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Published ahead of print on July 29, 2004, doi:10.1165/rcmb.2004-0105OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 565-572, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0105OC

Surfactant Protein D Binding to Terminal {alpha}1-3–Linked Fucose Residues and to Schistosoma mansoni

J. Koenraad van de Wetering, Alexandra van Remoortere, Arie B. Vaandrager, Joseph J. Batenburg, Lambert M. G. van Golde, Cornelis H. Hokke and Jaap J. van Hellemond

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Graduate School of Animal Health, Utrecht University, Utrecht; and Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.

Address correspondence to: Dr. J. J. van Hellemond, Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80176, 3508 TD, Utrecht, The Netherlands. E-mail: j.j.vanhellemond{at}vet.uu.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pulmonary surfactant protein (SP)-D is an important component of the innate immune system of the lung, which is thought to function by binding to specific carbohydrates on the surface of viruses and unicellular pathogens. SP-D has been shown to have a relatively high affinity for the monosaccharides mannose, glucose, and fucose. However, there is limited information on SP-D binding to complex carbohydrate structures, and binding of SP-D to fucose in the context of an oligosaccharide has not yet been investigated. In this study, we used surface plasmon resonance spectroscopy to examine the potential of SP-D to bind to various synthetic fucosylated oligosaccharides, and identified Fuc{alpha}1–3GalNAc and Fuc{alpha}1–3GlcNAc elements as strong ligands. These types of fucosylated glycoconjugates are presented at the surface of Schistosoma mansoni, a parasitic worm that, during development, transiently resides in the lung. In line with the findings by surface plasmon resonance, we found that SP-D can bind to larval stages of S. mansoni, demonstrating for the first time that SP-D interacts with multicellular lung pathogens.

Abbreviations: bronchoalveolar lavage, BAL • bovine serum albumin, BSA • carbohydrate recognition domain, CRD • ethylenediaminetetraacetic acid, EDTA • fluorescein isothiocyanate, FITC • Fuc{alpha}1–2Fuc{alpha}1–3GlcNAc, FF-Gn • Fuc{alpha}1–3GlcNAc, F-Gn • Fuc{alpha}1–3GalNAcß1–4GlcNAc, F-LDN • GlcNAc, Gn • human serum albumin, HSA • HEPES-buffered saline, HBS • immunoglobulin, Ig • GalNAcß1–4GlcNAc, LDN • {alpha}lNAcß1–4(Fuc{alpha}1–3)GlcNAc, LDN-F • Galß1–4(Fuc{alpha}1–3)GlcNAc, Lewis-X • mannan-binding lectin, MBL • phosphate-buffered saline, PBS • polyvinylidene fluoride, PVDF • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE • surfactant protein, SP • surface plasmon resonance, SPR • Tris-buffered saline and Tween, TBS-T


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Surfactant protein (SP)-D is a key component of the innate immunity in the alveoli of the lung (1, 2). SP-D belongs to the family of collectins, which includes, among others, SP-A, mannan-binding lectin (MBL), conglutinin, and CL-43. Collectins share some basic structural properties. They are multimers of polypeptides that each consist of a short, cysteine-rich N-terminal crosslinking domain, followed by a collagen domain, an {alpha}-helical neck domain, and, finally, a C-type lectin domain, also known as carbohydrate recognition domain (CRD) (3, 4). It has been shown that many pathogenic viruses and unicellular microorganisms, such as bacteria and fungi, can be bound by SP-D in vitro. Generally, SP-D interacts via its CRD with glycoconjugates present on the outside of the pathogens (2, 5, 6). As a result of this binding, microorganisms can be aggregated and/or opsonized, which leads, in many cases, to enhanced killing and clearance by phagocytic cells in the lung (4). In addition, SP-D also directly inhibits the growth of microorganisms (7, 8), which, in the case of Gram-negative bacteria, is the result of increased membrane permeability (8).

SP-D binds to a wide range of saccharides. As far as monosaccharides are concerned, it has moderate affinity for mannose, glucose, and fucose, whereas it has weak affinity for galactose, glucosamine, and N-acetylglucosamine (9, 10). However, binding of SP-D to monosaccharides is rather weak in general, and its binding to microbial surfaces occurs via the simultaneous binding of several of its CRDs to dense sugar arrays (4, 11) or polysaccharides (12), which are normally found on the surface of microorganisms. So far, a limited number of studies have been performed on SP-D binding to carbohydrate structures with a high content of glucose (12, 13) or mannose (14, 15). As regards glucose, it was found that nonterminal residues in a glucose polymer contribute to binding to SP-D, and that binding is dependent on the nature of the glycosidic linkage between monosaccharide units, as the hydroxyl groups on the 2- and 3-position, or on the 3- and 4-position, have to be available to dock on the CRD of SP-D (12, 13). As regards mannose, MBL recognizes equatorial hydroxy groups present at the 3- and 4-positions of the nonreducing terminal residues of polysaccharides (16). In view of the structural similarity between SP-D and MBL, this recognition may apply to SP-D as well. Binding of SP-D to fucose in the context of a polysaccharide has not yet been investigated, although fucose-containing glycoconjugates are abundantly present on the outer surface of a number of pathogens, of which Helicobacter pylori and, especially, Schistosoma mansoni are the best-studied examples (17, 18).

S. mansoni causes an important chronic disease called schistosomiasis, with an estimated 200 million humans infected world wide (19). S. mansoni adults live in the mesenteric veins of their mammalian hosts, where they produce eggs that penetrate the wall of the gut. These eggs are then excreted via the feces and, outside the body, they develop into miracidia, which subsequently infect a freshwater snail of the genus Biomphalaria. Subsequently, the snail releases cercariae that infect a human host by penetration of the skin, after which they quickly develop into schistosomula. These schistosomula then migrate via the heart to the lungs, where they reside for several days before migrating via the liver to the portal veins, where they sexually mature (20). Histologic studies have demonstrated many schistosomula in the extracellular space in the alveoli of the lung (21) and, therefore, this larval stage of S. mansoni can be considered as a transient and multicellular lung pathogen that it is likely to encounter SP-D in vivo.

