Carbohydrate-binding Ability Research Articles

Allosteric Regulation of the Carbohydrate-binding Ability of a Novel Conger Eel Galectin by d-Mannoside

Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of D-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that D-mannoside can act as a modulator of Con-P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type β-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties.

Fibrinogen-related proteins in ixodid ticks

BackgroundFibrinogen-related proteins with lectin activity are believed to be part of the tick innate immune system. Several fibrinogen-related proteins have been described and characterised mainly on the basis of their cDNA sequences while direct biochemical evidence is missing. One of them, the haemolymph lectin Dorin M from the tick Ornithodoros moubata was isolated and characterised in more depth.ResultsSeveral fibrinogen-related proteins were detected in the haemolymph of ixodid ticks Dermacentor marginatus, Rhipicephalus appendiculatus, R. pulchellus, and R. sanguineus. These proteins were recognised by sera directed against the tick lectin Dorin M and the haemagglutination activity of the ticks R. appendiculatus and D. marginatus. Cross-reactivity of the identified proteins with antibodies against the fibrinogen domain of the human ficolin was also shown. The carbohydrate-binding ability of tick haemolymph was confirmed by haemagglutination activity assays, and this activity was shown to be inhibited by neuraminic acid and sialylated glycoproteins as well as by N-acetylated hexosamines. The fibrinogen-related proteins were shown to be glycosylated and they were localised in salivary glands, midguts, and haemocytes of D. marginatus. Hemelipoglycoprotein was also recognised by sera directed against the fibrinogen-related proteins in all three Rhipicephalus species as well as in D. marginatus. However, this protein does not contain the fibrinogen domain and thus, the binding possibly results from the structure similarity between hemelipoglycoprotein and the fibrinogen domain.ConclusionsThe presence of fibrinogen-related proteins was shown in the haemolymph of four tick species in high abundance. Reactivity of antibodies directed against ficolin or fibrinogen-related proteins with proteins which do not contain the fibrinogen domain points out the importance of sequence analysis of the identified proteins in further studies. Previously observed expression of fibrinogen-related proteins in haemocytes together with the results of this study suggest involvement of fibrinogen-related proteins in tick immunity processes. Thus, they have potential as targets for anti-tick vaccines and as antimicrobial proteins in pharmacology. Research on fibrinogen-related proteins could reveal further details of tick innate immunity processes.

Catalytic and glycan-binding abilities of ppGalNAc-T2 are regulated by acetylation

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (“ricin-like”) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).

Rat recombinant β-defensin 22 is a heparin-binding protein with antimicrobial activity

Approximately 40-50 β-defensins are predominantly expressed in the male reproductive system of mammals. This selective expression raises the question as to the roles of these molecules in innate immunity and fertility in the male reproductive tract. Rat β-defensin 22 is an epididymis-specific β-defensin expressed in segments 12-14 of the epididymis. This protein contains both β-defensin and lectin signature sequences, yet its antimicrobial activity and carbohydrate-binding ability have not been shown. We herein demonstrated the antimicrobial activity of recombinant rat β-defensin 22 against Escherichia coli and Candida albicans. Its lectin-like activity was also investigated by demonstrating its binding ability with heparin beads. This heparin-binding activity implies some potential roles for this defensin in determining the fertilisation capabilities of sperm.

Human RegIV Protein Adopts a Typical C-Type Lectin Fold but Binds Mannan with Two Calcium-Independent Sites

Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of 15N relaxation parameters and simplified Carr–Purcell–Meiboom–Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract.

Inhibition of dextran sulfate sodium (DSS)-induced intestinal inflammation via enhanced IL-10 and TGF-β production by galectin-9 homologues isolated from intestinal parasites

