Enhanced anti-influenza activity of a surfactant protein D and serum conglutinin fusion protein

Kevan L. Hartshorn1, Kedarnath N. Sastry1, Donald Chang2, Mitchell R. White1, and Erika C. Crouch2

1 Departments of Medicine and Pathology, Boston University School of Medicine, Boston, Massachusetts 02118; and 2 Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that bovine serum conglutinin has markedly greater ability to inhibit influenza A virus (IAV) infectivity than other collectins. We now show that recombinant conglutinin and a chimeric protein containing the NH2 terminus and collagen domain of rat pulmonary surfactant protein D (rSP-D) fused to the neck region and carbohydrate recognition domain (CRD) of conglutinin (termed SP-D/Congneck+CRD) have markedly greater ability to inhibit infectivity of IAV than wild-type recombinant rSP-D, confirming that the potent IAV-neutralizing activity of conglutinin resides in its neck region and CRD. Furthermore, by virtue of incorporation of the NH2 terminus and collagen domain of SP-D, SP-D/Congneck+CRD caused substantially greater aggregation of IAV particles and enhancement of neutrophil binding of, and H2O2 responses to, IAV than recombinant conglutinin or recombinant rSP-D. Hence, SP-D/Congneck+CRD combined favorable antiviral and opsonic properties of conglutinin and SP-D. This study demonstrates an association of specific structural domains of SP-D and conglutinin with specific functional properties and illustrates that antimicrobial activities of wild-type collectins can be enhanced through recombinant strategies.

collectin; neutrophils; respiratory burst; virus; hemagglutination


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE COLLECTINS ARE A FAMILY of proteins present in mammalian serum and pulmonary surfactant that participate in innate immunity by binding to microbial carbohydrate structures (29). Collectins share a common molecular structure that includes NH2 terminus and collagen, neck, and carbohydrate recognition domains (CRD). The basic subunit of collectins is a trimer that has a globular COOH-terminal region with three carbohydrate recognition sites in association with a collagenous stalk. The neck region of the collectins appears to participate in stabilizing the trimer through formation of a three-stranded coiled coil structure (22). With the exceptions of the bovine serum collectin CL-43 and rodent mannose-binding protein C, the collectins form higher-order oligomers by association of trimers through disulfide bonds and possibly other interactions in the NH2 terminus. We have shown that both pulmonary surfactant collectins, surfactant proteins A and D (SP-A and SP-D), and the serum collectins bovine serum conglutinin and human serum mannose-binding lectin (MBL) inhibit infectivity and hemagglutination (HA) activity of influenza A viruses (IAVs) (13, 19-21). Among these collectins, conglutinin had the highest potency as a direct inhibitor of IAV infectivity. Conglutinin has been identified as the previously described bovine serum beta -inhibitor of influenza infectivity (12).

We hypothesized that the greater ability of conglutinin to inhibit infectivity of IAV compared with that of the other collectins results from distinctive properties of its CRD. The ability of SP-D, MBL, and conglutinin to inhibit HA activity or infectivity of IAV is mediated by the calcium-dependent lectin activity of these proteins (2, 12, 13, 19-22, 29). SP-A, in contrast, inhibits infectivity of IAV through an alternative mechanism that involves binding of IAV to N-linked oligosaccharide attachments on SP-A (5, 16). It is plausible to propose that the CRDs of collectins may have distinctive binding properties for oligosaccharides displayed on pathogens. There are subtle differences in binding specificities of the collectins for monosaccharides; e.g., conglutinin has greater affinity for binding N-acetylglucosamine than SP-D (28). However, even greater differences emerge between the collectins with respect to binding to oligosaccharides displayed on host, bacterial, or viral glycoproteins. For instance, conglutinin is able to bind to a high-mannose-type oligosaccharide on the complement protein iC3b, whereas MBL is not (despite high affinity of MBL for mannose) (30). In addition, we have found that conglutinin has a greatly reduced ability to bind to Escherichia coli compared with SP-D or MBL (unpublished observations). These diverse findings support the notion that the CRD of conglutinin has distinctive binding properties that may account for the greater ability of this collectin to inhibit infectivity of IAV.

In our studies, we also demonstrated that the collectins can act as opsonins for IAV, promoting binding and uptake of the virus by neutrophils. Furthermore, preincubation of IAV with the collectins caused a marked enhancement of the ability of IAV to act as a stimulus of neutrophil H2O2 production. In addition to acting as a stimulus for neutrophil activation (18), the virus depressed the ability of these cells to generate respiratory burst or degranulation responses to other stimuli (1, 17). Preincubation of IAV with SP-D, conglutinin, or MBL significantly protected neutrophils against this deactivating effect of the virus.

