Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin

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 approximately 60 kDa trimeric recombinant fragments of SP-D that are composed of the alpha-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.     

Collectin structure: a review

Collectins are animal calcium dependent lectins that target the carbohydrate structures on invading pathogens, resulting in the agglutination and enhanced clearance of the microorganism. These proteins form trimers that may assemble into larger oligomers. Each polypeptide chain consists of four regions: a relatively short N-terminal region, a collagen like region, an alpha-helical coiled-coil, and the lectin domain. Only primary structure data are available for the N-terminal region, while the most important features of the collagen-like region can be derived from its homology with collagen. The structures of the alpha-helical coiled-coil and the lectin domain are known from crystallographic studies of mannan binding protein (MBP) and lung surfactant protein D (SP-D). Carbohydrate binding has been structurally characterized in several complexes between MBP and carbohydrate; all indicate that the major interaction between carbohydrate and collectin is the binding of two adjacent carbohydrate hydroxyl group to a collectin calcium ion. In addition, these hydroxyl groups hydrogen bond to some of the calcium amino acid ligands. While each collectin trimer contains three such carbohydrate binding sites, deviation from the overall threefold symmetry has been demonstrated for SP-D, which may influence its binding properties. The protein surface between the three binding sites is positively charged in both MBP and SP-D.     

Enhanced antiviral and opsonic activity of a human mannose-binding lectin and surfactant protein D chimera

The carbohydrate recognition domains (CRDs) of human serum mannose-binding lectin (MBL) and pulmonary surfactant protein D (SP-D) have distinctive monosaccharide-binding properties, and their N-terminal and collagen domains have very different quaternary structures. We produced a chimeric protein containing the N terminus and collagen domain of human SP-D and the neck region and CRD of human MBL (SP-D/MBLneck+CRD) to create a novel human collectin. The chimera bound to influenza A virus (IAV), inhibited IAV hemagglutination activity and infectivity, and induced aggregation of viral particles to a much greater extent than MBL. Furthermore, SP-D/MBLneck+CRD caused much greater increases in neutrophil uptake of, and respiratory burst responses to, IAV than MBL. These results indicate that pathogen interactions mediated by the MBL CRD are strongly influenced by the N-terminal and collagen-domain backbone to which it is attached. The presence of the CRD of MBL in the chimera resulted in altered monosaccharide binding properties compared with SP-D. As a result, the chimera caused greater aggregation and neutralization of IAV than SP-D. Distinctive functional properties of collectin collagenous domains and CRDs can be exploited to generate novel human collectins with potential for therapy of influenza.     

Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.     

Pulmonary surfactant protein D binds MD-2 through the carbohydrate recognition domain

Pulmonary surfactant protein D (SP-D) is a member of the collectin family and plays crucial roles in the innate immunity of the lung. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with MD-2 and alters lipopolysaccharide signaling. In this study, we examined and characterized the binding of SP-D to MD-2 using a soluble form of recombinant MD-2 (sMD-2). SP-D bound in a concentration- and Ca(2+)-dependent manner to sMD-2 coated onto microtiter wells. Excess mannose abolished the binding of SP-D to sMD-2. In solution, SP-D cosedimented with sMD-2 in the presence of Ca(2+). The direct binding of SP-D to sMD-2 was confirmed by BIAcore analysis. Anti-SP-D monoclonal antibody that recognizes the carbohydrate recognition domain (CRD) of SP-D significantly inhibited the binding of SP-D to sMD-2, indicating the involvement of the CRD for the binding to sMD-2. Ligand blot analysis revealed that SP-D bound to N-glycopeptidase F-treated sMD-2. In addition, the biotinylated SP-D pulled down the mutant sMD-2 with Asn(26) --> Ala and Asn(114) --> Ala substitutions, which lacks the consensus for N-glycosylation. Furthermore, the sMD-2 mutant cosedimented SP-D. These results demonstrate that SP-D directly interacts with MD-2 through the CRD.     

