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Natural killer (NK) cells serve as a crucial first-line defense against tumors and virus-infected cells. We previously showed that lysis of influenza virus (IV)-infected cells is mediated by the interaction between the NK receptor, NKp46, and the IV hemagglutinin (HA) type 1 expressed by the infected cells. This interaction requires the presence of sialyl groups on the NKp46-T225 O-glycoforms. In the current study, we analyzed the O-glycan sequences that are imperative for the interaction between recombinant NKp46 (rNKp46) and IV H1N1 strains. We first showed that rNKp46 binding to IV H1N1 is not mediated by a glycoform unique to the Thr225 site. We then characterized the O-glycan sequences that mediate the interaction of rNKp46 and IV H1N1; we employed rNKp46s with dissimilar glycosylation patterns and IV H1N1 strains with different sialic acid α2,3 and α2,6 linkage preferences. The branched α2,3-sialylated O-glycoform Neu5NAcα2,3-Galβ1,4-GlcNAcβ1,6[Neu5NAcα2,3-Galβ1,3]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for α2,3 linkage. In contrast, the linear α2,3-sialylated O-glycoform Neu5NAcα2,3-Galβ1,3-GalNAc was not correlated with enhanced interaction between rNKp46 and IV H1N1 or a preference for α2,3 linkage. The branched α2,3- and α2,6-sialylated O-glycoform Neu5NAcα2,3-Galβ1,3[Neu5NAcα2,6]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for α2,6 linkage. Previous viral HA-binding-specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells. This study shed light on the O-glycan sequences involved in the interaction of glycoprotein and viral hemagglutinins and may help in the design of agents inhibitory to hemagglutinin for influenza treatment.
Hemagglutinin (HA) is the receptor-binding and membrane fusion protein of influenza virus (IV), as well as the target for infectivity-neutralizing antibodies (27). Terminal sialic acids of glycoproteins and glycolipids are the cellular receptors for the IV HA (27). Two major linkages between sialic acid and the penultimate galactose residues of carbohydrate side chains are found in nature, Neu5NAcα(2,3)-Gal and Neu5NAcα(2,6)-Gal (27); different HAs have different recognition specificities for these linkages and the sugar backbone beneath (23, 26, 30). However, all of the HA-binding specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells (8, 10, 20, 25). This could be sufficient for the design of IV-inhibitory agents, and yet, it contributes only partially to the understanding of the interaction of IV HAs with glycoproteins and glycolipids. We aimed to further explore the exact glycoform sequences conjugated to a specific glycoprotein's glycosylation site that is recognized by different IV strains.
For this purpose, we took advantage of our findings on the interaction of natural cytotoxicity receptors (NCRs) and IV HAs (2, 3, 13, 18, 19, 22, 34). We showed that the NKp44 and NKp46 NCRs but not the NKp30 NCR interact with IV HAs. This interaction requires the sialylation of NKp44 and NKp46 oligosaccharides, and the binding of these NCRs to viral HA is required for the lysis of virus-infected cells by NK cells (3, 13, 18). NKp46 displays two putative O-linked glycosylation sites at Thr125 and Thr225 and one N-linked glycosylation site at Asn216. In order to determine the specific sugar-carrying residue that is important for the HA1 recognition, site-directed mutagenesis of the three residues was performed to carry the glycan modifications. Only when Thr225 was replaced was a sharp decrease in the enhanced binding to IV HA1 and IV H1N1-infected cells observed (2). Therefore, for the NKp46 receptor, the interaction with IV HA1 is restricted to Thr225, one of its three glycosylation sites (2).
We already showed that producing recombinant NKp46 (rNKp46) in different cell lines resulted in dissimilar glycosylation patterns and had a strong effect on the binding to its ligands (11). Therefore, we analyzed the O-glycan patterns of rNKp46 produced from various cell lines and utilized the dissimilar glycosylation patterns to elucidate the NKp46 O-glycan sequences that mediate the interaction with IV H1N1 strains. To associate the results with the IV preference for sialic acid α2,3 and/or α2,6 linkages, we employed A/PR/8/34 (H1N1), A/NC/20/99 (H1N1), and A/Brisbane/59/2007 (H1N1) grown in either hen egg amnion or Madin-Darby canine kidney (MDCK) cells. Our results pointed to two branched O-glycan sequences that mediated the interaction of the NKp46 glycoprotein with IV H1N1 in correlation with the sialic acid linkage preference of the IV strain.
