Mechanism of multivalent glycoconjugate-lectin interaction: An update.

Lectins are predominantly oligomeric proteins with several binding sites per molecule. Glycoconjugates are their natural ligands, which often possess multiple binding epitopes. Thus, lectin-glycoconjugate interactions are mostly multivalent in nature. The mechanism of multivalent binding is fundamentally different from those described for monovalent interactions in textbooks and research papers. Over the years, binding studies that make use of different lectins and a variety of multivalent glycoconjugate ligands were conducted in order to understand the underlying principles of multivalency. Starting with seemingly simple synthetic multivalent analogs, systematic studies were carried out using natural glycoconjugate ligands with increasing valency and complexity. Those ligands included multivalent glycoproteins, polyvalent polysaccharides, including glycosaminoglycans, as well as supra-valent mucins and proteoglycans. Models and mechanisms of multivalent binding derived from quantitative data are summarized in the present updated review.

Selectively Modified Lactose and N-Acetyllactosamine Analogs at Three Key Positions to Afford Effective Galectin-3 Ligands.

Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((ß-D-galactopyranosyl)-(1→4)-ß-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-ß-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3' of each of the two sugars, resulting in a 3'-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl ß-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3' position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series.

Rapid Detection and Purification of Galectin-3 by the Capture and Release (CaRe) Method.

Specific interactions between lectins and glycoproteins determine the outcomes of numerous biological processes. To elucidate the roles of lectins and glycoproteins in those processes, it is essential to detect these proteins in biological samples and purify them to homogeneity. Conventional protein detection and purification techniques are multi-step, time-intensive, and expensive. They often require rigorous trial and error experimentations and fairly larger volumes of crude extracts. To minimize some of these challenges, we recently formulated a new method named Capture and Release (CaRe). This method is rapid, facile, precise, and inexpensive, and it works even when the sample volume is smaller. We developed this method to detect and purify recombinant human Galectin-3 and subsequently validated this method by purifying several other lectins. Besides lectins, CaRe is capable of detecting/purifying glycoproteins. In this method, targets (lectins and glycoproteins) are captured by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. CaRe can potentially be used as a tool to discover new lectins and glycoconjugates and elucidate their functions.

Revealing the Identity of Human Galectin-3 as a Glycosaminoglycan-Binding Protein.

Human galectin-3 (Gal-3) is a ß-galactoside-binding lectin. This multitasking protein preferentially interacts with N-acetyllactosamine moieties on glycoconjugates. Specific hydroxyl groups (4-OH, 6-OH of galactose, and 3-OH of glucose/N-acetylglucosamine) of lactose/LacNAc are essential for their binding to Gal-3. Through hemagglutination inhibition, microcalorimetry, and spectroscopy, we have shown that despite being a lectin, Gal-3 possesses the characteristics of a glycosaminoglycan (GAG)-binding protein (GAGBP). Gal-3 interacts with sulfated GAGs [heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC)] and chondroitin sulfate proteoglycans (CSPGs). Heparin, CSA, and CSC showed micromolar affinity for Gal-3, while the affinity of CSPGs for Gal-3 was much higher (nanomolar). Interestingly, CSA, CSC, and a bovine CSPG, not heparin and CSB, were multivalent ligands for Gal-3, and they formed reversible noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs to Gal-3 was completely inhibited when Gal-3 was preincubated with ß-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was also reversed by ß-lactose. These findings strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans. The GAG- and CSPG-binding properties of Gal-3 make the lectin a potential competitor/collaborator of other GAGBPs such as growth factors, cytokines, morphogens, and extracellular matrix proteins.

A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins.

Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).

A capture and release method based on noncovalent ligand cross-linking and facile filtration for purification of lectins and glycoproteins.

