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*
Department of Medicine, Boston University School of Medicine, Boston, MA 02118;
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110; and
Department of Pediatrics, Harvard Medical School, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The collectins can be divided into two families based on their quaternary structure. The quaternary structure of the collectins is determined by the N-terminal and collagenous domains, while carbohydrate binding is mediated by the globular carbohydrate recognition domain (CRD). All of the collectins initially form trimers. The globular CRD of all collectins identified so far is trimeric with three carbohydrate-binding sites. In the case of MBL, SP-D, SP-A, and bovine conglutinin, the basic trimers are associated together in higher-order multimeric structures. MBL and SP-A commonly are composed of six trimers in association (octadecamers) that form structures closely resembling that of C1q. In contrast, conglutinin and SP-D have much larger collagen domains than SP-A or MBL. Conglutinin and SP-D most commonly form dodecameric structures (i.e., with four trimeric globular CRDs) in which the distance between CRDs is much greater than for MBL or SP-A. SP-D also exists in vivo (or in recombinant preparations) as trimers and very high-order multimeric structures (i.e., containing up to 32 globular CRD heads in one molecule) (8, 9).
The collectins also differ in terms of the affinity for binding-specific monosaccharides. SP-D has a relatively stronger affinity for binding glucose or maltose and lower affinity for binding N-acetyl-D-glucosamine (GlcNAc), while the reverse pattern is observed for MBL and conglutinin. All of the collectins bind mannose with high affinity and have relatively low affinity for galactose. The significance of differences among collectins in monosaccharide affinity in terms of host defense functions has not been studied. All of the collectins have been shown to inhibit infectivity of influenza A viruses (IAVs) in vitro (10, 11, 12, 13). In the cases of SP-D, MBL, and conglutinin, this effect is mediated by attachment of their CRDs to virus-associated carbohydrates. In contrast, neutralization by SP-A is mediated by attachment of the viral hemagglutinin to sialylated carbohydrates on the SP-A CRD (13, 14). Murine studies also suggest that surfactant collectins play an important role in the initial containment of IAV (15), and there is evidence that impairment of clearance of IAV in diabetic mice results from interference with collectin-mediated host defense by glucose (16).
The collectins agglutinate both bacteria and IAV particles and promote uptake of bacteria and IAV by phagocytic cells (11, 12, 17, 18, 19, 20). Among the collectins, SP-D is most potent at aggregating IAV particles and enhancing neutrophil uptake of the virus. Considerably higher concentrations of MBL or SP-A were required to achieve effects comparable to SP-D in these assays (14). Based on functional studies of nondodecameric forms of natural SP-D and certain structural mutants, we have hypothesized that these effects are mediated by properties of the N-terminal and collagen domain of SP-D.
The goals of the current study were to determine the structural basis
for differences in viral aggregating and opsonic activity between SP-D
and MBL and to establish whether differences in monosaccharide binding
preferences between SP-D and MBL are of functional significance with
respect to interactions with IAV. Previous experiments have indicated
that isolated trimeric or monomeric CRD preparations would not be
useful for studies of viral-neutralizing or opsonic activities because
of the markedly diminished activity of these preparations (14, 19, 21). Therefore, we decided that the best method to compare
functional activities of the different collectin CRDs was to prepare
full-length, multimerized, chimeric collectins sharing the same
collagen and N-terminal domains and differing only in their CRDs. In
this paper, we report the production of a SP-D/MBL chimera and
demonstrate that this chimera has distinctive binding properties and
enhanced antiviral and opsonic activities compared with wild-type MBL
or SP-D. A brief summary of the collectin preparations used in this
study and of the results of various functional assays is provided in
Fig. 1
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640, sodium citrate, dextran, trypan blue stain, Wright-Giemsa stain, HRP-type II, and scopoletin were purchased from Sigma (St. Louis, MO). Dulbecco’s PBS with or without calcium and magnesium were purchased from Life Technologies (Grand Island, NY). Ficoll-Paque was obtained from Pharmacia Biotech (Piscataway, NJ).
Neutrophil preparation
Neutrophils from healthy volunteers were isolated to >95% purity by using dextran precipitation, followed by Ficoll-Paque gradient separation for the removal of mononuclear cells and then hypotonic lysis to eliminate any contaminating erythrocytes, as previously described (22). Cell viability was determined to be >98% by trypan blue staining.
