table II) calculated form the corrected distributions are: 1750 ± 240 M−1 (Ka,1), 800 ± 40 M−1 (Ka,2), 470 ± 30 M−1 (Ka,3) and 350 ± 190 M−1 (Ka,4) at 130 V and 1910 ± 260 M−1 (Ka,1), 1020 ± 150 M−1 (Ka,2), 760 ± 130 M−1 (Ka,3) and 760 ± 280 M−1 (Ka,4) at 150 V. The apparent increase in the stoichiometries and affinities with Cone voltage can be explained by the increased internal energy of the gaseous ions due to collisional heating in the source, which results in gas-phase (in-source) dissociation of the protein–oligosaccharides complexes. More pronounced in-source dissociation for the Pref-B type 1 tetrasaccharide complexes, which are kinetically less stable than the corresponding P dimer-B type 1 tetrasaccharide complexes, produces artificially high binding stoichiometries and affinities for the Saga P dimer following implementation of the Pref correction (Figure 4C and D). These results highlight the importance of appropriately ‘gentle’ instrumental conditions when implementing the Pref correction method. Evidence of P dimer conformational changes at high oligosaccharide concentrations The ESI-MS binding data reported here strongly suggest that HuNoV P dimers possess only two HBGA binding sites. These results are at odds with the conclusions drawn from the STD-NMR data (Mallagaray et al. 2015). The ‘step-like’ character of the STD-NMR binding isotherms were interpreted as originating from cooperative, stepwise binding events. However, the plateaus evident in the reported isotherms seem to be inconsistent with such a mechanism given that the extent of binding is relatively insensitive to ligand concentration in these ‘flat’ regions. Instead, the experimental observations are more suggestive of protein conformational changes induced by high concentrations of HBGA oligosaccharide. Conformational changes would be expected to change the chemical shifts of the protein protons, which in turn could lead to changes in saturation during the STD-NMR experiments (Jayalakshmi and Krishna 2002, 2004). Indirect evidence that the conformation of the Saga P dimer is sensitive to HBGA oligosaccharide concentration was obtained from IMS analysis of the gaseous P dimer ions produced from solutions containing varying concentrations of B trisaccharide. Shown in Figure 5 are the IMS arrival time distributions (ATDs) measured for P2Saga ions at +17 and +18 charge states produced from a 300 mM ammonium acetate solution (pH 7.0 and 25°C) of Saga P dimer (3 μM). For the +17 charge state, there is a dominant feature in the ATD centered at 16.4 ms, and a minor feature, corresponding to a less compact conformer (or group of conformers), appearing at longer arrival times (ATs). The ATD of the +18 charge state is similar—there is a dominant feature centered at 14.8 ms and a second distribution centered at 16.4 ms. The introduction of B trisaccharide to solution (at concentrations of 200 μM, 400 μM and 800 μM) resulted in changes in the ATDs of both charge states, (Figure 5). There is a small but significant shift to longer ATs with increasing B trisaccharide concentration. Additionally, the relative abundances of the minor conformer(s) for both charge states increases with the B trisaccharide concentration. IMS measurements carried out on the P2Saga ions in the presence of isomaltotriose, which is produced during starch digestion and does not interact specifically with the P dimer in solution (Supplementary data, Figure S6) gave similar results (Supplementary data, Figure S7). Taken together, these results, which indicate that the presence of high concentrations of B trisaccharide or isomaltotriose results in changes to the structures of the gaseous Saga P dimer ions, provide indirect support for the hypothesis that the conformation of the P dimer is sensitive to HBGA oligosaccharide concentration. Fig. 5. View largeDownload slide Ion mobility separation arrival time distributions measured for Saga P dimer (P2Saga) ions: (A) (P2Saga)17+ (m/z 4102.41), and (B) (P2Saga)18+ (m/z 3875.12). The ions were produced by ESI-MS from aqueous ammonium acetate (300 mM, pH 7.0 and 25°C) solutions containing P2Saga (3 μM) and B trisaccharide at concentrations ranging from 0 μM to 800 μM. Fig. 5. View largeDownload slide Ion mobility separation arrival time distributions measured for Saga P dimer (P2Saga) ions: (A) (P2Saga)17+ (m/z 4102.41), and (B) (P2Saga)18+ (m/z 3875.12). The ions were produced by ESI-MS from aqueous ammonium acetate (300 mM, pH 7.0 and 25°C) solutions containing P2Saga (3 μM) and B trisaccharide at concentrations ranging from 0 μM to 800 μM. Far-UV CD spectroscopy (190–250 nm) measurements performed on solutions of P dimer and varying concentrations of the B trisaccharide or isomaltotriose provide additional evidence of the influence of oligosaccharide concentration on the structure of the Saga P dimer. Shown in Figure 6 are CD spectra (superimposed in the 230–250 nm region) measured for solutions of Saga P dimer (12.6 μM) with B trisaccharide or isomaltotriose (0–100 mM) in phosphate buffer (100 mM, pH 7.0, 25°C). Inspection of the spectra reveals subtle changes in the secondary structure of the P dimer with increasing concentration of both oligosaccharides. Interestingly, both the ligand (B trisaccharide) and the nonbinder (isomaltotriose) resulted in decreased CD signal in the 205 –220 nm region and increased CD signal at ~195 nm (Figure 6). These observations suggest that secondary structure (i.e., α-helix and β-sheet) content is sensitive to oligosaccharide concentration (Prasad and Roy 2010; Das et al. 2016). Although it is not possible to derive any specific conclusions regarding the nature of the conformational changes, these results, suggest that the higher order structure of the Saga P dimer undergoes changes with increasing concentrations of oligosaccharide and that this phenomenon is not simply the result of specific oligosaccharide binding. Such changes could, in principle, be the origin of the ‘step-like’ character observed in the STD-NMR binding isotherms (Mallagaray et al. 2015). Future research will focus on elucidating more precisely the nature of the structural changes induced by high concentrations of oligosaccharide. Fig. 6. View largeDownload slide Influence of the B trisaccharide and isomaltotriose on the conformation of Saga P dimer analyzed by far-UV circular dichroism spectroscopy (190 –250 nm). Spectra were measured using aqueous phosphate buffer (100 mM, pH 7.0 and 25°C) solutions containing Saga P dimer (12.8 μM) alone and in the presence of 10 mM or 100 mM of (A) B trisaccharide or (B) isomaltotriose. Fig. 6. View largeDownload slide Influence of the B trisaccharide and isomaltotriose on the conformation of Saga P dimer analyzed by far-UV circular dichroism spectroscopy (190 –250 nm). Spectra were measured using aqueous phosphate buffer (100 mM, pH 7.0 and 25°C) solutions containing Saga P dimer (12.8 μM) alone and in the presence of 10 mM or 100 mM of (A) B trisaccharide or (B) isomaltotriose. Conclusions The results of a quantitative ESI-MS study of the interactions between the P dimers of the HuNoV Saga, Vietnam 026 and VA387 strains with the ethyl glycoside of the B trisaccharide and free B type 1 tetrasaccharide are reported. Following careful correction of the ESI mass spectra for the occurrence of nonspecific interactions (which formed in the ESI droplets due to solvent evaporation), all three P dimers were found to bind a maximum of two B trisaccharides at the highest ligand concentration investigated. The apparent affinities of B trisaccharide for the three P dimers are low (≤1,000 M−1). Moreover, the binding data suggest HBGA ligand binding exhibits positive cooperativity. Similar results were obtained for interactions between the B type 1 tetrasaccharide and Saga P dimer. Taken together, the results of this study strongly suggest that the P dimers of HuNoVs possess only two HBGA binding sites. It is also shown that harsh (energetic) ESI-MS ion source conditions can lead to apparent HuNoV–HBGA oligosaccharide binding stoichiometries and affinities that are artificially high. Finally, evidence that high concentrations of oligosaccharide can induce conformational changes in HuNoV P dimers is presented. Materials and methods