Importance of carbohydrate in the interaction of Tamm-Horsfall protein with complement 1q and inhibition of classical complement activation

Tamm-Horsfall protein (THP) binds strongly to complement 1q (C1q), a key component of the classical complement pathway. The goals of this study were to determine whether THP altered the activation of the classical complement pathway and whether the carbohydrate portion of THP was involved in this glycoprotein's binding to C1q and alteration of complement activation. The ability of THP to prevent complement activation in diluted serum or plasma incubated at 37 degrees C was assessed using both a haemolytic assay with antibody-sensitized sheep RBC and a C4d ELISA. Both these methods showed that THP inhibited activation of the classical complement pathway in a dose-dependent manner. Glycosidases were used to remove most of the carbohydrate from THP. This partially deglycosylated THP bound human IgG with a higher affinity (KD1 = 1.4 nmol/L; KD2 = 0.31 micromol/L) than did intact THP (KD1 = 33.4 nmol/L; KD2 = 31.0 micromol/L). An ELISA showed that removal of carbohydrate from THP reduced, but did not eliminate, the ability of this protein to inhibit binding of C1q to intact THP. Haemolysis assays using antibody-sensitized sheep RBC showed that removal of THP carbohydrate eliminated the ability of THP to protect against complement activation. In conclusion, THP inhibited the activation of the classical complement pathway that occurred in diluted serum or plasma. The carbohydrate moieties of THP appeared to be important in this inhibitory activity.     

Engineering of PA-IIL lectin from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> – Unravelling the role of the specificity loop for sugar preference

Background  

Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose – a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action.     

Mouse lymphoma cell lines resistant to pea lectin are defective in fucose metabolism

Two mutants of the BW5147 mouse lymphoma cell line have been selected for their resistance to the toxic effects of pea lectin. These cell lines, termed PLR1.3 and PHAR1.8 PLR7.2, have a decreased number of high affinity pea lectin-binding sites (Trowbridge, I.S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Intact cell labeling experiments using [2-3H]mannose indicated that PLR1.3 cells have a block in the conversion of GDP-[3H]mannose to GDP-[3H]fucose whereas PHAR1.8 PLR7.2 cells appear to be blocked in the transfer of fucose from GDP-[3H]fucose to glycoprotein acceptors. In vitro experiments with extracts of PLR1.3 cells confirmed the failure to convert GDP-mannose to GDP-fucose and indicated that the defect is in GDP-mannose 4,6-dehydratase (EC 4.2.1.47), the first enzyme in the conversion of GDP-mannose to GDP-fucose. The block in the PLR1.3 cells could be bypassed by growing the cells in the presence of fucose, demonstrating that an alternate pathway for the production of GDP-fucose presumably via fucose 1-phosphate is functional in this line. PLR1.3 cells grown in 10 mM fucose showed normal high affinity pea lectin binding. PHRA1.8 PLR7.2 cells synthesize GDP-fucose and have normal or increased levels of GDP-fucose:glycoprotein fucosyltransferase when assayed in vitro. The fucosyltransferases of this clone can utilize its own glycoproteins as fucose acceptors in in vitro assays. These findings indicate that this cell line fails to carry out the fucosyltransferase reaction in vivo despite the fact that it possesses the appropriate nucleotide sugar, glycoprotein acceptors, and fucosyltransferase. The finding of decreased glycoprotein fucose in two independent isolates of pea lectin-resistant cell lines and the restoration of high affinity pea lectin binding to PLR1.3 cells following fucose feeding strongly implicates fucose as a major determinant of pea lectin binding.     

Lectin Complement Protein Collectin 11 (CL-K1) and Susceptibility to Urinary Schistosomiasis

BackgroundUrinary Schistosomiasis is a neglected tropical disease endemic in many sub Saharan -African countries. Collectin Kidney 1 (CL-K1, encoded by COLEC11 on chromosome 2p25.3), a member of the vertebrate C-type lectin super family, has recently been identified as pattern-recognition molecule (PRR) of the lectin complement pathway. CL-K1 is preferentially expressed in the kidneys, but also in other organs and it is considered to play a role in host defense to some infectious agents. Schistosome teguments are fucosylated and CL-K1 has, through its collagen-like domain, a high binding affinity to fucose.ConclusionsIn agreement with findings from our earlier published study, our findings support the observation that CL-K1 and their functional variants may be host factors associated with protection in schistosomiasis and may be a useful marker for further investigations.     