Virus preparation
IAV was grown in the chorioallantoic fluid of 10-day-old chicken
eggs and purified on a discontinuous sucrose gradient as previously
described (22). The virus was dialyzed against PBS to
remove sucrose, aliquoted, and stored at -80°C until needed.
Philippines 82/H3N2 (Phil82) and Brazil 78/H1N1 (Braz78) strains and
their bovine serum ß-inhibitor-resistant variants (Phil82/BS and
Braz78/BS) were kindly provided by Dr. E. Margot Anders (University of
Melbourne, Melbourne, Australia). The A/Bangkok 79/H3N2 (Bangkok79)
strain was a generous gift of Dr. Robert Webster (St. Jude’s Hospital,
Memphis, TN). After thawing, the viral stocks contained 
5 x
108 PFU/ml.
Collectin preparations
Recombinant human MBL (rhMBL) was produced in murine Sp2 cells as described (10, 23). The rhMBL used was of the more common allelic variant (termed MBPG). As previously demonstrated, this rhMBL preparation is composed predominantly of multimers containing five or six trimers (i.e., octadecamers) in association (23). Recombinant human SP-D (rhSP-D) was produced in Chinese hamster ovary-K1 cells and purified as previously described (9).
A chimeric collectin containing the human SP-D N-terminal and collagen
domains and human MBL neck and CRD domains (called
SP-D/MBLneck+CRD) was constructed for these
studies. Fig. 2 provides a diagram of
this construct. To produce SP-D/MBLneck+CRD, two
PCR were initially performed. One reaction involved human SP-D cDNA as
the template and two primers, SP6 (vector) and MBLAS1. MBLAS1 contained
sequences both of SP-D and of MBL at the site fusion (i.e., beginning
of the MBL neck region, bp 849–820) as follows:
5'-ACTATCACCATCTGGAAGCCCACTTTCTCC-3'. A second reaction involved
human MBL cDNA as the template and SPDS and MBLAS2 primers. The
sequence of MBLAS2 (5'-GGAATTCCTGAGTGATATGACCCTTCAGATAG-3')
corresponded to bp 1299–1274 at the N terminus of human MBL, while
that of SPDS (5'-GTGGGCTTCCAGATGGTGATAGTAGCCTG-3') contained
overlapping sequences of SP-D and MBL at the site of fusion between the
two proteins (bp 826–855). PCR was conducted with an initial cycle of
1 min denaturation at 94°C. This was followed by five cycles of
amplification with 30 s denaturation at 94°C, 30 s
annealing at 45°C, and 2 min extension at 68°C. The next 25 cycles
were similar to earlier cycles, except the annealing temperature was
raised to 55°C. A final amplification at 68°C for 5 min was also
conducted. The products of the first PCR were run in 1% agarose gel
and recovered by using a QIAquick gel extraction kit (Qiagen,
Chatsworth, CA). A final PCR was conducted using 1 µl of each product
as templates and SP6 and MBLAS2 as primers using conditions described
above. The final PCR product of 1.3 kb was initially cloned into
pT7Blue vector (Novagen, Madison, WI), and after sequencing to
determine the orientation was subcloned into pEE14 in a directional
manner into HindIII-EcoRI sites. The construct in
pEE14 vector was mapped and sequenced completely to verify orientation
and to rule out errors during amplifications. One nucleotide change
from GGT to GGA was noted in the codon for the first Gly in the MBL
neck region (no change in predicted amino acid sequence).
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2 µg/ml of conditioned medium. The majority
of the recombinant protein was eluted from A15 M columns in the
position of human SP-D dodecamers, but a significant fraction was
eluted in the position of human SP-D trimers, as previously observed
for rhSP-D. For the studies described in this paper, dodecamer and trimeric fractions of the SP-D/MBLneck+CRD chimera were isolated as previously described (9, 24, 25). Briefly, the gel-filtration columns were initially calibrated by correlating the elution volume with the ultrastructural appearance of the protein in the corresponding column fractions (9). Human SP-D multimers reproducibly elute near the void volume and are resolved from the four-arm dodecamers. The position of elution of trimers was subsequently confirmed by reduction and alkylation of rat SP-D under nondenaturing conditions; this also corresponds to the position of elution of a trimeric rat SP-D mutant that lacks amino-terminal cysteine residues preventing the formation of dodecamers (21). When examined by SDS-PAGE, the purified dodecamers migrated near the position of SP-D trimers in the absence of reduction. However, the reduced monomer was seen to migrate slightly more slowly than the 43-kDa monomers of natural or recombinant SP-D.