Egg-adapted IV A/PR/8/34 (H1N1) was persistently grown in hen egg amnion as described previously (9) (titer of 1:1,024, determined by HA assay). For MDCK-grown A/PR/8/34 stock, egg-adapted virus was passaged twice in MDCK cells (titer of 1:128). Egg-adapted IV A/NC/20/99 (H1N1) was persistently grown in hen egg amnion as described previously (titer of 1:256) (1). MDCK-adapted A/NC/20/99 was persistently grown in MDCK cells (titer of 1:512). IV A/Brisbane/59/2007 (H1N1) was prepared from original 2007 WHO stock as follows: the original stock was passaged twice in hen egg amnion (egg-grown stock, titer of 1:512), and then the egg stock was passaged twice in MDCK cells (MDCK-grown stock, titer of 1:256). Five fresh clinical isolates (3 typed as A/Solomon Islands/3/2006 [H1N1] and 2 typed as A/Brisbane/59/2007 [H1N1]) were taken directly from positive MDCK cultures of clinical throat samples. MDCK cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 1% penicillin and streptomycin (Pen-Strep), 1% l-glutamine solution, and 6.25 U/ml nystatin. Cells were infected with MDCK-grown A/NC/20/99 when the monolayer reached a 50 to 60% confluence, as follows. The virus was diluted in DMEM-0% FCS containing 3.3 μg/ml trypsin type IX-S (catalog no. T0303; Sigma) to a final volume of 6 ml and incubated with the cells for 1.5 h at 37°C with 5% CO2. An additional 29 ml of DMEM-2% FCS plus trypsin medium was added to the cells, and the infected cells were further incubated for 3 to 5 days at 37°C with 5% CO2. Viral infectivity was determined by changes in cell morphology and mortality (60 to 80% cytopathic effect), and then, supernatant was collected and centrifuged at 2,500 rpm (40 min at 4°C) to remove cells. The supernatant containing the virus was further ultracentrifuged at 20,000 rpm for 6 h at 4°C (centrifuge head SW27) to form a pellet. The pellet was resuspended in saline or 1× phosphate-buffered saline (PBS) and stored in aliquots at −70°C.
Synthetic polyacrylamide (PAA)-based glycopolymers carrying multivalent displays of sialic acid residues were produced as described previously (6). The average molecular mass of the polymers was 30 kDa, and the sugar content was 20 mol%.
The receptor-Ig fusion proteins that we used in this work were mainly NKp46-Ig and its NKp46-T125A-Ig and NKp46-T225A-Ig mutants (2). The constructs were transiently transfected into African green monkey kidney fibroblast (COS) or human embryonic kidney 293T (HEK293T) cells or stably transfected into Chinese hamster ovary (CHO) cells. Medium containing the secreted fusion proteins was collected and purified on a HiTrap protein G column (5 ml; catalog no. 0405-01; Daniel Biotech, GE Healthcare) as previously described (2, 5, 12).
To examine the interactions of recombinant NKp46s and IV, plates were coated overnight at 4°C with 0 to 20 μg/ml of rNKp46 (produced in various cell lines and diluted in PBS to a final volume of 100 μl). Blocking buffer (PBS supplemented with 1% bovine serum albumin [BSA]; 200 μl/well) was applied for 2 h, and then the plates were washed with PBST (PBS with 0.05% Tween 20) and incubated with 100 μl of diluted A/PR/8/34 or A/NC/20/99 (H1N1) for 1 h at room temperature (RT). Following washing with PBST, a 1:200 or 1:250 dilution of MAb NA21C1 (kind gift of Jonathan W. Yewdell, National Institutes of Health) or anti-H1 MAb (catalog no. MAB8261; Chemicon International) (when using A/PR/8/34 or A/NC/20/99, respectively) was applied and incubated for 45 min at RT. After washing with PBST, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (product no. 115-035-062; Jackson ImmunoResearch) was added at a dilution of 1:1,500 and incubated for 45 min at RT. Following washing with PBST, 100 μl/well of tetramethylbenzidine (TMB) was added and color was allowed to develop at RT in the dark. The optical density was read at 650 nm (MRX microplate reader; Dynex Technologies). After the blocking stage, all dilutions were done in PBS supplemented with 0.2% BSA. For the A/Brisbane/59/2007 (H1N1) strain, wells were coated with the virus, followed by blocking and the application of 0 to 20 μg/ml of rNKp46. Detection was performed with HRP-conjugated goat anti-human IgG-Fcγ (product no. 109-036-098; Jackson ImmunoResearch), followed by TMB.