Proteins The P dimer of the VA387 strain (GII.4 genotype, MW 69,312 Da) was produced from the P domain of HuNoV VA387 VP1 (residues 225–539, GenBank accession number AY038600) and the P dimer of Vietnam 026 (GII.10 genotype) was produced from the P domain of HuNoV Vietnam 026 (residues 224–547, GenBank accession number AF504671). The methods used to prepare and purify the P dimers have been described elsewhere (Tan et al. 2004; Jin et al. 2015). Briefly, the P domains were expressed in Escherichia coli using pGEX-4T-1 vector through the glutathione S-transferase (GST) Gene Fusion System (GE Healthcare Life Science, Piscataway, NJ). The P domain-GST fusion proteins were purified using glutathione affinity chromatography (Glutathione Sepharose 4 Fast Flow, GE Healthcare Life Science) and then digested with thrombin (GE Healthcare Life Science) to remove the GST. The released P dimers were further purified by size-exclusion chromatography using a Superdex 200 size-exclusion column (GE Healthcare Life Science) and the corresponding fractions were collected. The P dimer of the Saga strain (GII.4 genotype, MW 69,734 Da) was produced from the P domain of HuNoV Saga as described elsewhere (Mallagaray et al. 2015). The codon-optimized DNA encoding residues 224–538 (GenBank accession number AB447457) was synthesized by GenScript (Piscataway, NJ) and cloned into a modified expression vector (pMal-c2X) at EcoRI and HindIII restriction sites. The vector was then transformed into E. coli BL21 cells. The transformed cells were grown at 30°C to an optical density (at 600 nm) of 0.5–0.6, and the protein expression was induced with isopropylthiogalactoside (0.3 mM) and further incubated at 20°C for 22 h with shaking. Cells were harvested by centrifugation and ruptured by lysis. The resulting His-tagged maltose binding protein (MBP)-P domain fusion protein was purified by affinity chromatography with a nickel column followed by digestion with HRV-3C protease (CEDARLANE, Burlington, Canada) overnight at room temperature. The cleaved P domain proteins were separated on the nickel column and further purified by size-exclusion chromatography with the Superdex 200 size-exclusion column (GE Healthcare Life Sciences) in the buffer containing 25 mM Tris-HCl and 300 mM NaCl (pH 7.4) and the corresponding fractions of Saga P dimer were collected. A single chain fragment (scFv, MW 26,539 Da) of the monoclonal antibody Se155−4 was produced using recombinant technology (Zdanov et al. 1994). Equine heart cytochrome c (cytoc, MW 12,384 Da) was purchased from Sigma-Aldrich Canada (Oakville, Canada). Each protein was concentrated and dialyzed into an aqueous 200 mM ammonium acetate solution (pH 7.0), unless otherwise indicated, using Amicon 0.5 mL microconcentrators (EMD Millipore, Billerica, MA) with a MW cutoff of 10 kDa or 30 kDa; protein concentrations were estimated by UV absorption (280 nm). All protein stock solutions were stored at −20°C until used. Carbohydrates The ethyl glycoside of the histo-blood group B trisaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-OC2H5, MW 516.49 Da) was a gift from Alberta Innovates Technology Futures (Alberta, Canada). The B type 1 tetrasaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-(1→3)-d-GlcNAc, MW 691.63 Da) was purchased from Elicityl SA (Crolles, France), and isomaltotriose (α-d-Glc-(1→6)-α-d-Glc-(1→6)-d-Glc, MW 504.44 Da) was purchased from Sigma-Aldrich Canada (Oakville, Canada). The structures are shown in Supplementary data, Figure S1. Stock solutions of each oligosaccharide were prepared by dissolving a known amount of solid in ultrafiltered Milli-Q water (EMD Millipore, MA); these were stored at −20°C until used. Mass spectrometry All ESI-MS measurements were carried out in positive ion mode on a Synapt G2S quadrupole–ion mobility separation–time of flight (Q-IMS-TOF) mass spectrometer (Waters, Manchester, UK) equipped with a nanoflow ESI (nanoESI) source. The ESI solutions were loaded into nanoESI tips, produced from borosilicate capillaries (1.0 mm o.d., 0.68 mm i.d.) using a P−1000 micropipette puller (Sutter Instruments, Novato, CA), with