Endotoxin assays and controls
The collectin preparations and buffers were assayed for
endotoxin using a quantitative assay (Limulus amebocyte
lysate; BioWhittaker, Walkersville, MD). Buffers and virus stocks
contained 
1 pg/ml (or 1 endotoxin units (EU)/ml) of endotoxin. The
stock preparations of collectins contained between 1.5 and 8 ng/ml of
endotoxin. After accounting for dilution of collectins for use in
antiviral or neutrophil function assays, the final concentrations of
endotoxin in samples containing the highest concentrations of
collectins were 20–100 pg/ml (or 6–12 EU/ml using internal assay
standard). Similar concentrations of Escherichia coli (K235
strain) or Salmonella minnesota (Re strain) LPS (kindly
provided by Dr. Douglas Golenboch, Boston University School of
Medicine, Boston, MA) had no significant effect on assays of viral
infectivity, hemagglutination activity, viral aggregation, neutrophil
uptake of virus, or neutrophil
H2O2 production in presence
or absence of SP-D (data not shown).
Assessment of binding of collectins to IAV
Binding of SP-D to IAV was tested using an ELISA in which about 1 µg/ml of IAV was allowed to dry onto 96-well plates, fixed on the plates with ethanol and methanol sequentially, followed by washing and incubating with biotinylated SP-D (26). The presence of bound, biotinylated SP-D was detected using streptavidin conjugated to HRP and tetramethylbenzidine substrate (Bio-Rad, Hercules, CA), and the reaction was stopped using 1 N sulfuric acid (H2SO4). The OD was measured on an ELISA plate reader at 450 nm wavelength. Each individual data point was performed in duplicate. Background binding levels were obtained by measuring OD450 in wells that contained no IAV that were treated with biotinylated collectins. These background binding levels were subtracted from values obtained for binding of the same concentration of collectin to IAV before statistical analysis.
Fluorescent focus assay of IAV infectivity
Viral samples were incubated with control buffer or collectins for 30 min at 37°C, followed by quantitation of infectious virus particles using a fluorescent focus assay as previously described (15, 24). Briefly, Madin-Darby canine kidney cell monolayers were infected with the viral samples for 30 min, followed by washing of the monolayer and further incubation for a total of 7 h in glucose-free (unless otherwise indicated) DMEM. Infected cells were detected with mAb A-3 directed against the influenza viral nucleoprotein (graciously provided by Nancy Cox, Influenza Branch, Centers for Disease Control, Atlanta, GA) and FITC-labeled goat anti-mouse IgG.
Measurement of aggregation of IAV particles
Aggregation of IAV particles was assessed following addition of various concentrations of collectins by monitoring changes in light transmission on a highly sensitive SLM/Aminco 8000C (Spectronic Instruments, Urbana, IL) spectrofluorometer as described (11). The aggregation of viral particles or liposomes is demonstrated by a decline in light transmission (i.e., increased turbidity).
Measurement of IAV binding to neutrophils
IAV was FITC-labeled, and aliquots were incubated with collectins for 30 min at 37°C, followed by incubation of neutrophils with these viral samples for 30 min at 4°C. The subsequent viral binding to neutrophils was measured by flow cytometry as previously described (11). Viral uptake by neutrophils was assessed using a previously described (14) modification of the binding assay. Neutrophils and viral samples were allowed to incubate for 30 min at 37°C, followed by addition of 0.2 mg/ml of trypan blue to quench extracellular fluorescence before measurement of neutrophil fluorescence using flow cytometry.
Measurement of neutrophil H2O2 production
H2O2 production was measured by assessing reduction in scopoletin fluorescence as previously described (27).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We compared the ability of SP-D/MBLneck+CRD
dodecamers and trimers, rhSP-D dodecamers, and rhMBL (predominantly
octadecamers) to inhibit hemagglutination (HA) activity of
representative wild-type strains of IAV. As shown in Table I, the dodecameric fraction of
SP-D/MBLneck+CRD was from 10- to 30-fold more
potent than rhMBL in these assays. HA inhibition was not observed when
assays were conducted in EDTA-containing buffer.