To examine the interaction of PAA-based glycopolymers and IV, plates were coated overnight at 37°C with 0 to 4 μg/ml of PAA-based glycopolymers (diluted in PBS to a final volume of 100 μl). Blocking, virus application, staining, and detection were performed as described above (32).
Glycoproteins were lyophylized and then cryogenically dried before hydrazinolysis. Samples were incubated with anhydrous hydrazine for 6 h at 60°C to release the O-linked glycan (glycan hydrazinolysis kit, catalog no. GK50202; Glyco/Prozyme). Excess hydrazine was removed by evaporation, and the glycans were re-N-acetylated with acetic anhydride in a saturated solution of sodium bicarbonate. Peptides were removed by descending paper chromatography on prewashed Whatmann 3-mm chromatography paper in butanol/ethanol/water (8:2:1 [vol/vol]) for 70 h. Glycans were recovered from the paper (1 to 3 cm from origin) by washing with 1.5 ml of water. A rotary evaporator was used to concentrate the samples, which were then stored at 20°C prior to labeling with 2AB (2-aminobenzamide).
Arrays of exoglycosidases were used in combination with normal phase (NP)-HPLC to determine the oligosaccharide sequence. Exoglycosidase sequencing was performed on the released, 2AB-labeled glycan solution containing enzymes at standard concentrations in the manufacturer's recommended buffers for 16 to 24 h at 37°C. Exoglycosidases were used at the following concentrations: Arthrobacter ureafaciens sialidase (sialidase A, cleaves both α2,3 and α2,6 sialic acid residues) (ABS, EC 188.8.131.52), 1 to 2 U/ml; bovine testis β-galactosidase (BTG, EC184.108.40.206), 2U/ml; and Streptococcus pneumoniae sialidase (sialidase S, cleaves only α2,3 sialic acid residues) (EC 220.127.116.11, Nan1), 1 U/ml (all enzymes were from Glyco/Prozyme). After digestion, samples were separated from the exoglycosidases before NP-HPLC analysis by allowing them to disperse into a protein-binding filter inset into a microcentrifuge tube (Pro-Spin 45-μm CN; R. B. Radley and Co., Ltd., Saffron Walden, Essex, United Kingdom) for 30 min before the glycans were eluted with 5% acetonitrile in water.
NP-HPLC was performed with the low-salt buffer system using a 4.6- by 250-mm GlycoSep N column (OGS) (Waters, Milford, MA). The solvents used were buffer B (50 mM ammonium formate, pH 4.4) and buffer C (acetonitrile). The glycans were eluted by linear gradient with buffer B, with initial conditions of 20% buffer B at a flow rate of 0.4 ml/min. The concentration of buffer B was changed from 35 to 53% over 132 min and then from 53 to 100% over the next 3 min, with a constant flow rate. The column was washed with 100% buffer B for 5 min at a flow rate of 1 ml/min before re-equilibration in the initial solvent system. Fluorescence was measured at 420 nm with an excitation of 330 nm (with 16-nm bandwidths). The system was calibrated using an external standard of hydrolyzed and 2AB-labeled glucose oligomers to create a dextran ladder. Briefly, the number of glucose residues in each dextran peak was plotted against the retention times of the peaks to obtain a standard curve using a fifth-order polynomial line fit. The retention time for individual glycans was converted to glucose units (GU) using this curve.
We previously reported that recombinant NKp46 interacts with IV HA1 and that the presence of sialic acid residues upon the NKp46 glycans is imperative for this interaction (2, 18, 19). The NKp46 ectodomain has one predicted N-glycosylation site (N216) and two predicted O-glycosylation sites (Thr125 and Thr225). We then showed that COS-produced rNKp46, rNKp46-T125A, and rNKp46-N216A recognized IV HA1, while COS-produced rNKp46-T225A did not (2). In agreement with the irrelevance of the N216 glycosylation site, peptide N-glycosidase F treatment of rNKp46 did not suppress the interaction with IV HA1 (data not shown). Therefore, the sialic acid-bearing glycans expressed on Thr225 of NKp46 are essential for the interaction with IV HA1.
