Nucleic Acid Is a Novel Ligand for Innate, Immune Pattern Recognition Collectins Surfactant Proteins A and D and Mannose-binding Lectin
2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês
10.1074/jbc.m403763200
ISSN1083-351X
AutoresNades Palaniyar, Jeya Nadesalingam, Howard Clark, Michael J. Shih, Alister W. Dodds, Kenneth B. M. Reid,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoCollectins are a family of innate immune proteins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). The CRDs of these proteins recognize various microbial surface-specific carbohydrate patterns, particularly hexoses. We hypothesized that collectins, such as pulmonary surfactant proteins (SPs) SP-A and SP-D and serum protein mannose-binding lectin, could recognize nucleic acids, pentose-based anionic phosphate polymers. Here we show that collectins bind DNA from a variety of origins, including bacteria, mice, and synthetic oligonucleotides. Pentoses, such as arabinose, ribose, and deoxyribose, inhibit the interaction between SP-D and mannan, one of the well-studied hexose ligands for SP-D, and biologically relevant d-forms of the pentoses are better competitors than the l-forms. In addition, DNA and RNA polymer-related compounds, such as nucleotide diphosphates and triphosphates, also inhibit the carbohydrate binding ability of SP-D, or ∼60 kDa trimeric recombinant fragments of SP-D that are composed of the α-helical coiled-coil neck region and three CRDs (SP-D(n/CRD)) or SP-D(n/CRD) with eight GXY repeats (SPD(GXY)8(n/CRD)). Direct binding and competition studies suggest that collectins bind nucleic acid via their CRDs as well as by their collagen-like regions, and that SP-D binds DNA more effectively than do SP-A and mannose-binding lectin at physiological salt conditions. Furthermore, the SP-D(GXY)8(n/CRD) fragments co-localize with DNA, and the protein competes the interaction between propidium iodide, a DNA-binding dye, and apoptotic cells. In conclusion, we show that collectins are a new class of proteins that bind free DNA and the DNA present on apoptotic cells by both their globular CRDs and collagen-like regions. Collectins may therefore play an important role in decreasing the inflammation caused by DNA in lungs and other tissues. Collectins are a family of innate immune proteins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). The CRDs of these proteins recognize various microbial surface-specific carbohydrate patterns, particularly hexoses. We hypothesized that collectins, such as pulmonary surfactant proteins (SPs) SP-A and SP-D and serum protein mannose-binding lectin, could recognize nucleic acids, pentose-based anionic phosphate polymers. Here we show that collectins bind DNA from a variety of origins, including bacteria, mice, and synthetic oligonucleotides. Pentoses, such as arabinose, ribose, and deoxyribose, inhibit the interaction between SP-D and mannan, one of the well-studied hexose ligands for SP-D, and biologically relevant d-forms of the pentoses are better competitors than the l-forms. In addition, DNA and RNA polymer-related compounds, such as nucleotide diphosphates and triphosphates, also inhibit the carbohydrate binding ability of SP-D, or ∼60 kDa trimeric recombinant fragments of SP-D that are composed of the α-helical coiled-coil neck region and three CRDs (SP-D(n/CRD)) or SP-D(n/CRD) with eight GXY repeats (SPD(GXY)8(n/CRD)). Direct binding and competition studies suggest that collectins bind nucleic acid via their CRDs as well as by their collagen-like regions, and that SP-D binds DNA more effectively than do SP-A and mannose-binding lectin at physiological salt conditions. Furthermore, the SP-D(GXY)8(n/CRD) fragments co-localize with DNA, and the protein competes the interaction between propidium