C-type Serum Lectin Research Articles

2600. Mannose-Binding Lectin Polymorphisms are Important Modulating Factors in Community- and Hospital-Acquired Pneumonia Caused by Legionella spp.

BackgroundCommunity-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP) are the leading causes of death from infection in developed countries. Mannose-binding lectin (MBL) is a C-type serum lectin that plays a central role in the innate pulmonary host defenses against respiratory pathogens. We hypothesized that MBL polymorphisms may increase the risk of developing Legionella pneumonia. Therefore, the aim of this study was to evaluate the role of MBL polymorphisms in susceptibility to CAP and HAP caused by Legionella spp.MethodsA total of 96 BAL and blood samples collected for routine microbiological analysis from Lithuanian patients presenting with CAP and HAP, were used for this study. MBL polymorphisms in the 54 codon (54 G/G, 54 G/A, and 54 A/A) were detected by BanIRFLP. Legionella spp. were detected by nested PCR.ResultsPolymorphisms in the 54 codon of MBL were determined in 96 patients. Among 96 patients with CAP and HAP, the most commonly observed 54 codon MBL variant was 54 G/G (69.8%, n = 67), followed by 54 G/A (21.9%, n = 21), and 54 A/A (8.3%, n = 8). By using nested PCR, Legionella pneumonia was detected among 15.6% (n = 15) of patients diagnosed with CAP and HAP. Legionella spp. were detected among 7 patients with MBL 54 codon G/G variant (10.4%), while 3 patients with MBL 54 G/A (14.3%), and 5 patients with MBL 54 codon A/A variant (62.5%). During this study Legionella spp. were detected more frequently (P < 0.05) among patients with the A/A variant of the 54 codon vs. those with other variants of MBL 54 codon. There were no significant differences found among those with Legionella infection and G/A (14.3%) or G/G (10.4%) variants of MBL gene 54 codon.Conclusion MBL gene 54 codon variant A/A polymorphisms may play an important role in increased susceptibility to lower respiratory infectionscaused by Legionella spp.Disclosures All authors: No reported disclosures.

Chicken mannose-binding lectin function in relation to antibacterial activity towards Salmonella enterica

Mannose-binding lectin (MBL) is a C-type serum lectin of importance in innate immunity. Low serum concentrations of MBL have been associated with greater susceptibility to infections. In this study, binding of purified chicken MBL (cMBL) to Salmonella enterica subsp. enterica (S. enterica) serotypes B, C1 and D was investigated by flow cytometry, and Staphylococcus aureus (S. aureus) was used for comparison. For S. enterica the C1 serotypes were the only group to exhibit binding to cMBL. Furthermore, functional studies of the role of cMBL in phagocytosis and complement activation were performed. Spiking with cMBL had a dose-dependent effect on the HD11 phagocytic activity of S. enterica subsp. enterica serovar Montevideo, and a more pronounced effect in a carbohydrate competitive assay. This cMBL dose dependency of opsonophagocytic activity by HD11 cells was not observed for S. aureus. No difference in complement-dependent bactericidal activity in serum with high or low cMBL concentrations was found for S. Montevideo. On the other hand, serum with high concentrations of cMBL exhibited a greater bactericidal activity to S. aureus than serum with low concentrations of cMBL. The results presented here emphasise that chicken cMBL exhibits functional similarities with its mammalian counterparts, i.e. playing a role in opsonophagocytosis and complement activation.

Mannan-Binding Protein, a C-Type Serum Lectin, Recognizes Primary Colorectal Carcinomas through Tumor-Associated Lewis Glycans

Mannan (mannose)-binding protein (MBP) is a C-type serum lectin that plays a key role in innate immunity. MBP forms large multimers (200-600 kDa) and exhibits broad specificity for mannose, N-acetylglucosamine, and fucose. MBP exhibits high affinity for unique oligosaccharides that have been isolated from human colorectal carcinoma (SW1116) cells and characterized as highly fucosylated high m.w. type 1 Lewis glycans. In this study, we first demonstrated that MBP recognizes human primary colorectal carcinoma tissues through tumor-associated MBP ligands. We performed fluorescence-based histochemistry of MBP in human colorectal carcinoma tissues and showed that MBP clearly stained cancer mucosae in a Ca(2+)-dependent manner. Coincubation with plant (Aleuria aurantia) lectin, but not Con A, blocked MBP staining, indicating that fucose, rather than mannose, is involved in this interaction. The expression of MBP ligands was detected in 127 of 330 patients (38.5%), whereas, most significantly, there was no expression in 69 nonmalignant tissues. The MBP-staining pattern in cancer mucosae significantly overlapped with that of Lewis b [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] staining, but the Lewis b staining in normal tissues was not associated with MBP staining. In addition, the MBP staining correlated inversely with the expression of CA19-9 Ag, and MBP stained 11 of 25 (44%) CA19-9 (sialyl Lewis a [NeuAc(α2-3)Galβ1-3(Fucα1-4)GlcNAc])(-) colorectal carcinoma tissues. We found a favorable prognosis in patients with MBP ligand(+) tumors. These results suggest that selective recognition of cancer cells by endogenous MBP seems to be associated with an antitumor effect and that tissue staining with MBP in combination with CA19-9 may serve as a novel indicator of colorectal carcinoma tissues.

