R-type Lectin Research Articles

Elucidating the glycan-binding specificity and structure of Cucumis melo agglutinin, a new R-type lectin.

Plant lectins have garnered attention for their roles as laboratory probes and potential therapeutics. Here, we report the discovery and characterization of Cucumis melo agglutinin (CMA1), a new R-type lectin from melon. Our findings reveal CMA1's unique glycan-binding profile, mechanistically explained by its 3D structure, augmenting our understanding of R-type lectins. We expressed CMA1 recombinantly and assessed its binding specificity using multiple glycan arrays, covering 1,046 unique sequences. This resulted in a complex binding profile, strongly preferring C2-substituted, beta-linked galactose (both GalNAc and Fuca1-2Gal), which we contrasted with the established R-type lectin Ricinus communis agglutinin 1 (RCA1). We also report binding of specific glycosaminoglycan subtypes and a general enhancement of binding by sulfation. Further validation using agglutination, thermal shift assays, and surface plasmon resonance confirmed and quantified this binding specificity in solution. Finally, we solved the high-resolution structure of the CMA1 N-terminal domain using X-ray crystallography, supporting our functional findings at the molecular level. Our study provides a comprehensive understanding of CMA1, laying the groundwork for further exploration of its biological and therapeutic potential.

The mannose-binding protein from Agaricus bisporus inhibits the growth of MDA-MB-231 spheroids.

A mannose-binding protein from the mushroom Agaricus bisporus (Abmb) inhibits the growth of MDA-MB-231 cells, which is of an aggressive breast cancer subtype. This ability was observed in a monolayer cell (2D) culture setup, which often is unable to capture changes in cell morphology, polarity and division. That shortcoming may overestimate Abmb potency for its development as a pharmaceutical agent and its use in a therapy. Hence, Abmb's inhibition to the cell growth was performed in the 3D cell (spheroid) culture, which is more representative to the situation in vivo. The result showed that, although the presence of Abmb at ~14.7 μM already disrupted the MDA-MB-231 cell morphology in the 2D culture, its presence at ~16.5 μM only ceased the growth of the MDA-MB-231 spheroid. Further, Abmb is unique because structurally it belongs to the R-type lectin (RTL) family; most of mannose-binding protein is of the C-type lectin (CTL). As the natural ligand of Abmb is unknown thus the mechanism of action is unclear, Abmb effect on the cancer cells was assessed via observation of the altered expression of genes involved in the Wnt/β-catenin signalling, which is one of the canonical pathways in the proliferation of cancer cells. The results suggested that Abmb did not alter the pathway upon exerting its anti-proliferative activity to the MDA-MB-231 cells.

In silico prediction of molecular mechanisms of toxicity mediated by the leptospiral PF07598 gene family-encoded virulence-modifying proteins.

Mechanisms of leptospirosis pathogenesis remain unclear despite the identification of a number of potential leptospiral virulence factors. We recently demonstrated potential mechanisms by which the virulence-modifying (VM) proteins-defined as containing a Domain of Unknown function (DUF1561), encoded by the PF07598 gene family-found only in group 1 pathogenic Leptospira-might mediate the clinical pathogenesis of leptospirosis. VM proteins belongs to classical AB toxin paradigm though have a unique AB domain architecture, unlike other AB toxins such as diphtheria toxin, pertussis toxin, shiga toxin, or ricin toxin which are typically encoded by two or more genes and self-assembled into a multi-domain holotoxin. Leptospiral VM proteins are secreted R-type lectin domain-containing exotoxins with discrete N-terminal ricin B-like domains involved in host cell surface binding, and a C-terminal DNase/toxin domain. Here we use the artificial intelligence-based AlphaFold algorithm and other computational tools to predict and elaborate on details of the VM protein structure-function relationship. Comparative AlphaFold and CD-spectroscopy defined the consistent secondary structure (Helix and ß-sheet) content, and the stability of the functional domains were further supported by molecular dynamics simulation. VM proteins comprises distinctive lectic family (QxW)3 motifs, the Mycoplasma CARDS toxin (D3 domain, aromatic patches), C-terminal similarity with mammalian DNase I. In-silico study proposed that Gln412, Gln523, His533, Thr59 are the high binding energy or ligand binding residues plausibly anticipates in the functional activities. Divalent cation (Mg+2-Gln412) and phosphate ion (PO4]-3-Arg615) interaction further supports the functional activities driven by C-terminal domain. Computation-driven structure-function studies of VM proteins will guide experimentation towards mechanistic understandings of leptospirosis pathogenesis, which underlie development of new therapeutic and preventive measures for this devastating disease.

