Abstract
Pigs can be infected with both human and avian influenza A virus (IAV) strains and are therefore considered to be important intermediates in the emergence of new IAV strains due to mixing of viral genes derived from human, avian, or porcine influenza viruses. These reassortant strains may have potential to cause pandemic influenza outbreaks in humans. The innate immune response against IAV plays a significant role in containment of IAV in the airways. We studied the interactions of IAV with porcine surfactant protein D (pSP-D), an important component of this first line defense system. Hemagglutination inhibition analysis shows that the distinct interactions of pSP-D with IAV mediated by the N-linked carbohydrate moiety in the carbohydrate recognition domain of pSP-D depend on the terminal sialic acids (SAs) present on this carbohydrate. Analysis by both lectin staining and by cleavage with linkage-specific sialidases shows that the carbohydrate of pSP-D is exclusively sialylated with α(2,6)-linked SAs, in contrast to surfactant protein A, which contains both α(2,3)- and α(2,6)-linked SAs on its N-linked carbohydrate. Enzymatic modification of the SA-linkages present on pSP-D demonstrates that the type of SA-linkage is important for its hemagglutination-inhibitory activity, and correlates with receptor-binding specificity of the IAV strains. The SAs present on pSP-D appear especially important for interactions with poorly glycosylated IAV strains. It remains to be elucidated to what extent the unique sialylation profile of pSP-D is involved in host range control of IAV in pigs, and whether it facilitates adaptation of avian or human IAV strains that can contribute to the production of reassortant strains in pigs.
Influenza A viruses (IAVs) are a major cause of respiratory disease and can infect a wide range of animal species including humans, pigs, and birds (1). IAV infection is initiated by the attachment of virus particles to target cells located in the upper respiratory tract. This process is facilitated through interaction of a viral spike glycoprotein, the hemagglutinin (HA), with the cell-surface glycoproteins and glycolipids, which contain terminal sialic acid (SA) residues (2). Variations in the binding specificity of the SA receptor, located on the head of the HA molecule, depend on the host species from which the virus is isolated (2, 3). The specificity of this receptor is characterized by differences in affinity for the SA species present on sialyloligosaccharides. These SA species can be either N-acetylneuraminic acid (Neu5Ac), or N-glycolylneuraminic acid (Neu5Gc), in which the N-acetyl-group of Neu5Ac is hydroxylated (Figure 1)
Figure 1. Structures of terminal SAs that display receptor binding specificity toward IAV. The structure of N-acetylneuraminic acid (Neu5Ac) is shown, present in either α(2,3)-linkage (top) or α(2,6)-linkage (bottom) to the penultimate galactose residues, as part of outer chain structures found in complex type sugar chains. Enzymatic hydroxylation of the methyl group of Neu5Ac (denoted with an asterisk), results in N-glycolylneuraminic acid (Neu5Gc), the other major SA species found in animals.
[More] [Minimize]The SA receptor of various IAV strains also differs in affinity to SAs terminally linked either α(2,3) or α(2,6) to the penultimate galactose (Gal) residues (Figure 1). Human IAVs preferentially recognize the α(2,6)-linkage, those isolated from birds and equines preferentially bind to α(2,3)-linkages, whereas swine IAVs recognize both linkages (4, 8–10). The receptor specificity for both SA species and SA linkages of IAVs isolated from the host animals correlates with the distribution of these SA species and linkages in various host animal species (4, 5, 7, 9, 11). The presence of both Neu5Ac and Neu5Gc SA species, and both α(2,3) and α(2,6) SA linkages in porcine pulmonary tissue, provides a molecular basis for efficient replication of human and avian IAVs in this host animal species. It was proposed by Scholtissek and coworkers (12), and supported by experimental infections in pigs (13, 14), that pigs may serve as “mixing vessels” where co-circulation of IAV may result in human–avian reassortant IAV strains. Therefore, pigs are an important source from which potentially hazardous IAV strains can be transmitted into the human population.
The host response against IAV involves specific as well as nonspecific immune mechanisms. Specific immune responses are needed for the eradication of the virus in established infection, in which protective immunity to IAV is acquired through the generation of antibodies that are directed against HA. However, especially during early stages of IAV infection, nonspecific immune responses play an essential role in limiting the extent of replication of IAV in the airway (15–18). Important components of this innate immune defense system in the lung are the surfactant proteins A (SP-A) and D (SP-D) (19, 20). These proteins are members of the collectin family, a group of collagenous carbohydrate binding proteins that are present as large, multimeric glycoproteins in alveolar lining fluid or serum. Neutralization of IAV by SP-A and SP-D has been demonstrated by in vitro and in vivo studies, and is obtained through direct interaction with IAV that results in inhibition of viral infectivity, formation of viral aggregates, and enhanced uptake of IAV by phagocytic cells (16, 17, 21, 22).