Abnormal processing of the modified oligosaccharide side chains of phytohemagglutinin in the presence of swainsonine and deoxynojirimycin

Phytohemagglutinin, the glycoprotein lectin of the common bean, Phaseolus vulgaris, has both high-mannose (Man_8-9GlcNAc₂) and modified oligosaccharide side chains. The modified side chains have glucosamine, mannose, fucose, and xylose in the molar ratios 2:3.8:0.6:0.5, and are resistant to hydrolysis by endoglycosidase H. Synthesis and processing of side chains in the presence of 1-deoxynojirimycin, an inhibitor of α-glucosidase, results in the formation of chains which are all alike. They are sensitive to endoglycosidase H, do not contain fucose, and are largely resistant to α-mannosidase. This indicates that they are probably high-mannose chains blocked by terminal glucose residues. Synthesis and processing of side chains in the presence of swainsonine, an inhibitor of α-mannosidase II, results in the formation of normal high-mannose chains, and of modified chains which contain fucose residues, are resistant to endoglycosidase H, and can be distinguished from normal modified chains only by the presence of extra mannose residues.

Processing of the phytohemagglutinin modified chains of PHA under normal conditions involves the attachment of peripheral N-acetylglucosamine residues in the Golgi complex and their subsequent removal in the protein bodies. The attachment of the N-acetylglucosamine residues is largely inhibited by deoxynojirimycin but still occurs in the presence of swainsonine. The results presented in this work show that processing of the asparagine-linked oligosaccharides is under the control of several glycosidases and glycosyltransferases and involves the formation of intermediate products.

     

The binding of fucose-containing glycoproteins by hepatic lectins. The binding specificity of the rat liver fucose lectin

The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA.     

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.     

Isolation and characterization of lectins specific for mannose/fucose/N-acetylglucosamine from rat peritoneal macrophages

Rat peritoneal macrophages were shown to have two distinct mannose/fucose/N-acetylglucosamine-specific lectins. The major lectin of 180 kDa, which is similar in size to the mannose receptor first isolated from alveolar macrophages (Wileman, T.E., Lennartz, M.R., & Stahl, P.D. (1986) Proc. Natl. Acad. Sci. U.S. 83, 2501-2505), was shown to occur as a dimer under nondenaturing conditions. The 29 and 32 kDa lectins were identified as members of the liver mannan-binding protein family on the basis of their immunochemical crossreactivity, collagenase sensitivity, and molecular sizes (Oka, S., Ikeda, K., Kawasaki, T., & Yamashina, I. (1988) Arch. Biochem. Biophys. 260, 257-266). Despite the similarity in the sugar binding specificity, these two types of lectin were clearly differentiated with regard to the binding to IgM molecules. The 29 and 32 kDa lectins bound to IgM most likely through high-mannose type oligosaccharides on IgM, whereas the 180 kDa lectin did not.     

Investigations on Collectin Liver 1

Collectins are pattern recognition molecules of the innate immune system showing binding to carbohydrate structures on microorganisms in a calcium-dependent manner. Recently, three novel collectins, collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1 and CL-11), and collectin placenta 1 (CL-P1), were discovered. The roles of these three collectins remain largely unknown. Here, we present a time-resolved immunofluorometric assay for quantification of CL-L1. The concentration of CL-L1 in donor plasma (n = 210) was distributed log-normally with a median value of 3.0 μg/ml (range 1.5–5.5 μg/ml). We observed on average 30% higher concentrations of CL-L1 in plasma as compared with serum. Size analysis by gel-permeation chromatography showed CL-L1 in serum to elute as large 700–800-kDa complexes and smaller 200–300-kDa complexes. CL-L1 showed specific binding to mannose-TSK beads in a Ca²⁺-dependent manner. This binding could be inhibited by mannose and glucose, but not galactose, indicating that CL-L1 binds via its carbohydrate-recognition domain and has ligand specificity similar to that of mannan-binding lectin. Western blot analysis of CL-L1 showed the presence of several oligomeric forms in serum. Ontogeny studies showed CL-L1 to be present at birth at near adult levels. CL-L1 levels exhibit low variation in healthy adults over a 1-year period. During acute-phase responses, the CL-L1 levels display only minor variations. In serum, CL-L1 was found in complexes with mannan-binding lectin-associated serine proteases, suggesting a role in the lectin pathway of complement activation. The presented data establish a basis for future studies on the biological role of CL-L1.     