The differential interaction with IV HA1 of the NKp46-T125A and -T225A mutants could be due to O-glycoforms unique for the NKp46-T225 position. We therefore compared the O-glycoforms of COS-produced rNKp46-T125 and rNKp46-T225 by studying the O-glycoforms of the rNKp46-T225A and the rNKp46-T125A mutant, respectively. This approach was plausible since the only two O-glycosylation sites on rNKp46 are Thr125 and Thr225, as there are no O-glycosylation sites on the Fc portion of rNKp46.
The NP-HPLC profiles of O-glycans released from COS-produced rNKp46 and the two mutants are shown in Fig. Fig.11 and Table Table1.1. The data reveal a similar pattern of the released O-glycans of rNKp46-T225A compared to those of rNKp46-T125A. We did not identify any glycoform that was unique for the Thr225 site. Moreover, the subtle differences in the proportions of the different glycoforms between the two glycosylation site mutants (Table (Table1)1) did not justify the published disparity in the interaction with IV HA1 (2). To summarize, the differences in IV HA1 interaction of the rNKp46-T125 and -T225 mutants could not be attributed to the presence of a unique O-glycoform at position Thr225.
To further explore the contribution of protein-specific O-glycoforms to the interaction with IV HA1, we analyzed the binding of rNKp46 produced in HEK293T, CHO, and COS cells to different IV strains. We employed the A/PR/8/34 (H1N1) strain persistently grown in hen eggs and the A/NC/20/99 (H1N1) strain persistently grown either in MDCK cells or in hen eggs. The MDCK stock of A/PR/8/34 was produced from egg stock by two successive passages in MDCK cells. Figure Figure22 shows the interaction of titrated amounts of the different rNKp46s with the different IV strains. HEK293T-produced NKp46 manifested the best binding to both hen egg-grown viruses (Fig. 2A and C), while the CHO-produced NKp46 showed a significantly lower level of binding to those viruses. The COS-produced NKp46 showed a moderate, in-between level of binding for both hen egg-grown viruses. The levels of interaction of the MDCK-passaged A/PR/8/34 with the three rNKp46s were similar, with the advantage to the HEK293T-produced NKp46 that manifested the highest binding, and yet, the difference was not statistically significant (Fig. (Fig.2B).2B). Interestingly, the interaction of the MDCK-grown A/NC/20/99 with the three rNKp46s showed a different pattern; COS-produced NKp46 showed a significantly higher level of binding than CHO- and HEK293T-produced NKp46, which bound similarly, with the advantage to the CHO-produced NKp46 (Fig. (Fig.2D2D).
We further studied the recently reported strain A/Brisbane/59/2007 (H1N1). The egg-grown virus was generated by 2 passages from WHO virus stock. The MDCK-grown virus was generated by 2 passages from the egg-grown virus. Interactions with HEK293T-produced and CHO-produced NKp46 were assayed. CHO-produced NKp46 showed a significantly higher level of binding than HEK293T-produced NKp46 for the MDCK-grown virus (Fig. (Fig.2F).2F). The same trend was observed with the egg-grown virus, but without a statistically significant difference (Fig. (Fig.2E).2E). In addition, we tested five clinical isolates (three typed as A/Solomon Islands/3/2006 [H1N1] and two typed as A/Brisbane/59/2007 [H1N1]) taken directly from positive MDCK cultures of clinical throat samples. The results match those for the MDCK-grown A/Brisbane/59/2007 (data not shown).
Therefore, rNKp46s produced in different cells exhibit different levels of interaction with IV HA1 that could be due to dissimilar repertoires of O-glycans (Fig. (Fig.2,2, all panels). In addition, the habitat in which the virus was grown had a strong effect on the binding preferences of different rNKp46s (Fig. 2C and D).