The collectins share a number of structural features, which include the presence of a collagenous region, an α-helical coiled-coil “neck” region, and a C-terminal Ca2+-dependent carbohydrate recognition domain (CRD) that can interact with glycoconjugates expressed at the surface of microorganisms. The basic structural unit of the collectins is a trimer, but differences are found in the degree of higher order oligomerization. SP-A is assembled as an octadecamer (hexamer of trimers), whereas SP-D is assembled into dodecamers, composed of four trimers, or higher order oligomeric complexes (23, 24). Among other structural variations, SP-A and SP-D also show differences in post-translational modifications. Human SP-A (hSP-A) contains a partly sialylated Asn187-linked complex type oligosaccharide in its CRD (25). Human SP-D is glycosylated in its collagen domain by O-linked glycosyl–galactosyl structures present on hydroxylated lysine residues, and by an Asn70-linked complex type glycosylation that is not sialylated (26). Interestingly, cDNA cloning of porcine SP-D (pSP-D) revealed unique features that are absent in SP-D from other species (27). These included the presence of an N-glycosylation motif sequence in the CRD (Asn323-Phe324-Thr325). Biochemical characterization of SP-D isolated from porcine lung lavage, has revealed that Asn323 is glycosylated with a complex type N-linked oligosaccharide. This carbohydrate is highly heterogeneous and remarkably rich in terminal SA residues (24). This structural feature has not been found in SP-Ds from any other animal species characterized to date.
SP-A and SP-D interact with IAV in different ways. SP-A binds in a Ca2+-independent fashion via SA residues, present on the N-linked carbohydrate in its CRD (22). It is likely that this interaction involves the SA receptor present on the HA of IAV. In contrast, SP-D binds via its CRD in a Ca2+-dependent manner to N-linked high-mannose carbohydrates present on the HA, and the other IAV envelope glycoprotein, neuraminidase (28). The activity of SP-D, especially that of the higher order multimeric form, is in general much greater than the activity of SP-A, as demonstrated by hemagglutination (HAA) inhibition and IAV aggregation experiments (29, 30).
Considering the important role of pigs in interspecies transmittance of IAV strains with birds and humans, we recently studied the anti-IAV activity of the porcine lung collectins SP-A (pSP-A) and pSP-D (31). HAA inhibition assays showed that both pSP-A and pSP-D display substantially greater inhibitory activity against a wide variety of IAV strains, isolated from human, swine, and horse, compared with SP-A and SP-D, respectively, from other animal species. It was also demonstrated by several functional assays, in which the A/Phillipines/82(H3N2) strain was used, that the N-linked oligosaccharide located in the CRD enhances the anti-influenza activity of pSP-D. HAA inhibition assays also showed that this carbohydrate moiety broadens the spectrum of IAV strains that can be inhibited by pSP-D. Although it was shown that the complex sugar in the CRD of pSP-D is highly sialylated, it remained unknown to what extent these SA moieties are responsible for the distinct interactions of pSP-D with IAV.
The present work shows that the contribution of the N-linked carbohydrate within the CRD in the activity of pSP-D against IAV is mediated via the terminal SA residues found on this oligosaccharide. Analysis and modification of the type of SA linkage of these SA residues, either α(2,3) or α(2,6) to Gal, shows that these SA linkages are important for the anti-IAV activity of pSP-D, and correlate with the receptor-binding specificity of IAV for type of SA linkage.
PBS and Dulbecco's PBS++ (PBS with 1 mM calcium and 0.5 mM magnesium) were purchased from Gibco BRL (Grand Island, NY). BSA and mannan-agarose were supplied by Sigma (St. Louis, MO).
Isolation of pSP-D from bronchoalveolar lavage fluid was performed by mannan affinity and gel filtration chromatography as previously described (24). Porcine SP-A was purified from the surfactant fraction using the procedure described for the isolation of rat SP-A (32) with the addition of a gel filtration purification step using a Sephacryl-300 High Resolution column equilibrated in 5 mM Tris-HCl (pH 7.4). Fractions were analyzed by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and pSP-A–positive fractions were pooled and stored in aliquots at –20°C. Human SP-A (hSP-A), isolated from bronchoalveolar lavage fluid of patients with alveolar proteinosis as described previously (32), was generously provided by Dr. J. F. van Iwaarden (Laboratory of Pediatrics, Erasmus University Rotterdam, Rotterdam, The Netherlands).
Recombinant rat SP-D and recombinant human SP-D, assembled as dodecamers, were both produced in CHO-K1 cells as described (29, 33) and generously provided by Dr. E. C. Crouch (Department of Pathology and Immunology, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, MO).
N-deglycosylated pSP-D (pSP-Ddeglyc) was obtained by treatment of pSP-D with recombinant N-glycanase (from Flavobacterium meningosepticum; Glyko Inc., Novato, CA) as described previously (31), and purified by affinity chromatography on mannan-sepharose according to the procedure described before (24), but without the gel filtration chromatography. Deglycosylation of pSP-A and hSP-A was performed with recombinant N-glycanase as described previously for pSP-A (31). Both N-deglycosylated pSP-A and N-deglycosylated hSP-A were purified by ultrafiltration using Microcon-100 filters (cutoff 100 kD), followed by repetitive washings with 5 mM Tris-HCl (pH 7.4) to remove enzyme and released glycans. N-deglycosylation was verified by Western blot analysis using previously published methods (24). In this analysis, polyclonal antibodies were used to detect SP-D or SP-A, whereas the presence or absence of glycoconjugates was determined by DIG Glycan staining (Roche Diagnostics GmbH, Mannheim, Germany).