In our studies, the ability of collectins to enhance neutrophil binding of, or respiratory burst responses to, IAV was associated with the ability of collectins to induce aggregation of viral particles (8, 13, 15, 19-21). Recombinant human SP-D (rhSP-D) was fractionated into trimeric, dodecameric, and highly multimerized preparations. The highly multimerized preparation had the greatest potency at inducing aggregation of IAV and was also most potent at enhancing neutrophil binding of, and respiratory responses to, IAV. In contrast, trimeric rhSP-D [or trimeric rat SP-D (rSP-D) produced by site-directed mutagenesis or NH2-terminal cysteines] caused minimal or no viral aggregation and did not alter interactions of IAV with neutrophils. Recombinant rSP-D (rrSP-D), like conglutinin, predominantly takes the form of dodecamers. Bovine serum conglutinin and rrSP-D were found to induce massive viral aggregation and to result in binding and uptake of large viral aggregates by neutrophils (15, 16). However, rhSP-D and rrSP-D induced maximal viral aggregation at somewhat lower concentrations than conglutinin in these experiments. In addition, the SP-Ds enhanced binding of IAV to neutrophils to a greater extent and at lower concentrations than conglutinin.

We questioned whether distinctive features of the NH2 terminus and collagen domain of SP-D could be responsible for the greater potency of SP-D at inducing viral aggregation and/or promoting IAV binding to neutrophils. As noted, SP-Ds (especially hSP-D) have a propensity to form higher-order multimers (i.e., greater than dodecamers), whereas conglutinin is predominantly present as dodecamers. The collagen domain of rrSP-D or rhSP-D is longer than that of conglutinin by six amino acids. Also, the collagen domain of SP-D does not have an interruption in the Gly-Xaa-Yaa sequence and, hence, lacks the kink found in the collagen domains of the other collectins (27). There is also an N-linked carbohydrate attachment within the collagen domain of SP-D that is not present in conglutinin. Finally, conglutinin undergoes limited proteolysis because of activity of endogenous serum serine proteases, leading to formation of truncated trimeric forms (24). This phenomenon reduces the conglutinating activity of the molecule and would be expected to reduce its viral aggregating activity as well. Such proteolysis has not been reported with SP-D. One or more of these structural differences could be responsible for the differences we observed in viral aggregating activity between conglutinin and SP-D.

As a first step, we decided to produce recombinant conglutinin and rrSP-D and to directly compare antiviral activity of these compounds. We chose rrSP-D for these comparisons because this molecule, like conglutinin, is predominantly present as dodecamers. We also reasoned that recombinant conglutinin might be less likely to undergo serum-dependent proteolysis than conglutinin derived from bovine serum. We then produced a recombinant fusion protein containing the neck region and CRD of conglutinin in association with the NH2 terminus and collagen domains of rrSP-D. This chimeric molecule was a markedly more potent inhibitor of IAV infectivity than rrSP-D and had greater aggregating and opsonic activity than recombinant bovine conglutinin.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Dextran, sodium citrate, RPMI 1640, propidium iodide, Dulbecco's PBS with calcium and magnesium, trypan blue stain, Wright's Giemsa stain, scopoletin, and horseradish peroxidase type II were purchased from Sigma (St. Louis, MO). Ficoll-Hypaque was obtained from Pharmacia Biotech. Dulbecco's PBS without calcium and magnesium was purchased from GIBCO BRL (Life Technologies, Grand Island, NY). Organic solvents were purchased from Fisher Scientific (Fair Lawn, NJ).

Neutrophil preparation. Neutrophils from healthy volunteer donors were isolated to >95% purity, as previously described, by dextran precipitation, then subjected to a Ficoll-Hypaque gradient separation for removal of mononuclear cells and hypotonic lysis to eliminate contaminating erythrocytes (17). Cell viability was >98%, as determined by trypan blue staining. Neutrophils were resuspended in the appropriate concentration in PBS with calcium and magnesium (also called control buffer), and cells were used within 5 h of isolation.

Virus preparation. IAV strain H3N3 A/Bangkok/79 (Bangkok 79) was a gracious gift of Robert Webster (St. Jude's Hospital, Memphis, TN). The H3N2 A/Philadelphia/82 (Philadelphia 82) and H1N1 Brazil/78 strains were the gracious gift from E. M. Anders (University of Melbourne, Melbourne, Australia). Virus was grown in the chorioallantoic fluid of 10-day-old embryonated hens' eggs and purified on a discontinuous sucrose density gradient, as previously described (17). Virus stock was dialyzed against PBS, divided into aliquots, and stored at -70°C until use. HA titer was determined by titration of virus samples in PBS followed by addition of thoroughly washed human type O red blood cells. Potency of virus stock was measured by HA assay and protein assay (Bio-Rad, Hercules, CA) after thawing.