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In summary, these results indicate that these collectins inhibit HA activity of these strains of IAV predominantly through calcium-dependent attachment to viral carbohydrates. The dodecameric fraction of the SP-D/MBLneck+CRD chimera has substantially greater HA-inhibitory activity than rhMBL against all strains tested. The dodecameric fraction of the chimera also had moderately greater inhibitory activity against several strains of IAV than rhSP-D dodecamers.
Inhibition of antiviral activity of collectins by monosaccharides reveals marked differences in monosaccharide preferences between rhSP-D and SP-D/MBLneck+CRD
Fig. 3 shows the effect of adding
increasing concentrations of monosaccharides on the ability of rhSP-D,
rhMBL, and SP-D/MBLneck+CRD to inhibit HA
activity of IAV. Markedly higher concentrations of rhSP-D were needed
to inhibit IAV HA activity in the presence of increasing concentrations
of glucose, while GlcNAc had minimal effect. The opposite pattern was
observed with rhMBL and SP-D/MBLneck+CRD.
Galactose did not significantly interfere with HA-inhibitory activity
of rhMBL or SP-D/MBLneck+CRD, but did interfere
to a modest (although statistically significant) extent with that of
rhSP-D. Concentrations as low as 21 mM glucose significantly interfered
with HA inhibition by rhSP-D. In contrast, 21–164 mM glucose did not
alter HA-inhibitory activity of SP-D/MBLneck+CRD.
Hence, exchanging the CRD of SP-D for that of MBL resulted in
alterations in the pattern of interference by specific
monosaccharides.
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SP-D/MBLneck+CRD has greater binding affinity for IAV than rhMBL
As measured by ELISA using biotinylated collectins (see Fig. 4),
SP-D/MBLneck+CRD dodecamers bound to the
Bangkok79 IAV with much greater affinity than rhMBL. The
multimerization state of the chimera was a strong determinant of
binding affinity. Binding of SP-D/MBLneck+CRD
trimers was similar to that of rhMBL.
SP-D/MBLneck+CRD dodecamers also bound to the
Bangkok79 strain of IAV with significantly greater affinity than rhSP-D
dodecamers. Similar results were obtained when testing the binding of
the lectins to the Phil 82 and Phil82/BS IAV strains (Table II).
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As shown in Fig. 5, SP-D/MBLneck+CRD dodecamers caused markedly
greater aggregation of IAV than rhMBL.
SP-D/MBLneck+CRD dodecamers caused maximal viral
aggregation at concentrations of 0.2 or 0.4 µg/ml. At these
concentrations rhMBL caused no detectable viral aggregation.
SP-D/MBLneck+CRD dodecamers also caused
significantly greater aggregation of the virus than rhSP-D dodecamers.
Similar results were obtained in four experiments using the Bangkok79
strain of IAV (e.g., 0.4 µg/ml of rhSP-D and
SP-D/MBLneck+CRD dodecamers reduced light
transmission through a suspension of Bangkok79 IAV to 97.4 ± 0.39
and 92 ± 0.33% of control, respectively, after 12 min;
p 0.001; n = 5). As expected, the
ability of the chimera to cause viral aggregation was strongly
dependent on multimerization (i.e., note the absence of aggregation in
response to SP-D/MBLneck+CRD trimers in Fig. 5).
HA titers were measured on all samples after completion of the
aggregation assay, and SP-D/MBLneck+CRD
dodecamers caused significantly greater reductions of HA activity than
any of the other collectins, including rhSP-D dodecamers (data not
shown).
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SP-D/MBLneck+CRD dodecamers were
significantly more effective at inhibiting the infection of Madin-Darby
canine kidney cells by the Phil82 strain of IAV at all concentrations
measured when compared with rhMBL (see Fig. 6A). Of note,
SP-D/MBLneck+CRD dodecamers were also
significantly more effective at inhibiting infectivity than rhSP-D
dodecamers. In addition, SP-D/MBLneck+CRD
dodecamers had much stronger activity in this assay than trimers (Fig. 6B). For comparative purposes, we tested the activity of a
preparation of SP-D/MBLneck+CRD purified using
anti-MBL Ab affinity chromatography. This preparation was composed
of a mixture of dodecamers and trimers and had intermediate
neutralizing activity between that of the purified dodecamer and trimer
preparations (Fig. 6B).