Many (fucose-containing) glycoconjugate structures are exposed on the surface of the cercariae and schistosomula of S. mansoni, and a significant number of them have been studied in detail. The glycoconjugate structures that appear to play a role in the immunology of schistosomiasis (18, 22–25) contain fucosylated oligosaccharide elements, such as GalNAcß1–4(Fuc{alpha}1–3)GlcNAc (LDN-F), Galß1–4(Fuc{alpha}1–3)GlcNAc (Lewis-X), Fuc{alpha}1–3GalNAcß1–4GlcNAc (F-LDN), Fuc{alpha}1–2Fuc{alpha}1–3GlcNAc (FF-Gn), and nonfucosylated variants thereof (Figure 1) (18, 26, and Refs. cited therein). In this study, we used neoglycoconjugates to investigate the importance of terminal fucose residues in glycoconjugates for binding to SP-D. Because terminal {alpha}1-3–linked fucose residues were detected as ligands for SP-D, and because these structures are exposed on the outer surface of larval S. mansoni that reside in the lung, we also investigated whether SP-D can bind to this multicellular lung pathogen. Although SP-D is known to bind a great variety of unicellular pathogens, binding to a multicellular pathogen has so far not been reported.



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  		Figure 1. Schematic diagram of the (synthetic) glycoconjugates tested for SP-D binding by SPR. Gal, D-galactose; GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glucosamine; Fuc, L-fucose; Man, D-mannose. Fucose residues are depicted in bold.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
(Bis)acrylamide for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was purchased from Bio-Rad (Hercules, CA). Nitrocellulose (Protran) and polyvinylidene fluoride (PVDF, Westran) blot membranes for Western blotting were obtained from Schleicher and Schuell (Dassel, Germany). RNase B from bovine pancreas was obtained from Sigma (St. Louis, MO). Unless otherwise indicated, all other chemicals came from Merck (Darmstadt, Germany) or Sigma.

Rabbit antiserum against human SP-D was produced as described previously (5). The immunoglobulin (Ig) G fraction was purified by affinity chromatography on protein A-Sepharose CL 4B. Western blot analysis using total bronchoalveolar lavage (BAL) fluid of patients with alveolar proteinosis showed only one band at the predicted position of human SP (hSP)-D (43 kD), demonstrating its specificity. Purified antibodies were stored at –20°C in 50% glycerol.

Purification of hSP-D
hSP-D was isolated from BAL fluid of patients suffering from alveolar proteinosis, as previously described (27). This procedure was approved by the ethics committee of the University of Amsterdam, The Netherlands, where the lavage was performed. Briefly, BAL fluid was centrifuged at 28,000 x g to collect the surfactant pellet, as the majority of SP-D is found in this fraction of the BAL fluid of patients with alveolar proteinosis. SP-D was eluted from the surfactant pellet by stirring the pellet in 50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 0.05% Tween-20, and 100 mM maltose. After centrifugation at 28,000 x g, the SP-D in the supernatant was further purified by gel filtration chromatography on a Bio-prep1000/17 column (Bio-Rad) in 50 mM Tris/HCl (pH 7.4) containing 150 mM NaCl and 5 mM ethylenediaminetetraacetic acid (EDTA) (28). Purity of the SP-D sample was assessed by SDS-PAGE and subsequent Coomassie blue staining and Western blotting. In addition, using the QCL-1000 Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD), it was demonstrated that only 15 pg of endotoxin was present per µg of SP-D. The concentration of SP-D in the final preparation was determined with Coomassie blue Plus-200 protein assay reagent (Pierce, Rockford, IL), as described by the manufacturer, using bovine serum albumin (BSA) as the standard. SP-D preparations were stored in 5 mM Tris/HCl (pH 7.4), 150 mM NaCl, and 5 mM EDTA, at –20°C, until use.

Neoglycoproteins
GalNAcß1–4GlcNAcß1-(CH2)8COOH (LDN-spacer), Galß1–4(Fuc{alpha}1–3)GlcNAcß1-(CH2)8COOH (Lewis-X-spacer), Fuc{alpha}1–3GalNAcß1–4GlcNAcß1–3Gal{alpha}1-(CH2)5NH2 (F-LDN-spacer), GalNAcß1–4(Fuc{alpha}1–3)GlcNAcß1–3Gal{alpha}1-(CH2)5NH2 (LDN-F-spacer), GlcNAcß1-(CH2)6NH2 (Gn-spacer), Fuc{alpha}1–3GlcNAcß1-(CH2)6NH2 (F-Gn-spacer), and Fuc{alpha}1–2Fuc{alpha}1–3GlcNAcß1-(CH2)6NH2 (FF-Gn-spacer) were synthesized and conjugated to BSA, as described elsewhere (25, 29–31, and Aguilera and Overkleeft, Leiden Institute of Chemistry, unpublished results). Matrix-assisted laser desorption ionization/time of flight mass spectrometry was used to analyze the BSA conjugates and confirmed the coupling of each of the different oligosaccharides in the range of 10–15 mol per mol BSA. Human serum albumin (HSA) conjugates, to which 25 mol Lewis-X oligosaccharides per mol HSA were coupled, were obtained from IsoSep (Lund, Sweden).

Surface Plasmon Resonance Spectroscopy
Surface plasmon resonance (SPR) spectroscopy was performed using a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). CM5 sensor chips, amino coupling kit, and surfactant P-20 were also obtained from Biacore AB. All buffers were filtered (0.2 µm) and degassed before use.

Binding studies were performed according to Van Remoortere and colleagues (25, 30). Briefly, the neoglycoconjugates and RNase B were immobilized at a flow of 5 µl/min in 10 mM sodium acetate (pH 4.0) onto a carboxylmethylated dextran CM5 sensor chip by covalent amine coupling, according to the manufacturer's instructions, until an increase of ~ 10,000 response units was observed by SPR. In this way, roughly equal amounts of each (neo)glycoprotein were loaded on the chips. The chips were used to study the binding of SP-D with the neoglycoproteins containing different sets of carbohydrate structures and with RNase B.

All analyses were performed at flow rates of 5 µl/min at 25°C using HEPES-buffered saline (HBS) buffer (pH 7.4) containing 0.005% P20 and 3 mM CaCl2 as eluent. Injection times of SP-D were 6 min followed by 10 min of buffer injection to allow dissociation. Surfaces were regenerated with HBS buffer containing 3 mM EDTA followed by 10 mM HCl. To correct for refractive index changes and nonspecific binding, BSA or HSA were used as blanks. Analysis of the data was performed using the Bia evaluation 3.2 software (Biacore AB).