Role of interaction of mannan-binding protein with meprins at the initial step of complement activation in ischemia/reperfusion injury to mouse kidney

Ischemia/reperfusion (I/R) is an important cause of acute renal failure. Recent studies have shown that the complement system mediated by the mannan-binding protein (MBP), which is a C-type serum lectin recognizing mannose, fucose and N-acetylglucosamine residues, plays a critical role in the pathogenesis of ischemic acute renal failure. MBP causes complement activation through the MBP lectin pathway and a resulting complement component, C3b, is accumulated on the brush borders of kidney proximal tubules in a renal I/R-operated mouse kidney. However, the initial step of the complement activation has not been studied extensively. We previously identified both meprins α and β, highly glycosylated zinc metalloproteases, localized on kidney proximal tubules as endogenous MBP ligands. In the present study, we demonstrated that serum-type MBP (S-MBP) and C3b were co-localized with meprins on both the cortex and the medulla in the renal I/R-operated mouse kidney. S-MBP was indicated to interact with meprins in vivo in the I/R-operated mouse kidney and was shown to initiate the complement activation through the interaction with meprins in vitro. Taken together, the present study strongly suggested that the binding of S-MBP to meprins triggers the complement activation through the lectin pathway and may cause the acute renal failure due to I/R on kidney transplantation and hemorrhagic shock.

Association of mannose-binding lectin gene polymorphisms with Kawasaki disease in the Japanese

Objective: Kawasaki disease (KD) is a systemic vasculitis in childhood; its etiology is unknown. The possibility that KD is an infectious disease has been discussed and investigated for decades, in light of the implication that infections are involved in the pathogenesis of KD. Young children rely on their innate immune system for protection against virus and micro‐organisms. Human mannose binding lectin (MBL) is a C‐type serum lectin synthesized by the liver as an acute phase protein and it plays an important role in the innate immune system. Here, we investigate the relationship between the MBL gene polymorphisms and the occurrence of KD in the Japanese population. Method: The frequencies of the genotypes, defined as mutations in codons 52, 54 and 57, and the functional promoter variants of the MBL were determined in 45 patients with KD. Results: The MBL codon‐54 polymorphism frequency of heterozygote (GGC/GAC) and promoter variants were significantly higher in the KD group than that in the control group (P < 0.05). Neither group showed codon 52 nor 57 polymorphisms. Conclusion: It is possible that mutations of the MBL gene might be related to the trigger of the pathogenesis of Kawasaki disease.

Protective role of mouse MBL-C on intestinal mucosa during Shigella flexneri invasion

Mannan-binding lectin (MBL) is a C-type serum lectin, which is believed to play an important role in the innate immunity against a variety of pathogens. MBL can bind to sugar determinants of a wide variety of microorganisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Given that small intestine is a predominant site of extrahepatic expression of MBL, here we addressed the question whether MBL is involved in mucosal innate immunity. The carbohydrate recognition domain (CRD) genes of mouse MBL-C (mMBL-C) were cloned and expressed in Escherichia coli. Recombinant mMBL-C-CRD binds to Shigella flexneri 2a in a calcium-dependent manner and that interaction could be blocked by the anti-mMBL-C-CRD antibody. mMBL-C-CRD protein could inhibit the adhesion of S. flexneri 2a to intestinal mucosa, while administration of anti-mMBL-C-CRD antibody caused an increased level of bacteria adhesion in vitro. Administration of recombinant mMBL-C-CRD protein reduced the secretion of IL-6 and monocyte chemoattractant protein 1 from primary intestinal epithelial cells stimulated with S. flexneri 2a. Furthermore, neutralization of MBL activity by anti-MBL-C-CRD resulted in a significant increase in the number of S. flexneri 2a that colonized the intestines of BALB/c mice and attenuated the severity of inflammation seen in the areas of bacterial invasion. These findings suggest that mMBL-C may protect host intestinal mucosa by directly binding to the bacteria.

Isolation, Cloning, and Characterization of a Novel Phosphomannan-binding Lectin from Porcine Serum

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.

Mannan-Binding Protein Blocks the Activation of Metalloproteases Meprin α and β

Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin alpha and beta (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases.

Characterization of Oligosaccharide Ligands Expressed on SW1116 Cells Recognized by Mannan-binding Protein: A HIGHLY FUCOSYLATED POLYLACTOSAMINE TYPE N-GLYCAN

Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.

Impact of mannose-binding lectin on susceptibility to infectious diseases.

When the adaptive immune response is either immature or compromised, the innate immune system constitutes the principle defense against infection. Mannose-binding lectin (MBL) is a C-type serum lectin that plays a central role in the innate immune response. MBL binds microbial surface carbohydrates and mediates opsonophagocytosis directly and by activation of the lectin complement pathway. A wide variety of clinical isolates of bacteria, fungi, viruses, and parasites are bound by MBL. Three polymorphisms in the structural gene MBL2) and 2 promoter gene polymorphisms are commonly found that result in production of low serum levels of MBL. Clinical studies have shown that MBL insufficiency is associated with bacterial infection in patients with neutropenia and meningococcal sepsis. Low MBL levels appear to predispose persons to HIV infection. Numerous other potential infectious disease associations have been described. Therapy to supplement low MBL levels is being explored using either plasma-derived or recombinant material.