iodide, a DNA-binding dye, and apoptotic cells. In conclusion, we show that collectins are a new class of proteins that bind free DNA and the DNA present on apoptotic cells by both their globular CRDs and collagen-like regions. Collectins may therefore play an important role in decreasing the inflammation caused by DNA in lungs and other tissues. Nucleic acids such as DNA and RNA of higher organisms and microbes are long, pentose sugar-based anionic phosphate polymers. Apoptotic cell death results in the fragmentation of DNA and its subsequent display on the surfaces of the cells as blebs (1Savill J. Fadok V. Nature. 2000; 407: 784-788Google Scholar). Inefficient removal of apoptotic cells leads to disintegration of their contents and the formation of necrotic cells. These leaky cells eventually release their intracellular contents, and many of these components elicit tissue inflammation. Furthermore, certain pulmonary pathogens, such as Pseudomonas aeruginosa, actively secrete DNA into the extracellular matrix to establish an active biofilm and chronic infections (2Whitchurch C.B. Tolker-Nielsen T. Ragas P.C. Mattick J.S. Science. 2002; 295: 1487Google Scholar). Removal of dying cells and their components, including DNA, is therefore essential to maintain inflammation-free tissues and to prevent autoimmune diseases (1Savill J. Fadok V. Nature. 2000; 407: 784-788Google Scholar). Although the multiple pathways and proteins involved in the apoptotic process have been studied in great detail, clearance of these cells and their debris is poorly understood. Collectins are a class of innate immune proteins that are often secreted into body fluids and extracellular cavities (3Palaniyar N. Nadesalingam J. Reid K.B. Immunobiology. 2002; 205: 575-594Google Scholar). Well-studied members of the collectin family include surfactant proteins (SPs) 1The abbreviations used are: SP, surfactant protein; MBL, mannose-binding lectin; CRD, carbohydrate-recognition domain; EMSA, electrophoretic mobility shift assay; SPR, surface plasmon resonance; FACS, fluorescence-activated cell sorting; SAP, serum amyloid component P; GlcNAc, N-acetyl glucosamine. A and D and mannose-binding lectin (MBL). These proteins, particularly SP-D, bind many of the inflammation-causing ligands derived from microbes (3Palaniyar N. Nadesalingam J. Reid K.B. Immunobiology. 2002; 205: 575-594Google Scholar), such as fungal cell wall glucans (4Allen M.J. Voelker D.R. Mason R.J. Infect. Immun. 2001; 69: 2037-2044Google Scholar), lipopolysaccharides (5Kuan S.F. Rust K. Crouch E. J. Clin. Investig. 1992; 90: 97-106Google Scholar), peptidoglycans and lipoteichoic acids (6van de Wetering J.K. van Eijk M. van Golde L.M. Hartung T. van Strijp J.A. Batenburg J.J. J. Infect. Dis. 2001; 184: 1143-1151Google Scholar), and lipids (7Ogasawara Y. Kuroki Y. Akino T. J. Biol. Chem. 1992; 267: 21244-21249Google Scholar). We have shown recently that SP-D also binds to proteoglycans with long glycosaminoglycan chains, such as decorin, via both protein-protein and protein-carbohydrate interactions, and that SP-D-decorin interactions may be related to reducing tissue-specific inflammation (8Nadesalingam J. Bernal A.L. Dodds A.W. Willis A.C. Mahoney D.J. Day A.J. Reid K.B. Palaniyar N. J. Biol. Chem. 2003; 278: 25678-25687Google Scholar). Collectins not only bind to these purified ligands but also recognize them on cell surfaces (3Palaniyar N. Nadesalingam J. Reid K.B. Immunobiology. 2002; 205: 575-594Google Scholar). Apoptotic cells, for example, contain carbohydrate polymers such as DNA on their surfaces, and accumulation of these dying cells, as well as excess free DNA, is known to cause inflammation and septic shock (9McLachlan G. Stevenson B.J. Davidson D.J. Porteous D.J. Gene Ther. 2000; 7: 384-392Google Scholar, 10Schwartz D.A. Quinn T.J. Thorne P.S. Sayeed S. Yi A.K. Krieg A.M. J. Clin. Investig. 