Isolation of Cercariae and Preparation of Schistosomula
S. mansoni cercariae were shed from infected Biomphalaria glabrata snails for 3 h at 28°C. After the contaminating snail debris had sedimented to the bottom, the supernatant was collected and cooled on ice to immobilize the cercariae. The cercariae then sedimented to the bottom, and the supernatant was removed to concentrate the cercariae. For the transformation of cercariae into schistosomula, cercariae were incubated for 3 h at 37°C in RPMI 1640 (Invitrogen, Breda, The Netherlands), containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 U/ml fungizone. Freshly transformed schistosomula sedimented to the bottom, and the supernatant was removed to concentrate the organisms.

Binding of SP-D and Concanavalin A to Cercariae and Schistosomula
Cercariae or schistosomula were fixed overnight using 2% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C, after which all subsequent incubations were performed at 22°C. In between incubation steps, the organisms were extensively washed using PBS containing 0.2% BSA and 1 mM CaCl2. Nonspecific binding was blocked by subsequent incubation of the organisms in 0.2% BSA in PBS for 1 h. Afterwards, cercariae or schistosomula were incubated for 1 h with SP-D (20 µg/ml) or fluorescein isothiocyanate (FITC)–conjugated concanavalin (Con) A (20 µg/ml) (EY laboratories, San Mateo, CA) or without lectins in PBS containing 0.2% BSA and 1 mM CaCl2. For each labeling reaction, ~ 750 organisms were used in a total reaction volume of 300 µl.

After washing, FITC-conjugated Con A–labeled organisms were ready for analysis by confocal fluorescence microscopy. To detect SP-D binding, organisms were subsequently incubated with rabbit anti–SP-D IgG (370 ng/ml), and Alexa-488–conjugated goat anti-rabbit antibody (Molecular Probes, Leiden, The Netherlands) (300 ng/ml). To minimize fading of fluorescent signals, the organisms were then embedded in FluorSave reagent (Calbiochem, San Diego, CA). Finally, the fluorescence signal was analyzed using a spectral confocal microscope (Leica TCS SP; Leica GmbH, Heidelberg, Germany). To examine the involvement of the CRD of SP-D in the interaction of SP-D with the parasites, the incubation of the cercariae or schistosomula with SP-D (20 µg/ml) was also performed in the presence of EDTA (2 mM) or maltose (20 mM). Detection of SP-D binding was subsequently performed as described above.

SP-D Lectin Blot
Cercariae and schistosomula were homogenized in a Dounce homogenizer at 4°C. Total protein content of the homogenates was determined using an adapted method of Lowry (32). Afterwards, proteins were concentrated using trichloroacetic acid precipitation and solubilized in Laemmli buffer (125 mM Tris/HCl [pH 6.8], 20% glycerol, 4% SDS, 2% ß-mercaptoethanol). Per sample 50 µg total protein was subjected to electrophoresis on a 7.5% SDS-polyacrylamide gel under reducing conditions. Thereafter, proteins were transferred to PVDF blotting membrane, according to standard procedures. All incubation steps (described below) were performed for 1 h at 22°C in the presence of 1 mM CaCl2. In between incubation steps, membranes were washed 3 times using 20 mM Tris/HCl pH 7.4, 500 mM NaCl, 0.05% Tween-20 (TBS-T) containing 1 mM CaCl2. Directly after blotting, PVDF membranes were blocked for 1 h in 0.3% Tween-20 in TBS and subsequently for 1 h in TBS-T containing 2% BSA. To investigate SP-D binding to immobilized proteins, blots were subsequently incubated overnight with or without 100 ng/ml SP-D in TBS-T containing 0.1% BSA and 1 mM CaCl2 at 4°C. SP-D binding was detected using rabbit anti–SP-D IgG (92 ng/ml), and, subsequently, a horseradish peroxidase–conjugated goat anti-rabbit antibody (2 µg/ml) (Nordic, Tilburg, The Netherlands). Reactive proteins were visualized using the pico range super signal west chemiluminiscent detection kit (Perbio, Rockford, IL). Parallel incubations in the presence of 2 mM EDTA were also performed to determine the requirement of divalent cations in the binding of SP-D to immobilized parasite proteins. Detection of SP-D binding was subsequently performed as described above.

Qualitative Analysis of Total Glycoprotein Content
For each parasite sample, 10 µg total protein was subjected to SDS-PAGE as described above. Subsequently, proteins were blotted onto nitrocellulose blotting membrane using standard procedures. Total glycoproteins were visualized using the DIG-glycan detection kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Blots were scanned with a CanoScan FB 630P scanner using CanoCraft CS-P 3.7 software (Canon, Hoofddorp, The Netherlands).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of Fucose Containing Glycoconjugate Ligands for SP-D
To investigate which fucose-containing carbohydrates are bound by SP-D, we used SPR analysis to detect the interaction of SP-D with a number of well defined synthetic oligosaccharides known to be exposed on the outer surface of schistosomes (Figure 1). Some of these oligosaccharides (F-LDN, LDN-F, FF-Gn, and Lewis-X) include a fucose residue, while others (LDN and Gn) do not. Together they constitute a broad panel of epitopes that is characteristic of the major schistosome glycoconjugates (18). All oligosaccharides were coupled to serum albumin as a carrier with similar oligosaccharide densities, as confirmed by matrix-assisted laser desorption ionization/time of flight mass spectrometry. Each of the synthetic glycoconjugates was immobilized on an SPR sensor chip channel with a comparable surface density of ~ 10,000 response units to permit direct comparison of the relative affinity of SP-D for the immobilized neoglycoconjugates. In addition to the panel of synthetic neoglycoconjugates, RNase B was used, which carries high-mannose-type glycans that are also present on schistosomes (26, 33).

SPR analysis revealed that 31 µg/ml SP-D showed a relatively high affinity for high-mannose–containing RNase B. In addition, high affinity of SP-D was also observed for the unbranched oligosaccharides F-LDN and F-Gn, both containing an {alpha}1-3–linked fucose residue at their nonreducing end (Figures 1 and 2A). Because of differences in presentation of the immobilized oligosaccharides, no direct comparison could be made between the relative affinity of SP-D for RNase B and the synthetic neoglycoproteins.



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  		Figure 2. Binding profiles of SP-D with different neoglycoconjugates and RNase B using SPR spectroscopy. Neoglycoconjugates coupled to BSA and RNase B were immobilized onto CM5 sensor chips at a density of 10,000 and 5,000 response units, respectively. Binding was analyzed by injection of SP-D (31.4 µg/ml) in the presence of 3 mM CaCl2 for 6 min followed by the injection of buffer without SP-D for 10 min. SP-D binding is expressed in arbitrary units. (A) The interaction of SP-D with (1) F-LDN, (2) F-Gn, (3) RNase B (containing a high-mannose–type N-glycan), (4) Gn, (5) LDN, (6) LDN-F, (7) Lewis-X, and (8) FF-Gn. (B) Binding profiles of the indicated SP-D concentrations to F-LDN.