Epithelial repair is inhibited by an alpha(1,6)-fucose binding lectin

The effective repair of damage to the airway epithelium is essential to maintain the ability to exclude airborne particulates and protect against potential pathogens. Carbohydrates on the cell surface have an important role in cell-cell and cell substrate interactions. Using a model of repair with airway epithelial-derived cells of the 16HBE 14o(-) cell line, we have examined the effect of the Aleuria aurantia lectin (AAL), which binds very selectively to alpha(1,6)-linked fucose residues. Addition of unconjugated or FITC-labeled AAL reduced the rate of epithelial repair to approximately one-third of control values as measured by image analysis while cell viability was maintained. Pulse labeling with AAL-FITC for 30 min followed by incubation in AAL-free medium caused similar inhibition of repair but could be reversed by addition of fucose up to 7 h after AAL removal. By confocal microscopy, AAL binding was found to be on the apical, but not basolateral, surfaces of cells, and internalization of the labeled lectin was seen. Preincubation of the lectin with fucose prevented this effect. Ulex europeaus I lectin, which is also fucose specific, resulted in similar binding to the cells and internalization, but it did not affect the speed of the repair process. We conclude that alpha(1,6)-fucose binding sites play an important role in epithelial repair. Better understanding of this process will provide a deeper insight into the crucial mechanisms of epithelial repair.     

The binding of fucose-containing glycoproteins by hepatic lectins. Purification of a fucose-binding lectin from rat liver

A lectin with a high affinity for binding ligands through fucose residues has been purified to homogeneity from rat liver. Affinity chromatography of the lectin on fucosyl-bovine serum albumin-agarose is the key step in the purification. Contaminating amounts of a previously described lectin that binds mannose and N-acetylglucosamine are removed from the fucose-binding lectin by either immunoadsorption on anti-mannose/N-acetylglucosamine lectin IgG-agarose or by specific elution of the fucose-binding lectin from fucosyl-bovine serum albumin-agarose. The pure fucose-binding lectin contains two polypeptide subunits with molecular weights of 88,000 and 77,000, respectively, as judged by gel electrophoresis. Peptide maps of the subunits, however, show that they are very similar structurally. In addition, peptide maps show that the fucose lectin is structurally distinct from other rat hepatic lectins. This is supported by the lack of cross-reaction among the different rat liver lectins and their specific antibodies and the inability of specific antibodies to the mannose/N-acetylglucosamine lectin to inhibit the binding of fucosyl-bovine serum albumin by the fucose lectin.     

Localization of fucosyl moieties in the mouse renal cortex by lectin histochemistry using the fucose binding lectins LTA and UEA I and by autoradiography using 3H-labelled fucose

Summary The binding patterns of the two fucose binding lectins, Lotus tetragonolobus (LTA) and Ulex europeus I (UEA I) were investigated using fluorescence lectin histochemistry on the unfixed renal cortex of the mouse (NMRI) embedded in LR-Gold. The fluorescence staining results were compared with the autoradiographic localization of the incorporation of radioactive fucose into the renal cortex. For this study the turnover of incorporated ³H-fucose in the renal cortex was investigated 30 min, 2 h and 8 h after application. The localization of the radioactive fucose within the renal cortex corresponded well to the labelling pattern observed for lecting histochemistry using LTA. In contrast, with UEA I, no binding sites for this lectin could be observed. The results of our investigation clearly showed that fucosyl moieties in the renal cortex of the NMRI mouse are recognized by the fucose binding lecting LTA, but not by UEA I and that postembedding fluorescence histochemistry with LTA on the LR-Gold embedded kidney is a suitable technique for the localization of fucosyl moieties at the light microscopical level.     

Evidence for glycoprotein components of the hepatocellular bile acid transporter

The hepatocellular transporter, responsible for the uptake of bile acids and some foreign substances, can be shown to contain carbohydrate moieties. The hepatocellular uptake of cholate and phallotoxin is immediately inhibited by addition of wheat-germ agglutinin. Concanavalin A and lentil lectin reduce the uptake in a time-dependent manner. Apparently sialic acids or N-acetylglucosamine residues are involved in the translocation process. Polypeptides (Mr 50,000, 54,000) of the above transport system, identified by affinity labeling with [3H]isothiocyanatobenzamido cholate and [3H2]diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid, are heterogenously glycosylated. Binding of 80-90% of the 54, 50 kDa polypeptides to all immobilized lectins tested suggests that both high-mannose and complex type oligosaccharides with fucose and terminal sialic acid residues occur as carbohydrate chains. A 67 kDa labeled polypeptide is not glycosylated. Pilot experiments for purification of the above glycosylated membrane proteins on concanavalin A, lentil lectin and wheat-germ lectin columns are described. However, lectin affinity chromatography is not suitable as a one-step purification procedure for the labeled polypeptides.     