an o.d. of ~5 μm. To carry out nanoESI, a platinum wire was inserted into the tip and a voltage of ~1.2 kV was applied. The Source temperature was 60°C. The Cone, Trap and Transfer voltages were 50 V, 5 V and 2 V, respectively, unless otherwise specified. For IMS, the wave height and wave velocity were set to 40 V and 4000 ms−1, respectively, while a helium flow rate of 180 mL min−1 was used in the helium cell and a nitrogen flow rate of 90 mL min−1 was used in the IMS cell. Data acquisition and processing were performed using MassLynx software (version 4.1) and DriftScope (version 2.5). Direct ESI-MS assay The direct ESI-MS assay was used to quantify the affinities of the B trisaccharide and B type 1 tetrasaccharide for HuNoV P dimers (referred to here as P). Measurements were carried out in triplicate. To analyze the data, the abundance (Ab) ratio (Rq) of ligand (L)-bound (q ligands) to free P (PLq and P, respectively) ions measured by ESI-MS (after correction for nonspecific L–P binding, vide infra) was taken to be equal to the equilibrium concentration ratio in solution, Eq. (1): Rq=Ab(PLq)Ab(P)=[PLq][P] (1) The fraction (f) of L-bound P was calculated using Eq. (2): f=∑q=1hqRq1+∑q=1hRq (2) The apparent (macroscopic) association constant (Ka,q) for each binding step can be expressed in terms of Rq and the initial P dimer ([P]0) and ligand ([L]0) concentrations, Eq. (3) (Kitova et al. 2012): Ka,q=Rq/Rq−1[L]0−[P]0∑q=1hqRq1+∑q=1hRq (3) Correction of ESI mass spectra for nonspecific binding As discussed in detail elsewhere, ESI-MS binding measurements performed using high concentrations of oligosaccharide ligand typically result in the formation of nonspecific L–P interactions in the ESI droplets as they evaporate to dryness (Kitova et al. 2012). Therefore, to obtain reliable information on P–L binding stoichiometry and affinity from ESI-MS measurements, the mass spectra need to be corrected for the formation of these nonspecific complexes. Two different methods were used to perform this correction in the present study. The reference protein (Pref) method involves the addition of a noninteracting Pref to the solution. The underlying assumption is that the distribution of nonspecifically bound L to Pref is identical to that of P. This assumption has been validated in numerous studies, which have shown that nonspecific glycan–protein binding during ESI is a random process and independent of the size and structure of the protein (Sun et al. 2006, 2010). A complete description of the implementation of the correction method can be found elsewhere (Sun et al. 2006; Kitova et al. 2012). Two different Pref were used in this study—scFv and cytoc. Cytoc is not a lectin and does not recognize HBGA oligosaccharides in solution. The scFv is derived from an anti-glycan antibody; however, the antibody is specific for glycans found in Salmonella typhimurium and it does not bind to human glycans (Zdanov et al. 1994). The ESI mass spectra were also corrected for nonspecific binding using a mathematical approach (referred to as Kns method, vide infra) developed by Sharon and coworker (Shimon et al. 2010). According to this method, all nonspecific protein–ligand interactions that form during the ESI process can be described using a single association constant (Kns). Further, it is assumed that the ligand concentration at equilibrium is similar to the initial concentration, i.e., [L] ≈ [L]0, which is reasonable in the case of low affinity interactions. Finally, the number of specific binding sites (h) must be known. The value of Kns is calculated by plotting the ratios of the apparent abundances (Abapp) of P ions bound to q and (q−1) where q>h, versus [L], Eq. (4): Kns[L]=Abapp(PLq)Abapp(PLq−1)=Rq,app (4) The ‘true’ abundance ratios (in solution) can be determined from Abapp, Kns and [L], Eq. (5a): Ab(PLq)Ab(P)=Abapp(PLq)Abapp(P)−Abapp(PLq−1)Abapp(P)Kns[L] (5a) Using the same notation as used to describe the Pref method, Eq. (5a) can be rewritten as: Rq=Rq,app−Rq−1,app⋅Kns[L] (5b) and Ka,q can be obtained from Eq. (3). Isothermal titration calorimetry The binding of B trisaccharide to