 
For all oligosaccharides interacting with SP-D, the observed binding was dependent on the concentration of SP-D used. As an example, the concentration dependency of SP-D binding to F-LDN is shown in Figure 2B. Furthermore, SP-D dissociated from these carbohydrate-coated chips during regeneration using 3 mM EDTA in HBS (data not shown), but not during the washout in the presence of 3 mM CaCl2, confirming that the presence of calcium was required for SP-D binding.

Under our experimental conditions, SP-D showed only weak interactions with the monosaccharide Gn BSA–conjugate, which is in agreement with other reports (34), and there was no detectable affinity for the schistosomal oligosaccharide surface epitopes LDN, Lewis-X, LDN-F, or FF-Gn. However, this does not exclude these oligosaccharides as potential SP-D ligands, as significant binding of SP-D to Lewis-X coupled to serum albumin was detected when the number of oligosaccharide groups was increased from 10 mol Lewis-X per mol of serum albumin to 25 mol of Lewis-X per mol of serum albumin (Figure 3). Because the amount of neoglycoconjugate coupled to the SPR chip was kept the same, the amount of oligosaccharide on the chip was 2.5-fold higher. The resultant signal of SP-D binding (Figure 3), however, increased by a factor of much more than 2.5. Hence, these results demonstrated that the binding of SP-D to weak ligands can be drastically enhanced by an increased density.



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  		Figure 3. Binding profiles of SP-D with different densities of immobilized Lewis-X using SPR spectroscopy. Lewis-X was coupled to serum albumin in a ratio of 10:1 (dashed line) or 25:1 (solid line) mol oligosaccharide per mol serum albumin, and subsequently immobilized onto CM5 sensor chips at a density of 10,000 response units. Binding was studied by the injection of 15.7 µg/ml SP-D in the presence of 3 mM CaCl2 for 6 min followed by the injection of buffer without SP-D for 10 min. SP-D binding is expressed in arbitrary units.

 
SP-D Binds to the Surface of S. mansoni Cercariae and Schistosomula
Because schistosomal glycoconjugate structures were detected as ligands for SP-D, we also investigated whether SP-D can bind to larval S. mansoni stages. SP-D showed specific binding to S. mansoni cercariae, as detected by confocal fluorescence microscopy (Figure 4A). The surface of the cercariae was not evenly labeled because a strong fluorescent signal was detected at the position of the oral sucker, in addition to a less intense spotted pattern at the cercarial tail (Figure 4A). In control experiments in which SP-D was omitted, no fluorescence higher than background levels was observed, indicating that the fluorescent signal was specific for SP-D (data not shown). Con A, a lectin recognizing high-mannose and, to a lesser extent, diantennary oligosaccharides, was used to show that the entire surface of the cercariae was covered with glycoconjugates (Figure 4E). This implies that SP-D only binds to a subset of these glycoconjugates at specific parts of the surface of cercariae.



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  		Figure 4. SP-D and Con A bind to the surface of cercariae. Confocal microscopy images of S. mansoni cercariae labeled with Con A–FITC (20 µg/ml) or SP-D (20 µg/ml). SP-D binding was detected using anti–SP-D IgG and subsequent Alexa-488–conjugated anti-rabbit antibodies. Fluorescence (A) and phase-contrast (B) image of cercariae incubated with SP-D in the presence of 1 mM CaCl2. Fluorescence (C) and phase-contrast (D) image of cercariae incubated with SP-D in the presence of 2 mM EDTA. Fluorescence (E) and phase contrast (F) image of cercariae incubated with Con A. Scale bars = 20 µm.

 
Schistosomula are a larval stage of S. mansoni that migrates to the lung, where it resides for several days. In the course of time, an increasing number of parasites enters the alveoli (21), where they are likely to encounter SP-D in vivo. In contrast to what was seen with cercariae, an intense fluorescent signal was detected by confocal microscopy on the entire surface of the SP-D–labeled schistosomula (Figure 5A). Again, in control experiments in which SP-D was omitted, no fluorescence higher than background levels could be detected (data not shown), demonstrating that the fluorescence is specific for SP-D. As reported before (23), labeling of schistosomula with Con A also resulted in an intense staining of the entire surface, which was comparable to the staining found for SP-D (Figures 5A and 5E).



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  		Figure 5. SP-D and Con A bind to S. mansoni schistosomula. Confocal microscopy images of in vitro transformed S. mansoni schistosomula, labeled with Con A–FITC (20 µg/ml) or SP-D (20 µg/ml). SP-D binding was detected using anti–SP-D IgG and subsequent Alexa-488–conjugated anti-rabbit antibodies. Fluorescence (A) and phase-contrast (B) image of schistosomula incubated with SP-D in the presence of 1 mM CaCl2. Fluorescence (C) and phase-contrast (D) image of schistosomula incubated with SP-D in the presence of 2 mM EDTA. Fluorescence (E) and phase-contrast (F) image of schistosomula incubated with Con A. Scale bars = 20 µm.

 
SP-D Binds to Cercariae and Schistosomula via Its CRD
From previous work, it is known that SP-D binds carbohydrates via its CRD in a calcium-dependent manner (6). To test whether this also holds true for the binding of SP-D to cercariae and schistosomula, this interaction was also studied in the presence of EDTA (2 mM), a known divalent cation chelator. Under these conditions, no attachment of SP-D was observed, neither to cercariae nor to schistosomula (Figures 4C and 5C). To further investigate the involvement of the CRD of SP-D in the binding to both stages of S. mansoni, it was tested whether the interaction could be blocked by competition with saccharides. For that purpose, 20 mM maltose was added as a competing sugar to the SP-D incubation buffer, and under these conditions no SP-D attached to the surface of cercariae or schistosomula (data not shown). Together, these data strongly suggest that the CRD domain of SP-D is involved in the calcium-dependent binding to both cercariae and schistosomula of S. mansoni.