1997; 100: 68-73Google Scholar). Fragments of bacterial DNA with specific CpG motifs, however, act as immunostimulatory molecules (11Krieg A.M. Yi A.K. Matson S. Waldschmidt T.J. Bishop G.A. Teasdale R. Koretzky G.A. Klinman D.M. Nature. 1995; 374: 546-549Google Scholar). Regulatory mechanisms and all the proteins that are involved in these pathways are not clearly identified. A typical collectin is composed of polypeptide chains containing a short, interchain, disulfide bond-forming N-terminal domain, a collagen-like region with Gly-X-Y repeats (where X is any amino acid and Y is often hydroxyproline or hydroxylysine), an α-helical hydrophobic neck region and a C-terminal globular carbohydrate recognition domain (CRD) (12Persson A. Chang D. Crouch E. J. Biol. Chem. 1990; 265: 5755-5760Google Scholar, 13Crouch E. Chang D. Rust K. Persson A. Heuser J. J. Biol. Chem. 1994; 269: 15808-15813Google Scholar). Three of these polypeptides form a trimeric subunit (14Palaniyar N. McCormack F.X. Possmayer F. Harauz G. Biochemistry. 2000; 39: 6310-6316Google Scholar, 15Head J.F. Mealy T.R. McCormack F.X. Seaton B.A. J. Biol. Chem. 2003; 278: 43254-43260Google Scholar, 16Hakansson K. Lim N.K. Hoppe H.J. Reid K.B. Structure Fold Des. 1999; 7: 255-264Google Scholar), which assembles to form higher order structures. The oligomeric assembly of SP-D (13Crouch E. Chang D. Rust K. Persson A. Heuser J. J. Biol. Chem. 1994; 269: 15808-15813Google Scholar, 17Lu J. Wiedemann H. Timpl R. Reid K.B. Behring Inst. Mitt. 1993; 93: 6-16Google Scholar) resembles that of an "X" (4 subunits) or "asterisk" (>10 subunits) with a prominent central "hub," whereas the other collectins SP-A and MBL (2-6 subunits) and a non-collectin, the complement system protein C1q (6 subunits), appear as a "bouquet of flowers" (3Palaniyar N. Nadesalingam J. Reid K.B. Immunobiology. 2002; 205: 575-594Google Scholar, 17Lu J. Wiedemann H. Timpl R. Reid K.B. Behring Inst. Mitt. 1993; 93: 6-16Google Scholar, 18Palaniyar N. Ridsdale R.A. Holterman C.E. Inchley K. Possmayer F. Harauz G. J. Struct. Biol. 1998; 122: 297-310Google Scholar). SP-A further assembles as unidimensional (19Palaniyar N. Ridsdale R.A. Hearn S.A. Heng Y.M. Ottensmeyer F.P. Possmayer F. Harauz G. Am. J. Physiol. 1999; 276: L631-L641Google Scholar) and multidimensional (20Palaniyar N. Ridsdale R.A. Possmayer F. Harauz G. Biochem. Biophys. Res. Commun. 1998; 250: 131-136Google Scholar) fibers. Although the trimeric subunits of the collectins have limited affinity (μm) for several carbohydrate targets, their oligomeric assembly provides high avidity so that these proteins bind to ligands selectively and with high affinity (nm to pm). Collectins bind microbes primarily via hexose sugars and enhance their phagocytosis (16Hakansson K. Lim N.K. Hoppe H.J. Reid K.B. Structure Fold Des. 1999; 7: 255-264Google Scholar), and whether these proteins can bind pentose sugars was unknown. We hypothesized that collectins could bind free RNA and DNA, as well as nucleic acid present on the apoptotic and necrotic cells, and promote their clearance. In this study, we used pentoses, ribonucleoside phosphates, deoxyribonucleoside phosphates, synthetic short oligonucleotide fragments, DNA isolated from bacteria and mouse cells, and the apoptotic forms of Jurkat cells and mouse alveolar macrophages. Evidence obtained by electrophoretic mobility shift assay (EMSA), electron microscopy, surface plasmon resonance (SPR), fluorescent-activated cell sorting (FACS), and confocal microscopy experiments show that collectins, particularly SP-D, bind DNA obtained from many sources. Furthermore, SP-D binds DNA both via their CRDs and