Diverse Localization Patterns of an R-Type Lectin in Marine Annelids.

Lectins facilitate cell–cell contact and are critical in many cellular processes. Studying lectins may help us understand the mechanisms underlying tissue regeneration. We investigated the localization of an R-type lectin in a marine annelid (Perinereis sp.) with remarkable tissue regeneration abilities. Perinereis nuntia lectin (PnL), a galactose-binding lectin with repeating Gln-X-Trp motifs, is derived from the ricin B-chain. An antiserum was raised against PnL to specifically detect a 32-kDa lectin in the crude extracts from homogenized lugworms. The antiserum detected PnL in the epidermis, setae, oblique muscle, acicula, nerve cord, and nephridium of the annelid. Some of these tissues and organs also produced Galactose (Gal) or N-acetylgalactosamine (GalNAc), which was detected by fluorescent-labeled plant lectin. These results indicated that the PnL was produced in the tissues originating from the endoderm, mesoderm, and ectoderm. Besides, the localizing pattern of PnL partially merged with the binding pattern of a fluorescent-labeled mushroom lectin that binds to Gal and GalNAc. It suggested that PnL co-localized with galactose-containing glycans in Annelid tissue; this might be the reason PnL needed to be extracted with haptenic sugar, such as d-galactose, in the buffer. Furthermore, we found that a fluorescein isothiocyanate-labeled Gal/GalNAc-binding mushroom lectin binding pattern in the annelid tissue overlapped with the localizing pattern of PnL. These findings suggest that lectin functions by interacting with Gal-containing glycoconjugates in the tissues.

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

NMR analysis of carbohydrate-binding interactions in solution: an approach using analysis of saturation transfer difference NMR spectroscopy.

One of the most commonly used ligand-based NMR methods for detecting ligand binding is saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. The STD NMR method is an invaluable technique for assessing carbohydrate-lectin interactions in solution, because STD NMR can be used to detect weak ligand binding (Kd ca. 10(-3)-10(-8) M). STD NMR spectra identify the binding epitope of a carbohydrate ligand when bound to lectin. Further, the STD NMR method uses 1H-detected NMR spectra of only the carbohydrate, and so only small quantities of non-labeled lectin are required. In this chapter, I describe a protocol for the STD NMR method, including the experimental procedures used to acquire, process, and analyze STD NMR data, using STD NMR studies for methyl-β-D-galactopyranoside (β-Me-Gal) binding to the C-terminal domain of an R-type lectin from earthworm (EW29Ch) as an example.

Identification and expression analysis of a novel R-type lectin from the coleopteran beetle, Tenebrio molitor

We have identified novel ricin-type (R-type) lectin by sequencing of random clones from cDNA library of the coleopteran beetle, Tenebrio molitor. The cDNA sequence is comprised of 495bp encoding a protein of 164 amino acid residues and shows 49% identity with galectin of Tribolium castaneum. Bioinformatics analysis shows that the amino acid residues from 35 to 162 belong to ricin-type beta-trefoil structure. The transcript was significantly upregulated after early hours of injection with peptidoglycans derived from Gram (+) and Gram (−) bacteria, beta-1, 3 glucan from fungi and an intracellular pathogen, Listeria monocytogenes suggesting putative function in innate immunity.

An acetylation site in lectin domain modulates the biological activity of polypeptide GalNAc-transferase-2

Polypeptide GalNAc-transferases (ppGalNAc-Ts) are a family of enzymes that catalyze the initiation of mucin-type O-glycosylation. All ppGalNAc-T family members contain a common (QXW)3 motif, which is present in the R-type lectin group. The acetylation site K521 is part of the QKW motif of β-trefoil in the lectin domain of ppGalNAc-T2. We used a combination of acetylation and site-directed mutagenesis approaches to examine the functional role of K521 in ppGalNAc-T2. Binding assays of non-acetylated and acetylated forms of the mutant ppGalNAc-T2K521Q to various naked and αGalNAc-glycosylated mucin peptides indicated that the degree of interaction of lectin domain with αGalNAc depends on the peptide sequence of mucin. Studies of the inhibitory effect of various carbohydrates on the interactions of ppGalNAc-T2 with MUC1αGalNAc indicate that point K521Q mutation enhance the carbohydrate specificity of lectin domain for αGalNAc. K521Q mutation resulted in an enzyme activity lower than that of the wild-type ppGalNAc-T2, similar to the acetylation of ppGalNAc-T2. We conclude that an acetylation site in the QKW motif of the lectin domain modulates carbohydrate recognition specificity and catalytic activity of ppGalNAc-T2 for partially preglycosylated acceptors and a certain naked peptide. Posttranslational modifications of ppGalNAc-Ts, such as acetylation, may play key roles in modulating the functions of the R-type lectin domains in cellular homeostasis.