Saga P dimer was measured at 25°C with a VP-ITC (MicroCal, Inc., Northampton, MA). For each experiment, the Saga P dimer solution (0.1 mM) in the sample cell was titrated with a solution of B trisaccharide (10 mM). Both the P dimer and B trisaccharide were prepared in the Tris buffer (25 mM Tris-HCl and 300 mM NaCl, pH 7.4) solutions. Circular dichroism spectroscopy Circular dichroism (CD) spectroscopy was performed at 25°C with an OLIS DSM CARY-17 spectrophotometer conversion and CD module (On-line Instrument Systems Inc., Bogart, GA) using a 0.2 mm path length quartz cuvette. Protein solutions were prepared in 100 mM phosphate buffer (pH 7.0) containing 0–100 mM B trisaccharide or isomaltotriose. Data were collected in scanning mode from 190 nm to 250 nm, and for each analysis, values from replicate (five) measurements were averaged and reported. Data were analyzed with OLIS Spectral Works (v4.3) and converted into molar ellipticity units. At each concentration, the CD spectrum of the corresponding solution of oligosaccharide alone was subtracted from the sample spectrum. Supplementary data Supplementary data is available at Glycobiology online. Acknowledgements The authors are grateful for financial support provided by the Alberta Glycomics Centre and the National Institutes of Health of the USA. Conflict of interest statement None declared. Abbreviations Ab, abundance; ATDs, arrival time distributions; ATs, arrival times; CD, circular dichroism; cytoc, cytochrome c; ESI-MS, electrospray ionization mass spectrometry; GST, glutathione S-transferase; HBGAs, histo-blood group antigens; HuNoVs, human noroviruses; IMS, ion mobility separation; ITC, isothermal titration calorimetry; Ka, association constant; MBP, maltose binding protein; m/z, mass-to-charge ratio; MW, molecular weight; nanoESI, nanoflow ESI; P2, P dimer; Pref, reference protein; scFv, single chain variable fragment; STD-NMR, saturation-transfer difference nuclear magnetic resonance; TOF, time of flight. References Cao S , Lou Z , Tan M , Chen Y , Liu Y , Zhang Z , Zhang XC , Jiang X , Li X , Rao Z . 2007 . Structural basis for the recognition of blood group trisaccharides by norovirus . J Virol . 81 : 5949 – 5957 . Google Scholar CrossRef Search ADS PubMed Chen Y , Tan M , Xia M , Hao N , Zhang XC , Huang P , Jiang X , Li X , Rao Z . 2011 . Crystallography of a Lewis-binding norovirus, elucidation of strain-specificity to the polymorphic human histo-blood group antigens . PLoS Path . 7 : e1002152 . Google Scholar CrossRef Search ADS Choi JM , Hutson AM , Estes MK , Prasad BVV . 2008 . Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus . Proc Natl Acad Sci U S A . 105 : 9175 – 9180 . Google Scholar CrossRef Search ADS PubMed Das M , Wilson CJ , Mei X , Wales TE , Engen JR , Gursky O . 2016 . Structural stability and local dynamics in disease-causing mutants of human apolipoprotein A-I: what makes the protein amyloidogenic? J Mol Biol . 428 : 449 – 462 . Google Scholar CrossRef Search ADS PubMed Donaldson EF , Lindesmith LC , LoBue AD , Baric RS . 2010 . Viral shape-shifting: norovirus evasion of the human immune system . Nat Rev Micro . 8 : 231 – 241 . Google Scholar CrossRef Search ADS Dutta AK , Rösgen J , Rajarathnam K . 2015 . Using isothermal titration calorimetry to determine thermodynamic parameters of protein–glycosaminoglycan interactions . Methods Mol Biol . 1229 : 315 – 324 . Google Scholar CrossRef Search ADS PubMed Freyer MW , Lewis EA . 2008 . Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions . Methods Cell Biol . 84 : 79 – 113 . Google Scholar CrossRef Search ADS PubMed Han L , Kitov PI , Kitova EN , Tan M , Wang L , Xia M , Jiang X , Klassen JS . 2013 . Affinities of recombinant norovirus P dimers for human blood group antigens . Glycobiology . 23 : 276 – 285 . Google Scholar CrossRef Search ADS PubMed Han L , Tan M , Xia M , Kitova EN , Jiang X , Klassen JS . 2014 . Gangliosides are ligands for human noroviruses . J Am Chem Soc . 136 : 12631 – 12637 . Google Scholar CrossRef Search ADS PubMed Hansman GS , Biertumpfel C , Georgiev I , McLellan JS , Chen L , Zhou T , Katayama K , Kwong PD . 