To better assess the contribution of the O-glycosylation patterns to the differential levels of interaction of the rNKp46s with the different IV H1N1 strains, we studied the O-glycans of the 3 rNKp46s (Fig. (Fig.11 and and33 and Table Table1).1). The O-glycan analysis revealed significant differences. O-Glycosylation from HEK293T-produced rNKp46 contained 38.81% glycoform 8 (hexamer, Neu5NAcα2,3-Galβ1,4-GlcNAcβ1,6[Neu5NAcα2,3-Galβ1,3]GalNAc). However, this glycoform accounted for just 7.04% or 0% of the O-glycans expressed on COS- and CHO-produced rNKp46s, respectively. Glycoform 6 (tetramer, Neu5NAcα2,3-Galβ1,3[Neu5NAcα2,6]GalNAc) was prominent in the O-glycosylation of all proteins (45.5% for HEK293T-, 39.55% for CHO-, and 66.8% for COS-produced rNKp46). Glycoform 3 (trimer, Neu5NAcα2,3-Galβ1,3-GalNAc) was prominent mostly in CHO-produced rNKp46 (5.58% for HEK293T-, 48.17% for CHO-, and 6.65% for COS-produced rNKp46). The obvious conclusion from these results is that the mechanism of interaction between the HA1 expressed by the various IV strains and the 3 rNKp46s relies in part on the broad O-glycosylation pattern presented by the NP-HPLC profile analysis.
The IV growth habitat also contributed to the observed differential levels of interaction (Fig. (Fig.2).2). Several publications have shown a correlation between the IV growth habitat and the sialic acid linkage (α2,3 or α2,6) preference of the IV HA1 (7, 10, 17, 31, 32). The A/PR/8/34 strain was grown consistently in hen eggs, and the A/NC/20/99 strain was grown either in MDCK cells or in hen eggs. To correlate the preference with the O-glycosylation patterns of the rNKp46s, we examined the binding of the different IV strains to two representative PAA-based glycopolymers, each containing the α2,3 or α2,6 Neu5NAc linkage. Figure Figure44 shows the different binding pattern manifested by each of these different IV strains. Egg-adapted A/PR/8/34 and A/NC/20/99 strains showed considerable binding to both the Neu5NAcα(2,3) and Neu5NAcα(2,6) linkages relative to their binding to the nonsialylated Galβ1,4-GlcNAcβ-PAA. The MDCK-adapted A/NC/20/99 and MDCK-grown A/Brisbane/59/2007 (WHO stock) strains preferentially bound the Neu5NAcα(2,6) linkage, in accordance with previous reports (7, 10, 17, 31, 32). Egg-adapted A/PR/8/34 passaged twice in MDCK still manifested considerable binding to both the Neu5NAcα(2,3) and Neu5NAcα(2,6) linkage (Fig. (Fig.4B).4B). Two egg amnion passages of WHO stock of A/Brisbane/59/2007 did not affect its preferential binding to the Neu5NAcα(2,6) linkage (Fig. (Fig.4E).4E). Clinical isolates of A/Brisbane/59/2007 taken directly from positive MDCK cultures of clinical throat samples manifested the same pattern as the MDCK-grown A/Brisbane/59/2007 stock (data not shown).
Most of the viral HA-binding-specificity studies were performed with glycopolymer conjugates or free synthetic sialo-oligosaccharides (8, 25). The basic idea behind these studies rises from the knowledge that viral HA plays a key role in cell recognition and infection initiation by binding sialic acid-containing receptors on the host cells, which then mediate the subsequent membrane fusion and viral entry. The use of glycopolymer conjugates or free synthetic sialo-oligosaccharides in IV infection studies could be sufficient for the design of IV-inhibitory agents, and yet, it contributes only partially to the understanding of the interaction of IV HAs with glycoproteins and glycolipids. An alternative approach is to study the binding of IV HAs to red blood cells by using specific sialidase and sialyltransferase treatments or to overexpress sialyltransferases in target infected cells (10, 20). However, most of those studies investigated the contribution of the N-linked glycosylations (28, 29, 33), and the involvement of the O-linked glycans of the glycoprotein receptor on the host cell in the interaction with IV HAs remains uninvestigated.