Glycan-binding proteins such as lectins are ubiquitous proteins that mediate many biological functions. To study their various biological activities and structure-function relationships, researchers must use lectins in their purest form. Conventional purification techniques, especially affinity column chromatography, have been instrumental in isolating numerous lectins and glycoproteins. These approaches, however, are time-consuming, consist of multiple steps, and often require extensive trial-and-error experimentation. Therefore, techniques that are relatively rapid and facile are needed. Here we describe such a technique, called capture and release (CaRe). The strength of this approach is rooted in its simplicity and accuracy. CaRe purifies lectins by utilizing their ability to form spontaneous noncovalently cross-linked complexes with specific multivalent ligands. The lectins are captured in the solution phase by multivalent capturing agents, released by competitive monovalent ligands, and then separated by filtration. CaRe does not require antibodies, solid affinity matrices, specialized detectors, a customized apparatus, controlled environments, or functionalization or covalent modification of reagents. CaRe is a time-saving procedure that can purify lectins even from a few milliliters of crude protein extracts. We validated CaRe by purifying recombinant human galectin-3 and five other known lectins and also tested CaRe's ability to purify glycoproteins. Besides purifying lectins and glycoproteins, CaRe has the potential to purify other glycoconjugates, including proteoglycans. This technique could also be used for nonlectin proteins that bind multivalent ligands. Given the ubiquity of glycosylation in nature, we anticipate that CaRe has broad utility.

Multitasking Human Lectin Galectin-3 Interacts with Sulfated Glycosaminoglycans and Chondroitin Sulfate Proteoglycans.

Glycosaminoglycan (GAG) binding proteins (GAGBPs), including growth factors, cytokines, morphogens, and extracellular matrix proteins, interact with both free GAGs and those covalently linked to proteoglycans. Such interactions modulate a variety of cellular and extracellular events, such as cell growth, metastasis, morphogenesis, neural development, and inflammation. GAGBPs are structurally and evolutionarily unrelated proteins that typically recognize internal sequences of sulfated GAGs. GAGBPs are distinct from the other major group of glycan binding proteins, lectins. The multifunctional human galectin-3 (Gal-3) is a ß-galactoside binding lectin that preferentially binds to N-acetyllactosamine moieties on glycoconjugates. Here, we demonstrate through microcalorimetric and spectroscopic data that Gal-3 possesses the characteristics of a GAGBP. Gal-3 interacts with unmodified heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC) as well as chondroitin sulfate proteoglycans (CSPGs). While heparin, CSA, and CSC bind with micromolar affinity, the affinity of CSPGs is nanomolar. Significantly, CSA, CSC, and a bovine CSPG were engaged in multivalent binding with Gal-3 and formed noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs was completely abolished when Gal-3 was preincubated with ß-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was reversed by ß-lactose. Both observations strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Hill plot analysis of calorimetric data reveals that the binding of CSA, CSC, and a bovine CSPG to Gal-3 is associated with progressive negative cooperativity effects. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans.

Glycan-Dependent Mutual and Reversible Sequestration of Two Thyroid Cancer Biomarkers.

BACKGROUND: Thyroglobulin (Tg), the major thyroidal protein, plays important roles in thyroid hormone biosynthesis and in autoimmune thyroid diseases (AITD). Tg also serves as a pre- and postoperative biomarker of differentiated thyroid cancer (DTC). The endogenous ß-galactoside binding lectin galectin-3 (Gal-3), secreted by transformed thyroid cells, has been shown to be another useful biomarker of DTC. Tg contains covalently linked complex-type glycans that can serve as binding epitopes of Gal-3. The objective of the study is to investigate the interaction between Tg and Gal-3 and discuss its potential consequences. METHODS: Binding interaction between Tg and Gal-3 was first studied by hemagglutination inhibition assays. Subsequently, a detailed analysis of binding thermodynamics was carried out by isothermal titration calorimetry. Quantitative precipitation was performed to study the complex formation between Tg and Gal-3 and to determine the binding stoichiometry. The concentration-dependent rate and amount of complex formation between Tg and Gal-3 was examined spectrophotometrically. A similar approach was taken to study the effect of free Tg and Gal-3 on preformed Tg-Gal-3 complex. RESULTS: Quantitative biochemical and biophysical data show that these two biomarkers produced by thyroid cancer cells interact with each other with submicromolar affinity and form an insoluble complex at their stoichiometric concentration. One Tg molecule could bind up to 14 molecules of Gal-3. Such complex formation mutually sequestered both Tg and Gal-3, decreasing the concentration of their freely available forms. Formation of the Tg-Gal-3 complex was reversible as the preformed complex was dissolved by free Tg as well as free Gal-3. While free Tg rapidly dissolved preformed Tg-Gal-3 complex in a concentration-dependent manner, Gal-3 was found to be much less efficient and slowly dissolved only a fraction of the preformed complex at a relatively higher Gal-3 concentration. CONCLUSIONS: Complex formation between Tg and Gal-3 through high affinity binding and the sensitivity of the complex to free Tg and Gal-3 can potentially influence their biological functions. Interactions between Tg and Gal-3 might also interfere with their clinical detection, the same way Tg autoantibody (TgAb) is reported to interfere with Tg assays. The data support a model of Gal-3-mediated homeostatic process of Tg.

