Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.     

Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene

CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata. This lectin shows very high N-acetylgalactosamine-binding specificity. We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells. Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps. The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca(2+)-concentrations, whereas it was weaker at lower Ca(2+)-concentrations due to decreased Ca(2+)-binding affinity. rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GalNAc-binding specificity, as examined by hemagglutination inhibition assay. Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Ca(2+). Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures. Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity. These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I. Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I.     

Evolutionary conservation of intron position in a subfamily of genes encoding carbohydrate-recognition domains

The structure of the gene encoding a chicken liver receptor, the chicken hepatic lectin, which mediates endocytosis of glycoproteins has been established. The coding sequence is divided into six exons separated by five introns. The first three exons correspond to separate functional domains of the receptor polypeptide (cytoplasmic tail, transmembrane sequence, and extracellular neck region), while the final three exons encode the Ca(2+)-dependent carbohydrate-recognition domain. These results, as well as computer-assisted multiple sequence comparisons, establish this receptor as the evolutionary homolog of the mammalian asialoglycoprotein receptors. It is interesting that the chicken receptor falls into a subfamily of proteins along with the mammalian asialoglycoprotein receptors, since the saccharide-binding specificity of the chicken receptor resembles more closely that of a different set of calcium-dependent animal lectins, which includes the mannose-binding proteins. The portions of the genes encoding the carbohydrate-recognition domains of these proteins lack introns. The results suggest that divergence of intron-containing and intron-lacking carbohydrate-recognition domains preceded shuffling events in which other functional domains were associated with the carbohydrate-recognition domains. This was followed by further divergence, generating a variety of saccharide-binding specificities.     

Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata

CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.     

A comparative study of bark lectins from three elderberry (Sambucus) species

Three elderberry lectins isolated from the bark of three different species of the genus Sambucus which are native to Europe (S. nigra), North America (S. canadensis), and Japan (S. sieboldiana) were studied comparatively with regard to their carbohydrate binding properties and some structural features. All three lectins contained two identical carbohydrate binding sites per molecule and showed a very high specificity for the Neu5Ac(alpha 2-6)-Gal/GalNAc sequence. However, relative affinities for various oligosaccharides were significantly different among them, suggesting differences in the detailed structure of the carbohydrate binding sites of these lectins. The three lectins were immunologically related, but not identical, and all were composed of hydrophobic and hydrophilic subunit regions, although the molecular sizes of these subunits were slightly different among the three lectins. N-terminal sequence analysis of the subunits of these lectins suggested that they have a very similar structure in this region but also indicated the occurrence of N-terminal processing such as the deletion of several amino acid residues at the N-termini for both hydrophobic and hydrophilic subunits of all three lectins. Tryptic peptide mapping of the three lectins showed a similar pattern for all of them but also showed the presence of some unique peptides for each lectin.     

Mannan-Binding Lectin of the Sea Urchin Strongylocentrotus nudus

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/β-protein with eight β-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.     

Caenorhabditis elegans galectins LEC-1-LEC-11: structural features and sugar-binding properties

Galectins form a large family of beta-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2-5 and 8-11) from a lambdaZAP cDNA library. Among them, lec-2-5 were found to encode 31-35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8-11 were found to encode 16-27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1-4, -6, and 8-10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various beta-galactosides, i.e., N-acetyllactosamine (Galbeta1-4GlcNAc), lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), and asialofetuin, demonstrated that LEC-1-4, -6, and -10 have a significant affinity for beta-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galbeta1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.     

Purification,characterization, molecular cloning,and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J. Smith (Polypodiaceae)

A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.     

The amino-acid sequence of the abalone (Haliotis laevigata) nacre protein perlucin. Detection of a functional C-type lectin domain with galactose/mannose specificity.

Perlucin isolated from abalone nacre consists of 155 amino acids including a glycosylated asparagine. The sequence of the first 130 amino acids shows a high similarity to the C-type carbohydrate-recognition domains of asialoglycoprotein receptors and other members of the group of C-type lectins but also a weaker similarity to related proteins without carbohydrate-binding activity. This C-type module is followed by a short C-terminal domain containing two almost identical sequence repeats with a length of 10 amino acids. Solid phase assays show a divalent metal ion-dependent binding of perlucin to (neo)glycoproteins containing D-galactose or D-mannose/D-glucose indicating that perlucin is a functional C-type lectin with broad carbohydrate-binding specificity. Our results also indicate that it may be difficult to predict carbohydrate-binding specificity and the occurrence of alternative binding configurations by amino-acid sequence comparisons and homology modeling.     

Minimal requirements for the binding of selectin ligands to a C-type carbohydrate-recognition domain.

The C-type carbohydrate-recognition domains of E-selectin and rat serum mannose-binding protein have similar structures. Selectin/mannose-binding protein chimeras created by transfer of key sequences from E-selectin into mannose-binding protein have previously been shown to bind the selectin ligand sialyl-Lewis(X) through a Ca(2+)-dependent subsite, common to many C-type lectins, and an accessory site containing positively charged amino acid residues. Further characterization of these chimeras as well as analysis of novel constructs containing additional regions of E-selectin demonstrate that selectin-like interaction with sialyl-Lewis(X) can be faithfully reproduced even though structural evidence indicates that the mechanisms of binding to E-selectin and the chimeras are different. Selectin-like binding to the nonfucosylated sulfatide and sulfoglucuronyl glycolipids can also be reproduced with selectin/mannose-binding protein chimeras that contain the two subsites involved in sialyl-Lewis(X) binding. These results indicate that binding of structurally distinct anionic glycans to C-type carbohydrate-recognition domains can be mediated by the Ca(2+)-dependent subsite in combination with a positively charged region that forms an ionic strength-sensitive subsite.     