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C).
This concentration of glucose caused relatively greater inhibition of
the neutralizing activity of rhSP-D than of rhMBL or
SP-D/MBLneck+CRD.
We also tested the ability of rhSP-D dodecamers,
SP-D/MBLneck+CRD dodecamers, and rhMBL to inhibit
infectivity of the Phil82/BS strain of IAV (Fig. 6D). As
expected, as compared with wild-type Phil82 IAV, this strain was highly
resistant to rhMBL, rhSP-D, or the
SP-D/MBLneck+CRD chimera. Nonetheless,
SP-D/MBLneck+CRD dodecamers inhibited Phil82/BS
to a significantly greater extent than rhSP-D dodecamers or rhMBL.
SP-D/MBLneck+CRD dodecamers had markedly greater opsonizing activity than rhMBL
We examined the effects of the collectins on the binding and
uptake of FITC-labeled IAV by neutrophils.
SP-D/MBLneck+CRD dodecamers caused much greater
enhancement of neutrophil binding of IAV than rhMBL or
SP-D/MBLneck+CRD trimers (Fig. 7), and significantly greater enhancement
of binding than rhSP-D dodecamers. Similar results were obtained in
four experiments assessing the effects of collectins on viral uptake
(e.g., 1.4 µg/ml of SP-D/MBLneck+CRD
dodecamers increased viral uptake to 209 ± 17% of control as
compared with 104 ± 4% for rhMBL; p <
0.01).
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). These results
suggest that factors other than enhanced neutrophil binding of IAV, per
se, are involved in the enhancement of
H2O2 production by these
collectins (i.e., since SP-D/MBLneck+CRD
dodecamers caused significantly greater enhancement of neutrophil
binding of IAV than rhSP-D dodecamers). In any case, preincubation of
IAV with SP-D/MBLneck+CRD dodecamers caused
markedly greater enhancement of IAV-induced neutrophil
H2O2 responses than either
rhMBL or SP-D/MBLneck+CRD trimers (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The collagen domain of SP-D differs from that of MBL in having much greater length and lacking an interruption or kink. These features result in a much greater separation between trimeric CRDs on a molecule of multimerized SP-D as compared with MBL, which could account for its greater ability to cross-link viral particles. In accordance with this hypothesis, the trimeric preparation of the SP-D/MBLneck+CRD chimera was almost devoid of aggregating activity. Based on ELISA, SP-D/MBLneck+CRD dodecamers bound to IAV with much higher affinity than rhMBL or SP-D/MBLneck+CRD trimers, suggesting that binding mediated by the MBL CRD is strongly affected by cooperative interactions among CRDs in a given molecule.
The changes introduced in MBL by replacing its collagen domain with
that of SP-D had a major impact on its antiviral activity and ability
to act as an opsonin. Fig. 1 summarizes results of various assays of
antiviral and opsonic activity examined in this paper.
SP-D/MBLneck+CRD dodecamers had markedly greater
HA-inhibitory and viral-neutralizing activity than rhMBL. This did not
appear to result from qualitative differences in the mechanism of
binding between the two preparations. HA inhibition mediated by the
chimera and rhMBL was calcium dependent, and both preparations had
reduced ability to bind to, or inhibit HA activity of, the
ß-inhibitor-resistant strains of IAV. These strains lack a
high-mannose oligosaccharide attachment on the viral HA. In addition,
the chimera and rhMBL showed similar patterns of monosaccharide
interference with HA-inhibitory activity (i.e., mannose and
GlcNAc interfered most strongly, while glucose and galactose
caused little or no detectable interference). Overall, these results
indicate that direct interactions of
SP-D/MBLneck+CRD dodecamers and rhMBL with IAV
are mediated largely by calcium-dependent binding of CRDs to
mannosylated viral carbohydrates. Interestingly, there was a
quantitative difference between SP-D/MBLneck+CRD
dodecamers and rhMBL in the concentration dependence of interference by
mannose or GlcNAc; much lower concentrations of these monosaccharides
were needed to interfere with HA inhibition by rhMBL. These results
(and results of ELISA) are consistent with enhanced binding affinity of
SP-D/MBLneck+CRD dodecamers (as compared with
rhMBL) for IAV-associated carbohydrates.
