Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin–N-glycan interactions

Background

Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed.

Methods

The roles of human and bovine lactoferrins involved in lectin–N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins.

Results and conclusions

Both native and asialo human/bovine lactoferrins reacted strongly with four Man-specific lectins — Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a LFuc>Man-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin-Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAc>Gal-specific lectin-Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four Gal>GalNAc-specific type-2 ribosome inactivating protein family lectins-ricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA.

General significance

This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin–N-glycan recognition processes.     

Purification and characterization of lectin from fruiting body of Ganoderma lucidum: lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5-9 and temperature up to 50 degrees C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantennary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

Nile Tilapia Neu3 sialidases: Molecular cloning,functional characterization and expression in Oreochromis niloticus

Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227 bp, 1194 bp and 1155 bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9 kDa, 44.4 kDa and 43.6 kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH 4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish.     

β-N-Acetylhexosaminidase: What's in a name…?

β-N-Acetylhexosaminidases (EC 3.2.1.52, belonging to CAZy GH families 3, 20 and 84) have recently gained a lot of attention, not only due to their implication in human physiology and disease, but also due to their great potential in the enzymatic synthesis of carbohydrates and glycomimetics. GH family 20 β-N-acetylhexosaminidases, and GH family 3 and 84 β-N-acetylglucosaminidases from all kinds of organisms have been intensively studied from the point of view of their physiological roles, reaction mechanisms, structure and inhibition. Thanks to their outstanding substrate promiscuity, extracellular β-N-acetylhexosaminidases from filamentous fungi are able to cleave and transfer substrates bearing various functionalities, ranging from carboxylates, sulfates, acylations to azides, and even 4-deoxy glycosides. Thus, they have proved to be versatile biosynthetic tools for the preparation of both natural and modified hexosaminides under mild conditions with good yields.     

Conformational change of a unique sequence in a fungal galectin from Agrocybe cylindracea controls glycan ligand-binding specificity

A fungal galectin from Agrocybe cylindracea (ACG) exhibits broad binding specificity for β-galactose–containing glycans. We determined the crystal structures of wild-type ACG and the N46A mutant, with and without glycan ligands. From these structures and a saccharide-binding analysis of the N46A mutant, we revealed that a conformational change of a unique insertion sequence containing Asn46 provides two binding modes for ACG, and thereby confers broad binding specificity. We propose that the unique sequence provides these two distinct glycan-binding modes by an induced-fit mechanism.     

Molecular Cloning and Crystal Structural Analysis of a Novel β-N-Acetylhexosaminidase from Paenibacillus sp. TS12 Capable of Degrading Glycosphingolipids

We report the molecular cloning and characterization of two novel β-N-acetylhexosaminidases (β-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two β-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with β-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 β-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the β-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-ΔC at a resolution of 1.8 Å; Hex1-ΔC in complex with β-N-acetylglucosamine at 1.6 Å; and Hex1-ΔC in complex with β-N-acetylgalactosamine at 1.9 Å. We made a docking model of Hex1-ΔC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the molecular cloning, characterization and X-ray structure of a procaryotic β-HEX capable of hydrolyzing glycosphingolipids.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc]₂ and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K_m and k_cat values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4–G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2–G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1–G5, and then the high-M_r intermediates (G3–G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.     

Biodegradation of phenanthrene and pyrene by Ganoderma lucidum

Biodegradation of two polycyclic aromatic hydrocarbons (PAHs), phenanthrene and pyrene, by a white rot fungus, Ganoderma lucidum, in broth cultures was investigated. It was found that the biomass of the organism decreased with the increase of PAH concentration in the cultures. In the cultures with 2 to 50 mg l⁻¹ PAHs, the degradation rate constants (k₁) increased with the PAH concentration, whereas, at the level of 100 mg l⁻¹, the degradation rate constants decreased. In the presence of 20 mg l⁻¹ PAHs, the highest degradation rates of both PAHs occurred in cultures with an initial pH of 4.0 at 30 °C. The addition of CuSO₄, citric acid, gallic acid, tartaric acid, veratryl alcohol, guaiacol, 2,2′-azino-bis-(3- ethylbenzothazoline-6-sulfonate) (ABTS) enhanced the degradation of both PAHs and laccase activities; whereas the supplement of oxalate, di-n-butyl phthalate (DBP), and nonylphenol (NP) decreased the degradation of both PAHs and inhibited laccase production. In conclusion, G. lucidum is a promising white rot fungus to degrade PAHs such as phenanthrene and pyrene in the environment.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