Increased antiviral and opsonic activity of a highly multimerized collectin chimera

Altering the carbohydrate binding properties of surfactant protein D (SP-D) [e.g., by replacing its carbohydrate recognition domain (CRD) with that of either mannose binding lectin (MBL) or conglutinin] can increase its activity against influenza A virus (IAV). The current study demonstrates that the degree of multimerization of SP-D is another independent determinant of antiviral activity. A chimeric collectin containing the N-terminus and collagen domain of human SP-D and the CRD of MBL formed high-molecular-weight multimers similar to those previously described for human SP-D. Using several complementary assays, and diverse viral strains, the chimeric multimers showed greater anti-IAV activity than similarly multimerized preparations of SP-D or incompletely oligomerized preparations of the chimera. More highly multimerized preparations of the chimera also caused greater increases in uptake of IAV by neutrophils. These studies may have implications for development of collectins as therapeutic agents and understanding of natural variations in susceptibility to IAV infection.     

Interaction of recombinant surfactant protein D with lipopolysaccharide: conformation and orientation of bound protein by IRRAS and simulations

Effective innate host defense requires early recognition of pathogens. Surfactant protein D (SP-D), shown to play a role in host defense, binds to the lipopolysaccharide (LPS) component of Gram-negative bacterial membranes. Binding takes place via the carbohydrate recognition domain (CRD) of SP-D. Recombinant trimeric neck+CRDs (NCRD) have proven valuable in biophysical studies of specific interactions. Although X-ray crystallography has provided atomic level information on NCRD binding to carbohydrates and other ligands, molecular level information about interactions between SP-D and biological ligands under physiologically relevant conditions is lacking. Infrared reflection-absorption spectroscopy (IRRAS) provides molecular structure information from films at the air/water interface where protein adsorption to LPS monolayers serves as a model for protein-lipid interaction. In the current studies, we examine the adsorption of NCRDs to Rd 1 LPS monolayers using surface pressure measurements and IRRAS. Measurements of surface pressure, Amide I band intensities, and LPS acyl chain conformational ordering, along with the introduction of EDTA, permit discrimination of Ca (2+)-mediated binding from nonspecific protein adsorption. The findings support the concept of specific binding between the CRD and heptoses in the core region of LPS. In addition, a novel simulation method that accurately predicts the IR Amide I contour from X-ray coordinates of NCRD SP-D is applied and coupled to quantitative IRRAS equations providing information on protein orientation. Marked differences in orientation are found when the NCRD binds to LPS compared to nonspecific adsorption. The geometry suggests that all three CRDs are simultaneously bound to LPS under conditions that support the Ca (2+)-mediated interaction.     

Complementation of pulmonary abnormalities in SP-D(-/-) mice with an SP-D/conglutinin fusion protein

Surfactant protein D (SP-D) and serum conglutinin are closely related members of the collectin family of host defense lectins. Although normally synthesized at different anatomic sites, both proteins participate in the innate immune response to microbial challenge. To discern the roles of specific domains in the function of SP-D in vivo, a fusion protein (SP-D/Cong(neck+CRD)) consisting of the NH(2)-terminal and collagenous domains of rat SP-D (rSP-D) and the neck and carbohydrate recognition domains (CRDs) of bovine conglutinin (Cong) was expressed in the respiratory epithelium of SP-D gene-targeted (SP-D(-/-)) mice. While SP-D/Cong(neck+CRD) fusion protein did not affect lung morphology and surfactant phospholipid levels in the lungs of wild type mice, the chimeric protein substantially corrected the increased lung phospholipids in SP-D(-/-) mice. The SP-D/Cong(neck+CRD) fusion protein also completely corrected defects in influenza A clearance and inhibited the exaggerated inflammatory response that occurs following viral infection. However, the chimeric protein did not ameliorate the ongoing lung inflammation, enhanced metalloproteinase expression, and alveolar destruction that characterize this model of SP-D deficiency. By contrast, a single arm mutant (RrSP-D(Ser15,20)) partially restored antiviral activity but otherwise failed to rescue the deficient phenotype. Our findings directly implicate the CRDs of both SP-D and conglutinin in host defense in vivo. Our findings also strongly suggest that the molecular mechanisms underlying impaired pulmonary host defense and abnormal lipid metabolism are distinct from those that promote ongoing inflammation and the development of emphysema.     