collagen-like regions. We used two recombinant fragments of SP-D in this study to further determine the roles of different domains of the protein: the SP-D(n/CRD) fragment, which lacks the N-terminal segment and the entire collagen-like region; and the SPD(GXY)8(n/CRD) fragment, which is similar to SP-D(n/CRD) except that it also contains eight of the Gly-X-Y repeats. Both of these proteins also recognize DNA. SP-D(GXY)8(n/CRD) binds nucleic acids present on apoptotic cells. Therefore, our findings establish a novel ligand for collectins and suggest that these proteins could enhance the clearance of nucleic acids and play an important role in limiting inflammation. DNA—Plasmid DNAs were purified by Midiprep procedures (Qiagen, West Sussex, England) and digested with EcoRI or BamHI (Promega, Southampton, England). Genomic DNA was isolated from the mouse lung by a DNeasy DNA isolation kit (Qiagen). Protein—SP-A (21Palaniyar N. Zhang L. Kuzmenko A. Ikegami M. Wan S. Wu H. Korfhagen T.R. Whitsett J.A. McCormack F.X. J. Biol. Chem. 2002; 277: 26971-26979Google Scholar) and SP-D (22Strong P. Kishore U. Morgan C. Lopez Bernal A. Singh M. Reid K.B. J. Immunol. Methods. 1998; 220: 139-149Google Scholar) were purified from therapeutic lung lavage fluid obtained from alveolar proteinosis patients as described previously. MBL (23Tan S.M. Chung M.C. Kon O.L. Thiel S. Lee S.H. Lu J. Biochem. J. 1996; 319 (Pt 2): 329-332Google Scholar) and C1q (24Reid K.B. Methods Enzymol. 1982; 82 (Pt A): 319-324Google Scholar) were purified from pooled human plasma (HD Supplies, Aylesbury, England). Recombinant preparations of the trimeric ∼60-kDa fragment composed of the neck and CRDs of SP-D, with and without eight Gly-X-Y repeats derived from the collagen-like region, were expressed in Escherichia coli and Pichia pastoris, respectively. As described previously, SP-D(GXY)8(n/CRD) and SP-D(n/CRD) were purified from inclusion bodies and cell culture media supernatant, respectively (16Hakansson K. Lim N.K. Hoppe H.J. Reid K.B. Structure Fold Des. 1999; 7: 255-264Google Scholar, 25Shrive A.K. Tharia H.A. Strong P. Kishore U. Burns I. Rizkallah P.J. Reid K.B. Greenhough T.J. J. Mol. Biol. 2003; 331: 509-523Google Scholar, 26Madan T. Kishore U. Singh M. Strong P. Clark H. Hussain E.M. Reid K.B. Sarma P.U. J. Clin. Investig. 2001; 107: 467-475Google Scholar). EMSA Assay—DNA-protein complexes were prepared for gel shift analyses by incubating 0.1-0.4 μg of linear plasmid DNA with protein in a 20-μl reaction containing 20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5 mm CaCl2, or 5 mm MgCl2, or 5 mm MnCl2, or 5 mm EDTA buffer as described previously (27Tseng M. Palaniyar N. Zhang W. Evans D.H. J. Biol. Chem. 1999; 274: 21637-21644Google Scholar). After 20-60 min at 37 °C, the DNA-protein complexes were fractionated by agarose gel (0.8 or 1%, w/v) electrophoresis in 40 mm Tris acetate (pH 8) buffer in the absence or presence of 1 mm EDTA and 0.5 μg/ml ethidium bromide and visualized by UV transilluminator as described previously (27Tseng M. Palaniyar N. Zhang W. Evans D.H. J. Biol. Chem. 1999; 274: 21637-21644Google Scholar, 28Palaniyar N. Gerasimopoulos E. Evans D.H. J. Mol. Biol. 1999; 287: 9-20Google Scholar). Protease Assay—SP-D (1.5 μg) and DNA (400 ng) were allowed to form complexes as described in EMSA and were treated either with 0.05% (w/v) SDS, 20 mm octyl glucoside, or 500 μg/ml proteinase K (Promega) in a 20-μl reaction for 1 h at 37 °C. In the collagenase experiment, SP-D (500 ng) was first treated with enzyme (40 μg, type 1 collagenase from Clostridium histolyticum, >90% pure; Calbiochem) in a 20-μl reaction for 1 h at 37 °C and then incubated with DNA (400 ng) for 1 h at 37 °C. In another reaction, SP-D (500 ng) was first incubated with DNA (400 