2011 . Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability . J Virol . 85 : 6687 – 6701 . Google Scholar CrossRef Search ADS PubMed Hansman GS , Shahzad-Ul-Hussan S , McLellan JS , Chuang GY , Georgiev I , Shimoike T , Katayama K , Bewley CA , Kwong PD . 2012 . Structural basis for norovirus inhibition and fucose mimicry by citrate . J Virol . 86 : 284 – 292 . Google Scholar CrossRef Search ADS PubMed Hao N , Chen Y , Xia M , Tan M , Liu W , Guan X , Jiang X , Li X , Rao Z . 2015 . Crystal structures of GI.8 Boxer virus P dimers in complex with HBGAs, a novel evolutionary path selected by the Lewis epitope . Protein Cell . 6 : 101 – 116 . Google Scholar CrossRef Search ADS PubMed Jayalakshmi V , Krishna NR . 2002 . Complete relaxation and conformational exchange matrix (CORCEMA) analysis of intermolecular saturation transfer effects in reversibly forming ligand–receptor complexes . J Magn Reson . 155 : 106 – 118 . Google Scholar CrossRef Search ADS PubMed Jayalakshmi V , Krishna NR . 2004 . CORCEMA refinement of the bound ligand conformation within the protein binding pocket in reversibly forming weak complexes using STD-NMR intensities . J Magn Reson . 168 : 36 – 45 . Google Scholar CrossRef Search ADS PubMed Jiang X , Wang M , Graham DY , Estes MK . 1992 . Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein . J Virol . 66 : 6527 – 6532 . Google Scholar PubMed Jin M , Tan M , Xia M , Wei C , Huang P , Wang L , Zhong W , Duan Z , Jiang X . 2015 . Strain-specific interaction of a GII.10 norovirus with HBGAs . Virology . 476 : 386 – 394 . Google Scholar CrossRef Search ADS PubMed Kitova EN , El-Hawiet A , Schnier PD , Klassen JS . 2012 . Reliable determinations of protein–ligand interactions by direct ESI-MS measurements. Are we there yet? J Am Soc Mass Spectrom . 23 : 431 – 441 . Google Scholar CrossRef Search ADS PubMed Koromyslova AD , Leuthold MM , Bowler MW , Hansman GS . 2015 . The sweet quartet: binding of fucose to the norovirus capsid . Virology . 483 : 203 – 208 . Google Scholar CrossRef Search ADS PubMed Koromyslova A , Tripathi S , Morozov V , Schroten H , Hansman GS . 2017 . Human norovirus inhibition by a human milk oligosaccharide . Virology . 508 : 81 – 89 . Google Scholar CrossRef Search ADS PubMed Kubota T , Kumagai A , Ito H , Furukawa S , Someya Y , Takeda N , Ishii K , Wakita T , Narimatsu H , Shirato H . 2012 . Structural basis for the recognition of Lewis antigens by genogroup I norovirus . J Virol . 86 : 11138 – 11150 . Google Scholar CrossRef Search ADS PubMed Lin H , Kitova EN , Klassen JS . 2014 . Measuring positive cooperativity using the direct ESI-MS assay. Cholera toxin B subunit homopentamer binding to GM1 pentasaccharide . J Am Soc Mass Spectrom . 25 : 104 – 110 . Google Scholar CrossRef Search ADS PubMed Liu W , Chen Y , Jiang X , Xia M , Yang Y , Tan M , Li X , Rao Z . 2015 . A unique human norovirus lineage with a distinct HBGA binding interface . PLoS Path . 11 : e1005025 . Google Scholar CrossRef Search ADS Mallagaray A , Lockhauserbaumer J , Hansman G , Uetrecht C , Peters T . 2015 . Attachment of norovirus to histo blood group antigens: a cooperative multistep process . Angew Chem, Int Ed . 54 : 12014 – 12019 . Google Scholar CrossRef Search ADS Oriol R . 1990 . Genetic control of the fucosylation of ABH precursor chains. Evidence for new epistatic interactions in different cells and tissues . J Immunogenet . 17 : 235 – 245 . Google Scholar CrossRef Search ADS PubMed Patel MM , Widdowson M-A , Glass RI , Akazawa K , Vinje J , Parashar UD . 2008 . Systematic literature review of role of noroviruses in sporadic gastroenteritis . Emerg Infect Dis . 14 : 1224 – 1231 . Google Scholar CrossRef Search ADS PubMed Prasad BVV , Hardy ME , Dokland T , Bella J , Rossmann MG , Estes MK . 1999 . X-ray crystallographic structure of the Norwalk virus capsid . Science . 286 : 287 – 290 . Google Scholar CrossRef Search ADS PubMed Prasad S , Roy I . 2010 . Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysis . Biotechnol Prog . 26 : 627 – 635 . Google Scholar CrossRef Search ADS PubMed Ravn V , Dabelsteen E . 