Search for fucose binding domains in recently sequenced hypothetical proteins using molecular modeling techniques and structural analysis

The crystal structure of a fucose-binding lectin from the bacteria Pseudomonas aeruginosa in complex with α-L-fucose has been recently determined. It is a tetramer; each monomer displays a nine-stranded, antiparallel, β-sandwiched arrangement and contains two calcium ions that mediate the binding of fucose in a recognition mode unique among protein-carbohydrate interactions. In search of this type of unique interactions in other newly discovered protein sequences, we have used molecular modeling techniques to predict and analyze the 3-D structures of some proteins, which exhibited reasonable degree of homology with the amino acid sequence of the bacterial protein. A BLAST search with the sequence of Pseudomonas aeruginosa as query in the non-redundant sequence database identified four proteins from different species, three organisms from bacteria and one from archaea. We have modeled the structures of these proteins as well as those of the complexes with carbohydrates and studied the nature of physicochemical forces involved in the complex formation both in presence and absence of calcium. The calcium-binding loops have been found to be highly conserved both in terms of primary and tertiary structures in these proteins, although a less acidic character is observed in Photorhabdus lectin due to the absence of two aspartic acid residues on the calcium-binding loop which also resulted in lower binding affinity. All these structures exhibited highly negative electrostatic environment in the vicinity of the calcium-binding loops which was essential for neutralizing the positive charges of two closely situated Ca⁺² ions. The comparison of the binding affinities of some monosaccharides other than fucose, e.g. mannose and fructose, showed higher binding energies confirming the fucose specificity of these proteins.     

Studies on phytohemagglutinins I. Some properties of the lectins of horse gram seeds (Dolichos biflorus L.)

The anti-A₁ lectin of Dolichos biflorus L. seeds has been isolated from the active protein fraction obtained by 40–60% saturation by ammonium sulfate of a saline extract (1 : 5) followed by successive chromatography on DEAE-cellulose, Sephadex G-100 and DEAE-cellulose. The lectin has a molecular weight of 122 000 and contains 1.25% neutral sugar, 0.8% glucosamine and only one terminal amino acid, alanine. Neutral sugar components comprise mannose, glucose, fucose and xylose.On the other hand, affinity chromatography of the same active protein fraction on O-(N-acetyl-α-d-galactosaminy;) polyacrylamide yields with different electrophoretic mobilities and with an overall neutral sugar content of 3.7%. Electrophoresis on polyacrylamide gel reveals a diffused broad band of unequal intensity. On ultracentrifugation the lectin shows a molecular weight of 124 000 with a tendency to form a dimer. Lectin isolated by affinity techniques contains 0.17% Ca, 0.11% Zn, 0.07% Mn and 0.01% Mg; lectin obtained by conventional chormatographic procedure shows a somewhat decreased content of Zn and Ca.Digestion with pepsin and pronase of the lectin isolated by conventional procedures yields a mixture of fragments from which three glycopeptides designated as G1, G2A and G2B can be isolated by chromatography on Sephadex G-25 and preparative paper electrophoresis. G1 contains aspartic acid, threonine, proline, glycine, alanine, glucose and fucose and its calculated minimal molecular weight is approximately 2800; glycopeptide G2A contains aspartic acid, serine, glucosamine, mannose and fucose and has a calculated minimal molecular weight of approximately 1800.The purified lectin isolated by either of the two methods is active in concentration of 10 μg/ml against A₁-erythrocytes. Its erythroagglutinating activity is enhanced by Ca²⁺, Co²⁺, Ni²⁺ and Mg²⁺ and inhibiited by both N-acetyl-d-galactosamine and EDTA. The mixture of glycopeptides G2 isoalted by pronase digestion shows erythroagglutinating activity at the same concentration, but is nonspecific.     

