Human mannose-binding lectin and L-ficolin function as specific pattern recognition proteins in the lectin activation pathway of complement

The innate immune response in vertebrates and invertebrates requires the presence of pattern recognition receptors or proteins that recognize microbial cell components including lipopolysaccharide, bacterial peptidoglycan (PGN), and fungal 1,3-beta-D-glucan. We reported previously that PGN and 1,3-beta-D-glucan recognition proteins from insect hemolymph were able to induce the activation of the prophenoloxidase-activating system, one of the major invertebrate innate immune reactions. The goal of this study was to characterize the biochemical properties and effects of the human counterparts of these molecules. Soluble pattern recognition proteins were purified from human serum and identified as human mannose-binding lectin (MBL) and L-ficolin. The use of specific microbial cell component-coupled columns demonstrated that MBL and L-ficolin bind to PGN and 1,3-beta-D-glucan, respectively. Purified MBL and L-ficolin were associated with MBL-associated serine proteases-1 and -2 (MASPs) and small MBL-associated protein as determined by Western blot analysis. Finally, the binding of purified MBL/MASP and L-ficolin/MASP complexes to PGN and 1,3-beta-D-glucan, respectively, resulted in the activation of the lectin-complement pathway. These results indicate that human PGN and 1,3-beta-D-glucan recognition proteins function as complement-activating lectins.     

Surface-bound myeloperoxidase is a ligand for recognition of late apoptotic neutrophils by human lung surfactant proteins A and D

Surfactant proteins A (SP-A) and D (SP-D), both members of the collectin family, play a well established role in apoptotic cell recognition and clearance. Recent in vitro data show that SP-A and SP-D interact with apoptotic neutrophils in a distinct manner. SP-A and SP-D bind in a Ca²⁺-dependent manner to viable and early apoptotic neutrophils whereas the much greater interaction with late apoptotic neutrophils is Ca²⁺-independent. Cell surface molecules on the apoptotic target cells responsible for these interactions had not been identified and this study was done to find candidate target molecules. Myeloperoxidase (MPO), a specific intracellular defense molecule of neutrophils that becomes exposed on the outside of the cell upon apoptosis, was identified by affinity purification, mass-spectrometry and western blotting as a novel binding molecule for SP-A and SP-D. To confirm its role in recognition, it was shown that purified immobilised MPO binds SP-A and SP-D, and that MPO is surface-exposed on late apoptotic neutrophils. SP-A and SP-D inhibit binding of an anti-MPO monoclonal Ab to late apoptotic cells. Fluorescence microscopy confirmed that anti-MPO mAb and SP-A/SP-D colocalise on late apoptotic neutrophils. Desmoplakin was identified as a further potential ligand for SP-A, and neutrophil defensin as a target for both proteins.     

Inosine-containing RNA is a novel innate immune recognition element and reduces RSV infection

During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p<0.01). Flow cytometry data revealed a corresponding 4-fold increase in influx of neutrophils into the lungs by ss-Ino-RNA treatment. In our in vitro experiments, treatment of epithelial cells with ss-Ino-RNA reduced replication of respiratory syncytial virus (RSV). Interestingly, RNA structural analysis showed that ss-Ino-RNA had increased formation of secondary structures. Our data further revealed that extracellular ss-Ino-RNA was taken up by scavenger receptor class-A (SR-A) which activated downstream MAP Kinase pathways through Toll-like receptor 3 (TLR3) and dsRNA-activated protein kinase (PKR). Our data suggests that ss-Ino-RNA is an as yet undescribed virus-associated innate immune stimulus.     

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.     

Peptidoglycan recognition proteins: a novel family of four human innate immunity pattern recognition molecules.

The innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Insects have a family of 12 peptidoglycan recognition proteins (PGRPs) that recognize peptidoglycan, a ubiquitous component of bacterial cell walls. We report cloning of three novel human PGRPs (PGRP-L, PGRP-Ialpha, and PGRP-Ibeta) that together with the previously cloned PGRP-S, define a new family of human pattern recognition molecules. PGRP-L, PGRP-Ialpha, and PGRP-Ibeta have 576, 341, and 373 amino acids coded by five, seven, and eight exons on chromosomes 19 and 1, and they all have two predicted transmembrane domains. All mammalian and insect PGRPs have at least three highly conserved C-terminal PGRP domains located either in the extracellular or in the cytoplasmic (or in both) portions of the molecules. PGRP-L is expressed in liver, PGRP-Ialpha and PGRP-Ibeta in esophagus (and to a lesser extent in tonsils and thymus), and PGRP-S in bone marrow (and to a lesser extent in neutrophils and fetal liver). All four human PGRPs bind peptidoglycan and Gram-positive bacteria. Thus, these PGRPs may play a role in recognition of bacteria in these organs.     