We isolated a galectin-9 (Gal-9) homologue gene (Tl-gal) from an adult worm of the canine gastrointestinal nematode parasite, Toxascaris leonina, via random cDNA library sequencing. The deduced amino acid sequence of the Tl-gal genes evidenced an identity of 89% with the galectin of Dirofilaria immitis, 87% identity with the galectin of Brugia malayi, and 35% identity with the human GAL-9 gene. To evaluate immune modulate function of Tl-GAL in host inflammatory response, we constructed recombinant Tl-GAL (rTl-GAL) using an Escherichia coli expression vector system and treated to intestinal inflammation mice. Although the carbohydrate-binding ability of rTl-GAL was less than that of rat galectin, we confirmed that recombinant rTl-GAL has carbohydrate-binding activity. The clinical symptoms of dextran sulfate sodium (DSS)-treated mice after rTl-GAL pre-treatment were found to be minimized, or less profound, as compared to those of the rTl-GAL untreated group. Additionally, the DSS-treated mice exhibited a significant shortening of the colon, but the large intestines of the rTl-GAL pre-treated mice were longer than those of the control group (P<0.05). Additionally, the rTl-GAL treated group exhibited significantly increased the levels of TGF-β and IL-10 (P<0.05). The production of these regulatory cytokines may ameliorate intestinal inflammation. These findings demonstrate that rTl-GAL could inhibit inflammation reactions via the inhibition of Th1 and Th2 cytokine production by increasing the production of TGF-β and IL-10 cytokines. The rTl-GAL may induce TGF-β expression, primarily via the activation of the p38 pathway. In conclusion, rTl-GAL may function like a host galectin, thus functioning as a regulatory molecule in the host immune system; rTl-GAL may prove useful in the design of novel therapeutic intervention strategies for the treatment of allergic and immune diseases.

Carbohydrate Recognition Properties of Human Ficolins: GLYCAN ARRAY SCREENING REVEALS THE SIALIC ACID BINDING SPECIFICITY OF M-FICOLIN

Ficolins are oligomeric innate immune recognition proteins consisting of a collagen-like region and a fibrinogen-like recognition domain that bind to pathogen- and apoptotic cell-associated molecular patterns. To investigate their carbohydrate binding specificities, serum-derived L-ficolin and recombinant H- and M-ficolins were fluorescently labeled, and their carbohydrate binding ability was analyzed by glycan array screening. L-ficolin preferentially recognized disulfated N-acetyllactosamine and tri- and tetrasaccharides containing terminal galactose or N-acetylglucosamine. Binding was sensitive to the position and orientation of the bond between N-acetyllactosamine and the adjacent carbohydrate. No significant binding of H-ficolin to any of the 377 glycans probed could be detected, providing further evidence for its poor lectin activity. M-ficolin bound preferentially to 9-O-acetylated 2-6-linked sialic acid derivatives and to various glycans containing sialic acid engaged in a 2-3 linkage. To further investigate the structural basis of sialic acid recognition by M-ficolin, point mutants were produced in which three residues of the fibrinogen domain were replaced by their counterparts in L-ficolin. Mutations G221F and A256V inhibited binding to the 9-O-acetylated sialic acid derivatives, whereas Y271F abolished interaction with all sialic acid-containing glycans. The crystal structure of the Y271F mutant fibrinogen domain was solved, showing that the mutation does not alter the structure of the ligand binding pocket. These analyses reveal novel ficolin ligands such as sulfated N-acetyllactosamine (L-ficolin) and gangliosides (M-ficolin) and provide precise insights into the sialic acid binding specificity of M-ficolin, emphasizing the essential role of Tyr(271) in this respect.

Distinct mutations in the glycogen debranching enzyme found in glycogen storage disease type III lead to impairment in diverse cellular functions

Glycogen storage disease type III (GSDIII) is a metabolic disorder characterized by a deficiency in the glycogen debranching enzyme, amylo-1,6-glucosidase,4-alpha-glucanotransferase (AGL). Patients with GSDIII commonly exhibit hypoglycemia, along with variable organ dysfunction of the liver, muscle or heart tissues. The AGL protein binds to glycogen through its C-terminal region, and possesses two separate domains for the transferase and glucosidase activities. Most causative mutations are nonsense, and how they affect the enzyme is not well understood. Here we investigated four rare missense mutations to determine the molecular basis of how they affect AGL function leading to GSDIII. The L620P mutant primarily abolishes transferase activity while the R1147G variant impairs glucosidase function. Interestingly, mutations in the carbohydrate-binding domain (CBD; G1448R and Y1445ins) are more severe in nature, leading to significant loss of all enzymatic activities and carbohydrate binding ability, as well as enhancing targeting for proteasomal degradation. This region (Y1445-G1448R) displays virtual identity across human and bacterial species, suggesting an important role that has been conserved throughout evolution. Our results clearly indicate that inactivation of either enzymatic activity is sufficient to cause GSDIII disease and suggest that the CBD of AGL plays a major role to coordinate its functions and regulation by the ubiquitin-proteasome system.

Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins

The extensive involvement of glycan-binding proteins (GBPs) as regulators in diverse biological phenomena provides a fundamental reason to investigate their glycan-binding specificities. Here, we developed a glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of GBPs. Eighty-nine selected multivalent glycoconjugates comprising natural glycoproteins, neo-glycoproteins, and polyacrylamide (PAA)-conjugated glycan epitopes were immobilized on an epoxy-activated glass slide. The GBP binding was monitored by an evanescent-field fluorescence-assisted scanner at equilibrium without washing steps. The detection principle also allows direct application of unpurified GBPs with the aid of specific antibodies. Model experiments using plant lectins (RCA120, ConA, and SNA), galectins (3 and 8), a C-type lectin (DC-SIGN) and a siglec (CD22) provided data consistent with previous work within 4 h using less than 40 ng of GBPs per analysis. As an application, serum profiling of antiglycan antibodies (IgG and IgM) was performed with Cy3-labeled secondary antibodies. Moreover, novel carbohydrate-binding ability was demonstrated for a human IL-18 binding protein. Thus, the developed glycan array is useful for investigation of various types of GBPs, with the added advantage of wash-free analysis.

S-Nitrosylation of mannose binding lectin regulates its functional activities and the formation of autoantibody in rheumatoid arthritis

The possibility of post-translational modifications of mannose binding lectin (MBL) leading to functional impairment of the MBL pathway and the presence of anti-MBL autoantibodies were reported earlier in rheumatoid arthritis (RA). MBL was observed to be S-nitrosylated ( S-nitrosated) in vitro. HepG2 cells were stimulated with 10% synovial fluid from RA patients to produce increased levels of MBL and nitric oxide. Under these experimental conditions MBL was observed to be S-nitrosated using biotin switch assay. The plasma of RA patients was also found to contain higher levels of S-nitrosylated MBL (SNO-MBL) in comparison to the healthy controls. Functional activities of SNO-MBL were compared with normal MBL. Mannan binding and C4 deposition ability of MBL was found to decrease after S-nitrosylation. It was also observed that S-nitrosylation of MBL leads to a decrease in the bacterial phagocytosis and apoptotic cell binding as measured by fluorescence microscopy and FACS analysis. These results indicate that the carbohydrate binding ability of MBL was affected by S-nitrosylation ( S-nitrosation). High levels of anti-MBL autoantibodies were detected against SNO-MBL in plasma of RA patients in comparison to normal MBL suggesting a role of SNO-MBL in generation of autoantibodies in RA patients.

Galectin-1 regulates neurogenesis in the subventricular zone and promotes functional recovery after stroke

Galectin-1 (Gal-1) has recently been identified as a key molecule that plays important roles in the regulation of neural progenitor cell proliferation in two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. To test the hypothesis that Gal-1 contributes to adult neurogenesis after focal ischemia, we studied the temporal profile of endogenous Gal-1 expression and the effects of human recombinant Gal-1 on neurogenesis and neurological functions in an experimental focal ischemic model. In the normal brain, Gal-1 expression was observed only in the SVZ. In the ischemic brain, Gal-1 expression was markedly upregulated in the SVZ and the area of selective neuronal death around the infarct in the striatum. The temporal profile of Gal-1 expression was correlated with that of neural progenitor cell proliferation in the SVZ of the ischemic hemisphere. Double-labeling studies revealed that Gal-1 was localized predominantly in both reactive astrocytes and SVZ astrocytes. Administration of Gal-1, which is known to have carbohydrate-binding ability, into the lateral ventricle increased neurogenesis in the ipsilateral SVZ and improved sensorimotor dysfunction after focal ischemia. By contrast, blockade of Gal-1 in the SVZ by the administration of anti-Gal-1 neutralizing antibody strongly inhibited neurogenesis and diminished neurological function. These results suggest that Gal-1 is one of the principal regulators of adult SVZ neurogenesis through its carbohydrate-binding ability and provide evidence that Gal-1 protein has a role in the improvement of sensorimotor function after stroke.

Expression and function of galectin-1 in adult neural stem cells.

Neural stem cells (NSCs) in the adult mammalian brain proliferate and continuously produce new neurons. To date, there has been little research into the functions of lectins in adult NSCs. Recently, we reported that a lectin, galectin-1, is expressed on adult NSCs and promotes their proliferation through its carbohydrate-binding ability. This evidence raises the possibility that glycans play roles in the proliferation of adult NSCs.

Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells

Galectins are implicated in a large variety of biological functions, many of which depend on their carbohydrate-binding ability. Fifteen members of the family have been identified in vertebrates based on binding to galactose (Gal) that is mediated by one or two, evolutionarily conserved, carbohydrate-recognition domains (CRDs). Variations in glycan structures expressed on glycoconjugates at the cell surface may, therefore, affect galectin binding and functions. To identify roles for different glycans in the binding of the three types of mammalian galectins to cells, we performed fluorescence cytometry at 4 degrees C with recombinant rat galectin-1, human galectin-3, and three forms of human galectin-8, to Chinese hamster ovary (CHO) cells and 12 different CHO glycosylation mutants. All galectin species bound to parent CHO cells and binding was inhibited >90% by 0.2 M lactose. Galectin-8 isoforms with either a long or a short inter-CRD linker bound similarly to CHO cells. However, a truncated form of galectin-8 containing only the N-terminal CRD bound only weakly to CHO cells and the C-terminal galectin-8 CRD exhibited extremely low binding. Binding of the galectins to the different CHO glycosylation mutants revealed that complex N-glycans are the major ligands for each galectin except the N-terminal CRD of galectins-8, and also identified some fine differences in glycan recognition. Interestingly, increased binding of galectin-1 at 4 degrees C correlated with increased propidium iodide (PI) uptake, whereas galectin-3 or -8 binding did not induce permeability to PI. The CHO glycosylation mutants with various repertoires of cell surface glycans are a useful tool for investigating galectin-cell interactions as they present complex and simple glycans in a natural mixture of multivalent protein and lipid glycoconjugates anchored in a cell membrane.

Functional Role Played by the Glycosylphosphatidylinositol Anchor Glycan of CD48 in Interleukin-18-induced Interferon-γ Production

Interleukin (IL)-18 induces T cells and natural killer cells to produce not only interferon-gamma but also other cytokines by binding to the IL-18 receptor (IL-18R) alpha and beta subunits. However, little is known about how IL-18, IL-18Ralpha, and IL-18Rbeta form a high-affinity complex on the cell surface and transduce the signal. We found that IL-18 and IL-18Ralpha bind to glycosylphosphatidylinositol (GPI) glycan via the third mannose 6-phosphate diester and the second beta-GlcNAc-deleted mannose 6-phosphate of GPI glycan, respectively. To determine which GPI-anchored glycoprotein is involved in the complex of IL-18 and IL-18Ralpha, IL-18Ralpha of IL-18-stimulated KG-1 cells was immunoprecipitated together with CD48 by anti-IL-18Ralpha antibody. More than 90% of CD48 was detected as beta-GlcNAc-deleted GPI-anchored glycoprotein, and soluble recombinant human CD48 without GPI glycan bound to IL-18Ralpha, indicating that CD48 is associated with IL-18Ralpha via both the peptide portion and the GPI glycan. To investigate whether the carbohydrate recognition of IL-18 is involved in physiological activities, KG-1 cells were digested with phosphatidylinositol-specific phospholipase C before IL-18 stimulation. Phosphatidylinositol-specific phospholipase C treatment inhibited the phosphorylation of tyrosine kinases and the following IL-18-dependent interferon-gamma production. These observations suggest that the complex formation of IL-18.IL-18Ralpha. CD48 via both the peptide portion and GPI glycan triggers the binding to IL-18Rbeta, and the IL-18.IL-18Ralpha.CD48.IL-18Rbeta complex induces cellular signaling.

The crystal structure of Ym1 at 1.31 Å resolution

Upon nematode infection, murine peritoneal macrophages synthesize and secrete large amounts of the Ym1 protein, which is a unique functional marker for alternatively activated macrophages in T H2-mediated inflammatory responses. Ym1 shares significant structural similarity to the family 18 chitinases. Previously, Ym1 has been studied with respect to its carbohydrate-binding ability and glycosyl hydrolysis activity and this has led to various inconclusive interpretations. Our present co-crystallization and soaking experiments with various glucosamine or N-acetylglucosamine oligomers yield only the uncomplexed Ym1. The refined Ym1 structure at 1.31 Å resolution clearly displays a water cluster forming an extensive hydrogen bond network with the “active-site” residues. This water cluster contributes notable electron density to lower resolution maps and this might have misled and given rise to a previous proposal for a monoglucosamine-binding site for Ym1. A structural comparison of family 18 glycosidase (-like) proteins reveals a lack of several conserved residues in Ym1, and illustrates the versatility of the divergent active sites. Therefore, Ym1 may lack N-acetylglucosamine-binding affinity, and this suggests that a new direction should be taken to unravel the function of Ym1.

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.

