Analyses of Pseudomonas aeruginosa Lectin Binding to α-Galactosylated Glycans

The specificity and binding capacity of the galactophilic lectin from the Gram negative bacterium Pseudomonas aeruginosa (PA-IL) was determined by solid phase measurements using galactosylated neoglycoproteins immobilized on microtiter plates. The bacterial lectin reacted with both short chain (monosaccharide) and long chain (pentasaccharide) glycoconjugates. Among the Galα1-XGal disaccharides, the highest affinity was observed towards the Galα1-3Gal structure. Raising the incubation temperature enhanced the lectin-polysaccharide agglutination, and it is suggested that binding to certain conformations of polysaccharides could vary between lectins with the same monocarbohydrate specificity and that this activity may, in part, be temperature dependent. Histochemical examination of lectin binding to different porcine tissues suggests a differential glycosylation of the carbohydrate antigens on endothelial cells in various parts of the vascular system. In the pancreas, PA-IL also adhered to the excretory ducts. These observations on PA-IL binding could be of importance both to determine infection foci in P. aeruginosa-mediated vacuities and to determine its role for pancreatic involvement in cystic fibrosis.     

Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives. From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Galalpha1-->6Glc. As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.      

Structural Basis for Multiple Sugar Recognition of Jacalin-related Human ZG16p Lectin

ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related β-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1–3Man and Glcβ1–3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using ¹⁵N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition.     

A Single Point Mutation in the Gene Encoding Gb3/CD77 Synthase Causes a Rare Inherited Polyagglutination Syndrome

Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR_int, and NOR2, in which Gal(α1–4), GalNAc(β1–3)Gal(α1–4), and Gal(α1–4)GalNAc(β1–3)Gal(α1–4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1–4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1–4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1–4)Gal and Gal(α1–4)GalNAc moieties.     

A Novel Core Fucose-specific Lectin from the Mushroom Pholiota squarrosa

Fucα1–6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1–6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH₂-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1–6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1–2-, α1–3-, and α1–4-fucosylated glycans. Furthermore, PhoSL bound to α1–6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1–6 fucosylation between primary and metastatic colon cancer tissues. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1–6 fucosylation and evaluating Fucα1–6 oligosaccharides as cancer biomarkers.     

Identification of Novel α1,3-Galactosyltransferase and Elimination of α-Galactose-containing Glycans by Disruption of Multiple α-Galactosyltransferase Genes in Schizosaccharomyces pombe

The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of d-galactose (Gal) in addition to d-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via α1,2- or α1,3-linkages. Previously we constructed and characterized a septuple α-galactosyltransferase disruptant (7GalTΔ) anticipating a complete lack of α-Gal residues. However, the 7GalTΔ strain still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating the presence of at least one more additional unidentified α1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1⁺–otg3⁺ (α one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalTΔ strain demonstrated that the resultant disruptant missing 10 α-galactosyltransferase genes, 10GalTΔ, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2⁺ gene product had Gal transfer activity toward a pyridylaminated Man₉GlcNAc₂ oligosaccharide and pyridylaminated Manα1,2-Manα1,2-Man oligosaccharide. In addition, the otg3⁺ gene product exhibited Gal transfer activity toward the pyridylaminated Man₉GlcNAc₂ oligosaccharide. Generation of an α1,3-linkage was confirmed by HPLC analysis, α-galactosidase digestion analysis, ¹H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in α1,3-galactosylation of S. pombe oligosaccharides.     