Curculin,a sweet-tasting and taste-modifying protein,is a non-functional mannose-binding lectin

A three-dimensional model of curculin, a sweet-tasting and taste-modifying protein from the fruits of Curculigo latifolia, was built from the X-ray coordinates of GNA, a mannose-binding lectin from snowdrop (Galanthus nivalis). The three mannose-binding sites present in GNA were found in curculin but are devoid of mannose-binding activity as shown by docking experiments performed with mannose. Some regions well exposed on the surface of the three-dimensional model of curculin could act as epitopes responsible for the sweet-tasting properties of this protein.     

High-resolution structural insights into ligand binding and immune cell recognition by human lung surfactant protein D

Lung surfactant protein D (SP-D) can directly interact with carbohydrate residues on pulmonary pathogens and allergens, stimulate immune cells, and manipulate cytokine and chemokine profiles during the immune response in the lungs. Therapeutic administration of rfhSP-D, a recombinant homotrimeric fragment of human SP-D comprising the alpha-helical coiled-coil neck plus three CRDs, protects mice against lung allergy and infection caused by the fungal pathogen Aspergillus fumigatus. The high resolution crystal structures of maltose-bound rfhSP-D to 1.4A, and of rfhSP-D to 1.6A, define the fine detail of the mode and nature of carbohydrate recognition and provide insights into how a small fragment of human SP-D can bind to allergens/antigens or whole pathogens, and at the same time recruit and engage effector cells and molecules of humoral immunity. A previously unreported calcium ion, located on the trimeric axis in a pore at the bottom of the funnel formed by the three CRDs and close to the neck-CRD interface, is coordinated by a triad of glutamate residues which are, to some extent, neutralised by their interactions with a triad of exposed lysine residues in the funnel. The spatial relationship between the neck and the CRDs is maintained internally by these lysine residues, and externally by a glutamine, which forms a pair of hydrogen-bonds within an external cleft at each neck-CRD interface. Structural links between the central pore and the cleft suggest a possible effector mechanism for immune cell surface receptor binding in the presence of bound, extended natural lipopolysaccharide and phospholipid ligands. The structural requirements for such an effector mechanism, involving both the trimeric framework for multivalent ligand binding and recognition sites formed from more than one subunit, are present in both native hSP-D and rfhSP-D, providing a possible explanation for the significant biological activity of rfhSP-D.     

Polysaccharide recognition by surfactant protein D: novel interactions of a C-type lectin with nonterminal glucosyl residues.

Surfactant protein D (SP-D), a C-type lectin, is an important pulmonary host defense molecule. Carbohydrate binding is critical to its host defense properties, but the precise polysaccharide structures recognized by the protein are unknown. SP-D binding to Aspergillus fumigatus is strongly inhibited by a soluble beta-(1-->6)-linked but not by a soluble beta-(1-->3)-linked glucosyl homopolysaccharide (pustulan and laminarin, respectively), suggesting that SP-D recognizes only certain polysaccharide configurations, likely through differential binding to nonterminal glucosyl residues. In this study we have computationally docked alpha/beta-D-glucopyranose and alpha/beta-(1-->2)-, alpha/beta-(1-->3)-, alpha/beta-(1-->4)-, and alpha/beta-(1-->6)-linked glucosyl trisaccharides into the SP-D carbohydrate recognition domain. As with the mannose-binding proteins, we found significant hydrogen bonding between the protein and the vicinal, equatorial OH groups at the 3 and 4 positions on the sugar ring. Our docking studies predict that alpha/beta-(1-->2)-, alpha-(1-->4)-, and alpha/beta-(1-->6)-linked but not alpha/beta-(1-->3)-linked glucosyl trisaccharides can be bound by their internal glucosyl residues and that binding also occurs through interactions of the protein with the 2- and 3-equatorial OH groups on the glucosyl ring. By using various soluble glucosyl homopolysaccharides as inhibitors of SP-D carbohydrate binding, we confirmed the interactions predicted by our modeling studies. Given the sequence and structural similarity between SP-D and other C-type lectins, many of the predicted interactions should be applicable to this protein family.     