Introduction of N-linked glycans in the lectin domain of surfactant protein D: impact on interactions with influenza A viruses

Porcine surfactant protein D (pSP-D) displays distinctively strong, broad-range inhibitory activity against influenza A virus (IAV). N-Linked glycosylation of the carbohydrate recognition domain (CRD) of pSP-D contributes to the high affinity of this collectin for IAV. To investigate the role of the N-linked glycan further, HEK293E protein expression was used to produce recombinant pSP-D (RpSP-D) that has similar structural and antiviral properties as NpSP-D. We introduced an additional N-linked glycan in the CRD of RpSP-D but this modification did not alter the antiviral activity. Human SP-D is unglycosylated in its CRD and less active against IAV compared with pSP-D. In an attempt to modify its antiviral properties, several recombinant human SP-D (RhSP-D) mutants were constructed with N-linked glycans introduced at various locations within its CRD. To retain lectin activity, necessary for the primary interactions between SP-D and IAV, N-linked glycosylation of RhSP-D was shown to be restricted to the corresponding position in the CRD of either pSP-D or surfactant protein A (SP-A). These N-glycosylated RhSP-D mutants, however, did not show increased neutralization activity against IAV. By developing RhSP-D mutants that also have the pSP-D-specific Ser-Gly-Ala loop inserted in the CRD, we could demonstrate that the N-linked glycan-mediated interactions between pSP-D and IAV involves additional structural prerequisites of the pSP-D CRD. Ultimately, these studies will help to develop highly effective SP-D-based therapeutic and prophylactic drugs against IAV.     

Isolation and partial characterization of lumican and decorin from adult chicken corneas. A keratan sulfate-containing isoform of decorin is developmentally regulated.

The proteoglycans extracted from adult chicken were initially purified by DEAE-chromatography. Digestion of these proteoglycans with chondroitinase ABC generated a single 40-kDa core protein while digestion with keratanase generated a single 52-kDa core protein. Digestion with both enzymes combined, however, increased the amount of 40-kDa core protein produced. This suggested that the 40-kDa core protein exists with chondroitin/dermatan sulfate (C/DS) side chains alone and with both C/DS and keratan sulfate (KS) side chains. The proteoglycan fraction was initially digested with chondroitinase ABC, and the M(r) = 40,000 core protein derived from proteoglycans containing C/DS side chains alone was isolated. Amino-terminal sequencing showed it to be the chick cognate of decorin. The remaining proteoglycans were then digested with keratanase, and both the 40-kDa core protein and the 52-kDa core proteins derived from KS-containing proteoglycans were purified. The M(r) = 40,000 core protein derived from proteoglycans containing both C/DS and KS side chains had the same amino-terminal sequence as decorin and cross-reacted with antibodies to decorin. Sequence from the 52-kDa core protein derived from KS-containing proteoglycans showed it to be lumican. The results of this study suggest that adult chick corneas contain two isoforms of decorin: one containing C/DS side chains and the other, a hybrid, containing both C/DS and KS side chains. Embryonic corneas did not contain the hybrid isoform of decorin. These results suggest that different post-translational modifications occur to the decorin gene product during corneal development and maturation.     

Interaction of decorin with CNBr peptides from collagens I and II. Evidence for multiple binding sites and essential lysyl residues in collagen.

Decorin is a small leucine-rich chondroitin/dermatan sulfate proteoglycan reported to interact with fibrillar collagens through its protein core and to localize at d and e bands of the collagen fibril banding pattern. Using a solid-phase assay, we have determined the interaction of peptides derived by CNBr cleavage of type I and type II collagen with decorin extracted from bovine tendon and its protein core and with a recombinant decorin preparation. At least five peptides have been found to interact with all three decorin samples. The interaction of peptides with tendon decorin has a dissociation constant in the nanomolar range. The triple helical conformation of the peptide trimeric species is a necessary requisite for the binding. All positive peptides have a region within the d and e bands of collagen fibrils. Two chemical derivatives of collagens and of positive peptides were prepared by N-acetylation and N-methylation of the primary amino group of Lys/Hyl side chains. Chemical modifications performed in mild conditions do not significantly alter the thermal stability of peptide trimeric species whereas they affect the interaction with decorin: N-acetylation eliminates both the positive charge and the binding to decorin, whereas N-methylation preserves the cationic character and modulates the binding. We conclude that decorin makes contacts with multiple sites in type I collagen and probably also in type II collagen and that some collagen Lys/Hyl residues are essential for the binding.     