Genomic analysis of C-type lectins

Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell-cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.     

Use of a library of mutated Maackia amurensis hemagglutinin for profiling the cell lineage and differentiation

Thirty-five variant lectins were prepared by mutations of two amino acids within the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH). Each lectin showed unique carbohydrate specificity according to their bindings to soluble polyacrylamide with various mono- and oligosaccharides and to glycophorin A. The relative intensity of the bindings of carcinoma, myeloid, fibroblastic, and melanoma cells to immobilized MAH variant lectins was examined. Each cell line showed distinct profiles regarding the number of cells bound to wild-type and 35 MAH variants and the differences and the similarities in these binding profiles were quantitatively documented by the cluster analysis. The cell lines were classified into several groups and these groups surprisingly corresponded to the lineage of the cells. These results indicated that a library of mutated MAH is useful as a tool for the profiling of various cells based on the variations of the surface glycans.     

The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose

We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC(50) for mannose of approximately 20 mM compared to an IC(50) of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man(9)GlcNAc(2)). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.     

Ligand-binding characteristics of rat serum-type mannose-binding protein (MBP-A). Homology of binding site architecture with mammalian and chicken hepatic lectins

Sugar-binding characteristics of rat serum mannose-binding protein (MBP) were studied using the carbohydrate-recognition domain of this protein expressed from a cloned cDNA. To assess the binding affinity of various test compounds, they were added as inhibitors in a binding assay in which 125I-MBP was incubated with yeast cells and the extent of binding was estimated from the radioactivity associated with the pelleted cells. The results of such inhibition assays suggest that MBP has a small binding site which is probably of the trough-type. The 3- and 4-OH of the target sugar are indispensable, while the 6-OH is not required. These characteristics are shared by the rat hepatic lectin and chicken hepatic lectin, both of which are C-type lectins containing carbohydrate-recognition domains highly homologous to that of MBP. Apparently, the related primary structures of these lectins give rise to similar gross architecture of their binding sites, despite the fact that each exhibits different sugar binding specificities.     

Identification, cloning, and characterization of two N-acetylgalactosamine-binding lectins from the albumen gland of Helix pomatia

Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant production of HPA, is required. A combination of isoelectric focusing, amino acid sequence analysis, and cloning revealed two polypeptides in native HPA preparations (HPAI and HPAII), both consistent with GalNAc-binding lectins of the H-type family. Pairwise sequence alignment showed that HPAI and HPAII share 54% sequence identity whereas molecular modeling using SWISS-MODEL suggests they are likely to adopt similar tertiary structure. The inherent heterogeneity of native HPA highlighted the need for production of functional recombinant protein; this was addressed by preparing His-thioredoxin-tagged fusion products in Escherichia coli Rosetta-gami B (DE3) cells. The recombinant lectins agglutinated human blood group A erythrocytes whereas their oligosaccharide specificity, evaluated using glycan microarrays, showed that they predominantly bind glycans with terminal α-GalNAc residues. Surface plasmon resonance with immobilized GalNAc-BSA confirmed that recombinant HPAI and HPAII bind strongly with this ligand (K(d) = 0.60 nm and 2.00 nm, respectively) with a somewhat higher affinity to native HPA (K(d) = 7.67 nm). Recombinant HPAII also bound the breast cancer cells of breast cancer tissue specimens in a manner similar to native lectin. The recombinant HPA described here shows important potential for future studies of cancer cell glycosylation and as a reagent for cancer prognostication.     

Cloning a rat meprin cDNA reveals the enzyme is a heterodimer.

The structure of the kidney microvillar membrane metallopeptidase meprin (EC 3.4.24.18) from rats has been examined. Previously reported to be a homotetramer, we demonstrate that the enzyme is composed of two similar but distinct subunits through tryptic peptide mapping and the sequencing of peptides of the papain solubilized form of the enzyme. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the native rat meprin tetramer is dissociated by detergent into disulfide-linked heterodimers. A full-length cDNA clone encoding one of the meprin subunits has been isolated and sequenced. The cDNA contains an open reading frame of 668 amino acids, coding for a polypeptide of molecular weight 75,054. The enzyme contains the zinc binding sequence HEFLH and a potential membrane-spanning region near its amino terminus. Comparison of this clone with peptide sequences from mouse meprins A and B shows that the clone is a B type or beta subunit. Northern blot analysis is consistent with the existence of two distinct subunits and further indicates that rat meprin subunits may be differentially expressed in various rat tissues.     

The crystal structure of the carbohydrate-recognition domain of the glycoprotein sorting receptor p58/ERGIC-53 reveals an unpredicted metal-binding site and conformational changes associated with calcium ion binding

p58/ERGIC-53 is a calcium-dependent animal lectin that acts as a cargo receptor, binding to a set of glycoproteins in the endoplasmic reticulum (ER) and transporting them to the Golgi complex. It is similar in structure to calcium-dependent leguminous lectins. We have determined the structure of the carbohydrate-recognition domain of p58/ERGIC-53 in its calcium-bound form. The structure reveals localized but large conformational changes in relation to the previously determined metal ion-free structure, mapping mostly to the ligand-binding site. It reveals the presence of two calcium ion-binding sites located 6A apart, one of which has no equivalent in the plant lectins. The second metal ion-binding site present in that class of lectins, binding Mn(2+), is absent from p58/ERGIC-53. The absence of a short loop in the ligand-binding site in this protein suggests that it has adapted to optimally bind the high-mannose Man(8)(GlcNAc)(2) glycan common to glycoproteins at the ER exit stage.     

Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism

The seeds of jack fruit (Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the beta-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post-translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 A resolution, respectively. The structure of the lectin complexed with methyl-alpha-mannose, has also been determined at 2.9 A resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the specificities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the beta-prism I fold.