Desialylated pSP-D (pSP-Ddesial) was obtained by treatment of pSP-D with a nonspecific recombinant sialidase (from Arthrobacter ureafaciens; Glyko Inc.) which releases terminal α(2,3/6/8/9)-linked SAs from complex carbohydrates. Purified pSP-D (60 μg/200 μl), dissolved in 50 mM sodium phosphate pH 6.0, was mixed with 100 mU sialidase and incubated at 37°C for 1 h. Sham-treated pSP-D (pSP-Dcon) was obtained by incubation in the absence of sialidase. After incubation, pSP-Ddesial and pSP-Dcon were purified by mannan affinity chromatography as described for pSP-Ddeglyc. Efficiency of desialylation of pSP-D was determined, after SDS-PAGE and transfer to nitrocellulose, by detection with a DIG Glycan Differentiation Kit (Roche Diagnostics GmbH), which is described in SA Linkage Analysis below.
Resialylation of pSP-Ddesial (10 μg/50 μl) was performed in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 5 mM EDTA, in the presence of 1.6 mU of either α2,3-(N)-sialyltransferase or α2,6-(N)-sialyltransferase (34) and 1.5 mM CMP-N-acetylneuraminic acid (Calbiochem Biochemicals, San Diego, CA). The mixture was incubated for 2 h at 37°C, after which α(2,3)-resialylated pSP-D (pSP-Dα2,3TF) or α(2,6)-resialylated pSP-D (pSP-Dα2,6TF) were purified by mannan affinity chromatography as described for pSP-Ddeglyc. SA linkages present on resialylated pSP-Ds were determined by DIG-conjugated lectin detection as described in SA Linkage Analysis below.
IAV was grown in the chorioallantoic fluid of 10-d-old chicken eggs and purified on a discontinuous sucrose gradient as described previously (35). Virus stocks were dialysed against PBS, aliquoted and stored at -70°C. A/Phillipines/82(H3N2) (Phil) and the bovine serum β-inhibitor-resistant variant, PhilBS were provided by Dr. E. M. Anders (Department of Microbiology, University of Melbourne, Melbourne, Australia). The A/Puerto Rico/8/34(H1N1) (PR-8) strain was provided by Dr. J. Abramson (Department of Pediatrics, Bowman Gray School of Medicine, Wake Forest University, Winston Salem, NC). HAA titres of each strain were determined by titration of virus samples in PBS++ with thoroughly PBS-washed human type O, Rh(-) red blood cells as described (36).
The IAV strains were tested for the presence of N-linked high-mannose oligosaccharides by reducing SDS-PAGE analysis of 5 μg IAV extract, followed by Western blotting and staining with DIG-conjugated Galanthus nivalis agglutinin, a lectin which specifically recognizes ‘high-mannose’ N-glycan chains. The procedure was performed according to the instructions supplied by the manufacturer (DIG Glycan Differentiation Kit, Roche Diagnostics GmbH).
HAA inhibition was measured by serially diluting collectin preparations in round-bottom 96-well plates (Serocluster U-Vinyl plates; Costar, Cambridge, MA) using PBS++ as diluent (25 μl/well). The highest SP-D concentration tested for HAA inhibitory activity against the Phil and PhilBS strains was 1,620 ng/ml, and that against the PR-8 strain 2,800 ng/ml. After adding 25 μl IAV solution, giving a final concentration of 40 HAA U/ml or 4 HAA U/well, the IAV/collectin mixture was preincubated for 15 min, followed by addition of 50 μl human erythrocyte suspension in PBS++. The entire procedure was performed at room temperature. The minimal concentration of a collectin, required to fully inhibit the HAA caused by the virus, was determined by reading the plates after 2 h. HAA inhibition was detected as the formation of a pellet of red blood cells. To enable graphical comparisons of HAA inhibition results, data were mathematically converted and expressed as the number of HAA units inhibited by 100 ng of collectin.
Presence and linkage patterns of terminally linked SAs on various collectin preparations were analyzed by reducing SDS-PAGE and Western blotting, followed by detection with digoxigenin-labeled lectins according to the instructions supplied by the manufacturer (DIG Glycan Differentiation Kit; Roche Diagnostics GmbH). A control gel was included as reference (Coomassie stain). DIG-conjugated lectins used in this analysis, and a description of their carbohydrate binding specificity, were as follows: Datura stramonium agglutinin recognizes unsubstituted galactose-β(1,4)-N-acetylglucosamine; Maackia amurensis agglutinin recognizes SA residues terminally linked α(2,3) to galactose; and Sambucus nigra agglutinin recognizes SA residues terminally linked α(2,6) to galactose or N-acetylgalactosamine. N-deglycosylated collectin preparations were also tested to distinguish between SAs present on either N-linked glycans or O-linked glycans. Lectin binding was detected by anti-digoxigenin-alkaline phosphatase, followed by a staining reaction. Control glycoproteins were included to assess lectin-binding efficiency and specificity.
The linkage profile of terminally linked SA residues present on pSP-D and pSP-A was also determined by digestion with linkage-specific sialidases, followed by two-dimensional IEF/SDS-PAGE analysis. Purified pSP-D and pSP-A (20 μg/60 μl) dissolved in 50 mM sodium phosphate buffer (pH 6.0) was incubated for 2 h at 37°C in the presence of 35 mU of one of the following linkage-specific cleaving recombinant sialidases: α(2,3)-specific (from Streptococcus pneumoniae), α(2,3/6)-specific (from Clostridium perfringens) or α(2,3/6/8)-specific (from Arthrobacter ureafaciens; all sialidases purchased from Glyko Inc.). Sham-treated pSP-D and pSP-A was obtained by incubation in the absence of enzyme (control). From each incubation mixture, 30 μl was taken for 2D IEF/SDS-PAGE analysis which was performed as described previously (24), using Immobiline DryStrips, pH 3–10 linear (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) for isoelectric focusing in the first dimension. Analysis in the second dimension was performed by SDS-PAGE using 11% (wt/vol) acrylamide gels. After transfer to nitrocellulose, pSP-D or pSP-A was detected by immunostaining using rabbit anti-porcine SP-D or rabbit anti-porcine SP-A antibodies, respectively.
