No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Carbohydrate binding properties of the stinging nettle (Urtica dioica) rhizome lectin
Abstract
The interaction of the stinging nettle rhizome lectin (UDA) with carbohydrates was studied by using the techniques of quantitative precipitation, hapten inhibition, equilibrium dialysis, and uv difference spectroscopy. The Carbohydrate binding site of UDA was determined to be complementary to an N,N',N"-triacetylchitotriose unit and proposed to consist of three subsites, each of which has a slightly different binding specificity. UDA also has a hydrophobic interacting region adjacent to the carbohydrate binding site. Equilibrium dialysis and uv difference spectroscopy revealed that UDA has two carbohydrate binding sites per molecule consisting of a single polypeptide chain. These binding sites either have intrinsically different affinities for ligand molecules, or they may display negative cooperativity toward ligand binding.
... Urtica dioica agglutinin is a small (8.5 kDa) monomeric protein with high contents of glycine, cysteine, tryptophan and has a very low specific agglutination activity when it is compared to other plant lectins [11]. UDA is a quite stable protein consisting of a single polypeptide chain [12]. UDA with a small-molecular-weight, extracted from stinging nettle rhizomes, induces murine cell proliferation [13]. ...
... These protein structures induce a cascade of events at the end leading to cell activation processes, including cell proliferation, production of lymphokines, and cell differentiation [15]. Together with mentioned above, UDA since the name itself gives a hint that it agglutinates erythrocytes nonspecifically irrespective of blood group type and also induces the production of gamma interferon human lymphocytes [12]. Despite the fact that most well-characterized plant lectins, in particular those of legumes, as it may refer to the metabolism and physiological mission of these carbohydrate-binding proteins, the quantity of plant lectins extracted from the other parts of the plant has turned into a great interest [12]. ...
... Together with mentioned above, UDA since the name itself gives a hint that it agglutinates erythrocytes nonspecifically irrespective of blood group type and also induces the production of gamma interferon human lymphocytes [12]. Despite the fact that most well-characterized plant lectins, in particular those of legumes, as it may refer to the metabolism and physiological mission of these carbohydrate-binding proteins, the quantity of plant lectins extracted from the other parts of the plant has turned into a great interest [12]. Again, the question arises whether proteins with an entirely discrete configurations and sugar-binding specificity would accomplish alike in other words parallel physiological roles [10]. ...
... UDA is a chitin and is a GlcNAc oligomer specific lectin derived from plants. 128,129 UDA is speculated to be antifungal and insecticidal in nature. 130,131 Binding experiments suggest that the lectin has two binding sites with a preference for (GlcNAc) 5 , with an affinity of −5.9 kcal mol −1 that decreases to −3.9 kcal mol −1 upon a reduction in chain length to (GlcNAc) 2 . ...
... While solution properties of the glycans are important, their interactions with other biomolecules are equally critical. The binding of N-glycans to lectins, carbohydrate binding proteins that make specific interactions with terminal sugars of glycan chains, 123,128,130,[132][133][134][135][136]154 is crucial in many biological phenomena. Thus, optimizing the binding properties of these glycans with proteins is important for their applicability in multiprotein complexes, and reproduction of experimental affinities of glycans with proteins represents a useful way of optimizing the nonbonded parameters, as shown here. ...
Glycans play a vital role in a large number of cellular processes. Their complex and flexible nature hampers structure-function studies using experimental techniques. Molecular dynamics (MD) simulations can help in understanding dynamic aspects of glycans if the forcefield parameters used can reproduce key experimentally observed properties. Here, we present optimized coarse-grained (CG) Martini forcefield parameters for N-glycans, calibrated against experimentally derived binding affinities for lectins. The CG bonded parameters were obtained from atomistic (ATM) simulations for different glycan topologies including high mannose and complex glycans with various branching patterns. In the CG model, additional elastic networks are shown to improve maintenance of the overall conformational distribution. Solvation free energies and octanol-water partition coefficients were also calculated for various n-glycan disaccharide combinations. When using standard Martini non-bonded parameters, we observed that glycans spontaneously aggregated in the solution and required down-scaling of their interactions for reproduction of ATM model radial distribution functions. We also optimised the non-bonded interactions for glycans interacting with seven lectin candidates and show that a relatively modest scaling down of the glycan-protein interactions can reproduce free energies obtained from experimental studies. These parameters should be of use in studying the role of glycans in various glycoproteins, carbohydrate binding proteins as well as their complexes, while benefiting from the efficiency of CG sampling.
... are three conserved glycine residues and one conserved serine residue in all the hevein like domains. The sequence data on UDA clearly show the existence of two similar but not exactly identical sites raising the possibility that it could have 2 types of sites [17]. It is also noteworthy that the hinge region between the two domains is four residues longer than those between the cereal lectins and forms a more flexible connection. ...