Cytotoxic triterpenoids from Ganoderma lucidum

A systematic study of the metabolites in Ganoderma lucidum led to isolation of 43 triterpenoids, six of them (1-6) are hitherto unknown. The structures of the latter were elucidated on the basis of spectroscopic studies and comparison with the known related compounds. All of the compounds were assayed for their inhibitory activities against human HeLa cervical cancer cell lines. Some compounds exhibit significant cytotoxicity, and their structure-activity relationships are discussed.     

Allelopathy is involved in the formation of pure colonies of the fern Gleichenia japonica

The fern Gleichenia japonica is one of the most widely distributed fern and occurs throughout East to South Asia. The species often dominates plant communities by forming large monospecific colonies. However, the potential mechanism for this domination has not yet been described. The objective of this study was to test the hypothesis that allelochemicals are involved in the formation of G. japonica colonies. An aqueous methanol extract of G. japonica inhibited the growth of seedlings of garden cress (Lepidium sativum), lettuce (Lactuca sativa), ryegrass (Lolium multiflorum) and timothy (Phleum pratense). Increasing extract concentration increased the inhibition. These results suggest that G. japonica contain allelopathic substances. The extract was then purified by several chromatographies with monitoring the inhibitory activity and two growth inhibitory substances causing the allelopathic effect were isolated. The chemical structures of the two substances were determined by spectral data to be a novel compound 3-O-β-allopyranosyl-13-O-β-fucopyranosyl-3β-hydroxymanool (1) and 18-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-13-epitorreferol (2). These compounds inhibited the shoot and root growth of garden cress, lettuce, alfalfa (Medicago sativa), timothy, ryegrass and barnyardgrass (Echinochloa crus-galli) at concentrations greater than 0.1–1.0 mM. The concentrations required for 50% growth inhibition of root and shoot growth of these test plants ranged from 0.72 to 3.49 mM and 0.79 to 3.51 mM for compounds 1 and 2, respectively. Concentration of compounds 1 and 2 in soil under the pure colony of G. japonica was 4.9 and 5.7 mM, respectively, indicating concentrations over those required for 50% growth inhibition are potentially available under monocultural stands of these ferns. Therefore, these compounds may contribute to the allelopathic effects caused by presence of G. japonica and may thus contribute to the establishment of monocultural stands by this fern.     

Antitumor activity of sulfated extracellular polysaccharides of Ganoderma lucidum from the submerged fermentation broth

Water-soluble extracellular polysaccharides are known to possess weak or no in vitro antitumor activity. In this experiment, a mixture of extracellular Ganoderma lucidum polysaccharides (GLP) from the submerged fermentation broth was sulfated and studied on their antitumor activity. The sulfated GLP performed significant inhibition on the proliferation of assayed carcinoma cells in a dose-dependent manner, and present a degree of substitution-dependent suppressing to HepG2 cells. Meanwhile, the sulfated GLP presented remarkable but not dose-dependent inhibition on Heps hepatona in mice. With same degree of substitution, the sulfation protocol with aminosulfonic acid-pyridine yielded GLP sulfates with higher activity on HepG2 cells. In comparison, the native GLP showed no or little antitumor activity on the assayed cell lines but remarkable inhibition on suppressing the proliferation of rat Heps. The highest in vivo inhibition rate of 55.5% provided by sulfated GLP was observed on suppressing the proliferation of rat Heps.     