Collectin preparations. rrSP-D and rhSP-D were expressed in Chinese hamster ovary K1 cells (American Type Culture Collection) and purified using maltosyl-agarose affinity chromatography and gel filtration, as previously described (9). Using the cDNA of bovine conglutinin (26), we prepared recombinant bovine conglutinin and a hybrid collectin containing the neck domain and CRD of conglutinin coupled to the collagen domain and NH2 terminus of rrSP-D (termed SP-D/Congneck+CRD) by similar methods. The method used for constructing the SP- D/Congneck+CRD hybrids is depicted in Fig. 1. The recombinant collectin cDNA constructs were made by a PCR-based approach that involves amplification of cDNA corresponding to the NH2 or COOH terminus of the proteins, with oligonucleotides designed in such a manner as to have overlapping regions.


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Fig. 1.   PCR methodology for construction of SP-D/Congneck+CRD hybrid collectin. N-ter, NH2 terminus; CRD, carbohydrate recognition domain; SP-D, surfactant protein D.

Two PCRs were initially performed: one reaction utilized rSP-D cDNA template and two primers, RSPDH and BCRSPDAS. RSPDH corresponded to bp 407-428 of rrSP-D (sequence 5'-ATA GGA CCC CAA GGC AA CCA G-3'). BCRSPDAS corresponded to bp 702-667 of the chimera and contained sequences derived from conglutinin and rrSP-D as follows: 5'-AG AGC ATT GAC CTC TGG AAG TCC ACT TTC GCC TTT G-3'. The PCR conditions involved one 10-min cycle of 95°C for denaturation, 1 min at 51°C for annealing, and 3 min at 72°C for extension, 30 cycles of 94°C (1 min), 51°C (1 min), and 72°C (3 min), and, finally, 7 min at 72°C for extension. The second PCR involved bovine conglutinin cDNA with pBluescript KS(+) vector as template and two additional primers, RSPDBCS (derived from sequences of rrSP-D and conglutinin as follows: 5'-GAA AGT GGA CTT CCA GAG GTC AAT GCT CTC AAG CAG-3') and T3 promoter primer (derived from vector sequences 5'-AATTAACCCTCACTAAAGGG-3'). The conditions for PCR were identical to those described above. The products from the two PCRs were purified from the primers by running a 1% low-melting-point agarose gel. The two products were combined in a third reaction in which PCR conditions outlined above were used and RSPDH and T3 primers were utilized. The resulting ~1-kb PCR product was digested with Sac I and cloned into the pUC19 vector. After digestion of the construct with Pst I and religation, the ~260-bp Sac I-Pst I fragment was sequenced to confirm that no errors were present. The ~600-bp EcoR I-Sac I fragment from rSP-D cDNA was cloned to the Sac I-Pst I fragment toward the 5'-end. Next, the pUC19 vector with EcoR I-Pst I fragment was digested with Bgl II and Hind III, and an ~530-bp Bgl II-Hind III fragment from the 3'-end of bovine conglutinin cDNA was subcloned toward the 3'-end. Overall, this method was employed to minimize the amount of PCR-derived DNA in the final construct (to reduce chance of errors). The hybrid cDNA was isolated as an EcoR I fragment and subcloned into the pEE14 vector, and the orientation was confirmed by restriction digestion as well as sequencing.

The resulting SP-D/Congneck+CRD protein was expressed in Chinese hamster ovary cells as described for SP-D (13), except N-acetylglucosamine affinity chromatography was used in purification instead of maltose affinity chromatography. Conglutinin has the highest affinity for N-acetylglucosamine, whereas SP-D has very low affinity for this sugar. Such a difference in affinity has been used for separating conglutinin and MBL in bovine serum (3). Additionally, a monoclonal anti-conglutinin antibody graciously provided by Dr. E. M. Anders was helpful in identifying the protein. The reason for use of rSP-D, rather than hSP-D cDNA, was that rSP-D largely occurs as dodecamers with a lesser population of high-order multimers (9), whereas the hSP-D has a greater proportion of higher-order multimers (see above). Because conglutinin (whether native or recombinant) exists predominantly as dodecamers, this will allow the most accurate comparison of the functional activities of recombinant conglutinin and the chimera. As expected, SP-D/Congneck+CRD hybrid eluted in the expected position of authentic rSP-D dodecamers by gel filtration and comigrated with rrSP-D by SDS-PAGE (Figs. 2 and 3).