Our prior studies have indicated that the ability of collectins to enhance binding or uptake of IAV by neutrophils, or to potentiate IAV-induced neutrophil respiratory burst responses, correlates with their ability to cause viral aggregation. We have demonstrated that pretreatment of IAV with SP-D or conglutinin causes the binding and internalization of massive aggregates of viral particles to neutrophils (14, 19). This is quite distinct from the binding and internalization of individual viral particles observed in the absence of these collectins. By contrast, MBL and SP-A cause the binding of much smaller viral aggregates to neutrophils (14). We now demonstrate that the ability of MBL to enhance neutrophil binding and uptake of, and respiratory burst responses to, IAV is greatly enhanced by substituting the SP-D N terminus and collagen domain, resulting in the assembly of "macroagglutinating" dodecamers with the carbohydrate-binding specificities of MBL. Results obtained with the SP-D/MBLneck+CRD trimers clearly confirm the importance of higher-order multimerization in mediating viral binding, neutralization, aggregating, and opsonic activity with respect to neutrophils. Overall, our results are consistent with the hypothesis that viral aggregation is an important determinant of opsonic activity. Restrepo et al. (20) have found that SP-D stimulates phagocytosis of Pseudomonas aeruginosa by rat alveolar macrophages through a mechanism that does not involve aggregation. Therefore, it may be that various mechanisms of opsonization exist depending on the type of collectin, phagocyte, or pathogen.
In summary, our studies demonstrate that functional activities of the MBL CRD are strongly influenced by the properties of the N-terminal and collagen-domain backbone. Prior reports have indicated that IAV-neutralizing activity of MBL is potentiated in the presence of complement and serum (31). Because complement fixation by MBL depends on interaction of its collagen domain with MBL-associated serine proteases, it appears unlikely that the SP-D/MBLneck+CRD chimera would be able to fix complement. Future studies will address this question. Studies involving further modifications of the SP-D/MBLneck+CRD collagen domain to assess the effect on interactions with pathogens or phagocytic cells are underway.
Another major conclusion is that the SP-D and MBL CRD domains have distinctive functional properties with respect to IAV. The SP-D/MBLneck+CRD construct allows us to make direct functional comparisons of the MBL and SP-D CRDs having controlled for potential contributions of the SP-D N terminus and collagen domain. As demonstrated by the results obtained with SP-D/MBLneck+CRD trimers, it is critical to use similarly multimerized fractions of SP-D in these comparisons because the degree of multimerization strongly impacts on binding and other functional properties of the CRD. The presence of the MBL CRD in the chimera caused a clear cut change in the profile of monosaccharide interference with HA-inhibitory or viral-neutralizing activity as compared with SP-D. An important additional finding was that HA-inhibitory activity of SP-D/MBLneck+CRD was much less susceptible to interference by monosaccharides than that of SP-D, suggesting that the chimera had greater binding affinity for IAV-associated carbohydrates. This finding may explain the further observations that SP-D/MBLneck+CRD dodecamers caused significantly greater aggregation and neutralization of IAV than the similarly multimerized fraction of rhSP-D. We have reported similar results comparing antiviral activities of a recombinant rat SP-D/bovine conglutinin chimera with those of wild-type recombinant rat SP-D (24). The MBL and conglutinin CRDs have much higher affinity for GlcNAc than SP-D. Because GlcNAc is a common component of IAV-associated carbohydrates (32), it could be that enhanced binding to GlcNAc increases IAV-neutralizing activity. Alternatively, it could be that MBL and conglutinin have a greater ability to bind to mannosylated carbohydrates on IAV than SP-D. Site-directed modifications of CRDs to increase affinity for specific monosaccharides could address these questions.
Serum levels of MBL are highly variable but are 1.5 µg/ml in
subjects who do not have MBL deficiency (3). Although MBL
is not normally present in airway secretions in mice, low levels
(100 µg/ml) were detected after experimental IAV infection
(15). Levels of SP-D in bronchoalveolar lavage fluids
range from 100 ng/ml to 500 ng/ml (12, 15). In
IAV-infected mice, levels of SP-D increase severalfold (i.e., to levels
>1 µg/ml) (15). Hence, the concentrations of collectins
used in our experiments are within physiological ranges. Further
studies will be needed to determine whether the
SP-D/MBLneck+CRD chimera can enhance clearance of
IAV in vivo or be useful as a therapeutic agent (e.g., via instillation
into the airway during IAV infection).