Halogranum salarium sp. nov., a halophilic archaeon isolated from sea salt

Three halophilic archaea, strains B-1^T, B-3 and B-4, were isolated from evaporitic salt crystals from Namhae, Korea. Cells of the strains were Gram-stain-negative, motile and pleomorphic, and colonies were red-pigmented. The three isolates had identical 16S rRNA gene sequences and formed a tight phylogenetic clade with Halogranum rubrum RO2-11^T in the genus Halogranum, showing 99.5% sequence similarity. The next most closely related species were Halogranum amylolyticum and Halogranum gelatinilyticum (97.4 and 96.3% similarity to the respective type strains). The phylogeny based on the full-length RNA polymerase subunit B′ gene (rpoB′) was in agreement with the 16S rRNA gene sequence analysis, but allowed better discrimination. DNA-DNA hybridization between a representative strain (B-1^T) and the type strains of Hgn. rubrum, Hgn. amylolyticum and Hgn. gelatinilyticum revealed less than 40% relatedness. Polar lipid analysis showed that the three isolates contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and three glycolipids. Combined genotypic and phenotypic data supported the conclusion that strains B-1^T, B-3 and B-4 represent a novel species of the genus Halogranum, for which the name Halogranum salarium sp. nov. is proposed. The type strain is B-1^T (=KCTC 4066^T = DSM 23171^T).     

Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in β-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of β1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAcβ1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.     

Genome-wide analysis of simple sequence repeats in the model medicinal mushroom Ganoderma lucidum

Simple sequence repeats (SSRs) or microsatellites are one of the most popular sources of genetic markers and play a significant role in gene function and genome organization. We identified SSRs in the genome of Ganoderma lucidum and analyzed their frequency and distribution in different genomic regions. We also compared the SSRs in G. lucidum with six other Agaricomycetes genomes: Coprinopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium, Postia placenta, Schizophyllum commune and Serpula lacrymans. Based on our search criteria, the total number of SSRs found ranged from 1206 to 6104 and covered from 0.04% to 0.15% of the fungal genomes. The SSR abundance was not correlated with the genome size, and mono- to tri-nucleotide repeats outnumbered other SSR categories in all of the species examined. In G. lucidum, a repertoire of 2674 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. The highest SSR relative abundance was found in introns (108 SSRs/Mb), followed by intergenic regions (84 SSRs/Mb). A total of 684 SSRs were found in the protein-coding sequences (CDSs) of 588 gene models, with 81.4% of them being tri- or hexa-nucleotides. After scanning for InterPro domains, 280 of these genes were successfully annotated, and 215 of them could be assigned to Gene Ontology (GO) terms. SSRs were also identified in 28 bioactive compound synthesis-related gene models, including one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), three polysaccharide biosynthesis genes and 24 cytochrome P450 monooxygenases (CYPs). Primers were designed for the identified SSR loci, providing the basis for the future development of SSR markers of this medicinal fungus.     

Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose–agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 °C, retained partial activity by treatment at 70 °C, and was fully inactivated at 90 °C. On western blot analysis, SauFBP showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, the similarity of the N-terminal sequence and a partial coding domain to teleost F-type lectins suggests that SauFBP32 is a member of this emerging family of lectins.     

Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: A study involving site-directed mutagenesis,HPLC, and real-time ESI-MS

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5–10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of + 1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC₅₀ value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from − 3 to − 1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.     

Purification and characterization of a lectin from endophytic fungus Fusarium solani having complex sugar specificity

A lectin from the mycelial extract of an endophytic strain of Fusarium solani was purified. Its hemagglutinating activity was inhibited by glycoproteins possessing N-linked as well as O-linked glycans. The thermodynamics and kinetics of binding of glycans and glycoproteins to F. solani lectin was studied using surface plasmon resonance. The lectin showed high affinity for asialofetuin, asialomucin, asialofibrinogen, and thyroglobulin; and comparatively low affinity for mucin, fetuin, fibrinogen, and holotransferrin. Glycoproteins showed several fold higher affinity than their corresponding glycans with significant contribution from enthalpy and positive entropy, suggesting the involvement of non-polar protein-protein interaction. Moreover, the higher affinity of the glycoproteins was due to their faster association rates and low activation energy.