Elucidation of a novel Vibrio cholerae lipid A secondary hydroxy-acyltransferase and its role in innate immune recognition

Similar to most Gram-negative bacteria, the outer leaflet of the outer membrane of Vibrio cholerae is comprised of lipopolysaccharide. Previous reports have proposed that V. cholerae serogroups O1 and O139 synthesize structurally different lipid A domains, which anchor lipopolysaccharide within the outer membrane. In the current study, intact lipid A species of V. cholerae O1 and O139 were analysed by mass spectrometry. We demonstrate that V. cholerae serogroups associated with human disease synthesize a similar asymmetrical hexa-acylated lipid A species, bearing a myristate (C14:0) and 3-hydroxylaurate (3-OH C12:0) at the 2'- and 3'-positions respectively. A previous report from our laboratory characterized the V. cholerae LpxL homologue Vc0213, which transfers a C14:0 to the 2'-position of the glucosamine disaccharide. Our current findings identify V. cholerae Vc0212 as a novel lipid A secondary hydroxy-acyltransferase, termed LpxN, responsible for transferring the 3-hydroxylaurate (3-OH C12:0) to the V. cholerae lipid A domain. Importantly, the presence of a 3-hydroxyl group on the 3'-linked secondary acyl chain was found to promote antimicrobial peptide resistance in V. cholerae; however, this functional group was not required for activation of the innate immune response.     

ULBP4 is a novel ligand for human NKG2D

The ULBPs are a family of MHC class I-related molecules. We have previously shown that ULBPs 1, 2, and 3 are functional ligands of the NKG2D/DAP10 receptor complex on human natural killer (NK) cells. Here, we describe a new member of the ULBP family, ULBP4, which contains predicted transmembrane and cytoplasmic domains, unlike the other ULBPs, which are GPI-linked proteins. Transduction of ULBP4 into EL4 cells confers the ability to bind recombinant NKG2D and mediates increased cytotoxic activity by human NK cells, consistent with the role of ULBPs as ligands for the NKG2D/DAP10 activating receptors. Tissue expression of ULBP4 differs from other members of the family, in that it is expressed predominantly in the skin.     

Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor gamma chain to induce innate immune responses

Antigen presenting cells recognize pathogens via pattern recognition receptors (PRR), which upon ligation transduce intracellular signals that can induce innate immune responses. Because some C-type lectin-like receptors (e.g. dectin-1 and DCSIGN) were shown to act as PRR for particular microbes, we considered a similar role for dectin-2. Binding assays using soluble dectin-2 receptors showed the extracellular domain to bind preferentially to hyphal (rather than yeast/conidial) components of Candida albicans, Microsporum audouinii, and Trichophyton rubrum. Selective binding for hyphae was also observed using RAW macrophages expressing dectin-2, the ligation of which by hyphae or cross-linking with dectin-2-specific antibody led to protein tyrosine phosphorylation. Because dectin-2 lacks an intracellular signaling motif, we searched for a signal adaptor that permits it to transduce intracellular signals. First, we found that the Fc receptor gamma (FcRgamma) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcRgamma, activation of NF-kappaB, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor alpha and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcRgamma chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcRgamma to induce innate immune responses.     

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature.     

The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A

Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional domains.     

A macrophage protein, Ym1, transiently expressed during inflammation is a novel mammalian lectin

Oral infections of mice with Trichinella spiralis induce activation of peritoneal exudate cells to transiently express and secrete a crystallizable protein Ym1. Purification of Ym1 to homogeneity was achieved. It is a single chain polypeptide (45 kDa) with a strong tendency to crystallize at its isoelectric point (pI 5.7). Co-expression of Ym1 with Mac-1 and scavenger receptor pinpoints macrophages as its main producer. Protein microsequencing data provide information required for full-length cDNA cloning from libraries constructed from activated peritoneal exudate cells. A single open reading frame of 398 amino acids with a leader peptide (21 residues) typical of secretory protein was deduced and later deposited in GenBank (accession number M94584) in 1992. By means of surface plasmon resonance analyses, Ym1 has been shown to exhibit binding specificity to saccharides with a free amine group, such as GlcN, GalN, or GlcN polymers, but it failed to bind to other saccharides. The interaction is pH-dependent but Ca2+ and Mg2+ ion-independent. The binding avidity of Ym1 to GlcN oligosaccharides was enhanced by more than 1000-fold due to the clustering effect. Specific binding of Ym1 to heparin suggests that heparin/heparan sulfate may be its physiological ligand in vivo during inflammation and/or tissue remodeling. Although it shares approximately 30% homology with microbial chitinases, no chitinase activity was found associated with Ym1. Genomic Southern blot analyses suggest that Ym1 may represent a member of a novel lectin gene family.     