ng) in a 20-μl volume for 1 h at 37 °C and then treated with enzyme for 1 h at 37 °C. Dot Blot—SP-D (370 ng) and linear pUC18 DNA (150 ng and 2-fold dilutions) were carefully dotted (1 μl) onto Hybond-C Extra nitrocellulose membrane discs (Amersham Biosciences) and allowed to dry in a Petri dish. Membranes were incubated with 2% (w/v) bovine serum albumin in PBS, 0.02% (v/v) Tween (PBST) buffer for 18 h at 4 °C. Remaining procedures were carried out at 23 °C. Membranes were washed three times with 20 ml of PBST for 5-10 min on a shaker. Membranes were either incubated with 10 ml of PBST or PBST with SP-D (0.5 μg/ml) for 1 h. The membranes were washed as above and incubated further with 10 ml of biotinylated rabbit polyclonal anti-human SP-D(GXY)8(n/CRD) antibody (1 μg/ml) for 1 h. Antibody was washed away as described above, and the membranes were incubated with horseradish peroxidase-conjugated streptavidin (1:10,000) for 20 min. SP-D complexes were detected by ECL reagents (Amersham Biosciences). Western Blot—Proteins were separated in a gradient polyacrylamide gel (4-20% (w/v); Bio-Rad), soaked in transfer buffer (49 mm Tris-HCl, 39 mm glycine, 0.0375% (w/v) SDS, pH 8.3) for 0.5 h, and transferred to polyvinylidene difluoride Immobilon P membrane (Millipore, Dorset, England) by a semidry transfer apparatus (Biometra, Goettingen, Germany) at 0.8 mA/cm2 for 3 h. The membranes were blocked with 2% (w/v) bovine serum albumin in PBST, and the SP-D present on the membranes was detected as described for dot blots. Electron Microscopy—Mouse lung DNA and proteins were incubated in the DNA binding buffer, spread in a thin layer on carbon-coated specimen grids, negatively stained with 2% (w/v) uranyl acetate, and examined using a Jeol JEM 1010 or 100 CX electron microscope (Leica UK Ltd., Cambridge, England) (18Palaniyar N. Ridsdale R.A. Holterman C.E. Inchley K. Possmayer F. Harauz G. J. Struct. Biol. 1998; 122: 297-310Google Scholar, 27Tseng M. Palaniyar N. Zhang W. Evans D.H. J. Biol. Chem. 1999; 274: 21637-21644Google Scholar). Biotinylation of Carbohydrate Ligands—Yeast mannan was biotinylated as described previously (8Nadesalingam J. Bernal A.L. Dodds A.W. Willis A.C. Mahoney D.J. Day A.J. Reid K.B. Palaniyar N. J. Biol. Chem. 2003; 278: 25678-25687Google Scholar). Briefly, mannan (1 mg/ml) was reacted with 1.5 mm NaIO4 in 100 mm sodium acetate buffer (pH 5.5) for 30 min on ice, and the reaction was stopped with 15 mm glycerol. The mixture was dialyzed into 10 mm sodium acetate buffer (pH 5.5) for 18 h at 4 °C and reacted with 5 mm biotin-LC-hydrazide (Pierce, Perbio Science, Cheshire, England) for 2 h at 23 °C in the same buffer. The mixture was dialyzed for 18 h at 4 °C to remove non-reacted biotin and stored at the same temperature. Oligonucleotides, 5′-GTAATACGACTCACTATAGGGC-3′ (22-mer; Stratagene, Cedar Creek, TX) and 5′-TCCATGAGCTTCCTGAGTCT-3′ (20-mer; Applied Biosciences) that contained 5′ biotin anchor were purchased. SPR Analyses—Biotinylated oligonucleotides (2.5 μg/ml) or mannan (200 μg/ml) were immobilized on a streptavidin SA chip (BIAcore 2000, Herts, England). The biotinylated ligands were diluted in 10 mm NaOAc (pH 5.5) buffer and immobilized on individual streptavidin-coated chips by injecting them at 5 μl/min for 5-10 min. Leaving flow cell 1 as blank, the next three flow cells were used for immobilization of ligands. Free streptavidin in all flow cells was blocked with biotin, and the flow cell surface was normalized with 50% (v/v) glycerol solution. Binding of proteins (0-50 μg/ml) to the oligonucleotides was analyzed in 10 mm HEPES-NaOH (pH 7.4), 5 mm CaCl2, and 0 or 150 mm NaCl buffer at a flow rate of 10 μl/min. In competition assays, SP-D (1 or 5 μg/ml) or SP-D(GXY)8(n/CRD) (200 μg/ml) was allowed to bind (10 μl/min) to immobilized mannan in the presence of different concentrations of competitors (0-80 mm). The SPR response obtained in the absence of any competitor was considered as 100%, and the relative binding was calculated for each competitor concentration. Binding of SP-D(G-XY)8(n/CRD) to Apoptotic Cells—Nicked DNA of apoptotic Jurkat cells (Apo-direct, BD PharMingen) was labeled with Cy3-dUTP by deoxynucleotidyl transferase as described by the manufacturers (Apo-direct, BD PharMingen). These cells were incubated with FITC-labeled SP-D(GXY)8(n/CRD) fragment in the presence of 2 mm CaCl2 for 20-30 min at 23 °C, fixed with 0.5% (v/v) paraformaldehyde/PBS, washed, and resuspended in PBS. Distribution of the labels was determined by FACS (Becton Dickinson, Oxford, England) and confocal microscopy (Zeiss, Welwyn Garden City, England). In FACS experiments involving apoptotic mouse alveolar macrophages (29Clark H. Palaniyar N. Strong P. Edmondson J. Hawgood S. Reid K.B. J. Immunol. 2002; 169: 2892-2899Google Scholar), different concentrations (0, 4, and 20 μg/ml) of SP-D(GXY)8(n/CRD) were incubated with the cells, followed by staining with propidium iodide. Detection of Collectin Binding to Bacterial DNA by EMSA—To determine whether collectins bind DNA, we incubated linear plasmid DNA with collectins and related proteins and analyzed the complexes in agarose gels. This EMSA showed that native forms of SP-D, MBL, and C1q, but not SP-A, shifted the migration pattern of the DNA in the gel (Fig. 1A). SP-D(n/CRD), a yeast-expressed, recombinant ∼60 kDa trimeric subunit fragment of SP-D that contains only the coiled-coil neck and the CRDs, as well as a known DNA-binding pentraxin, serum amyloid component P (SAP), did not shift the DNA bands in EMSA. Because both SAP (30Hind C.R. Collins P.M. Renn D. Cook R.B. Caspi D. Baltz M.L. Pepys M.B. J. Exp. Med. 1984; 159: 1058-1069Google Scholar) and CRDs of surfactant proteins have lectin domains and bind agarose to varying degrees, the DNA binding ability of these proteins might partly be competed by the matrix. These experiments showed that in addition to the non-collectin, subcomponent C1q of the classical complement pathway (31Jiang H. Cooper B. Robey F.A. Gewurz H. J. Biol. Chem. 1992; 267: 25597-25601Google Scholar), innate immune collectins could bind DNA. These EMSA results were consistent with the interpretation that, similar to the collagen-like regions of C1q (31Jiang H. Cooper B. Robey F.A. Gewurz H. J. Biol. Chem. 1992; 267: 25597-25601Google Scholar), SP-D and MBL bound DNA via their collagen-like regions, or that oligomerization of the proteins was required for the formation of large complexes. Interestingly, SP-D bound DNA even at 1 m NaCl concentration, indicating that this collectin strongly interacted with DNA (Fig. 1B). Because calcium and other divalent cations affect the structure and function of collectins (16Hakansson K. Lim N.K. Hoppe H.J. Reid K.B. Structure Fold Des. 1999; 7: 255-264Google Scholar), we examined the effect of these ions on collectin-DNA interactions. SP-D bound more effectively to DNA in the presence of 5 mm calcium, magnesium, and manganese than EDTA (Fig. 1C). The ion-dependent increase in the binding of SP-D to DNA was detectable only with supercoiled but not linearized DNA. Hence, these results suggest that the structure of DNA is important for SP-D-DNA interactions, and that both lectin and non-lectin types of binding may be involved in the formation of these complexes. Determination of the Nature of SP-D-DNA Complexes—To determine the types of complexes formed by SP-D, we incubated SP-D (1.5 μg) with linear pUC18 DNA (400 ng) in the standard DNA-binding buffer for 1 h at 37 °C and added either SDS (0.05%, w/v), octyl glucoside (20 mm), or proteinase