Significant other half of a glycoconjugate: contributions of scaffolds to lectin-glycoconjugate interactions.

The glycan epitopes of natural and synthetic glycoconjugates exist as covalent attachments of well-defined inner structures or scaffolds. Macromolecules such as proteins, peptides, lipids, and saccharides and synthetic structures serve as scaffolds of glycoconjugates. It is generally perceived that the biological activities of glycoconjugates are determined mainly by the attached glycans, while the seemingly inert inner scaffolds play a passive role by providing physical support to the attached glycan epitopes. However, our data show that scaffolds actively influence lectin recognition and can potentially modulate lectin-mediated signaling properties of glycoconjugates. Through in vitro experiments, we found that the scaffolds significantly altered the thermodynamic binding properties of the covalently attached glycan epitopes. When a free glycan was attached to a scaffold, its lectin binding entropy became more positive. The level of positive entropic gain was dependent on the types of scaffolds tested. For example, protein scaffolds of glycoproteins were found to generate more positive entropy of binding than synthetic scaffolds. Certain scaffolds were found to have limiting effects on glycoconjugate affinity. We also found that scaffold-bearing glycans with a similar affinity or an identical valence demonstrated different kinetics of lattice formation with lectins, when the scaffold structures were different. Our data support the view that scaffolds of glycoconjugates (i) help the covalently attached glycans become more spontaneous in lectin binding and (ii) help diversify the lattice forming or cross-linking properties of glycoconjugates.

Implication of the VirD4 coupling protein of the Lvh type 4 secretion system in virulence phenotypes of Legionella pneumophila.

The genome of the Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires' disease, encodes two virulence-associated type 4 secretion systems (T4SSs), the Dot/Icm type 4B (T4BSS) and the Lvh type 4A (T4ASS). Broth stationary-phase cultures of most dot/icm mutants are defective in entry and evasion of phagosome acidification. However, those virulence defects can be reversed by incubating broth cultures of dot/icm mutants in water, termed water stress (WS). WS reversal requires the lvh T4ASS locus, suggesting an interaction between the two T4SSs in producing Legionella virulence phenotypes. In the current work, the loss of WS reversal in a dotA Δlvh mutant of strain JR32 was shown to be attributable to loss of the lvh virD4 gene, encoding the putative coupling protein of the T4ASS. Transformation of a dotA Δlvh mutant with virD4 also reversed entry and phagosome acidification defects in broth cultures. In addition, broth cultures of Δlvh and ΔvirD4 mutants, which were dot/icm(+), showed 5-fold and >6-fold increases in translocation of the Dot/Icm translocation substrates, proteins RalF and SidD, respectively. These data demonstrate that the Lvh T4ASS functions in both broth stationary-phase cultures conventionally used for infection and cultures exposed to WS treatment. Our studies in a dotA Δlvh mutant and in a dot/icm(+) background establish that VirD4 and the Lvh T4ASS contribute to virulence phenotypes and are consistent with independent functioning of Dot/Icm and Lvh T4SSs or functional substitution of the Lvh VirD4 protein for a component(s) of the Dot/Icm T4BSS.