SP-D Recognizes a Specific Subset of Schistosomal Glycoproteins
The glycoproteins present in either cercariae or schistosomula were analyzed by separation of total protein samples on SDS-PAGE, subsequent blotting, and glycan detection. Both cercarial and schistosomal stages contained many glycoproteins, because a smear of glycoproteins was detected by glycoprotein staining (Figure 6B). In contrast, lectin blot analysis revealed that SP-D bound only to a subset of these glycoproteins (Figure 6A). Furthermore, SP-D binding to these immobilized glycoproteins was found to be specific, as no signal was found when SP-D was omitted (Figure 6A). In addition, the interaction was shown to be dependent on divalent cations because, in the presence of EDTA, no binding was detected (Figure 6A), again suggesting that the CRD domain of SP-D is involved in the binding.



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  		Figure 6. SP-D binds to a subset of glycoproteins of cercariae and schistosomula on lectin blot. Homogenates of cerceriae (cerc) or schistosomula (schis) were subjected to reducing SDS-PAGE and transferred to PVDF (A) or nitrocellulose (B) as described in MATERIAL AND METHODS section. Afterwards, blots were incubated with (+) or without (–) SP-D in the presence of 1 mM CaCl2 or 2 mM EDTA, as indicated (A). Bound SP-D was detected using anti–SP-D IgG and subsequent horseradish peroxidase–conjugated anti-rabbit antibodies. Total glycoproteins were visualized using the DIG-glycan detection kit (B). The position of molecular mass markers are indicated in kDa. Shown is a representative example of a triplicate experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although SP-D is known to bind relatively well to the monosaccharide fucose, binding of SP-D to fucose residues in complex oligosaccharides has not been investigated yet. Therefore, we investigated SP-D binding to a set of oligosaccharides with and without fucose residues, and to the glycoprotein RNase-B. The synthetic oligosaccharides we used represent glycans expressed on the outside of various developmental schistosomal stages, as part of glycoproteins as well as glycolipids (18, 22, 35). RNase B contains high-mannose glycans (Man6-9GlcNAc2), known to be present on the surface of many eukaryotes and viruses (4, 36), including S. mansoni schistosomula (26). The interaction of SP-D that we observed with RNase B by SPR analysis (Figure 2) is in line with the known high affinity of SP-D for mannan, another carbohydrate structure that contains multiple mannose residues. This observation is supportive of our SPR-analysis strategy for detection of ligands for SP-D. Our SPR experiments further showed that SP-D had relatively high affinity for two synthetic fucosyl oligosaccharides, F-LDN and F-Gn. SP-D did not bind to LDN, and bound only weakly to Gn, indicating that the Fuc{alpha}1–3GalNAc and Fuc{alpha}1–3GlcNAc moieties in F-LDN and F-Gn, respectively, were responsible for the observed binding of SP-D. It is interesting to note that LDN-F, although also carrying an {alpha}1-3–linked fucose residue, was not bound under the same experimental conditions. The inability of SP-D to bind to LDN-F could be the result of sterical hindrance by the GalNAc residue, which is in close proximity to the {alpha}1-3–linked fucose residue. Moreover, in Lewis-X, which is also bound very poorly by SP-D, the fucose residue has a similar presentation as that in LDN-F (Figure 1). This suggests that the presence of an additional monosaccharide linked to a carbon atom adjacent to the carbon to which the fucose is linked sterically interferes with the binding of SP-D to the fucose residue.

Although FF-Gn is a linear trisaccharide with a terminal fucose residue, SP-D did not bind to this glycoconjugate. This might be explained by the fact that the terminal fucose residue is {alpha}1-2–linked, and is thus presented differently from a terminal {alpha}1-3–linked fucose residue. It is thought that the monosaccharide fucose is bound by SP-D via interactions with equatorial hydroxyl groups present on the 2- and 3-positions of its sugar ring (12). Computational modeling of FF-Gn using Sweet (program for constructing 3D models of polysaccharides; http://www.dkfz-heidelberg.de/spec/sweet2/) and RasMol version 2.6-ß-2 (http://www.umass.edu/microbio/rasmol/) (37) suggests that these hydroxyl groups of the terminal fucose residue in FF-Gn are not readily accessible to SP-D, because these hydroxyl groups are in close proximity to the linkage site of the protein-spacer conjugate at the glucosamine residue (see online supplement). The hydroxyl groups present on the 2- and 3-positions of the terminal fucose residue in F-LDN are presented opposite to the linkage site of the conjugate, suggesting that these hydroxyl groups are readily accessible to SP-D. However, these modeling studies should be interpreted with caution, because the sugar moieties were modeled without their spacer and protein elements. The presence of these elements may have significant effects on the sterical presentation of the glycoconjugates. In conclusion, our SPR experiments confirmed that not only the presence of specific terminal sugar residues, but also their sterical presentation and clustering, are of crucial importance for SP-D binding to complex oligosaccharide ligands, which is in agreement with the binding characteristics reported for collectins (12, 38, 39).

Our observation that significant SP-D binding to Lewis-X could only be detected using chips coated with serum albumin to which a large number of Lewis-X epitopes was coupled suggests that SP-D binds with multiple CRD domains to glycoconjugates in a cooperative manner (Figure 3). Clustering of monosaccharides on BSA has also been shown to dramatically increase the affinity of MBL for these monosaccharides (38). Therefore, it is thought that simultaneous binding of several CRDs is needed for high-affinity interaction between the collectin and pathogens.

Our SPR analysis demonstrated that SP-D has a relatively high affinity for glycoconjugate structures present on the outer surface of schistosomula of the parasitic worm S. mansoni. Because these schistosomula are known to mature in the lung, and because histologic studies have detected multiple schistosomula in the alveoli (21), it is likely that schistosomula encounter SP-D in vivo. Although numerous studies have reported that SP-D interacts with a wide variety of unicellular microorganisms ranging from viruses to fungi (40), the present observation that SP-D binds to the outer surface of S. mansoni schistosomula demonstrates for the first time that SP-D can also bind multicellular pathogens. SP-D was also found to interact in a calcium-dependent manner via its CRD to specific parts of the surface of S. mansoni cercariae, although carbohydrates were shown to be present on the entire cercarial surface (Figure 4). Therefore, SP-D bound only to a specific set of carbohydrate structures on the surface of the cercariae. In contrast to SP-D, MBL has been shown to bind to the entire surface of cercariae (41), which indicates that distinct oligosaccharide structures are recognized by these different collectins, despite the great homology of their CRDs.

Which glycoconjugate structures are involved in SP-D binding to cercariae and schistosomula? Lectin blots clearly identified glycoproteins as SP-D binding sites. However, it cannot be excluded that specific glycolipids are also bound by SP-D. Schistosome glycolipids, in particular those from eggs, have been shown to contain the F-LDN element (35) that we show here to be recognized by SP-D, but it is not known whether schistosomula express F-LDN-containing glycolipids as well.