... The AH 1 , and TAS 1 values for high affinity site are close to that of WGA, suggesting that the binding mode of the two lectins are similar [22]. However, the low affinity site in UDA binds with fivefold lower affinity which is in qualitative agreement with the values reported using an indirect binding assay [17]. A plot of –AH versus –TAS, yields a straight line with the slope greater than unity (1.06; correlation coefficient 0.99) which indicates that the reactions for the most part are driven enthalpically. ...
UDA (Urtica dioica agglutinin) contains two hevein like domains with two non-identical interacting sites and is specific for chitooligosaccharides. The binding of chitooligosaccharides to UDA was studied by Isothermal Titration Calorimetry. Each site is composed of three subsites, each binding to a sugar residue. Thermodynamic parameters obtained show that while chitobiose has two independent non-interacting sites, chitotriose, chitotetrose and chitopentose have two interacting sites on each monomer of UDA. Values of binding enthalpy (H) increase almost by a factor of 7 in going from chitobiose to chitotriose indicating the existence of three subsites in the combining site of UDA. The binding constant for chitotetrose and chitopentose increase without any further enhancement in the values of H indicating that for oligomers larger than chitotriose interaction is favoured entropically.
... Urtica dioica agglutinin (UDA), on the other hand, is a tiny 8.7 kDa lectin isolated from the Nettle (Urtica dioica L.), with a specificity for N, N′, N′′-tri-acetyl chitotriose (polymer of acetylated acetylglucosamines). This peptide lectin has a hydrophobic binding region adjacent to its sugar-binding site in addition to its sugar-binding site [48]. It stops SARS-CoV from replicating by interfering with viral attachment to the host cell, most likely by binding to N-acetylglucosamine (GlcNAc) units in the spike protein [49,50]. ...
Lectins are non-immune carbohydrate-binding proteins/glycoproteins that are found everywhere in nature, from bacteria to human cells. They have also been a valuable biological tool for the purification and subsequent characterisation of glycoproteins due to their carbohydrate binding recognition capacity. Antinociceptive, antiulcer, anti-inflammatory activities and immune modulatory properties have been discovered in several plant lectins, with these qualities varying depending on the lectin carbohydrate-binding site. The Coronavirus of 2019 (COVID-19) is a respiratory disease that has swept the globe, killing millions and infecting millions more. Despite the availability of COVID-19 vaccinations and the vaccination of a huge portion of the world's population, viral infection rates continue to rise, causing major concern. Part of the reason for the vaccine's ineffectiveness has been attributed to repeated mutations in the virus's epitope determinant elements. The surface of the Coronavirus envelope is heavily glycosylated, with approximately sixty N-linked oligomannose, composite, and hybrid glycans covering the core of Man3GlcNAc2Asn. Some O–linked glycans have also been discovered. Many of these glyco-chains have also been subjected to multiple mutations, with only a few remaining conserved. As a result, numerous plant lectins with specificity for these viral envelope sugars have been discovered to interact preferentially with them and are being investigated as a potential future tool to combat coronaviruses such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by preventing viral attachment to the host. The review will discuss the possible applications of plant lectins as anti-coronaviruses including SARS-CoV-2, antinociceptive, anti-inflammation and its immune modulating effect.
... Isolariciresinol, pinoresinol, neoolivil, secoisolariciresinol, dehydrodiconiferyl alcohol, and 3,4-divanillyltetrahydrofuran (Sch€ ottner et al., 1997;Chaurasia and Wichtl, 1986) are lignans found in the root (Sch€ ottner et al., 1997;Chaurasia and Wichtl, 1987). U. dioica agglutinin (UDA), a single-chain polypeptide having 89 amino acids and rich in glycines, cysteines, and tryptophans, is found in the root of U. dioica (Van Damme et al., 1988;Shibuya et al., 1986). Phytosterols are found in the root, including stigmasterol, campesterol, stigmast-4-en-3-on, hecogenin, and sitosterol (Seliya and Kothiyal, 2014;Chaurasia and Wichtl, 1987). ...
Stinging nettle (Urtica dioica L.) is a wild herbaceous perennial blooming plant that is commonly known as
stinging nettle. It’s a common, multi-purpose crop that’s sometimes overlooked. Europe, Asia, North Africa, and
North America are all home to stinging nettle. It is a plant that’s edible and has nutritional and medicinal
properties. Young leaves can be used to make curries, herb soups, and sour soups. The root of the stinging nettle is
used to treat mictional difficulties associated with benign prostatic hyperplasia, while the leaves are used to treat
arthritis, rheumatism, and allergic rhinitis. Its leaves are abundant in fiber, minerals, vitamins, and antioxidant
compounds like polyphenols and carotenoids, as well as antioxidant compounds like polyphenols and carotenoids.