Recognition by TNF-α of the GPI-anchor glycan induces apoptosis of U937 cells

Tumor necrosis factor-α (TNF-α) binds to TNF-α receptors (TNFR) to produce a hexameric (TNF-α) 3–(TNFR) 3 structure that stimulates apoptosis. We found by using ELISA that TNF-α binds to the glycosylphosphatidylinositol (GPI) anchor glycans of carcinoembryonic antigen, human placental alkaline phosphatase (hAP), and Tamm–Horsfall glycoprotein. These binding abilities were inhibited by 10 −6 M mannose-6-phosphate. Treatment of hAP with mild acid and phosphatase, which releases the N-acetylglucosamine (GlcNAc) β1 → phosphate → 6 residue from the GPI-anchor glycan of hAP, abrogated the binding of TNF-α to hAP. Thus, TNF-α binds to the GlcNAcβ1 → phosphate → 6Man residue in GPI-anchor glycans. To investigate whether the carbohydrate-binding ability of TNF-α is related to its physiological functions, human lymphoma U937 cells were used. TNF-α stimulates U937 cell apoptosis in a dose-dependent manner and the presence of mannose-6-phosphate inhibited this. TNF-α-dependent tyrosine phosphorylation of several proteins in U937 cells was also diminished by mannose-6-phosphate. Phosphatidylinositol-specific phospholipase C-pretreatment also inhibited this tyrosine phosphorylation. These data suggest that TNF-α stimulates U937 cell apoptosis by forming a high-affinity nanomeric (TNF-α) 3–(TNFR) 3–(GPI-anchored glycan) 3 complex. The GPI-anchored glycoprotein involved remains to be identified.

Characterization of functional domains of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata.

CEL-III is a Ca(2+)-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectin isolated from the marine invertebrate Cucumaria echinata. This lectin exhibits strong hemolytic activity and cytotoxicity through pore formation in target cell membranes. The amino acid sequence of CEL-III revealed the N-terminal two-thirds to have homology to the B-chains of ricin and abrin, which are galactose-specific plant toxic lectins; the C-terminal one-third shows no homology to any known proteins. To examine the carbohydrate-binding ability of the N-terminal region of CEL-III, the protein comprising Pyr1-Phe283 was expressed in Escherichia coli cells. The expressed protein showed both the ability to bind to a GalNAc-immobilized column as well as hemagglutinating activity for rabbit erythrocytes, confirming that the N-terminal region has binding activity for specific carbohydrates. Since the C-terminal region could not be expressed in E. coli cells, a fragment containing this region was produced by limited proteolysis of the native protein by trypsin. The resulting C-terminal 15 kDa fragment of CEL-III exhibited a tendency to self-associate, forming an oligomer. When mixed with erythrocytes, the oligomer of the C-terminal fragment caused hemagglutination, probably due to hydrophobic interaction with cell membranes, while the monomeric fragment did not. Chymotryptic digestion of the preformed CEL-III oligomer induced upon lactose binding also yielded an oligomer of the C-terminal fragment comprising six molecules of the 16 kDa fragment. These results suggest that after binding to cell surface carbohydrate chains, CEL-III oligomerizes through C-terminal domains, leading to the formation of ion-permeable pores by hydrophobic interaction with the cell membrane.

Mannan-binding lectin and its role in innate immunity.

Mannan-binding lectin (MBL) is a plasma collectin (C-type lectin with a collagen-like domain) and is considered an important component of innate immunity. Circulating MBL is genetically determined for the major part, but plasma concentration is also markedly influenced by nongenetic factors. The carbohydrate-binding ability of MBL can be inhibited by simple sugars like mannose, fucose and N-acetylglucosamine, but its greatest avidity appears to be for repeating mannose-based structural patterns typical of microbial surfaces. By this means, MBL can bind to a wide variety of bacteria and other microbes, neutralizing them and/or opsonizing them by activating complement using the recently discovered lectin pathway of complement activation. Individual humans differ 1000-fold in MBL concentration, and individuals with low circulating MBL appear to be more vulnerable to infections in a number of clinical settings, especially when combined with secondary immune deficiency. The best evidence that MBL deficiency or insufficiency is physiologically relevant comes from a rapidly expanding literature of clinical studies. MBL insufficiency appears to be a significant risk factor for infections in infants, and for individuals of any age undergoing chemotherapy or post-transplant immunosuppression. Moreover, MBL appears to have a significant influence on the course of certain chronic diseases like rheumatoid arthritis and cystic fibrosis. Replacement therapy with a plasma-derived product is safe and seems promising, while recombinant MBL provides hope for large-scale therapeutic applications. Randomized clinical trials of MBL therapy, which are now on the horizon, should provide unambiguous evidence for the physiological significance of MBL in innate immunity.