K (500 μg/ml) and incubated for an additional 1 h at 37 °C. DNA-protein complexes were size-fractionated in an agarose gel, and the DNA present in each complex was visualized with ethidium bromide staining and UV light. Linear DNA (Fig. 2A, lane 2) migrated into the gel, and no noticeable amount of DNA was present in SP-D preparations (Fig. 2A, lane 3). SP-D retarded the mobility of the DNA, and the DNA complexes were detected in the wells (Fig. 2A, lane 4). Although SDS did not alter the migration of DNA in the gel (Fig. 2A, lane 5), this strong anionic detergent disassociated the DNA from the complexes (Fig. 2A, lane 6). Hence, SP-D-DNA complexes were formed by noncovalent interactions. A non-ionic mild detergent, octyl glucoside, neither affected the DNA (Fig. 2A, lane 7) nor SP-DDNA complexes (Fig. 2A, lane 8). Therefore, SP-D-DNA complexes were strong and likely involved charge interactions. Digestion of these complexes with proteinase K resulted in the release of DNA, indicating that they were formed by protein-DNA interactions (Fig. 2A, lanes 9 and 10). To further show that these complexes were formed by SP-D, we treated the complexes with collagenase enzyme and analyzed the samples by agarose gels, SDS-PAGE under reducing and denaturing conditions, and Western blots to specifically visualize the DNA, proteins, and SP-D, respectively (Fig. 2, B-D). Linear pUC18 DNA contained no aggregated complexes (Fig. 2, B-D, lanes 2). The 110-kDa collagenase enzyme neither showed any aggregates (Fig. 2, B-D, lanes 3) nor affected the mobility of DNA (data not shown). SP-D itself did not contain any significant amount of DNA or DNA-protein complexes when 500 ng of protein was used (Fig. 2B, lane 4). Under reducing and denaturing conditions, SP-D migrated at about 43 kDa (Fig. 2C, lane 4) and was detected by anti-SP-D antibody (Fig. 2D, lane 4). The agarose gel showed that SP-D formed complexes and shifted the DNA to the wells (Fig. 2B, lane 5). Under reducing and denaturing conditions, SP-D separated away from DNA without any noticeable change to its structure (Fig. 2, C and D, lanes 5). When the SP-D was digested with collagenase for 1 h and then incubated with DNA, no shift in the mobility of DNA was noticed (Fig. 2B, lane 6). SDS-PAGE and Western blots showed that the collagen-like region of SP-D was digested by the collagenase (Fig. 2, C and D, lanes 6). The calculated molecular mass of the collagenase-resistant fragment of SP-D was ∼16 kDa, and this was consistent with the protein detected by SDS-PAGE and Western blot after the enzyme digestion. Interestingly, when the collagenase was added after the formation of SP-D-DNA complexes, the enzyme did not completely eliminate these complexes (Fig. 2B, lane 7) suggesting that bound DNA may protect the collagen-like region from collagenase digestion. SDS-PAGE and Western blots showed that the collagen-like region of approximately half of the SP-D molecules was intact (Fig. 2, C and D, lanes 7). In the absence of DNA, collagenase completely digested the collagen-like regions of SP-D (Fig. 2, B-D, lanes 8). These results established that SP-D bound DNA, and that the collagen-like region of the protein was important for the formation of large SP-D-DNA complexes and subsequent gel shift. Binding of SP-D to Immobilized DNA—To directly show that SP-D could bind DNA, we first immobilized linearized plasmid DNA on nitrocellulose membranes and incubated the membranes with buffer or SP-D (0.5 μg/ml). After washing the membranes, SP-D was detected by anti-SP-D antibody and ECL reagents. SP-D antibody detected the 370 ng of SP-D spotted on both membranes equally well (Fig. 3, A and B, lanes 1). The polyclonal antibody did not show any significant binding to DNA in the absence of SP-D (Fig. 