Fibulin-1 is a ligand for the C-type lectin domains of aggrecan and versican.

The aggregating proteoglycans (aggrecan, versican, neurocan, and brevican) are important components of many extracellular matrices. Their N-terminal globular domain binds to hyaluronan, but the function of their C-terminal region containing a C-type lectin domain is less clear. We now report that a 90-kDa protein copurifies with recombinant lectin domains from aggrecan and versican, but not from the brain-specific neurocan and brevican. Amino acid sequencing of tryptic peptides from this protein identified it as fibulin-1. This extracellular matrix glycoprotein is strongly expressed in tissues where versican is expressed (blood vessels, skin, and developing heart), and also expressed in developing cartilage and bone. It is thus likely to interact with these proteoglycans in vivo. Surface plasmon resonance measurements confirmed that aggrecan and versican lectin domains bind fibulin-1, whereas brevican and neurocan do not. As expected for a C-type lectin, the interactions with fibulin-1 are Ca2+-dependent, with KD values in the low nanomolar range. Using various deletion mutants, the binding site for aggrecan and versican lectin domains was mapped to the epidermal growth factor-like repeats in domain II of fibulin-1. No difference in affinity was found for deglycosylated fibulin-1, indicating that the proteoglycan C-type lectin domains bind to the protein part of fibulin-1.     

Biological activities of human mannose-binding lectin bound to two different ligand sugar structures, Lewis A and Lewis B antigens and high-mannose type oligosaccharides.

The biological activities of mannose-binding lectin (MBL) which binds to different ligands on mammalian cells were examined using two types of Colo205 cells, a human colon adenocarcinoma cell line: one naturally expressing Lewis A and Lewis B antigens as ligands for MBL (NT-Colo205), and the other modified to express high-mannose type oligosaccharides by treatment with benzyl-2-acetamide-2-deoxy-alpha-galactopyranoside and 1-deoxymannojirimycin (Bz+dMM-Colo205). Although the final lysis was not observed, the deposition of C4 and C3 was observed on both types of Colo205 cells after treatment with MBL and complements as a result of complement activation by MBL. MBL bound to Bz+dMM-Colo205 could also activate human peripheral blood leukocytes and induce superoxide production; however, MBL bound to NT-Colo205 could not. This may be explained by the lower affinity of MBL to Lewis A and Lewis B antigens than to high-mannose type oligosaccharides under physiological conditions, since MBL bound to NT-Colo205 was more easily released from the cell surface than that bound to Bz+dMM-Colo205 at 37 degrees C. These findings suggest that the difference in the affinity of MBL to its ligands could influence the expression of some biological activities of MBL.     

Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells

Pattern recognition receptors, non-clonal immune proteins recognizing common microbial components, are critical for non-self recognition and the subsequent induction of Rel/NF-kappaB-controlled innate immune genes. However, the molecular identities of such receptors are still obscure. Here, we present data showing that Drosophila possesses at least three cDNAs encoding members of the Gram-negative bacteria-binding protein (DGNBP) family, one of which, DGNBP-1, has been characterized. Western blot, flow cytometric, and confocal laser microscopic analyses demonstrate that DGNBP-1 exists in both a soluble and a glycosylphosphatidylinositol-anchored membrane form in culture medium supernatant and on Drosophila immunocompetent cells, respectively. DGNBP-1 has a high affinity to microbial immune elicitors such as lipopolysaccharide (LPS) and beta-1,3-glucan whereas no binding affinity is detected with peptidoglycan, beta-1,4-glucan, or chitin. Importantly, the overexpression of DGNBP-1 in Drosophila immunocompetent cells enhances LPS- and beta-1,3-glucan-induced innate immune gene (NF-kappaB-dependent antimicrobial peptide gene) expression, which can be specifically blocked by pretreatment with anti-DGNBP-1 antibody. These results suggest that DGNBP-1 functions as a pattern recognition receptor for LPS from Gram-negative bacteria and beta-1, 3-glucan from fungi and plays an important role in non-self recognition and the subsequent immune signal transmission for the induction of antimicrobial peptide genes in the Drosophila innate immune system.     

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D₃ and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.