Could the observed binding of SP-D to the schistosomal surface with SP-D play a role in the reported clearance of schistosomula from the lung? Several reports have indicated that the lung is the most important site for elimination of this parasite in vaccinated as well as in naive mammalian hosts (42–47), suggesting that innate immunity is involved in killing of the schistosomes (41, 48). SP-D, which is an important component of the local innate defense system of the lung, could function in this reported elimination in several manners. The reported trapping of schistosomula inside alveoli (21) might be explained by the SP-D–dependent cross-linking of the schistosomula to other compounds or cells present in the alveoli, thereby preventing further schistosomal migration. The observation that SP-D at a concentration of 20 µg/ml was able to aggregate live schistosomula in vitro (J. K. van de Wetering, unpublished observation) indicates that the amount of SP-D bound to this parasite is large enough to cause SP-D–dependent cross-linking of the schistosomula. Furthermore, binding of SP-D could result in direct tegumental damage, in analogy to the effect found for Con A on schistosomula (49) and the reported induction of increased membrane permeability in Gram-negative bacteria by SP-D (8). Another way in which SP-D might lead to enhanced elimination of schistosomula is by the attraction of effector immune cells, as it has previously been suggested that SP-D has chemotactic properties for immune cells, such as macrophages (50) and neutrophils (51). SP-D might also be involved in stimulation of the adaptive immune response toward schistosomes, as SP-D has been shown to augment antigen presentation by dendritic cells in vitro (52). Furthermore, SP-D could modulate pathogen interactions with phagocytic cells by altered oxidant responses (53). Some of these potential effects of SP-D binding on parasite survival are under current investigation.

Is the terminal {alpha}1-3–linked fucose residue a physiologically important ligand for SP-D for its functioning in the innate immunity of the lung? So far, fucose-containing glycoconjugates have been observed on the outer surface of a limited number of pathogens. H. pylori, a human pathogen that adheres to the gastric mucosa, is the only prokaryotic pathogen known to expose oligosaccharides containing {alpha}1-3–linked fucose residues (54). Recently, SP-D has been shown to bind directly to intact H. pylori. This binding could be inhibited by Helicobacter-specific lipopolysaccharide structures that contain {alpha}1–3 linked fucose-residues in their O-antigens (55). In addition to S. mansoni, {alpha}1–3 linked fucose residues are also present in oligosaccharides found on the outer surface of several parasitic worms, such as Haemonchus contortus and Dictyocaulus viviparus (17, 18, 56). Although the presence or absence of fucosylation in pathogens has only been studied for a limited number of species, it appears that terminal {alpha}1-3–linked fucose residues do not occur in a widespread manner on lung pathogens, which suggests that binding of lung pathogens by SP-D is not often facilitated by this ligand. However, many distinct fucose-containing oligosaccharides, including Lewis-X and sialyl–Lewis-X, are exposed on the surface of mammalian leukocytes where they are involved in inflammatory processes (57, 58). In view of our data, it is not likely that Lewis-X or sialylated/sulphated variants thereof are physiological high-affinity ligands for SP-D. However, because it is known that SP-D can bind to neutrophils and monocytes in a carbohydrate-dependent manner (59), it is conceivable that other fucose- or oligomannose-containing glycoconjugates serve as endogenous ligands on leukocytes for SP-D. To address this question, it will be of interest to further explore the SPR approach used in this study to specifically test the binding of SP-D to leukocyte-derived glycoconjugates.


   Acknowledgments
 
The authors thank their colleagues in the Department of Pulmonology (Academic Medical Center, University of Amsterdam) for providing lavage fluid from patients with alveolar proteinosis. They thank Begona Aguilera and Hermen Overkleeft (Leiden Institute of Chemistry) for providing F-Gn and FF-Gn, and Johannis P. Kamerling (Bijvoet Center, Utrecht University) for providing F-LDN and LDN-F. They also thank Aloysius G. M. Tielens (Department of Biochemistry and Cell biology, Faculty of Veterinary Medicine, Utrecht University) for critical reading of the manuscript.


   Footnotes
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: J.K.V. has no declared conflicts of interest; A.V. has no declared conflicts of interest; A.B.V. has no declared conflicts of interest; J.J.B. has no declared conflicts of interest; L.M.G.V. has no declared conflicts of interest; C.H.H. has no declared conflicts of interest; and J.J.V. has no declared conflicts of interest.