The receptor specificity of the IAV strains used in this study was determined by the use of modified erythrocytes, essentially as described by Rogers and Paulson (10). The HAA activity of the IAV strains was tested against enzymatically modified human erythrocytes, composed of SA moieties with a defined α(2,3)/α(2,6)-linkage pattern, and compared with the activity against native cells.
Fresh human erythrocytes were prewashed twice in PBS++ (adjusted to pH 5.0) and 40 μl erythrocyte pellet, obtained by centrifugation at 500 × g for 2 min, was incubated in PBS++ (adjusted to pH 6.0) in a final volume of 100 μl for 16 h at room temperature in the presence of 60 mU of recombinant sialidases cleaving either α(2,3), α(2,3/6), or α(2,3/6/8/9) terminally linked SA residues. Sham-treated erythrocytes were obtained by incubation in the absence of enzyme and were used as control. After incubation, the cells were washed three times with PBS++ and stored at 4°C. The cells treated with α(2,3/6/8/9)sialidase were used for resialylation with either α(2,3)-linked or α(2,6)-linked SAs. Resialylation was performed by diluting 100 μl erythrocyte pellet with 100 μl PBS++ (adjusted to pH 6.0) and addition of 1.5 mM CMP-N-acetylneuraminic acid and 4 mU of either α2,3-(N)-sialyltransferase or α2,6-(N)-sialyltransferase (Calbiochem Biochemicals). The cell suspensions were incubated under continuous rotation for 3 h at room temperature, followed by three washings with PBS++. Sham-treated erythrocytes were obtained by incubation of desialylated erythrocytes in the absence of sialyltransferase and were used for comparison with resialylated erythrocytes.
HAA activity of IAV was measured by mixing 50 μl erythrocyte suspension with 50 μl IAV suspension (final concentration of 80 HAA U/ml when control erythrocytes were used). HAA caused by the virus was determined by reading the plates after 2 h. Loss of HAA activity was detected as the formation of a pellet of red blood cells.
Where multiple comparisons were made with a control condition, statistical analysis was performed by repeated-measures ANOVA. In those cases in which the F-test indicated that there was a significant difference (P < 0.05) among groups, comparisons with the control condition were made with Dunnett's test. In those cases where only 2 means were compared, statistical analysis was performed by Student's t test.
To determine whether the SA moieties present on the N-linked oligosaccharide in the CRD of pSP-D participate in the distinct interactions of pSP-D with IAV, we compared the inhibitory activity of sham-treated pSP-D (pSP-Dcon, sialidase protocol) with both N-deglycosylated pSP-D (pSP-Ddeglyc) and desialylated pSP-D (pSP-Ddesial) against HAA by the Phil, PhilBS, and PR-8 strain. Desialylation of pSP-D was verified by SDS-PAGE followed by Western blotting and DIG-lectin staining which recognize SAs. As shown in Figure 6, lane 2, the repurified pSP-Ddesial was practically free of SAs, although a faint signal for α(2,6)-linked SAs was still present.
The HAA inhibitory activity of pSP-Dcon, pSP-Ddeglyc, and pSP-Ddesial was tested against the human wild-type Phil strain; its variant, the β-inhibitor–resistant mutant strain PhilBS; and PR-8. The HAA inhibitory activity of pSP-Dcon against these strains decreased in the order: Phil > PhilBS > PR-8 (Figure 2)
Figure 2. Effect of N-deglycosylation versus desialylation on inhibition by pSP-D of HAA by various IAV strains. IAV strains (final concentration of 40 HAA U/ml) were preincubated for 15 min at room temperature with pSP-Dcon (open bars), pSP-Ddeglyc (filled bars), or pSP-Ddesial (shaded bars) preparations, serially diluted in PBS++. HAA inhibition was determined and data were analyzed as described in Materials and Methods. Results are expressed as number of HAA units that can be inhibited by 100 ng of collectin. Values are mean ± SEM of three experiments. *P < 0.05; **P < 0.01 compared with pSP-Dcon. Statistical analysis was conducted by the Student t test.
[More] [Minimize]The SA linkages present on pSP-D were analyzed and compared with those present in other mammalian collectins by reducing SDS-PAGE, followed by Western blotting and staining with SA linkage–specific DIG-conjugated lectins (Figure 3)
Figure 3. Linkage analysis of terminal sialic acids present on lung collectins. Purified SP-D, SP-A, and N-deglycosylated products were subjected to reducing SDS-PAGE (12%). A control gel was Coomassie stained and included as reference (A). After blotting onto nitrocellulose, proteins were detected by the following DIG-conjugated lectins, with their carbohydrate binding specificity indicated: Datura stramonium agglutinin, galactose-β(1,4)-N-acetylglucosamine (Gal-β(1,4)-GlcNAc, B); Maackia amurensis agglutinin, terminal SAs α(2,3)-linked to galactose (C); and Sambucus nigra agglutinin, terminal SAs α(2,6)-linked to galactose or N-acetylgalactosamine (D). hSP-Adeglyc, N-deglycosylated hSP-A; pSP-Adeglyc, N-deglycosylated pSP-A; rhSP-D, recombinant human SP-D; rrSP-D, recombinant rat SP-D.