Detection of a high affinity binding site in recombinant <Emphasis Type="Italic">Aleuria aurantia</Emphasis> lectin

Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K (d) values in the micromolar range. This would make mushroom lectins ideal candidates to study protein-carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K (d) values in the nanomolar range. This site could bind to oligosaccharides with fucose linked alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.     

Differential ability to resist to complement lysis and invade host cells mediated by MBL in R4 and 860 strains of Trypanosoma cruzi

To produce an infection Trypanosoma cruzi must evade lysis by the complement system. During early stages of infection, the lectin pathway plays an important role in host defense and can be activated by binding of mannan-binding lectin (MBL) to carbohydrates on the surface of pathogens. We hypothesized that MBL has a dual role during parasite-host cell interaction as lectin complement pathway activator and as binding molecule to invade the host cell. We used two polarized strains of T. cruzi, R4 (susceptible) and 860 (resistant) strains, to investigate the role of MBL in complement-mediated lysis. Interestingly R4, but not 860 metacyclic strain, markedly increases the invasion of host cells, suggesting that MBL drives the invasion process while the parasite deactivates the Lectin complement pathway.     

Collectin 11 (CL-11, CL-K1) is a MASP-1/3-associated plasma collectin with microbial-binding activity

Collectins play important roles in the innate immune defense against microorganisms. Recently, a new collectin, collectin 11 (CL-11 or CL-K1), was identified via database searches. In present work, we characterize the structural and functional properties of CL-11. Under nonreducing conditions, in gel permeation chromatography recombinant CL-11 forms disulfide-linked oligomers of 100 and 200 kDa. A mAb-based ELISA estimates the concentration of CL-11 in plasma to be 2.1 μg/ml, and the presence of CL-11 in plasma was further verified by Western blotting and mass spectrometry. Mannan-binding lectin-associated serine protease 1 (MASP-1) copurified with CL-11 and the interaction in plasma with MASP-1 and/or MASP-3 was further demonstrated using ELISA. We identified the adrenal glands, the kidneys, and the liver as primary sites of expression. CL-11 lectin activity was demonstrated by ELISA and showed that CL-11 has preference for l-fucose and d-mannose. We finally show that CL-11 binds to intact bacteria, fungi, and viruses and that CL-11 decreases influenza A virus infectivity and forms complexes with DNA. On the basis of the significant concentration of CL-11 in circulation and CL-11's interaction with various microorganisms and MASP-1 and/or MASP-3, it is conceivable that CL-11 plays a role in activation of the complement system and in the defense against invading microorganisms.     

Unusual entropy-driven affinity of Chromobacterium violaceum lectin CV-IIL toward fucose and mannose

The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.     

The distribution and localization of the fucose-binding lectin in rat tissues and the identification of a high affinity form of the mannose/N-acetylglucosamine-binding lectin in rat liver

A small-scale affinity chromatographic procedure was developed to screen for the presence of fucose and mannose/N-acetylglucosamine-binding lectins in small amounts of rat tissues. Of all tissues examined, only the liver contained the fucose-binding lectin, whereas both liver and blood serum contained the mannose/N-acetylglucosamine lectin. By means of immunocytological methods using antibodies to hepatic lectins, the fucose lectin was shown to be uniquely present in Kupffer cells and absent in all other types of rat macrophages examined. The binding and uptake of different neoglycoproteins by nonparenchymal cell fractions of liver indicated that the fucose-binding lectin was either not responsible for the uptake or that more than one lectin was acting. With the identification of another lectin (Mr = 180,000) by the above screening procedure for hepatic lectins and the results of studies in the following paper (Haltiwanger, R.S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7440-7444) two lectins appear to be involved. A small amount of the hepatic mannose/N-acetylglucosamine lectin was found by the above screening procedure to have a higher affinity for L-fucosyl-bovine serum albumin-Sepharose than the majority of the lectin in hepatocytes. This lectin, called the high affinity form, was purified and its properties examined. On a weight basis the high affinity form bound 7-12 times more ligand than the normal form. Its Ka for L-fucosyl-bovine serum albumin was 2.3 X 10(9) M-1 compared to 3.5 X 10(8) M-1 for the normal form. Moreover, the concentrations of monosaccharides required to inhibit the high affinity form were about 3 times less than those required to inhibit binding of the normal form. The two forms, however, have identical molecular weights (32,000) under reducing and nonreducing conditions, bind anti-lectin antibodies in the same way, and give identical peptide maps after V-8 protease digestion. The structural basis for the different binding affinities of the two forms remains unknown.