Received in original form March 28, 2004

Received in final form July 19, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. van de Wetering, J. K., L. M. G. van Golde, and J. J. Batenburg. 2004. Collectins: players of the innate immune system. Eur. J. Biochem. 271:1229–1249.[Medline]
  2. Lawson, P. R., and K. B. M. Reid. 2000. The roles of surfactant proteins A and D in innate immunity. Immunol. Rev. 173:66–78.[CrossRef][Medline]
  3. Kuroki, Y., and D. R. Voelker. 1994. Pulmonary surfactant proteins. J. Biol. Chem. 269:25943–25946.[Free Full Text]
  4. Holmskov, U., S. Thiel, and J. C. Jensenius. 2003. Collectins and ficolins: humoral lectins of the innate immune defense. Annu. Rev. Immunol. 21:547–578.[CrossRef][Medline]
  5. van de Wetering, J. K., M. van Eijk, L. M. G. van Golde, T. Hartung, J. A. G. van Strijp, and J. J. Batenburg. 2001. Characteristics of surfactant protein A and D binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of Gram-positive bacteria. J. Infect. Dis. 184:1143–1151.[CrossRef][Medline]
  6. Crouch, E. C., K. Hartshorn, and I. Ofek. 2000. Collectins and pulmonary innate immunity. Immunol. Rev. 173:52–65.[CrossRef][Medline]
  7. van Rozendaal, B. A. W. M., A. B. van Spriel, J. G. J. van de Winkel, and H. P. Haagsman. 2000. Role of pulmonary surfactant protein D in innate defense against Candida albicans. J. Infect. Dis. 182:917–922.[CrossRef][Medline]
  8. Wu, H., A. Kuzmenko, S. Wan, L. Schaffer, A. Weiss, J. H. Fisher, K. S. Kim, and F. X. McCormack. 2003. Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability. J. Clin. Invest. 111:1589–1602.[CrossRef][Medline]
  9. Håkansson, K., and K. B. M. Reid. 2000. Collectin structure: A review. Protein Sci. 9:1607–1617.[Abstract]
  10. Crouch, E. C. 1998. Structure, biologic properties, and expression of surfactant protein D (SP-D). Biochim. Biophys. Acta 1408:278–289.[Medline]
  11. Lu, J., C. Teh, U. Kishore, and K. B. M. Reid. 2002. Collectins and ficolins: sugar pattern recognition molecules of the mammalian innate immune system. Biochim. Biophys. Acta 1572:387–400.[Medline]
  12. Allen, M. J., A. Laederach, P. J. Reilly, and R. J. Mason. 2001. Polysaccharide recognition by surfactant protein D: novel interactions of a C-type lectin with nonterminal glucosyl residues. Biochemistry 40:7789–7798.[CrossRef][Medline]
  13. Allen, M. J., D. R. Voelker, and R. J. Mason. 2001. Interactions of surfactant proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus. Infect. Immun. 69:2037–2044.[Abstract/Free Full Text]
  14. Sahly, H., I. Ofek, R. Podschun, H. Brade, Y. He, U. Ullmann, and E. C. Crouch. 2002. Surfactant protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing mannose-rich O-antigens. J. Immunol. 169:3267–3274.[Abstract/Free Full Text]
  15. Hartshorn, K. L., M. R. White, D. R. Voelker, J. Coburn, K. Zaner, and E. C. Crouch. 2000. Mechanism of binding of surfactant protein D to influenza A viruses: importance of binding to haemagglutinin to antiviral activity. Biochem. J. 351:449–458.
  16. Weis, W. I., K. Drickamer, and W. A. Hendrickson. 1992. Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature 360:127–134.[CrossRef][Medline]
  17. Staudacher, E., F. Altmann, I. B. Wilson, and L. Marz. 1999. Fucose in N-glycans: from plant to man. Biochim. Biophys. Acta 1473:216–236.[Medline]
  18. Hokke, C. H., and A. M. Deelder. 2001. Schistosome glycoconjugates in host–parasite interplay. Glycoconj. J. 18:573–587.[CrossRef][Medline]
  19. Butterworth, A. E., A. J. Fulford, D. W. Dunne, J. H. Ouma, and R. F. Sturrock. 1988. Longitudinal studies on human schistosomiasis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 321:495–511.[CrossRef][Medline]
  20. Sher, A., S. L. James, A. J. Simpson, J. K. Lazdins, and M. S. Meltzer. 1982. Macrophages as effector cells of protective immunity in murine schistosomiasis: III. Loss of susceptibility to macrophage-mediated killing during maturation of S. mansoni schistosomula from the skin to the lung stage. J. Immunol. 128:1876–1879.[Abstract]
  21. Crabtree, J. E., and R. A. Wilson. 1986. The role of pulmonary cellular reactions in the resistance of vaccinated mice to Schistosoma mansoni. Parasite Immunol. 8:265–285.[Medline]
  22. Cummings, R. D., and A. K. Nyame. 1999. Schistosome glycoconjugates. Biochim. Biophys. Acta 1455:363–374.[Medline]
  23. Samuelson, J. C., J. P. Caulfield, and J. R. David. 1982. Schistosomula of Schistosoma mansoni clear concanavalin A from their surface by sloughing. J. Cell Biol. 94:355–362.[Abstract/Free Full Text]
  24. Hayunga, E. G., and M. P. Sumner. 1986. Expression of lectin-binding surface glycoproteins during the development of Schistosoma mansoni schistosomula. J. Parasitol. 72:913–920.[Medline]
  25. Remoortere, A., C. H. Hokke, G. J. van Dam, I. van Die, A. M. Deelder, and D. H. van den Eijnden. 2000. Various stages of Schistosoma express Lewis(x), LacdiNAc, GalNAcbeta1–4 (Fucalpha1–3)GlcNAc and GalNAcbeta1–4(Fucalpha1–2Fucalpha1–3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins. Glycobiology 10:601–609.[Abstract/Free Full Text]
  26. Nyame, K., R. D. Cummings, and R. T. Damian. 1988. Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula. J. Parasitol. 74:562–572.[CrossRef][Medline]
  27. Crouch, E. C., A. Persson, and D. Chang. 1993. Accumulation of surfactant protein D in human pulmonary alveolar proteinosis. Am. J. Pathol. 142:241–248.[Abstract]
  28. Persson, A., D. Chang, K. Rust, M. Moxley, W. Longmore, and E. C. Crouch. 1989. Purification and biochemical characterization of CP4 (SP-D), a collagenous surfactant-associated protein. Biochemistry 28:6361–6367.[CrossRef][Medline]
  29. Vermeer, J. H., K. M. Alkes, J. A. van Kuik, J. P. Kamerling, and J. F. Vliegenthart. 2000. Synthesis and conjugation of oligosaccharide fragments related to the immunologically reactive part of the circulating anodic antigen of the parasite Schistosoma mansoni. J. Chem. Soc. Perkin Trans. 14:2249–2263.[CrossRef]
  30. Van Remoortere, A., G. J. van Dam, C. H. Hokke, D. H. van den Eijnden, I. van Die, and A. M. Deelder. 2001. Profiles of immunoglobulin M (IgM) and IgG antibodies against defined carbohydrate epitopes in sera of Schistosoma-infected individuals determined by surface plasmon resonance. Infect. Immun. 69:2396–2401.[Abstract/Free Full Text]