Implication of proteins containing tetratricopeptide repeats in conditional virulence phenotypes of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a >10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ΔlpnE or the ΔenhC mutant required that the other TPR-containing protein be present. The single and double ΔlpnE and ΔenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ΔlpnE single and the ΔlpnE ΔenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates.

Environmental mimics and the Lvh type IVA secretion system contribute to virulence-related phenotypes of Legionella pneumophila.

Legionella pneumophila, the causative organism of Legionnaires' disease, is a fresh-water bacterium and intracellular parasite of amoebae. This study examined the effects of incubation in water and amoeba encystment on L. pneumophila strain JR32 and null mutants in dot/icm genes encoding a type IVB secretion system required for entry, delayed acidification of L. pneumophila-containing phagosomes, and intracellular multiplication when stationary-phase bacteria infect amoebae and macrophages. Following incubation of stationary-phase cultures in water, mutants in dotA and dotB, essential for function of the type IVB secretion system, exhibited entry and delay of phagosome acidification comparable to that of strain JR32. Following encystment in Acanthamoeba castellanii and reversion of cysts to amoeba trophozoites, dotA and dotB mutants exhibited intracellular multiplication in amoebae. The L. pneumophila Lvh locus, encoding a type IVA secretion system homologous to that in Agrobacterium tumefaciens, was required for restoration of entry and intracellular multiplication in dot/icm mutants following incubation in water and amoeba encystment and was required for delay of phagosome acidification in strain JR32. These data support a model in which the Dot/Icm type IVB secretion system is conditionally rather than absolutely required for L. pneumophila virulence-related phenotypes. The data suggest that the Lvh type IVA secretion system, previously thought to be dispensable, is involved in virulence-related phenotypes under conditions mimicking the spread of Legionnaires' disease from environmental niches. Since environmental amoebae are implicated as reservoirs for an increasing number of environmental pathogens and for drug-resistant bacteria, the environmental mimics developed here may be useful in virulence studies of other pathogens.

Icm/dot-independent entry of Legionella pneumophila into amoeba and macrophage hosts.

Legionella pneumophila, the causative agent of Legionnaires' disease, expresses a type IVB secretion apparatus that translocates bacterial proteins into amoeba and macrophage hosts. When stationary-phase cultures are used to infect hosts, the type IVB apparatus encoded by the icm/dot genes is required for entry, delay of phagosome-lysosome fusion, and intracellular multiplication within host cells. Null mutants with mutations in icm/dot genes are defective in these phenotypes. Here a new model is described in which hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.5 buffer. This model is called Ers treatment because it enhances the resistance to acid, hydrogen peroxide, and antibiotic stress beyond that of stationary-phase cultures. Following Ers treatment entry into amoeba and macrophage hosts does not require dotA, which is essential for Legionella virulence phenotypes when hosts are infected with stationary-phase cultures, dotB, icmF, icmV, or icmX. Defective host entry is also suppressed for null mutants with mutations in the KatA and KatB catalase-peroxidase enzymes, which are required for proper intracellular growth in amoeba and macrophage hosts. Ers treatment-induced suppression of defective entry is not associated with increased bacterial adhesion to host cells or with morphological changes in the bacterial envelope but is dependent on protein expression during Ers treatment. By using proteomic analysis, Ers treatment was shown to induce a protein predicted to contain eight tetratricopeptide repeats, a motif previously implicated in enhanced entry of L. pneumophila. Characterization of Ers treatment-dependent changes in expression is proposed as an avenue for identifying icm/dot-independent factors that function in the entry of Legionella into amoeba and macrophage hosts.

Legionella pneumophila catalase-peroxidases are required for proper trafficking and growth in primary macrophages.

Legionella pneumophila, a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of Legionella-containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in katA and katB, encoding the two Legionella catalase-peroxidases, were analyzed to evaluate the hypothesis that L. pneumophila must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing katA or katB mutant Legionella colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in dotA and dotB, encoding components of the Type IV secretion system. Quantitative similarity of the katA/B phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H(2)O(2) compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that dotA and dotB mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba Acanthamoeba castellanii, indicating that certain dotA/B functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent Legionella phagosomes in macrophages.