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

We previously demonstrated that bovine serum conglutinin has markedly greater ability to inhibit influenza A virus (IAV) infectivity than other collectins. We now show that recombinant conglutinin and a chimeric protein containing the NH(2) terminus and collagen domain of rat pulmonary surfactant protein D (rSP-D) fused to the neck region and carbohydrate recognition domain (CRD) of conglutinin (termed SP-D/Cong(neck+CRD)) have markedly greater ability to inhibit infectivity of IAV than wild-type recombinant rSP-D, confirming that the potent IAV-neutralizing activity of conglutinin resides in its neck region and CRD. Furthermore, by virtue of incorporation of the NH(2) terminus and collagen domain of SP-D, SP-D/Cong(neck+CRD) caused substantially greater aggregation of IAV particles and enhancement of neutrophil binding of, and H(2)O(2) responses to, IAV than recombinant conglutinin or recombinant rSP-D. Hence, SP-D/Cong(neck+CRD) combined favorable antiviral and opsonic properties of conglutinin and SP-D. This study demonstrates an association of specific structural domains of SP-D and conglutinin with specific functional properties and illustrates that antimicrobial activities of wild-type collectins can be enhanced through recombinant strategies.     

Collectin surfactant protein D binds antibodies and interlinks innate and adaptive immune systems

Innate immune collectins, such as surfactant protein D (SP-D), contain fibrillar collagen-like regions and globular carbohydrate-recognition domains (CRDs). SP-D recognizes carbohydrate arrays present on microbial surfaces via its CRDs, agglutinates microbes and enhances their phagocytosis. In contrast, adaptive immune proteins such as immunoglobulins (Igs) recognize pathogens via binding to specific antigens. Here we show that: SP-D binds various classes of immunoglobins, including IgG, IgM, IgE and secretory IgA, but not serum IgA; the globular domains of SP-D bind both the Fab and Fc domains of IgG; SP-D recognizes IgG via calcium-dependent protein-protein interactions, aggregates IgG-coated beads and enhances their phagocytosis by murine macrophage RAW 264.7 cells. Therefore, we propose that SP-D effectively interlinks innate and adaptive immune systems.     

Endocytosis of different members of the small chondroitin/dermatan sulfate proteoglycan family.

The family of small interstitial chondroitin/dermatan sulfate proteoglycans consists of at least three different molecular species: biglycan (proteoglycan I), decorin (proteoglycan II), and proteoglycan-100, which has a glycosylated core protein of about 100 kDa. The core protein of decorin has been shown to be responsible for receptor-mediated endocytosis of this proteoglycan species by a variety of mesenchymal cells. It is now demonstrated that skin fibroblasts and articular chondrocytes endocytose biglycan with an efficiency similar to that of decorin. Uptake of biglycan is also mediated by its core protein and can be inhibited by decorin in a partially competitive manner. In human fibroblasts, endosomal proteins of 51 and 26 kDa, which are known to bind decorin core protein, also interact with biglycan. This interaction can be inhibited by decorin. Bovine articular chondrocytes contained binding proteins of 48 and 25 kDa. Proteoglycan-100 can be distinguished from biglycan and decorin by its low clearance rate, which however, exceeds the rate of fluid phase endocytosis.     

Aerosolized endotoxin is immediately bound by pulmonary surfactant protein D in vivo.

Collectins are carbohydrate binding proteins that are implicated in innate host defense. The lung collectins, surfactant proteins A and D (SP-A and SP-D), bind a variety of pathogens in vitro and influence phagocytosis by alveolar macrophages. In this report we show that SP-D binds endotoxin (lipopolysaccharide, LPS) in vivo in a rat model of acute respiratory distress syndrome (ARDS). Intratracheal aerosolization of LPS in rats resulted in the typical features of human ARDS. Total amounts of SP-D, as well as the carbohydrate binding properties of SP-D were measured in lung lavage as a function of time. The amount of SP-D did not change during 24 h. Interestingly, SP-D in lung lavage isolated from rats during the first 2 h after LPS treatment, was not able to bind to carbohydrate. Further analysis revealed that the carbohydrate binding sites of SP-D were occupied by LPS, suggesting that SP-D is an LPS scavenging molecule in vivo. Electron microscopic analysis indicated that, 1 h after LPS aerosolization, aggregates of SP-D with LPS were found in lysosomal structures in alveolar macrophages. We conclude that the lung collectin SP-D binds inhaled endotoxin in vivo, which may help to protect the lung from endotoxin-induced disease.     

CL-46, a novel collectin highly expressed in bovine thymus and liver

Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via alpha-helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the alpha-helical neck region and the C-type lectin domain binds preferential N-acetyl-D-glucoseamine and N-acetyl-D-mannoseamine. The gene encoding CL-46 spans approximately 10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5'-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development.     