NMR structure and dynamics of the C-terminal domain of R-type lectin from the earthwormLumbricus terrestris

The C-terminal domain (Ch; C-half) of the R-type earthworm 29-kDa lectin (EW29), isolated from the earthworm Lumbricus terrestris, has two sugar-binding sites, in subdomains α and γ, and the protein uses the two sugar-binding sites for its function as a single domain-type haemagglutinin. Our previous NMR titration experiments showed that the α sugar-binding site is a high-affinity site and the γ sugar-binding site is a low-affinity site. However, it remains unclear why the α sugar-binding site of EW29Ch binds to lactose much more strongly because the crystal structure of lactose-bound EW29Ch showed that the interaction between the α sugar-binding site and lactose was almost same as that between the γ sugar-binding site and lactose. In the present study, we have determined the NMR structure of EW29Ch in the sugar-free state and performed (15)N relaxation experiments for EW29Ch in both the sugar-free state and the lactose-bound states. The conformation of EW29Ch in the sugar-free state was similar to that of EW29Ch in complex with lactose. Conformational changes upon binding of lactose were observed only for the α sugar-binding site. By contrast, the (15)N relaxation experiments revealed a conformational exchange at the α sugar-binding site in the sugar-free state, which was suppressed in the lactose-bound state. The conformational exchange phenomenon observed for the α sugar-binding site was not observed for the γ sugar-binding site. Differences in the conformational change and the backbone dynamics between subdomains α and γ may be associated with the difference of the sugar-binding modes between the two sugar-binding sites. Structural data for the NMR structure of EW29Ch in the sugar-free state have been deposited in the Protein Data Bank database under accession number 2RST.

Protective immunity to ricin toxin conferred by antibodies against the toxin's binding subunit (RTB)

The B subunit (RTB) of ricin toxin is a galactose-/N-acetyl galactosamine-specific lectin that promotes attachment and entry of ricin into host cells. RTB is also the archetype of the so-called R-type lectin family, whose members include haemagglutinins of botulinum neurotoxin (BoNT) progenitor toxins, as well as the binding subunits of cytolethal distending toxins. Although RTB is an appealing subunit vaccine candidate, as well as a potential target for immunotherapeutics, the degree to which RTB immunization elicits protective antibodies against ricin toxin remains unresolved. To address this issue, groups of mice were immunized with RTB and then challenged with 5×LD(50)s of ricin administered intraperitoneally. Despite high RTB-specific serum antibody titers, groups of RTB immunized mice were only partially immune to ricin challenge. Analysis of a collection of RTB-specific B cell hybridomas suggested that only a small fraction of antibodies against RTB have demonstrable neutralizing activity. Two RTB-specific neutralizing monoclonal IgG(1) antibodies, 24B11 and SylH3, when passively administered to mice, were sufficient to protect the animals against a 5×LD(50) dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to the surfaces of bone marrow-derived macrophages (BMM), suggesting that they function by steric hindrance and recognize epitopes located on RTB's carbohydrate recognition sub-domains (1α or 2γ). These data raise the possibility of using specific RTB sub-domains, rather than RTB itself, as antigens to more efficiently elicit neutralizing antibodies and protective immunity against ricin.

Catalytic and glycan-binding abilities of ppGalNAc-T2 are regulated by acetylation

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (“ricin-like”) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.

Role of the mannose receptor (CD206) in ricin toxin pathogenesis (99.16)

Abstract The entry of ricin toxin into macrophages and other cell types is proposed to occur through two distinct pathways. The first involves the toxin’s B subunit (RTB), an R-type lectin that binds to cell-surface glycolipids and glycoproteins bearing terminal β1,4-linked galactose residues. The second involves recognition of ricin’s mannose side chains by the mannose receptor (MR; CD206), a C-type lectin that functions in both scavenging of serum glycoproteins and uptake of microbial pathogens. While much is known about the role of RTB in ricin uptake, much less is understood about the contribution of the MR in ricin pathogenesis. In this study, we demonstrate using the human monocyte cell line, THP-1 that the MR does indeed mediate toxin uptake and contribute to ricin-induced cell killing in vitro. To determine the role of the MR in ricin toxicity in vivo, MR knock-out mice (MR-/-) or age-matched wild type controls were challenged with 2.5 or 5 lethal dose 50s (LD50s) of ricin, administered by intraperitoneal injection. We found that mice lacking the MR were more susceptible to toxin-induced death than control animals, suggesting that the MR promotes toxin clearance rather than productive internalization, as has been proposed. These findings have implications for the development of biodefense therapeutics aimed at rapidly ridding the toxin from circulation.