[More] [Minimize]Analysis of pSP-D showed that exclusively α(2,6)-linked SAs were present (Figure 3D, lane 1). Nonsialylated oligosaccharide terminii or α(2,3)-linked SAs could not be detected (Figures 3B and 3C, lane 1). The pSP-Ddeglyc preparation still contained α(2,6)-linked SAs, although the signal was substantially decreased (Figure 3D, lane 2). With regard to the other SP-Ds, recombinant human SP-D and recombinant rat SP-D, no staining was observed by any of the DIG-lectins used in this experiment (Figure 3, lanes 7 and 8).
Both pSP-A and hSP-A were positively stained after exposure to each of the three DIG-lectins used (Figures 3B–3D, lanes 3 and 5), although pSP-A gave only a weak signal for α(2,6)-linked SAs (Figure 3D, lane 3). No differences were found in the SA linkage pattern between monomeric and nonreducible, dimeric hSP-A. No staining was observed for both N-deglycosylated SP-A preparations after detection with any of the DIG-lectins, although weak staining by all DIG-lectins was present in the N-deglycosylated pSP-A preparation due to incomplete digestion by N-glycanase (Figures 3B–3D, lanes 4 and 6). This indicates that terminal SAs are only present on the N-linked carbohydrate moiety of SP-A.
The SA linkages present on pSP-D and pSP-A were also determined by analysis of their differently charged isoforms before and after treatment with linkage-specific cleaving sialidases (Figures 4A–4D
Figure 4. Effect of linkage-specific cleaving sialidase treatment on 2D IEF/SDS-PAGE patterns of porcine SP-D and SP-A. Purified pSP-D (A–D) and pSP-A (E–H) was incubated in the absence (control, A and E) or presence of sialidases with cleaving specificity for either α(2,3)-linkages (B and F), α(2,3/6)-linkages (C and G) or α(2,3/6/8)-linkages (D and H). After incubation, the mixtures were subjected to isoelectric focusing in the first dimension using Immobiline DryStrips (Amersham Pharmacia Biotech), followed by SDS-PAGE analysis in the second dimension using 11% (wt/vol) acrylamide gels. After blotting onto nitrocellulose, proteins were immunostained using rabbit polyclonal antibodies against either pSP-D or pSP-A.
[More] [Minimize]Digestion with α(2,3)sialidase led to a pI shift of most pSP-A isoforms toward pI 4.5 (Figure 4F versus 4E), whereas no change was observed in the charge pattern of the pSP-D isoforms (Figure 4B versus 4A). Treatment with α(2,3/6)sialidase caused a major shift of most pSP-D isoforms to a more basic pH and the appearance of a major isoform was detected at pI 10 (Figure 4C). Digestion of pSP-A with the same enzyme only caused a shift of a minor fraction of pSP-A isoforms as compared with the α(2,3)sialidase-treated pSP-A (Figure 4G versus 4F). Treatment with α(2,3/6/8)sialidase did not result in any changes of the 2D-IEF/SDS-PAGE patterns of pSP-D and pSP-A, as compared with the result obtained after α(2,3/6)sialidase treatment (Figures 4D and 4H versus 4C and 4G).
The Phil, PhilBS, and PR-8 strains used in this study were analyzed for their binding specificity to either α(2,3)-linked or α(2,6)-linked terminal SA residues. This was determined by measuring changes in the HAA activity of these strains (80 HAA U/ml) on human erythrocytes treated with linkage-specific cleaving sialidases (Figure 5A
Figure 5. Receptor specificity and high-mannose oligosaccharide analysis of IAV strains. The preferential binding of Phil, PhilBS, and PR-8 for either α(2,3)- or α(2,6)-linked SAs was determined by HAA tests using modified human erythrocytes. HAA activity of IAV was measured by mixing 50 μl erythrocyte suspension with 50 μl IAV suspension (final concentration of 80 HAA U/ml when control erythrocytes were used). HAA activity of viral strains against sham-treated erythrocytes was compared with the HAA activity against erythrocytes treated with sialidases with cleavage-specificity for either α(2,3)-linked SAs, α(2,3/6)-linked SAs, or α(2,3/6/8/9)-linked SAs. In a second approach, desialylated erythrocytes, obtained after treatment with α(2,3/6/8/9)sialidase, were subjected to linkage-specific resialylation by treatment with either α2,3-(N)-sialyltransferase or α2,6-(N)-sialyltransferase. Sham-resialylation was included as control. Plus signs, HAA activity detected; minus signs, no HAA activity detected (A). The IAV strains were analyzed for the presence of high-mannose oligosaccharides by reducing SDS-PAGE analysis, followed by Western blotting and staining with DIG-conjugated Galanthus nivalis agglutinin. Arrow indicates location of the HA1 domain of IAV (B).
[More] [Minimize]HAA activity of the Phil strain was not affected by changes in the number of α(2,3)-linked SAs present on the erythrocytes. However, specific removal of α(2,3/6)-linked SAs, or reattachment of α(2,6)-linked SAs to desialylated erythrocytes, resulted in complete loss and restoration, respectively, of the HAA activity by Phil.