In our study, we aimed to further explore the exact O-glycoform sequences of specific NKp46 glycosylation sites that are recognized by different IV strains. Previously published work from our laboratory showed that the NKp44 and NKp46 NCRs interact with IV HAs and that this interaction requires the sialylation of NKp46 oligosaccharides (2, 4, 18, 19). As reported, NKp46 displays two putative O-linked glycosylation sites at Thr125 and Thr225 and one N-linked glycosylation site at Asn216, all located within the second domain. In order to further investigate the contribution of each glycosylation site, site-directed mutagenesis of the three residues was performed to abolish the glycan modifications. It was shown in this work that only a mutation in Thr225 caused a sharp decrease in the enhanced binding of rNKp46 to viral HAs (2).
The O-glycans expressed on the two NKp46 mutants manifested similar profiles, with similar percentages of the various glycoform sequences (Fig. (Fig.11 and Table Table1).1). Thus, we assume that the differences between the ability of the two glycosylation sites to interact with IV HAs does not derive from unique sialo-oligosaccharide sequences but from the location of the glycosylation site in the protein structure. Thr125 is located within domain 2 of NKp46, while Thr225 is located within the unstructured hinge region (approximately 35 amino acids) that connects domain 2 of NKp46 to the cell membrane. This NKp46-derived sequence connects the rNKp46 to the Fc backbone. Therefore, the Thr225 glycans might be more accessible than the Thr125 glycans to the HA. Similarly, the glycosylated hinge region of NKp44 mediates the interaction with IV HA (A. Porgador, unpublished results). In addition, structurally close amino acids with a positively charged group might interact with the glycans' terminal sialic acids and influence the interaction with the viral HA. No such amino acid was found around Thr225; however, positively charged amino acids are found in close proximity to Thr125, e.g., Arg145 (data not shown).
In contrast to common studies that investigate IV sialo-oligosaccharide recognition, this work is one of the few studies that characterize the interaction of viral HA with soluble glycoprotein receptors. The similarity in NKp46-Ig recognition of egg-adapted A/PR/8/34 and A/NC/20/99 (Fig. (Fig.2)2) can be easily explained by the two main glycoforms expressed on HEK293T-produced rNKp46: a hexamer (glycoform 8) containing two α2,3 linkages (Fig. (Fig.33 and Table Table1),1), Neu5NAcα2,3-Galβ1,4-GlcNAcβ1,6[Neu5NAcα2,3-Galβ1,3]GalNAc (38.81%), and a tetramer (glycoform 6) containing α2,3 and α2,6 linkages, Neu5NAcα2,3-Galβ1,3[Neu5NAcα2,6]GalNAc (45.5%). The dominant presence of glycoform 8 is unique to the HEK293T-produced rNKp46 and can explain the efficient binding of egg-adapted viruses which are more compatible with the α2,3 linkage (Fig. (Fig.4).4). Furthermore, we found that egg-adapted A/PR/8/34 and A/NC/20/99 manifest moderate binding to COS-produced rNKp46, which expressed mostly glycoform 6 (66.8%) (Fig. (Fig.11 and Table Table1),1), a result which conforms with the dual affinity for α2,3 and α2,6 linkages shown by the results in Fig. Fig.4.4. Interestingly, CHO-produced rNKp46 had the lowest affinity for binding by the egg-adapted viruses, in spite of the fact that it expresses 39.55% glycoform 6 (Fig. (Fig.33 and Table Table1).1). Moreover, CHO-produced rNKp46 contains 48.17% glycoform 3, a trimer with one α2,3 residue, Neu5NAcα2,3-Galβ1,3-GalNAc. The observation that egg-adapted A/PR/8/34 and A/NC/20/99 have significantly lower levels of binding to CHO-produced rNKp46 (which has 0% glycoform 8 and 48.17% glycoform 3) than to HEK293T-produced rNKp46 (with 38.81% glycoform 8 and 5.58% glycoform 3) indicates that branched and not linear α2,3 linkages are required for a potent interaction of egg-adapted HA with glycoprotein-conjugated O-glycans. Therefore, a potent inhibitory agent for viruses grown in hen egg can be achieved by synthesizing branched sialo-oligosaccharide residues which terminate with an α2,3 Neu5NAc linkage in each branch. Consequently, glycoform 8 should be considered as an inhibitory agent for avian IV infections.