  31. Agoston, K., J. Kerekgyarto, J. Hajko, G. Batta, D. J. Lefeber, J. P. Kamerling, and J. F. Vliegenthart. 2002. Synthesis of fragments of the glycocalyx glycan of the parasite Schistosoma mansoni. Chemistry 8:151–161.[CrossRef][Medline]
  32. Bensadoun, A., and D. Weinstein. 1976. Assay of proteins in the presence of interfering materials. Anal. Biochem. 70:241–250.[CrossRef][Medline]
  33. Nyame, K., R. D. Cummings, and R. T. Damian. 1988. Characterization of the high mannose asparagine-linked oligosaccharides synthesized by Schistosoma mansoni adult male worms. Mol. Biochem. Parasitol. 28:265–274.[CrossRef][Medline]
  34. Persson, A., D. Chang, and E. C. Crouch. 1990. Surfactant protein D is a divalent cation-dependent carbohydrate-binding protein. J. Biol. Chem. 265:5755–5760.[Abstract/Free Full Text]
  35. Wuhrer, M., S. R. Kantelhardt, R. D. Dennis, M. J. Doenhoff, G. Lochnit, and R. Geyer. 2002. Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(alpha1–3)GalNAc-, GalNAc(beta1–4)[Fuc(alpha1–3)] GlcNAc- and Gal(beta1–4)[Fuc(alpha1–3)]GlcNAc- (Lewis X) terminal structures. Eur. J. Biochem. 269:481–493.[Medline]
  36. Drickamer, K., and M. E. Taylor. 1998. Evolving views of protein glycosylation. Trends Biochem. Sci. 23:321–324.[CrossRef][Medline]
  37. Sayle, R. A., and E. J. Milner-White. 1995. RASMOL: biomolecular graphics for all. Trends Biochem. Sci. 20:374–376.[CrossRef][Medline]
  38. Lee, R. T., Y. Ichikawa, M. Fay, K. Drickamer, M. C. Shao, and Y. C. Lee. 1991. Ligand-binding characteristics of rat serum-type mannose-binding protein (MBP-A): homology of binding site architecture with mammalian and chicken hepatic lectins. J. Biol. Chem. 266:4810–4815.[Abstract/Free Full Text]
  39. Epstein, J., Q. Eichbaum, S. Sheriff, and R. A. Ezekowitz. 1996. The collectins in innate immunity. Curr. Opin. Immunol. 8:29–35.[CrossRef][Medline]
  40. Crouch, E., and J. R. Wright. 2001. Surfactant proteins A and D and pulmonary host defense. Annu. Rev. Physiol. 63:521–554.[CrossRef][Medline]
  41. Klabunde, J., J. Berger, J. C. Jensenius, M. Q. Klinkert, U. E. Zelck, P. G. Kremsner, and J. F. J. Kun. 2000. Schistosoma mansoni: adhesion of mannan-binding lectin to surface glycoproteins of cercariae and adult worms. Exp. Parasitol. 95:231–239.[CrossRef][Medline]
  42. Ratcliffe, E. C., and R. A. Wilson. 1992. The role of mononuclear-cell recruitment to the lungs in the development and expression of immunity to Schistosoma mansoni. Parasitology 104:299–307.
  43. Dean, D. A., and B. L. Mangold. 1992. Evidence that both normal and immune elimination of Schistosoma mansoni take place at the lung stage of migration prior to parasite death. Am. J. Trop. Med. Hyg. 47:238–248.
  44. Kamiya, H., S. R. Smithers, and D. J. McLaren. 1987. Schistosoma mansoni: autoradiographic tracking studies of isotopically-labelled challenge parasites in naive and vaccinated CBA/Ca mice. Parasite Immunol. 9:515–529.[Medline]
  45. Wilson, R. A., P. S. Coulson, and B. Dixon. 1986. Migration of the schistosomula of Schistosoma mansoni in mice vaccinated with radiation-attenuated cercariae, and normal mice: an attempt to identify the timing and site of parasite death. Parasitology 92:101–116.
  46. Dean, D. A., B. L. Mangold, J. R. Georgi, and R. H. Jacobson. 1984. Comparison of Schistosoma mansoni migration patterns in normal and irradiated cercaria-immunized mice by means of autoradiographic analysis: evidence that worm elimination occurs after the skin phase in immunized mice. Am. J. Trop. Med. Hyg. 33:89–96.
  47. Von Lichtenberg, F., A. Sher, and S. McIntyre. 1977. A lung model of schistosome immunity in mice. Am. J. Pathol. 87:105–123.[Abstract]
  48. Vignali, D. A., Q. D. Bickle, M. G. Taylor, G. Tennent, and M. B. Pepys. 1988. Comparison of the role of complement in immunity to Schistosoma mansoni in rats and mice. Immunology 63:55–61.[Medline]
  49. Simpson, A. J. G., and D. J. McLaren. 1982. Schistosoma mansoni: tegumental damage as a consequence of lectin binding. Exp. Parasitol. 53:105–116.[Medline]
  50. Cai, G. Z., G. L. Griffin, R. M. Senior, W. J. Longmore, and M. A. Moxley. 1999. Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils. Am. J. Physiol. 276:L131–L136.
  51. Tino, M. J., and J. R. Wright. 1999. Surfactant proteins A and D specifically stimulate directed actin-based responses in alveolar macrophages. Am. J. Physiol. 276:L164–L174.
  52. Brinker, K. G., E. Martin, P. Borron, E. Mostaghel, C. Doyle, C. V. Harding, and J. R. Wright. 2001. Surfactant protein D enhances bacterial antigen presentation by bone marrow–derived dendritic cells. Am. J. Physiol. 281:L1453–L1463.
  53. van Iwaarden, J. F., H. Shimizu, P. H. van Golde, D. R. Voelker, and L. M. G. van Golde. 1992. Rat surfactant protein D enhances the production of oxygen radicals by rat alveolar macrophages. Biochem. J. 286:5–8.
  54. Aspinall, G. O., and M. A. Monteiro. 1996. Lipopolysaccharides of Helicobacter pylori strains P466 and MO19: structures of the O antigen and core oligosaccharide regions. Biochemistry 35:2498–2504.[CrossRef][Medline]
  55. Murray, E., W. Khamri, M. M. Walker, P. Eggleton, A. P. Moran, J. A. Ferris, S. Knapp, Q. N. Karim, M. Worku, P. Strong, K. B. M. Reid, and M. R. Thursz. 2002. Expression of surfactant protein D in the human gastric mucosa and during Helicobacter pylori infection. Infect. Immun. 70:1481–1487.[Abstract/Free Full Text]
  56. Haslam, S. M., G. C. Coles, H. R. Morris, and A. Dell. 2000. Structural characterization of the N-glycans of Dictyocaulus viviparus: discovery of the Lewis(x) structure in a nematode. Glycobiology 10:223–229.[Abstract/Free Full Text]
  57. Becker, D. J., and J. B. Lowe. 2003. Fucose: biosynthesis and biological function in mammals. Glycobiology 13:41R–53R.[Abstract/Free Full Text]
  58. McEver, R. P. 1997. Selectin–carbohydrate interactions during inflammation and metastasis. Glycoconj. J. 14:585–591.[CrossRef][Medline]
  59. Crouch, E. C., A. Persson, G. L. Griffin, D. Chang, and R. M. Senior. 1995. Interactions of pulmonary surfactant protein D (SP-D) with human blood leukocytes. Am. J. Respir. Cell Mol. Biol. 12:410–415.[Abstract]




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