Removal of either α(2,3)-linked or α(2,3/6)-linked SAs did not alter the HAA activity of this strain. Treatment with α(2,3/6/8/9)sialidase, however, resulted in complete loss of HAA activity. Reattachment of either α(2,3)-linked SAs or α(2,6)-linked SAs to desialylated erythrocytes facilitated in both cases in restoration of the HAA activity by PhilBS.
No HAA activity was detectable after treatment of erythrocytes with either α(2,3)sialidase or α(2,3/6)sialidase. Absence of HAA activity on desialylated erythrocytes was only restored after treatment of these cells with α(2,3)-(N)-sialyltransferase.
It is concluded from these data that Phil specifically recognizes α(2,6)-linked SAs, whereas PR-8 interacts only with α(2,3)-linked SAs. The SA receptor of the PhilBS mutant strain is less specific and may interact with SAs present in various types of linkage.
High-mannose oligosaccharides present on the HA of IAV are important for its interactions with the CRD of SP-D. High-mannose glycans were abundantly present on the HA of both Phil and PhilBS, in contrast to the HA of PR-8, which completely lacks these glycans (Figure 5B).
To determine whether changes in the SA linkages present on pSP-D can affect its neutralizing activity against IAV, pSP-D preparations containing either α(2,3)-linked SAs or α(2,6)-linked SAs on their N-linked oligosaccharides were produced (38). First, pSP-Ddesial was produced as described in Materials and Methods, and the absence of SAs was confirmed by DIG-conjugated lectin detection (Figure 6
Figure 6. Analysis of terminal SAs present on pSP-D after enzymatic modifications. Purified pSP-D was incubated in the absence (pSP-Dcon, lane 1) or presence of α(2,3/6/8/9)sialidase (pSP-Ddesial, lane 2). pSP-Ddesial was repurified and incubated in the presence of either α2,3-(N)-sialyltransferase (pSP-Dα2,3TF, lane 3), or α2,6-(N)-sialyltransferase (pSP-Dα2,6TF, lane 4), and CMP-N-acetylneuraminic acid as a substrate, according to procedures as described in Materials and Methods. Samples were subjected to reducing SDS-PAGE (12%) and, after blotting onto nitrocellulose, proteins were detected by DIG-conjugated lectins with carbohydrate binding specificity for either terminal SAs α(2,3)-linked to galactose (Maackia amurensis agglutinin, B) or terminal SAs α(2,6)-linked to galactose or N-acetylgalactosamine (Sambucus nigra agglutinin, C). A control gel was Coomassie stained and included as reference (A).
[More] [Minimize]HAA inhibition of Phil, PhilBS, and PR-8 by the modified pSP-D preparations characterized above, showed that for all IAV strains, HAA inhibition by pSP-Ddesial was substantially lower compared with the unmodified, sham-treated pSP-Dcon preparation (Figure 7)
Figure 7. Effect of SA linkage modification of pSP-D on HAA by various IAV strains. IAV strains (final concentration of 40 HAA U/ml) were preincubated for 15 min at room temperature with pSP-Dcon (open bars), pSP-Ddesial (filled bars), pSP-Dα2,3TF (lightly shaded bars), or pSP-Dα2,6TF (darkly shaded bars) preparations, serially diluted in PBS++. HAA inhibition was determined and data were analyzed as described in Materials and Methods. Results are expressed as percentage of HAA inhibitory activity for pSP-Ddesial, pSP-Dα2,3TF, and pSP-Dα2,6TF as compared with pSP-D (100%), for each individual IAV strain tested. Values are mean ± SEM of at least three experiments. *P < 0.05; **P < 0.01 compared with pSP-Dcon. Statistical analysis was performed by repeated-measures ANOVA.
[More] [Minimize]Recent in vitro studies revealed that lung collectin–mediated defense against IAV in pigs, as part of a first line barrier against infection by IAV, is more efficient compared with that of other animal species. Notably, the HAA inhibitory activity of porcine SP-D (pSP-D) is substantially enhanced by virtue of a terminally sialylated, asparagine-linked oligosaccharide located in the CRD of pSP-D (31). Because terminal SAs are the cellular receptor determinants for IAV, we determined whether the N-glycosylation–dependent activity of pSP-D is mediated via its SAs and, to what extent the SA–Gal linkages present on pSP-D are important for interactions with IAV.
In this study three different human IAV strains were used to study the role of SAs present on pSP-D. These strains exhibit differences in resistance against inhibition by pSP-D (Figure 2), in accordance with earlier HAA inhibition studies (31). The differences in inhibitory activity correlate with variations in the degree of glycosylation of the HA of IAV that affect the CRD-mediated interactions with SP-D (28). The wild-type human Phil strain (H3N2), which carries ten potential glycosylation sites on its HA (39), is highly susceptible to inhibition by pSP-D, whereas its variant PhilBS, in which the high-mannose oligosaccharide overlying the SA receptor-binding site of the HA molecule is absent (40), shows markedly increased resistance. The third strain, PR-8, a mouse-adapted H1N1-subtype strain that completely lacks high-mannose oligosaccharides on its HA (41), is by far the most resistant to inhibition by pSP-D. The differences in degree of HA glycosylation are illustrated by SDS-PAGE/Western blot analysis of the three IAV strains, followed by detection for high-mannose oligosaccharides (Figure 5B). However, despite these differences in the degree of HA glycosylation, HAA inhibition assays showed a marked decrease in inhibitory activity against the three IAV strains analyzed after desialylation of pSP-D compared with sham-treated pSP-D. This decrease was similar to the drop in activity observed after N-deglycosylation of pSP-D. Therefore, it is concluded that the contribution of the N-linked carbohydrate present in the CRD to the inhibitory activity of pSP-D is due to the presence of the SA residues on this carbohydrate. It should be mentioned that although pSP-D has a second, conserved Asn70-linked oligosaccharide present in its collagen domain, which also may contain SAs, its contribution to viral neutralization is probably of minor importance. Earlier studies with recombinant rat SP-D, in which the Asn70-carbohydrate was deleted by site-directed mutagenesis, showed that this carbohydrate located in the collagen domain did not contribute to the anti-IAV activity of SP-D (30). This might be due to the poor accessibility of this carbohydrate moiety, located in the interior of the pSP-D multimer.