Molecular Clock Regulates Daily α1–2-Fucosylation of the Neural Cell Adhesion Molecule (NCAM) within Mouse Secondary Olfactory Neurons

The circadian clock regulates various behavioral and physiological rhythms in mammals. Circadian changes in olfactory functions such as neuronal firing in the olfactory bulb (OB) and olfactory sensitivity have recently been identified, although the underlying molecular mechanisms remain unknown. We analyzed the temporal profiles of glycan structures in the mouse OB using a high-density microarray that includes 96 lectins, because glycoconjugates play important roles in the nervous system such as neurite outgrowth and synaptogenesis. Sixteen lectin signals significantly fluctuated in the OB, and the intensity of all three that had high affinity for α1–2-fucose (α1–2Fuc) glycan in the microarray was higher during the nighttime. Histochemical analysis revealed that α1–2Fuc glycan is located in a diurnal manner in the lateral olfactory tract that comprises axon bundles of secondary olfactory neurons. The amount of α1–2Fuc glycan associated with the major target glycoprotein neural cell adhesion molecule (NCAM) varied in a diurnal fashion, although the mRNA and protein expression of Ncam1 did not. The mRNA and protein expression of Fut1, a α1–2-specific fucosyltransferase gene, was diurnal in the OB. Daily fluctuation of the α1–2Fuc glycan was obviously damped in homozygous Clock mutant mice with disrupted diurnal Fut1 expression, suggesting that the molecular clock governs rhythmic α1–2-fucosylation in secondary olfactory neurons. These findings suggest the possibility that the molecular clock is involved in the diurnal regulation of olfaction via α1–2-fucosylation in the olfactory system.     

Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase⁺ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca²⁺ and Mg²⁺) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.     

Computational and Experimental Studies on β-Sheet Breakers Targeting Aβ1–40 Fibrils

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ_1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ_1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH₂ and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH₂) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH₂). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ_1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH₂ β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ_1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ_1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ_1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH₂ β-sheet breaker.     

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from Phanerochaete chrysosporium

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln²⁶³ serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.     

X-ray Structures of Human Galectin-9 C-terminal Domain in Complexes with a Biantennary Oligosaccharide and Sialyllactose

Galectin-9, a tandem-repeat-type β-galactoside-specific animal lectin with two carbohydrate recognition domains (CRDs) at the N- and C-terminal ends, is involved in chemoattraction, apoptosis, and the regulation of cell differentiation and has anti-allergic effects. Its ability to recognize carbohydrates is essential for its biological functions. Human galectin-9 (hG9) has high affinity for branched N-glycan-type oligosaccharides (dissociation constants of 0.16–0.70 μm) and linear β1–3-linked poly-N-acetyllactosamines (0.09–8.3 μm) and significant affinity for the α2–3-sialylated oligosaccharides (17–34 μm). Further, its N-terminal CRD (hG9N) and C-terminal CRD (hG9C) differ in specificity. To elucidate this unique feature of hG9, x-ray structures of hG9C in the free form and in complexes with N-acetyllactosamine, the biantennary pyridylaminated oligosaccharide, and α2–3-sialyllactose were determined. They are the first x-ray structural analysis of C-terminal CRD of the tandem-repeat-type galectin. The results clearly revealed the mechanism by which branched and α2–3-sialylated oligosaccharides are recognized and explained the difference in specificity between hG9N and hG9C. Based on structural comparisons with other galectins, we propose that the wide entrance for ligand binding and the shallow binding site of hG9C are favorable for branched oligosaccharides and that Arg²²¹ is responsible for recognizing sialylated oligosaccharides.     

Characterization of Five β-Glycoside Hydrolases from Cellulomonas fimi ATCC 484

The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.     

Molecular Cloning of Pigeon UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase and UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Gal��1�C4Gal��1�C4Gal��1�C4GlcNAc Sequence

We previously found that pigeon IgG possesses unique N-glycan structures that contain the Galα1–4Galβ1–4Galβ1–4GlcNAc sequence at their nonreducing termini. This sequence is most likely produced by putative α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1–4Gal and Galβ1–4Gal sequences on the N-glycans, respectively. Because no such glycan structures have been found in mammalian glycoproteins, the biosynthetic enzymes that produce these glycans are likely to have distinct substrate specificities from the known mammalian GalTs. To study these enzymes, we cloned the pigeon liver cDNAs encoding α4GalT and β4GalT by expression cloning and characterized these enzymes using the recombinant proteins. The deduced amino acid sequence of pigeon α4GalT has 58.2% identity to human α4GalT and 68.0 and 66.6% identity to putative α4GalTs from chicken and zebra finch, respectively. Unlike human and putative chicken α4GalTs, which possess globotriosylceramide synthase activity, pigeon α4GalT preferred to catalyze formation of the Galα1–4Gal sequence on glycoproteins. In contrast, the sequence of pigeon β4GalT revealed a type II transmembrane protein consisting of 438 amino acid residues, with no significant homology to the glycosyltransferases so far identified from mammals and chicken. However, hypothetical proteins from zebra finch (78.8% identity), frogs (58.9–60.4%), zebrafish (37.1–43.0%), and spotted green pufferfish (43.3%) were similar to pigeon β4GalT, suggesting that the pigeon β4GalT gene was inherited from the common ancestors of these vertebrates. The sequence analysis revealed that pigeon β4GalT and its homologs form a new family of glycosyltransferases.     