Two MDCK passages of egg-adapted A/PR/8/34 did not switch linkage affinity as assayed by using PAA-conjugated synthetic trisaccharides (Fig. 4A and B). This is in accordance with previous reports that only a few passages did not suffice to switch linkage affinity (14, 15). Yet, the interaction of MDCK-passaged A/PR/8/34 with glycoprotein-conjugated O-glycans was more sensitive than the assay with the synthetic trisaccharides; the significant interaction of HEK293T-produced NKp46 is reduced to a nonsignificant trend (Fig. 2A and B). MDCK-grown stock of a recent human virus, A/Brisbane/59/2007, manifested clear interaction with synthetic trisaccharides terminating with an α2,6 linkage (Fig. (Fig.4F).4F). Two egg passages did not induce a significant change (Fig. (Fig.4E).4E). And yet, the assay with the glycoprotein-conjugated O-glycans was again more sensitive. CHO-produced NKp46 manifested significantly higher levels of interaction with MDCK-grown A/Brisbane/59/2007 than HEK293T-produced NKp46 (Fig. (Fig.2F);2F); however, two passages in eggs reduced the difference to a nonsignificant trend (Fig. (Fig.2E).2E). Clinical isolates of A/Brisbane/59/2007 and A/Solomon Islands/3/2006 behave similarly to MDCK-grown A/Brisbane/59/2007, showing significant levels of interaction with the CHO-produced NKp46 (data not shown).
Integrating the results shown in Fig. Fig.2,2, ,3,3, and and44 and those for the clinical isolates suggests that glycoform 6 and not glycoforms 8 and 3 could be the focal point for the interaction of human H1N1 with glycoprotein-conjugated O-glycans. Egg adaptation or passage, which does not mimic human H1N1 linkage preferences, favors or improves interaction with HEK293T-produced NKp46, which has nearly 39% glycoform 8. MDCK adaptation (A/NC/20/99 and A/Brisbane/59/2007) and clinical isolates, which mimic human H1N1 linkage preferences, favor or improve interactions with CHO- and COS-produced NKp46s that have 0 and 7% glycoform 8, respectively. Therefore, glycoform 8 cannot mediate this interaction. Glycoform 3, which is expressed by CHO NKp46 (48%), is not a likely candidate due to the low to no interaction of MDCK-adapted viruses with similar synthetic trisaccharides. The above-described exclusion of glycoforms 8 and 3 points to glycoform 6 as the plausible frequent sialylated candidate to mediate the better interaction of CHO and COS NKp46s with MDCK-adapted and clinical isolate H1N1 viruses. The imperfection in this conclusion is the high level of expression of glycoform 6 by HEK293T NKp46. Factors like the reduced glycan occupancy of position Thr225 and/or asymmetric distribution of glycoform 6 between Thr125 and Thr225 of HEK293T NKp46 (though not shown for COS NKp46) could explain this flaw. Nevertheless, the reasonable removal of glycoforms 8 and 3 from the candidate list leaves glycoform 6 as the better candidate to mediate the interaction of human H1N1 with glycoprotein-conjugated O-glycans.
To conclude, synthetic linear trisaccharides terminating with α2,3 linkages were explored as inhibitory agents for IV HA (21, 31, 32). Our study suggests that these linear trisaccharides might not be a potent O-glycoform that mediates hemagglutinin interactions in human IV-H1N1 infections. Branched 7-mer saccharides terminating with α2,3 and α2,6 linkages were also suggested as inhibitory agents for human IV H1N1 infections (e.g., patent IPN WO2003/074570). However, our findings indicate that when synthesizing a potent inhibitory agent for viruses grown in MDCK cells, which are naturally more important and relevant than egg-grown viruses for human IV infections, the tetrameric sialo-oligosaccharide residues Neu5NAcα2,3-Galβ1,3[Neu5NAcα2,6]GalNAc should be considered.
In summary, we characterized the carbohydrates presented on rNKp46s from various cell line production sources and their involvement in the viral recognition of HA1. The combination of IV H1N1 grown in different sources and the varied NKp46 O-linked glycans revealed a broad repertoire of HA-glycoprotein interaction affinities. These results can contribute to a better understanding of the carbohydrate involvement in IV infections and may help in the improvement of clinical therapeutic aspects.
This work was supported by a joint Croatia-Israel research grant and by a grant from the European Commission (GLYFOIS contract no. 03766).
Published ahead of print on 10 February 2010.