Considering the importance of SA residues present on pSP-D (this study) and SP-A (22) for their interaction with IAV, the SA residues on both pSP-D and SP-A were subjected to further investigation in which, as a first step, the type of linkage of these SAs was determined. Western blot analysis with SA linkage–specific staining DIG-lectins revealed remarkable differences between SA linkages present on the N-linked oligosaccharides of pSP-D and those present on pSP-A or hSP-A (Figure 3). Both SP-A preparations are partially endcapped with SAs, which show heterogeneity in their linkage to the penultimate galactose: both α(2,3)- and α(2,6)-linkages were detected, in accordance with earlier observations for hSP-A (25, 42). With regard to pSP-A, the ratio of α(2,3)/α(2,6)-linked SAs appears higher compared with that of hSP-A. This dissimilarity might play a role in the stronger HAA inhibitory activity against IAV observed for pSP-A compared with hSP-A (31).
In contrast to SP-A, the pSP-D preparation was fully sialylated, because no unsubstituted galactose-β(1,4)-N-acetylglucosamine residues could be detected (Figure 3). This finding is in accordance with FACE analysis performed on the released N-glycosylation present in the CRD of pSP-D, which also indicated that all termini of the oligosaccharide are occupied by SA residues (24). Interestingly, the SAs present on pSP-D are exclusively linked in a α(2,6) manner: no α(2,3)-linked SAs could be detected. Analysis of pSP-Ddeglyc showed that α(2,6)-linked SAs were still present, although the signal was considerably weaker compared with that of pSP-D. This could imply that a small fraction of the SAs present on pSP-D is not attached to the N-linked glycans, but associated with other modifications, e.g., O-linked sugars present in the collagen domain. However, HAA inhibition analysis performed with all three IAV strains revealed no significant differences in activity between pSP-Ddeglyc and pSP-Ddeglyc after treatment with nonspecific sialidase (data not shown). This further emphasizes, in addition to the studies performed with the Asn70-carbohydrate deletion mutant as mentioned earlier, that only the SAs present on the N-linked oligosaccharide located in the CRD of pSP-D are involved in interactions with IAV. With regard to the recombinant human and rat SP-D preparations, no terminal SAs could be detected.
Additional evidence for the existence of differences between the linkage patterns of the SAs present on pSP-D and those present on pSP-A was provided by analysis of their differently charged isoforms, as a function of linkage-specific sialidase treatment of both collectins (Figure 4). These results further support our findings with Western blot analysis (Figure 3), and show that pSP-D is only sialylated with α(2,6)-linked SAs and that pSP-A primarily contains α(2,3)-linked SAs. About the mechanism by which differences in terminal sialylation between pSP-D and pSP-A are brought about we can only speculate. Transfer of SA residues to newly synthesized glycoproteins has been shown to be controlled by the expression and activity of various sialyltransferases, which is regulated in a cell type– and development-specific manner (43, 44). The type of SA linkage produced by a cell may therefore result from the ratio of the two sialyltransferases expressed by a particular cell (45). The relative production of SP-A and SP-D by the pulmonary cells producing these collectins, the alveolar type II cells and the nonciliated bronchiolar epithelial cells, could be different (46, 47). Therefore, differences in terminal sialylation between SP-A and pSP-D may reflect cell type specific expression of the corresponding sialyltransferases. Termination of oligosaccharide chains in either α(2,3)-linked or α(2,6)-linked SAs is also controlled by the complexity of the oligosaccharide, e.g., degree of branching (48), which may well be different for the N-linked glycan structures present in pSP-A and pSP-D. Finally, differences in cellular recycling (mechanisms) between SP-A and SP-D might also lead to variations in de- and resialylation processes as part of postbiosynthetic adaptation (49).
Because the N-linked oligosaccharide in the CRD of pSP-D is exclusively endcapped with SAs that are α(2,6)-linked to Gal, we investigated whether this structural feature may have a distinctive effect on the interactions of pSP-D with IAV strains, which preferentially recognize either α(2,3)-linked or α(2,6)-linked SA residues. Therefore, the SA receptors of the three IAV strains used in this study were analyzed for their SA linkage preference. The HAA activity of the Phil strain proved to be dependent on the presence of α(2,6). This finding matches receptor specificity analysis that was performed on other human IAV strains circulating at the same period of time (7, 9, 50–52). In contrast, the receptor of the PR-8 strain preferentially recognizes α(2,3)-linked SAs, in accordance with data previously published (7, 51). Receptor-binding specificity of the mutant PhilBS strain is less clear-cut in recognizing a particular type of SA linkage, in contrast to the parent Phil strain. The HA of the PhilBS strain is characterized by a single amino acid substitution (T167L) that results in loss of the high-mannose oligosaccharide at Asn165 located near the SA-binding pocket (40). It can be assumed that either loss of the carbohydrate moiety itself (53), or conformational changes of the SA receptor that result from the single amino acid mutation (52), give rise to the difference in receptor-binding specificity that was observed between PhilBS and Phil.