Host-Specific Enzyme-Substrate Interactions in SPM-1 Metallo-β-Lactamase Are Modulated by Second Sphere Residues

Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.     

Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.     

Galectin-3 Regulates Desmoglein-2 and Intestinal Epithelial Intercellular Adhesion

The desmosomal cadherins, desmogleins, and desmocollins mediate strong intercellular adhesion. Human intestinal epithelial cells express the desmoglein-2 isoform. A proteomic screen for Dsg2-associated proteins in intestinal epithelial cells identified a lectin referred to as galectin-3 (Gal3). Gal3 bound to N-linked β-galactosides in Dsg2 extracellular domain and co-sedimented with caveolin-1 in lipid rafts. Down-regulation of Gal3 protein or incubation with lactose, a galactose-containing disaccharide that competitively inhibits galectin binding to Dsg2, decreased intercellular adhesion in intestinal epithelial cells. In the absence of functional Gal3, Dsg2 protein was internalized from the plasma membrane and degraded in the proteasome. These results report a novel role of Gal3 in stabilizing a desmosomal cadherin and intercellular adhesion in intestinal epithelial cells.     

High Mannose-Binding Antiviral Lectin PFL from Pseudomonas fluorescens Pf0-1 Promotes Cell Death of Gastric Cancer Cell MKN28 via Interaction with α2-Integrin

Novel anti-HIV lectin family which shows a strict binding specificity for high mannose glycans has been found in lower organisms. The bacterial orthologue has been identified in the genome of Pseudomonas fluorescens Pf0-1 and the gene coding a putative lectin was cloned, expressed in Escherichia coli and purified by one step gel filtration. Glycan array screening of the recombinant lectin, termed PFL, has revealed that PFL preferentially recognizes high mannose glycans with α1-3 Man that was highly exposed at the D2 position. In contrast, masking of this α1-3 Man with α1-2 Man dramatically impaired lectin-carbohydrate interactions. Reducing terminal disaccharide, GlcNAc-GlcNAc of high mannose glycans was also essential for PFL-binding. PFL showed a potent anti-influenza virus activity by inhibiting the virus entry into cells at doses of low nanomolar concentration. At micromolar concentration or higher, PFL showed a cytotoxicity accompanying loss of the cell adhesion against human gastric cancer MKN28 cells. The cell surface molecule to which PFL bound was co-precipitated with biotin-labeled PFL and identified as integrin α2 by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Intriguingly, upon treatment with exogenous PFL, integrin α2 on the cell surface underwent rapid internalization to the cytoplasm and accumulated to perinuclear region, together with the bound PFL. The resulting loss of cell adherence would trigger a signaling pathway that induced anoikis-like cell death. These events were effectively inhibited by pretreatment of PFL with mannnan, indicating the involvement of high mannose glycans on PFL-induced cell death that was triggered by PFL-integrin α2 interactions.     

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

A novel lectin structure was found for a 17-kDa α-d-galactose-binding lectin (termed “MytiLec”) isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45–52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1–4Galβ1–4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.     

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding

To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K_d) of polyamides 1 and 2 were 1.1 (± 0.3) × 10^–6 M and 1.7 (± 0.4) × 10^–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (^mCs), the K_d value of polyamide 2 increased to 5.8 (± 0.7) × 10^–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.