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Fig. 2.   SDS-PAGE of recombinant rat SP-D (rrSP-D) and chimeric protein containing NH2 terminus and collagen domain of rat pulmonary SP-D fused to neck region and CRD of conglutinin (SP-D/Congneck+CRD). Left: SDS-PAGE of 14C-labeled, affinity-purified rrSP-D (lanes 2 and 5) and SP-D/Congneck+CRD (lanes 3 and 6) in presence and absence of sulfhydryl (dithiothreitol) reduction (+DTT). Lanes 4 and 7, SP-D/Congneck+CRD after further purification by gel filtration (see Fig. 3). Expected migration of trimers and monomers is indicated by T and M, respectively. Lane 1, molecular-weight standards. Right: effect of N-glycanase treatment on rrSP-D (lanes 2 and 3) and SP-D/Congneck+CRD chimera (lanes 4 and 5). rrSP-D lanes were overloaded relative to SP-D/Congneck+CRD lanes. Small amounts of additional breakdown products are evident in rrSP-D lanes after overnight incubation at 37°C (right, lanes 2 and 3).



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Fig. 3.   Gel-filtration analysis of SP-D/Congneck+CRD chimera, recombinant bovine conglutinin (rbCong), and recombinant human SP-D (rhSP-D). Expected elution positions from A-15M fractions of trimers, dodecamers, and higher-molecular-weight multimers are indicated (T, D, and M, respectively). Vo, void volume. Left lanes of SP-D/Congneck+CRD (chimera) and recombinant bovine conglutinin show starting material. rhSP-D was included to demonstrate presence of significant proportion of high-molecular-weight multimers, as previously described. rrSP-D (data not shown) eluted with a pattern similar to SP-D/Congneck+CRD chimera and to recombinant bovine conglutinin (e.g., predominantly in region of dodecamers).

Assessment of binding of collectins to IAV. Binding of collectins to IAV was also tested using an ELISA in which suspensions (~1 µg/ml) of the organisms were allowed to dry onto 96-well plates, fixed on the plates with methanol, and then washed and incubated with biotinylated collectins (4). Before addition of SP-D, plates were blocked with BSA and gelatin. The presence of bound collectins was detected using streptavidin conjugated to horseradish peroxidase and TMB substrate (Bio-Rad). The reaction was stopped with 1 N H2SO4. Optical density was measured with an ELISA reader. Each individual data point was performed in triplicate. There was minimal background binding of the biotinylated collectins to wells not containing IAV. The collectins were biotinylated by incubation at a 2:1 ratio by weight with NHS-LC-biotin (Pierce, Rockford, IL) for 2 h at room temperature and dialysis overnight against PBS.

Fluorescent focus assay of IAV infectivity. Madin-Darby canine kidney monolayers were prepared in 96-well plates. The layers were then incubated with IAV preparations diluted in PBS containing 2 mM calcium for 45 min at 37°C, and the monolayers were washed three times in virus-free DMEM containing 1% penicillin and streptomycin. The monolayers were then incubated for 7 h at 37°C in DMEM and repeatedly washed, and the cells were fixed with 80% (vol/vol) acetone for 10 min at -20°C. The monolayers were then incubated with monoclonal antibody directed against IAV nucleoprotein (monoclonal antibody A-3; a gracious gift from Dr. Nancy Cox, Influenza Branch, Centers for Disease Control, Atlanta, GA) and then with rhodamine-labeled goat anti-mouse IgG. Fluorescent foci were counted directly under fluorescent microscopy. Initially, various dilutions of virus were tested to determine a concentration of virus that resulted in formation of ~500 fluorescent foci per well. These foci appeared to be single infected cells in general. Then the effect of preincubating this concentration of IAV with various concentrations of the collectins was determined.

Measurement of aggregation of IAV particles. Aggregation of IAV particles was assessed by monitoring changes in light transmission through stirred suspensions of IAV after addition of various concentrations of the collectins with a highly sensitive spectrofluorometer (model SLM/Aminco 8000C, SLM Instruments, Urbana, IL), as described previously (20). Aggregation of viral particles or liposomes is manifested in the assay by a decline in light transmission (i.e., increased turbidity) (20).

Measurement of IAV binding to neutrophils. IAV was labeled with FITC, aliquots of the labeled virus were incubated with neutrophils for 15 min at 4°C, and viral binding to neutrophils was measured by flow cytometry, as previously described (19). To test the effect of collectins on IAV binding, aliquots of the virus were preincubated with various concentrations of the collectins for 30 min at 37°C, then aliquots of these samples were incubated with neutrophils.