Data obtained from HAA inhibition experiments with pSP-A correlate well with the receptor specificity determined for the Phil (α2,6) and PR-8 (α2,3) strains. The inhibitory activity of pSP-A, which predominantly contains α(2,3)-linked SAs, against PR-8 is relatively strong, whereas it is undetectable for Phil (31). As regards pSP-D, the role of the SAs appears more complicated. Interactions of pSP-D via its CRD with glycoconjugates present on IAV appear to be crucial because interactions between pSP-D and IAV, like those for SP-Ds from other species, require the presence of calcium (28–31). Therefore, the SA-mediated interactions of pSP-D can be considered as a secondary mode of interaction, which cannot take place in the absence of CRD-dependent interactions. However, another possible explanation for this phenomenon could be that calcium affects the conformation of the CRD in such a way that the SAs present on the N-linked glycan in the CRD are more accessible for interactions with the HA receptor. Furthermore, removal of the SAs from pSP-D (which are entirely α(2,6)-linked) decreases the HAA inhibition of IAVs with either α(2,6)- or α(2,3)-recognizing IAV receptors, e.g., Phil and PR-8, respectively (Figure 2). This suggests that interactions of the α(2,6)-linked SAs found on natural pSP-D with IAV are not restricted to those strains with SA receptors that show preferential binding to α(2,6)-linked SAs.
To further elucidate the mechanism of its SA-mediated interaction with IAV, pSP-D was subjected to a two-step enzymatic modification in which first all SA residues present on native pSP-D were removed, after which the resulting pSP-Ddesial was repurified and resialylated with either α(2,3)-linked SA residues (therefore resulting in an “SP-A-like” linkage pattern), or, as a control, with α(2,6)-linked SA residues, resembling the original native preparation (Figure 6). When used in an HAA inhibition assay against Phil and PhilBS, the change of SA linkage composition introduced did not affect the inhibitory activity of pSP-D. However, replacing the α(2,6)-linkages of native pSP-D with α(2,3)-linked SAs, resulted in a dramatic increase of HAA inhibitory activity against the PR-8 strain. It is of interest that relatively simple modification of SA determinants on pSP-D can render it a very potent inhibitor of an otherwise largely SP-D–resistant viral strain. Considering that PR-8 preferentially recognizes α(2,3)-linkages, this result clearly proves that for pSP-D, not only the presence of SA residues in the CRD as such, but also the type of linkage in which they are attached to the N-linked carbohydrate, can have a profound effect on its interactions with IAV. Therefore, this interaction may discriminate between IAV strains that exhibit different SA receptor–binding specificities. Furthermore, the striking difference in activity between α(2,3)- and α(2,6) resialylated pSP-D is only observed for the poorly glycosylated PR-8 strain. This may imply that the role of the SAs in IAV neutralization by pSP-D is more pronounced for IAV strains with less extensive carbohydrate modification, which can escape from the primary, CRD-mediated interactions with SP-D in other animal species than the pig.
In conclusion, the data presented in this article show that the binding of pSP-D to IAV does not only involve the “usual” interactions of the CRD with glycoconjugates expressed by IAV but, in addition, involves interactions of the SA residues present on the N-linked oligosaccharide in the CRD of pSP-D, with the SA receptor of IAV. The importance of this SA-mediated, secondary mode of interaction appears to depend on the receptor-binding specificity and the degree of HA glycosylation, as illustrated by SA linkage–specific interactions between pSP-D and PR-8. Due to the heterogeneity of SA linkages and SA species expressed on the epithelial surface of this animal species, pigs are relatively tolerant to infection by avian as well as human IAVs. It remains unclear to what extent the distinct SA α(2,6)-linkage pattern expressed by pSP-D contributes to selective immunologic pressure in this host by acting as a natural inhibitor of the binding of IAV receptors to cell-surface sialoglycoconjugates. The SA linkage pattern of pSP-D might be especially important for infections by IAV strains that do not have many glycosylation sites. Comparison studies on the distribution of glycosylation sites on HA1 subunits of duck, swine, and human H1 isolates showed that duck and swine viruses do not contain any conserved glycosylation sites at the tip of their HA. This is in contrast to human H1 viruses, which contain two glycosylation sites or more, which suggests that host specificity of IAV may also be determined by HA glycosylation characteristics (54). The SA-mediated interactions of SP-D can only occur in pigs, due to the unique glycosylation profile of SP-D in this animal species. It remains to be investigated to what extent these interactions affect host range control of such H1 IAV strains with potentially high virulence for humans. We speculate that the distinct lung collectin-mediated interactions with IAV in pigs may alter the adaptive immune response that is essential for the ultimate elimination of IAV. This could give rise to conditions in which IAV infection can persist, and contribute to the role of pigs as a source of potentially hazardous IAVs that can generate human influenza pandemics.
This work was supported by the Netherlands Organization for Scientific Research grant R92–227 (M.v.E.), and NIH grant HL-69031 (K.L.H.). J.J.B. and H.P.H. received support from the European Commission (contract no. QLK2-CT-2000-00325).
The authors thank Dr. E. C. Crouch for critical reading of the manuscript.
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