Significant Elevations in Serum Mannose-Binding Lectin Levels in Patients with Chronic Renal Failure

Background/Aims: Mannose-binding lectin (MBL), a liver-derived C-type serum lectin, activates the complement cascade through the lectin pathway. Since the complement system contributes to the host defense against infections and mediates inflammatory processes including atherosclerosis, and since chronic renal failure (CRF) patients are prone to the development of infectious complications and cardiovascular disease, we focused on serum MBL levels in CRF patients who were either uremic, or who were receiving hemodialysis treatment. Methods: MBL levels were measured in the sera of subjects with CRF before they began dialysis treatment (pre-HD patients; n = 23) and in the sera of subjects who were receiving maintenance hemodialysis (HD patients; n = 178), by ELISA using polyclonal anti-rabbit IgG and a monoclonal antibody directed against MBL (3E7). Results: Mean levels (± SD) of serum MBL were significantly higher in pre-HD subjects (4.343 ± 2.533 µg/ml, p < 0.05) and in HD subjects (8.897 ± 4.920 µg/ml, p < 0.05), than in healthy controls (1.452 ± 0.692 µg/ml). Levels were also significantly higher in HD subjects than in pre-HD subjects (p < 0.05). Conclusions: Elevated serum MBL levels in patients with CRF might have significantly influence pathologic conditions such as alterations of the immune system and acceleration of atherogenesis.

Human mannan-binding lectin inhibits the infection of influenza A virus without complement.

Mannan-binding lectin (MBL) is a C-type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagen-like sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro-organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV-A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti-human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non-damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.

Sialic‐acid‐binding lectin from the slug Limax flavus

A cDNA library of Limax flavus was constructed and screened for sialic-acid-specific lectins. Complementary DNA clones were categorized into seven groups corresponding to closely related but different sequences. Group 1 clones contained an ORF encoding 199 amino acids including a sequence identical to the partial amino acid sequence obtained from the lectin protein. Within its 1074-bp 3' untranslated region, ten closely related 60-bp sequence repeats were found. Group 2 clones contained an ORF encoding a polypeptide chain of the same number of amino acid residues, with 89.1% overall identity to that of the group 1 and eight 60-bp repeat sequences in the 3' untranslated region. The remaining groups of clones contained ORF with highly similar full or partial sequences, with or without 60 bp repeats in the 3' untranslated region. The large number of closely related but different cDNA clones obtained indicated that the slug sialic-acid-specific lectin gene is a member of a multigene family. The lectin amino acid sequence showed significant similarity with the fibrinogen domain of human tenascin-C, with a human C-type serum lectin, and with pig ficolin. Immunostaining analysis of slug tissue for the lectin indicated that it is present primarily on the epidermal surface and in mucous glands. Recombinant slug lectin protein lacking the 20-amino-acid N-terminal signal sequence produced in a bacterial expression system from a group-1 clone accumulated as aggregates in inclusion bodies, suggesting that large-scale production of the active agglutinin may be possible.

Role of the Collagen-like Domain of the Human Serum Mannan-Binding Protein in the Activation of Complement and the Secretion of This Lectin

Human mannan-binding protein (MBP) is a C-type serum lectin Involved In an Immunoglobulin-independent host defense. Recently, a common defect of immune opsonin was found to be associated with very low serum levels of MBP. This deficiency was thought to be an effect of a single base substitution in the MBP gene. which resulted in the conversion of G1y54 to Asp. In this study, three mutant MBPs were expressed in COS-1 cells and the effects of these mutations were studied. Gly54Asp-MBP and Gly57Asp-MBP were secreted into the medium almost at the same levels as the wild type MBP, but the formation of higher multimers and the activation of complement were interfered with significantly. The other mutant, in which Leu was Inserted into the G1y63-Gln-Gly sequence to restore the collagen motif of the Gly-Xaa-Yaa repeat, was secreted at levels similar to that of the wild type, formed higher multimers and activated complement in a manner not significantly altered from that of the wild type. These results are discussed with regard to the molecular basis of patients with opsonic defects.

Distinct and overlapping functions of allelic forms of human mannose binding protein.

Human mannose binding protein (MBP) is a C-type serum lectin involved in first-line host defense against a variety of bacterial, fungal and viral pathogens. Recently an association was found between low levels of serum MBP and an increased frequency of recurrent infections in infants. A particular genotype, in which glycine is substituted by aspartic acid at codon 54 of MBP in the fifth collagen repeat, shows apparent concordance with the clinical phenotype. We report, however, that this genotype occurs in 5% of the population and encodes a functional protein. Our results indicate that the Gly54Asp allele does not account for a deficiency state, but instead suggest that MBP may have two predominant allelic forms that have overlapping function and differ only in their ability to activate the classical pathway of complement.