Measurement of neutrophil H2O2 production. H2O2 production was measured by assessing reductions in scopoletin fluorescence, as previously described (18).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biochemical characterization of recombinant bovine conglutinin and the chimeric fusion protein SP-D/Congneck+CRD. As shown in Fig. 2, SP-D/Congneck+CRD migrated at similar locations on reduced and unreduced SDS-PAGE as rrSP-D monomers or trimers, respectively. The chimera showed glycosylation similar in extent to that of rrSP-D on the basis of change in molecular weight after N-glycanase treatment (Fig. 2). Figure 3 shows the results of gel filtration analysis of SP-D/Congneck+CRD, recombinant bovine conglutinin, and rrSP-D, which confirm that the protein preparations were predominantly composed of dodecamers. Neither recombinant bovine conglutinin nor SP-D/Congneck+CRD showed any component eluting at the position of trimers or high-molecular-weight multimers under these conditions. The gel filtration profile of rhSP-D is shown for comparison (note high-molecular-weight multimers designated M). Dodecameric fractions of recombinant bovine conglutinin, rrSP-D, and SP-D/Congneck+CRD were pooled and used in the experiments described here.

Binding of recombinant bovine conglutinin, SP-D, and SP-D/Congneck+CRD to IAV. As shown in Fig. 4, recombinant bovine conglutinin and SP-D/Congneck+CRD bound avidly to IAV, as assessed by ELISA. Binding of SP-D/Congneck+CRD to IAV was significantly greater than binding of recombinant bovine conglutinin or rrSP-D.


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Fig. 4.   ELISA of binding of rrSP-D, SP-D/Congneck+CRD, and recombinant bovine conglutinin (rbCong) to influenza A virus (IAV). Microtiter plates were coated with IAV, biotinylated collectins were added, and bound collectins were measured by ELISA. Values are means ± SE expressed as optical density (OD) at 450 nm of >= 3 experiments. Although all collectins showed significant binding to IAV (P equal 0.05), binding of SP-D/Congneck+CRD to IAV was significantly greater than that of recombinant bovine conglutinin (*) or than both recombinant bovine conglutinin and rrSP-D (**).

Effect of recombinant bovine conglutinin, rrSP-D, and SP-D/Congneck+CRD infectivity of IAV. We used a fluorescent focus assay to determine the effect of the collectins on ability of IAV to infect monolayers of Madin-Darby canine kidney cells. As shown in Fig. 5, recombinant bovine conglutinin caused markedly greater inhibition of viral infectivity than rrSP-D in this assay. Higher concentrations of rrSP-D than those shown (e.g., ~270 ng/ml) were required to cause 50% inhibition of infectious focus formation (data not shown). Recombinant bovine conglutinin caused ~50% inhibition at 2.8 ng/ml (Fig. 5). Hence, recombinant bovine conglutinin was ~100-fold more potent than rrSP-D at inhibiting infectivity in this assay. Modification of rrSP-D by replacing its neck domain and CRD with that of conglutinin markedly enhanced its ability to inhibit infectivity of IAV (Fig. 5). Like recombinant bovine conglutinin, SP-D/Congneck+CRD was markedly more potent than rrSP-D at inhibiting infectious focus formation. SP-D/Congneck+CRD also caused significantly greater inhibition of infectivity than recombinant bovine conglutinin (Fig. 5). Similar results were obtained with the Bangkok 79 IAV strain (data not shown).


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Fig. 5.   Effect of SP-D/Congneck+CRD, rrSP-D, and recombinant bovine conglutinin on infectivity of IAV. Infectivity was assessed by a fluorescent focus assay. Viral samples were preincubated with collectins, and samples were added to monolayers of Madin-Darby canine kidney cells. After incubation for 45 min at 37°C, monolayers were washed and incubated for 7 h at 37°C, fixed with acetone, and labeled with monoclonal antibody to IAV nucleoprotein and FITC-labeled goat anti-mouse IgG. Number of infectious foci were counted under fluorescent microscopy and expressed as percentage of control foci (e.g., number of foci in collectin-treated samples/number in control samples × 100). Values are means ± SE of 3 separate experiments. Recombinant bovine conglutinin and SP-D/Congneck+CRD caused significant inhibition of infectious focus formation compared with untreated or rrSP-D-treated samples at all concentrations tested (P < 0.05). Only highest concentration of rrSP-D (45 ng/ml) caused significant inhibition of infectivity. At 0.35, 2.8, 5.6, and 45 ng/ml, SP-D/Congneck+CRD caused significantly greater inhibition of infectivity than recombinant bovine conglutinin.