SP-D/MBLneck+CRD has greater promise as a
therapeutic agent than the previously reported
SP-D/conglutininneck+CRD chimera
(24) because it is composed entirely of human components.
The relative resistance of the IAV-neutralizing activity of
SP-D/MBLneck+CRD to interference by glucose
suggests a potential for therapeutic application in IAV-infected
diabetics.
In summary, our findings add important new insights regarding the contributions of specific collectin domains to functional activities relevant to host defense and demonstrate that modification of collectins through recombinant techniques can result in enhanced anti-microbial activity. Further development and in vivo testing of such constructs holds promise as a strategy for elucidation of collectin biology and possibly for development of new therapeutic agents.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kevan L. Hartshorn, Boston University School of Medicine, Evans Biomedical Research Center, Room 414, 650 Albany Street, Boston, MA 02118.
3 Abbreviations used in this paper: MBL, mannose-binding lectin; SP-D, surfactant protein D; IAV, influenza A virus; CRD, carbohydrate recognition domain; HA, hemagglutination; SP-A, surfactant protein A; rhMBL, recombinant human MBL; rhSP-D, recombinant human SP-D; Phil82, Philippines 82/H3N2 strain; Bangkok79, A/Bangkok 79/H3N2 strain; GlcNAc, N-acetyl-D-glucosamine.
Received for publication March 8, 2000. Accepted for publication June 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R. Leth-Larsen, P. Garred, H. Jensenius, J. Meschi, K. Hartshorn, J. Madsen, I. Tornoe, H. O. Madsen, G. Sorensen, E. Crouch, et al. A Common Polymorphism in the SFTPD Gene Influences Assembly, Function, and Concentration of Surfactant Protein D J. Immunol., February 1, 2005; 174(3): 1532 - 1538. [Abstract] [Full Text] [PDF]
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 K. L. Hartshorn, M. R. White, and E. C. Crouch Contributions of the N- and C-Terminal Domains of Surfactant Protein D to the Binding, Aggregation, and Phagocytic Uptake of Bacteria Infect. Immun., November 1, 2002; 70(11): 6129 - 6139. [Abstract] [Full Text] [PDF]
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 Y. He and E. Crouch Surfactant Protein D Gene Regulation. INTERACTIONS AMONG THE CONSERVED CCAAT/ENHANCER-BINDING PROTEIN ELEMENTS J. Biol. Chem., May 24, 2002; 277(22): 19530 - 19537. [Abstract] [Full Text] [PDF]
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K. J. Haley, A. Ciota, J. P. Contreras, M. R. Boothby, D. L. Perkins, and P. W. Finn Alterations in lung collectins in an adaptive allergic immune response Am J Physiol Lung Cell Mol Physiol, March 1, 2002; 282(3): L573 - L584. [Abstract] [Full Text] [PDF]
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J. S. Ferguson, D. R. Voelker, J. A. Ufnar, A. J. Dawson, and L. S. Schlesinger Surfactant Protein D Inhibition of Human Macrophage Uptake of Mycobacterium tuberculosis Is Independent of Bacterial Agglutination J. Immunol., February 1, 2002; 168(3): 1309 - 1314. [Abstract] [Full Text] [PDF]
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V. C. Huber, J. M. Lynch, D. J. Bucher, J. Le, and D. W. Metzger Fc Receptor-Mediated Phagocytosis Makes a Significant Contribution to Clearance of Influenza Virus Infections J. Immunol., June 15, 2001; 166(12): 7381 - 7388. [Abstract] [Full Text] [PDF]
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P. Zhang, A. McAlinden, S. Li, T. Schumacher, H. Wang, S. Hu, L. Sandell, and E. Crouch The Amino-terminal Heptad Repeats of the Coiled-coil Neck Domain of Pulmonary Surfactant Protein D Are Necessary for the Assembly of Trimeric Subunits and Dodecamers J. Biol. Chem., June 1, 2001; 276(23): 19862 - 19870. [Abstract] [Full Text] [PDF]
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). SP-D has one N-linked oligosaccharide as shown.
The CRD and the linking 
-helical neck region (L) were derived from
human MBL. The diagram depicts a single monomeric subunit of SP-D. Such
monomers assemble into trimers and then into higher-order structures
through disulfide bond interactions among N-terminal cysteines.