Engineering a versatile tandem repeat-type α2-6sialic acid-binding lectin

Previously, we developed an α2-6-sialic acid (Sia)-specific lectin (SRC) starting from an R-type galactose-specific lectin C-terminal domain. However, it showed relatively low affinity because of its monovalency. Here, we engineered a tandem repeat construct (SRC2) showing substantial affinity for α2,6-sialylated N-glycans (in the order of 10 −6 M in K d), almost comparable to a natural α2-6Sia-specific lectin from Sambucus sieboldiana (SSA). Notably, its binding to branched N-glycans was found to be more selective than SSA. Nevertheless, SRC2 showed no apparent hemagglutinating activity, while it exerted strong erythrocyte-binding activity. This unique feature will help flow cytometry analysis, where usual lectins including SSA agglutinate cells. Some other biochemical properties investigated for SRC2, e.g., high productivity in bacteria and easy release of captured glycoproteins with lactose have demonstrated versatility of this mutant protein as a powerful tool for sialoglycomics.

NMR studies on the interaction of sugars with the C‐terminal domain of an R‐type lectin from the earthworm Lumbricus terrestris

The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14,500 Da) showing 27% identity with each other. The C-terminal domain (Ch; C-half) of EW29 (EW29Ch) has two sugar-binding sites in subdomains alpha and gamma, and the protein uses these sugar-binding sites for its function as a single-domain-type hemagglutinin. In order to determine the sugar-binding ability and specificity for each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-shift changes in EW29Ch were monitored using (1)H-(15)N HSQC spectra as a function of increasing concentrations of lactose, melibiose, D-galactose, methyl alpha-D-galactopyranoside and methyl beta-D-galactopyranoside. Shift perturbation patterns for well-resolved resonances confirmed that all of these sugars associated independently with the two sugar-binding sites of EW29Ch. NMR titration experiments showed that the sugar-binding site in subdomain alpha had a slow or intermediate exchange regime on the chemical-shift timescale (K(d) = 10(-2) to 10(-1) mM), whereas that in subdomain gamma had a fast exchange regime for these sugars (K(d) = 2-6 mM). Thus, our results suggest that the two sugar-binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10-fold lower than that of the whole protein because EW29Ch has two sugar-binding sites in the same molecule, one of which has a weak binding mode.

Sugar-complex structures of the C-half domain of the galactose-binding lectin EW29 from the earthwormLumbricus terrestris

R-type lectins are one of the most prominent types of lectin; they exist ubiquitously in nature and mainly bind to the galactose unit of sugar chains. The galactose-binding lectin EW29 from the earthworm Lumbricus terrestris belongs to the R-type lectin family as represented by the plant lectin ricin. It shows haemagglutination activity and is composed of a single peptide chain that includes two homologous domains: N-terminal and C-terminal domains. A truncated mutant of EW29 comprising the C-terminal domain (rC-half) has haemagglutination activity by itself. In order to clarify how rC-half recognizes ligands and shows haemagglutination activity, X-ray crystal structures of rC-half in complex with D-lactose and N-acetyl-D-galactosamine have been determined. The structure of rC-half is similar to that of the ricin B chain and consists of a beta-trefoil fold; the fold is further divided into three similar subdomains referred to as subdomains alpha, beta and gamma, which are gathered around the pseudo-threefold axis. The structures of sugar complexes demonstrated that subdomains alpha and gamma of rC-half bind terminal galactosyl and N-acetylgalactosaminyl glycans. The sugar-binding properties are common to both ligands in both subdomains and are quite similar to those of ricin B chain-lactose complexes. These results indicate that the C-terminal domain of EW29 uses these two galactose-binding sites for its function as a single-domain-type haemagglutinin.

Crystallization and preliminary X-ray crystallographic studies of the C-terminal domain of galactose-binding lectin EW29 from the earthworm Lumbricus terrestris.

The galactose-binding lectin EW29 from the earthworm Lumbricus terrestris is composed of two homologous domains, both of which are members of the R-type lectin family. The truncated mutant rC-half comprising the C-terminal domain was crystallized by the hanging-drop vapour-diffusion method. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 61.2, c = 175.6 A, and diffracted to beyond 1.9 A resolution. Matthews coefficient calculations suggested that this crystal contained two molecules per asymmetric unit.