Effect of rrSP-D, recombinant bovine conglutinin, and SP-D/Congneck+CRD on IAV HA activity. Results obtained in HA inhibition assays were similar to those obtained using the infectious focus assays. We compared the HA inhibitory activity of the collectins against limiting dilutions of IAV (e.g., 40 HA units/ml), as shown in Table 1. In addition to testing activity of the collectins against the Bangkok 79 and Philadelphia 82 H3N2 strains of IAV, activity against a representative H1N1 strain (the other common circulating subtype of IAV) was tested as well. Recombinant bovine conglutinin and SP-D/Congneck+CRD inhibited HA activity of these IAV strains at significantly lower concentrations than rrSP-D or rhSP-D dodecamers.

                              
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Table 1.   Comparison of HA inhibitory activity of rrSP-D, recombinant bovine conglutinin, and SP-D/Congneck+CRD

We also tested HA inhibitory activity of the collectins against the Philadelphia 82/BS and Brazil 78/BS variant strains of IAV. These strains were selected in ovo for resistance to inhibition by bovine serum beta -inhibitor (i.e., conglutinin) and differ from their respective parent strains by virtue of loss of a single N-linked carbohydrate attachment site on the HA molecule (12). As expected, these strains were moderately (Brazil 78/BS) or markedly (Philadelphia 82/BS) resistant to conglutinin-mediated HA inhibition. Of interest, however, SP-D/Congneck+CRD had substantially greater HA inhibitory activity against these strains than conglutinin.

Aggregation of IAV particles by rrSP-D, recombinant bovine conglutinin, and SP-D/Congneck+CRD. Figure 6 shows the comparative ability of rrSP-D, recombinant bovine conglutinin, and SP-D/Congneck+CRD to aggregate IAV particles. The results obtained with recombinant bovine conglutinin were very similar to those we previously reported with bovine serum conglutinin (20). SP-D/Congneck+CRD was significantly more potent at inducing viral aggregation than recombinant bovine conglutinin. Similar results were obtained in three experiments in which a different strain of IAV (Philadelphia 82) was used (data not shown). Viral aggregates became visible in samples treated with 0.8 µg/ml of SP-D/Congneck+CRD, whereas no such aggregates were observed in samples treated with rrSP-D or recombinant bovine conglutinin (data not shown). When the concentration of SP-D/Congneck+CRD was increased to 1.6 µg/ml, the viral aggregates became large enough to precipitate out of solution, resulting in a paradoxical rise in light transmission through the suspension (data not shown). This phenomenon was not observed with recombinant bovine conglutinin or rrSP-D at concentrations up to 3.2 µg/ml.


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Fig. 6.   Comparison of ability of rrSP-D, recombinant bovine conglutinin, and SP-D/Congneck+CRD to aggregate IAV particles. Light transmission through stirred suspensions of IAV (Bangkok 79 strain) particles was monitored after addition of 0.8 µg/ml rrSP-D, recombinant bovine conglutinin, or SP-D/Congneck+CRD, as described previously (20). Values are means ± SE of 3 experiments. Degree of aggregation induced by SP-D/Congneck+CRD was significantly greater (* P equal 0.05) than that induced by recombinant bovine conglutinin or rrSP-D. SP-D/Congneck+CRD also caused significantly greater aggregation of IAV than other collectins at 0.4 µg/ml (data not shown).

We also tested HA activity of these samples after aggregation assay. The concentration of IAV in the starting suspension was ~100-fold higher (i.e., ~5,000 HA units/ml) in these assays than in those shown in Table 1. SP-D/Congneck+CRD had substantially greater ability to reduce HA activity than rrSP-D or recombinant bovine conglutinin. At concentrations of 0.4 and 0.8 µg/ml of SP-D/Congneck+CRD, the HA titers of the viral suspensions were reduced to 1,600 ± 0 and 450 ± 50 compared with 2,800 ± 230 and 1,500 ± 100 for the corresponding concentrations of recombinant bovine conglutinin or 3,600 ± 1,000 and 1,840 ± 526 for rrSP-D, respectively (n = 3, P equal 0.05).

Effect of recombinant bovine conglutinin and SP-D/Congneck+CRD on neutrophil binding of IAV. As shown in Fig. 7, SP-D/Congneck+CRD caused significantly greater enhancement of neutrophil binding of the Bangkok 79 or Philadelphia 82 strain of IAV than recombinant bovine conglutinin. Results obtained with SP-D/Congneck+CRD were similar to those obtained with rrSP-D in this assay (Fig. 7A). All the collectins significantly enhanced IAV binding compared with untreated virus. Examination of plastic adherent neutrophils under fluorescent microscopy confirmed the presence of fluorescent aggregates associated with neutrophils treated with FITC-IAV that had been preincubated with SP-D/Congneck+CRD (data not shown). HA assays were performed on aliquots of the collectin-treated virus samples used in the Philadelphia 82 binding experiments shown in Fig. 7. Again, SP-D/Congneck+CRD caused significantly greater inhibition of viral HA activity than recombinant bovine conglutinin or rrSP-D (data not shown).