3A, lane 2). The dot blot showed that SP-D bound to the DNA present on the membrane in a concentration-dependent manner (Fig. 3B, lane 2). These results showed that SP-D directly interacted with DNA. Visualization of SP-D-Genomic DNA Complexes by Electron Microscopy—To obtain further confirmatory evidence of SP-D interacting with DNA, we examined the binding of SP-D to mouse lung genomic DNA by electron microscopy (Fig. 4). DNA was detected as long strands on the carbon support film (Fig. 4, A and E). SAP appeared as pentamers (Fig. 4B), which avidly bound DNA (Fig. 4F). When SP-D(n/CRD) was incubated with DNA, it coated the entire DNA (Fig. 4, C and G). Similar complexes were obtained when SP-D(GXY)8(n/CRD) was incubated with the DNA (Fig. 4, D and H). Interestingly, these complexes were very different compared with the native SP-D-DNA assemblies. Native SP-D, which contained long collagen-like fibrillar regions, efficiently spooled the DNA into aggregates (Fig. 5, A and B). The hub and nearby collagen-like regions preferentially bound DNA (Fig. 5C), suggesting that this collectin also interacted with the ligand probably via polyionic interactions. These images (Figs. 4 and 5) suggested that both globular and collagen-like regions of the collectin interacted effectively with DNA. SPR Analysis of Interaction between Collectins and Oligonucleotides—To characterize collectin-DNA interaction using a more sensitive system, we allowed the proteins to bind to short oligonucleotides (22-mer) that were immobilized on a BIAcore chip. The SPR response showed that although SP-A bound oligonucleotides in a low ionic strength buffer, it progressively lost DNA-binding ability as the ionic strength increased (Fig. 6, A and B). In contrast, SP-D bound to DNA in 150 mm NaCl buffer (Fig. 6C). Hence, SP-D, but not SP-A, interacted with the oligonucleotides under physiological salt concentrations (Fig. 6). MBL showed a moderate level of binding under physiological salt conditions (data not shown). We conducted similar SPR experiments with another biotinylated oligonucleotide (20-mer). Similar levels of SP-D binding were observed for both of those oligonucleotides (data not shown). Although the SPR response of SP-D-oligonucleotide (Fig. 6) was relatively small compared with SP-D-mannan ligand (8Nadesalingam J. Bernal A.L. Dodds A.W. Willis A.C. Mahoney D.J. Day A.J. Reid K.B. Palaniyar N. J. Biol. Chem. 2003; 278: 25678-25687Google Scholar), these results suggested that all three collectins bound single-stranded short oligonucleotides. SPR Analyses of Competition of Collectin-Carbohydrate Interactions—To directly investigate whether collectins could bind pentoses, the carbohydrate moieties present in nucleic acids, we allowed SP-D to bind immobilized mannan present on a BIAcore chip in the presence of different concentrations of hexoses or pentoses (Figs. 7 and 8). BIAcore assays behaved similarly to the conventional ELISA system (32Holmskov U.L. APMIS Suppl. 2000; 100: 1-59Google Scholar), and maltose, mannose, myo-inositol, and glucose inhibited the interaction between SP-D and mannan with relatively low IC50 values compared with those of d-fucose, d-galactose, and GlcNAc (Fig. 7). We conducted competition experiments under identical conditions for pentoses (Fig. 8). d-Arabinose and glucose inhibited binding with a similar IC50 value (Figs. 7A and 8A). Furthermore, d-ribose was a better competitor than d-2-deoxyribose, and they competed with IC50 values comparable with those of d-galactose, d-fucose, and GlcNAc (Figs. 7A and 8A). Interestingly, the d-forms of all three of these pentoses inhibited better than their l-forms of the respective sugars (Fig. 8, A and B). Because nucleic acids contain
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