2000 . Tissue distribution of histo-blood group antigens . APMIS . 108 : 1 – 28 . Google Scholar CrossRef Search ADS PubMed Shanker S , Choi JM , Sankaran B , Atmar RL , Estes MK , Prasad BV . 2011 . Structural analysis of histo-blood group antigen binding specificity in a norovirus GII.4 epidemic variant: implications for epochal evolution . J Virol . 85 : 8635 – 8645 . Google Scholar CrossRef Search ADS PubMed Shanker S , Czako R , Sankaran B , Atmar RL , Estes MK , Prasad BV . 2014 . Structural analysis of determinants of histo-blood group antigen binding specificity in genogroup I noroviruses . J Virol . 88 : 6168 – 6180 . Google Scholar CrossRef Search ADS PubMed Shimon L , Sharon M , Horovitz A . 2010 . A method for removing effects of nonspecific binding on the distribution of binding stoichiometries: application to mass spectroscopy data . Biophys J . 99 : 1645 – 1649 . Google Scholar CrossRef Search ADS PubMed Siebenga JJ , Vennema H , Zheng DP , Vinje J , Lee BE , Pang XL , Ho EC , Lim W , Choudekar A , Broor S et al. . 2009 . Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001–2007 . J Infect Dis . 200 : 802 – 812 . Google Scholar CrossRef Search ADS PubMed Singh BK , Leuthold MM , Hansman GS . 2015 . Human noroviruses’ fondness for histo-blood group antigens . J Virol . 89 : 2024 – 2040 . Google Scholar CrossRef Search ADS PubMed Sun J , Kitova EN , Wang W , Klassen JS . 2006 . Method for distinguishing specific from nonspecific protein–ligand complexes in nanoelectrospray ionization mass spectrometry . Anal Chem . 78 : 3010 – 3018 . Google Scholar CrossRef Search ADS PubMed Sun N , Soya N , Kitova EN , Klassen JS . 2010 . Nonspecific interactions between proteins and charged biomolecules in electrospray ionization mass spectrometry . J Am Soc Mass Spectrom . 21 : 472 – 481 . Google Scholar CrossRef Search ADS PubMed Tan M , Hegde RS , Jiang X . 2004 . The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors . J Virol . 78 : 6233 – 6242 . Google Scholar CrossRef Search ADS PubMed Tan M , Jiang X . 2005 . Norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle . Trends Microbiol . 13 : 285 – 293 . Google Scholar CrossRef Search ADS PubMed Tan M , Jiang X . 2010 . Norovirus gastroenteritis, carbohydrate receptors, and animal models . PLoS Pathog . 6 : e1000983 . Google Scholar CrossRef Search ADS PubMed Tan M , Jiang X . 2011 . Norovirus–host interaction: multi-selections by human histo-blood group antigens . Trends Microbiol . 19 : 382 – 388 . Google Scholar CrossRef Search ADS PubMed Tan M , Jiang X . 2014 . Histo-blood group antigens: a common niche for norovirus and rotavirus . Expert Rev Mol Med . 16 : e5 . Google Scholar CrossRef Search ADS PubMed Wegener H , Mallagaray A , Schone T , Peters T , Lockhauserbaumer J , Yan H , Uetrecht C , Hansman GS , Taube S . 2017 . Human norovirus GII.4 (MI001) P dimer binds fucosylated and sialylated carbohydrates . Glycobiology . 27 : 1027 – 1037 . Google Scholar CrossRef Search ADS PubMed Weichert S , Koromyslova A , Singh BK , Hansman S , Jennewein S , Schroten H , Hansman GS . 2016 . Structural basis for norovirus inhibition by human milk oligosaccharides . J Virol . 90 : 4843 – 4848 . Google Scholar CrossRef Search ADS PubMed Zdanov A , Li Y , Bundle DR , Deng SJ , Mackenzie CR , Narang SA , Young NM , Cygler M . 1994 . Structure of a single-chain antibody variable domain (fv) fragment complexed with a carbohydrate antigen at 1.7-angstrom resolution . Proc Natl Acad Sci U S A . 91 : 6423 – 6427 . Google Scholar CrossRef Search ADS PubMed Zheng DP , Ando T , Fankhauser RL , Beard RS , Glass RI , Monroe SS . 2006 . Norovirus classification and proposed strain nomenclature . Virology . 346 : 312 – 323 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
TI - Quantifying the binding stoichiometry and affinity of histo-blood group antigen oligosaccharides for human noroviruses
JF - Glycobiology
DO - 10.1093/glycob/cwy028
DA - 2018-03-19
UR - https://www.deepdyve.com/lp/oxford-university-press/quantifying-the-binding-stoichiometry-and-affinity-of-histo-blood-0L5m8032yQ
SP - 1
EP - 498
VL - Advance Article
IS - 7
DP - DeepDyve
ER -