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Fig. 7.   Effect of recombinant bovine conglutinin and SP-D/Congneck+CRD on binding of IAV to neutrophils. Binding of Philadelphia 82 (A) or Bangkok 79 (B) IAV strain to neutrophils was measured by FITC-labeled IAV and flow cytometry. Values are means ± SE expressed as neutrophil fluorescence (n = 4 for Bangkok 79 results and n = 7 for Philadelphia 82). SP-D/Congneck+CRD enhanced neutrophil binding of either strain of IAV to a significantly greater extent than other collectins.

Effect of recombinant bovine conglutinin, rrSP-D, and SP-D/Congneck+CRD on IAV-induced neutrophil H2O2 responses. As shown in Fig. 8, IAV that had been preincubated with rrSP-D, SP-D/Congneck+CRD, or recombinant bovine conglutinin stimulated a greater degree of neutrophil H2O2 production than untreated IAV. SP-D/Congneck+CRD was more potent than rrSP-D or recombinant bovine conglutinin at enhancing IAV-induced neutrophil H2O2 production.


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Fig. 8.   Effect of rrSP-D, recombinant bovine conglutinin, and SP-D/Congneck+CRD on IAV-stimulated neutrophil H2O2 production. Neutrophil H2O2 production was measured using fluorescent scopoletin assay after addition of IAV preparations to neutrophil suspensions. IAV samples were aliquots of collectin-treated samples shown in Fig. 6 (taken after completion of aggregation assay). * SP-D/Congneck+CRD significantly increased H2O2 production compared with untreated IAV or IAV preincubated with same concentration of recombinant bovine conglutinin or rrSP-D. All collectins significantly increased H2O2 production compared with response elicited by untreated IAV.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously found that bovine serum conglutinin has the greatest potency among the collectins at inhibiting IAV infectivity (13). Here we report production of recombinant conglutinin and of a chimeric protein containing the NH2 terminus and collagen domain of SP-D fused to the neck and CRD of conglutinin, SP-D/Congneck+CRD. These proteins formed dodecamers (similar to bovine serum conglutinin or rSP-D). An infectious focus assay showed that recombinant bovine conglutinin and SP-D/Congneck+CRD caused markedly greater inhibition of infectivity of IAV than wild-type rrSP-D. SP-D/Congneck+CRD also caused a modestly greater degree of inhibition of infectivity and HA activity of IAV than recombinant bovine conglutinin.

Eda et al. (10, 11) found that a recombinant preparation of conglutinin, which lacks the NH2 terminus and collagen domain of the molecule, retained the ability to inhibit IAV HA activity, whereas an analogous preparation of SP-D did not. We have found that similar preparations of SP-D (i.e., containing only the CRD and neck domains of the molecule) have greatly reduced HA inhibitory activity compared with wild-type SP-D (15, 16). These findings, coupled with the findings presented here, strongly suggest that distinctive properties of the conglutinin CRD are responsible for the particular potency of conglutinin at inhibiting IAV infectivity. We have found that conglutinin and SP-D/Congneck+CRD have a reduced ability to bind to or aggregate common bacteria compared with SP-Ds (unpublished observations). SP-D/Congneck+CRD has, therefore, been an effective tool for determining which properties of conglutinin or SP-D reside specifically in their CRD. It will be of interest to compare interactions of SP-D/Congneck+CRD and rrSP-D with lipids, in light of the probable role of SP-D in surfactant lipid homeostasis (6, 25).

Some of our results also indicate, however, that the binding properties of the conglutinin CRD may be modified in SP-D/Congneck+CRD compared with recombinant bovine conglutinin. SP-D/Congneck+CRD had enhanced ability to bind to IAV compared with rrSP-D or recombinant bovine conglutinin. In addition, SP-D/Congneck+CRD was able to inhibit HA activity of the bovine serum beta -inhibitor (i.e., conglutinin)-resistant strains of IAV (e.g., Philadelphia 82/BS and Brazil 78/BS) to a considerably greater extent than recombinant bovine conglutinin. Further study is needed to clarify the mechanism of these effects; however, it does appear that the presence of the collagen domain and NH2 terminus of SP-D in SP-D/Congneck+CRD modifies the ability of the molecule to bind to some strains of IAV.