The carbohydrate recognition domain of collectins

Collectins are effector molecules of the innate immune system that play an important role in the first line of defence against bacteria, viruses and fungi. Most of their interactions with microorganisms are mediated through their carbohydrate recognition domain (CRD), which binds in a Ca(2+)-dependent manner to glycoconjugates. This domain is a well-known structure that is present in a larger group of proteins comprising the C-type lectin domain family. Collectins form a subgroup within this family based on the presence of a collagen domain and the trimerization of CRDs, which are essential for the ligand-binding properties of these proteins. The ligand specificity among the nine collectin members is significantly different as a result of both the structural organization of the trimers and specific sequence changes in the binding pocket of the CRD. In addition, some collectin members have additional features, such as N-linked glycosylation of CRD residues and additional loop structures within the CRD that have a large impact on their interaction with the glycoconjugates present on microorganisms or host cells. The availability of crystal structures of three members of the collectin family (surfactant proteins A and D and mannan-binding protein) provides an important tool for addressing the impact of these CRD differences on ligand binding. In this review, the structural differences and similarities between the CRDs of collectins are summarized and their relationship with their ligand-binding characteristics is discussed.     

Porcine lung surfactant protein D: complementary DNA cloning, chromosomal localization, and tissue distribution

Porcine organs and lung surfactant have medically important applications in both xenotransplantation and therapy. We have started to characterize porcine lung surfactant by cloning the cDNA of porcine surfactant protein D (SP-D). SP-D and SP-A are important mediators in innate immune defense for the lung and possibly other mucosal surfaces. Porcine SP-D will also be an important reagent for use in existing porcine animal models for human lung infections. The complete cDNA sequence of porcine SP-D, including the 5' and 3' untranslated regions, was determined from two overlapping bacteriophage clones and by PCR cloning. Three unique features were revealed from the porcine sequence in comparison to SP-D from other previously characterized species, making porcine SP-D an intriguing species addition to the SP-D/collectin family. The collagen region contains an extra cysteine residue, which may have important structural consequences. The other two differences, a potential glycosylation site and an insertion of three amino acids, lie in the loop regions of the carbohydrate recognition domain, close to the carbohydrate binding region and thus may have functional implications. These variations were ruled out as polymorphisms or mutations by confirming the sequence at the genomic level in four different pig breeds. Porcine SP-D was shown to localize primarily to the lung and with less abundance to the duodenum, jejunum, and ileum. The genes for SP-D and SP-A were also shown to colocalize to a region of porcine chromosome 14 that is syntenic with the human and murine collectin loci.     

Surfactant protein D binds to Mycobacterium tuberculosis bacilli and lipoarabinomannan via carbohydrate-lectin interactions resulting in reduced phagocytosis of the bacteria by macrophages.

Surfactant protein-D (SP-D) is a collectin produced in the distal lung airspaces that is believed to play an important role in innate pulmonary immunity. Naive immunologic responses to Mycobacterium tuberculosis (M.tb) are especially important in the lung, since entry of this inhaled pathogen into the alveolar macrophage is a pivotal event in disease pathogenesis. Here we investigated SP-D binding to M.tb and the effect of this binding on the adherence of M. tb to human macrophages. These studies demonstrate specific binding of SP-D to M.tb that is saturable, calcium dependent, and carbohydrate inhibitable. In addition to purified SP-D, SP-D in bronchoalveolar lavage fluids from healthy donors and patients with alveolar proteinosis also binds to M.tb. Incubation of M.tb with SP-D results in agglutination of the bacteria. In contrast to its binding to M.tb, SP-D binds minimally to the avirulent Mycobacterium smegmatis. SP-D binds predominantly to lipoarabinomannan from the virulent Erdman strain of M.tb, but not the lipoarabinomannan from M. smegmatis. The binding of SP-D to Erdman lipoarabinomannan is mediated by the terminal mannosyl oligosaccharides of this lipoglycan. Incubation of M.tb with subagglutinating concentrations of SP-D leads to reduced adherence of the bacteria to macrophages (62.7% of control adherence +/- 3.3% SEM, n = 8), whereas incubation of bacteria with surfactant protein A leads to significantly increased adherence to monocyte-derived macrophages. These data provide evidence for specific binding of SP-D to M. tuberculosis and indicate that SP-D and surfactant protein A serve different roles in the innate host response to this pathogen in the lung.