SP-D/Congneck+CRD showed other distinctive properties compared with recombinant bovine conglutinin that appear to relate to the presence of the SP-D NH2 terminus and collagen domain regions in the molecule. SP-D/Congneck+CRD had greater IAV-aggregating capacity than recombinant bovine conglutinin. We have also found that SP-D/Congneck+CRD had greater ability to aggregate E. coli than recombinant bovine conglutinin (unpublished data). Unexpectedly, SP-D/Congneck+CRD had greater ability to aggregate IAV than rrSP-D as well (Fig. 6). This may reflect enhanced ability of SP-D/Congneck+CRD to bind to IAV compared with rrSP-D. In any case, consistent with our prior findings (15), this greater aggregating activity was also associated with greater activity of SP-D/Congneck+CRD in promoting binding of IAV to neutrophils and enhancing neutrophil respiratory burst responses to the virus than recombinant bovine conglutinin (or rrSP-D).

As noted above, certain features distinguish the collagen domain of SP-D from that of conglutinin (i.e., presence of N-linked carbohydrate attachment, greater length, lack of kink, and tendency to form higher-order multimers), which may contribute to interactions with IAV. It is unlikely that differences in the degree of multimerization were responsible for the greater aggregating capacity of SP-D/Congneck+CRD, since SP-D/Congneck+CRD and recombinant bovine conglutinin had essentially identical profiles on gel filtration. N-linked glycosylation of the collectins may play a role in their functional activity. A single N-linked carbohydrate attachment on the SP-A CRD has been implicated as a binding site for IAV and herpes simplex virus (5, 16, 23). rSP-D and hSP-D have a single N-linked attachment located in the collagen domain. Results of N-glycanase treatment of SP-D/Congneck+CRD indicate a similar degree of glycosylation. We have found, however, that deletion of the N-linked carbohydrate from normally multimerized SP-D does not substantially alter structure (7) nor does it reduce the ability of SP-D to aggregate IAV or inhibit viral HA activity (16). In fact, our recent results indicate that removal of the N-linked carbohydrate on SP-D actually leads to enhanced ability of SP-D to inhibit viral infectivity and induce viral or bacterial aggregation (14; unpublished data). Hence, it is unlikely that the presence of the N-linked carbohydrate on SP-D/Congneck+CRD alone accounts for the findings that it inhibits IAV HA activity or infectivity to a greater extent than recombinant bovine conglutinin. Further studies are clearly needed to determine which features of the SP-D NH2 terminus and collagen domain contribute to the greater ability of SP-D/Congneck+CRD than of conglutinin to induce viral and bacterial aggregation.

We believe that the findings reported here may be of therapeutic significance. SP-D/Congneck+CRD or other similar molecules may have usefulness as therapeutic agents against IAV or other pathogens. Recombinant bovine conglutinin and SP-D/Congneck+CRD have striking activity as direct inhibitors of IAV infectivity and could be expected to add to the activity of SP-D present in the airway. SP-D/Congneck+CRD appears to have further advantages compared with conglutinin, especially with regard to viral aggregation and promotion of binding to phagocytes. These properties may be important in aiding clearance of virus from the airway, especially during peak viral replication. Finally, the presence of the SP-D NH2 terminus and collagen domain may make SP-D/Congneck+CRD (or a similar molecule containing these domains of hSP-D) less antigenic or more likely to interact with other elements in the airway than recombinant bovine conglutinin.

A variety of avenues of further research are opened by findings reported here. Studies of interactions of SP-D/Congneck+CRD with other pathogens, phagocytic cells, complement, or lipids are planned. We also plan to generate further chimeric or specifically mutated molecules (e.g., in residues conferring carbohydrate specificity in the CRD) to further modify SP-D/Congneck+CRD (e.g., by removing the N-linked carbohydrate attachment site) to further characterize contribution of certain structural features to functional properties of collectins and also to further strengthen antiviral or antibacterial properties of these molecules. Future studies of the in vivo antiviral activities of SP-D/Congneck+CRD or other constructs will be of importance as well.


    ACKNOWLEDGEMENTS

This work was supported by National Institute on Aging Grants AI-348897 (to K. L. Hartshorn) and AI-33130 (to K. N. Sastry); National Heart, Lung, and Blood Institute Grant HL-29594 (to E. C. Crouch); and the Boston City Hospital Fund for Excellence (K. L. Hartshorn).


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. L. Hartshorn, Boston University School of Medicine, Section of Hematology-Oncology, 80 East Concord St., Boston, MA 02118 (E-mail: khartsho{at}bu.edu).

Received 17 March 1999; accepted in final form 11 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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