Stinging nettle has antiproliferative, anti-inflammatory, antioxidant, analgesic, anti-infectious, hypotensive, and
antiulcer characteristics, as well as the ability to prevent cardiovascular disease, in all parts of the plant (leaves,
stems, roots, and seeds). Stinging nettle improves fish reproductive performance, making it a cost-effective
aquaculture plant. Fertilizer and insecticides can be made from the plants. This review examines the nutritional and pharmacological aspects of stinging nettle, as well as its possible health advantages. Scientists, farmers,
and academicians interested in stinging nettle collecting, cultivation, research, and development would find this
review useful.
... Isolariciresinol, pinoresinol, neoolivil, secoisolariciresinol, dehydrodiconiferyl alcohol, and 3,4-divanillyltetrahydrofuran (Sch€ ottner et al., 1997;Chaurasia and Wichtl, 1986) are lignans found in the root (Sch€ ottner et al., 1997;Chaurasia and Wichtl, 1987). U. dioica agglutinin (UDA), a single-chain polypeptide having 89 amino acids and rich in glycines, cysteines, and tryptophans, is found in the root of U. dioica (Van Damme et al., 1988;Shibuya et al., 1986). Phytosterols are found in the root, including stigmasterol, campesterol, stigmast-4-en-3-on, hecogenin, and sitosterol (Seliya and Kothiyal, 2014;Chaurasia and Wichtl, 1987). ...
Stinging nettle (Urtica dioica L.) is a wild herbaceous perennial blooming plant that is commonly known as stinging nettle. It’s a common, multi-purpose crop that’s sometimes overlooked. Europe, Asia, North Africa, and North America are all home to stinging nettle. It is a plant that’s edible and has nutritional and medicinal properties. Young leaves can be used to make curries, herb soups, and sour soups. The root of the stinging nettle is used to treat mictional difficulties associated with benign prostatic hyperplasia, while the leaves are used to treat arthritis, rheumatism, and allergic rhinitis. Its leaves are abundant in fiber, minerals, vitamins, and antioxidant compounds like polyphenols and carotenoids, as well as antioxidant compounds like polyphenols and carotenoids. Stinging nettle has antiproliferative, anti-inflammatory, antioxidant, analgesic, anti-infectious, hypotensive, and antiulcer characteristics, as well as the ability to prevent cardiovascular disease, in all parts of the plant (leaves, stems, roots, and seeds). Stinging nettle improves fish reproductive performance, making it a cost-effective aquaculture plant. Fertilizer and insecticides can be made from the plants. This review examines the nutritional and pharmacological aspects of stinging nettle, as well as its possible health advantages. Scientists, farmers, and academicians interested in stinging nettle collecting, cultivation, research, and development would find this review useful.
... On the other hand, Urtica dioica agglutinin (UDA); the lectin purified from the Nettle (Urtica dioica L.), is a small 8.7 kDa protein with a specificity directed towards N, N 0 , N 00 -tri-acetylchitotriose (polymer of acetylated acetylglucosamines). This peptide lectin besides its sugar-binding site also had a hydrophobic binding region adjacent to its carbohydrate-binding site (Shibuya et al. 1986). It inhibits the SARS-CoV replication cycle by interfering with the viral attachment to the host cell probably through binding to spike protein N-acetylglucosamine (GlcNAc) units (Kumaki et al. 2011;Saul et al. 2000). ...
Lectins are defined as carbohydrate-binding proteins/glycoproteins of none immune origin, they are ubiquitous in nature, exist from bacteria to human cells. And due to their carbohydrate-binding recognition capacity, they have been a useful biological tool for the purification of glycoproteins and their subsequent characterization. Some plant lectins have also been revealed to own antinociceptive, antiulcer, and anti-inflammatory properties, where these features, in many instances, depending on the lectin carbohydrate-binding site. Coronavirus disease of 2019 (COVID-19) is a respiratory disease that struck the entire world leaving millions of people dead and more infected. Although COVID-19 vaccines have been made available, and quite a large number of world populations have already been immunized, the viral infection rates remained in acceleration, which continues to provoke major concern about the vaccines' efficacy. The belief in the ineffectiveness of the vaccine has been attributed in part to the recurrent mutations that occur in the epitope determinant fragments of the virus. Coronavirus envelope surface is extensively glycosylated being covered by more than sixty N-linked oligomannose, composite, and hybrid glycans with a core of Man3GlcNAc2Asn. In addition some O–linked glycans are also detected. Of these glyco-chains, many have also been exposed to several mutations, and a few remained conserved. Therefore, numerous plant lectins with a specificity directed towards these viral envelope sugars have been found to interact preferentially with them and are suggested to be scrutinized as a possible future tool to combat coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through blocking the viral attachment to the host cells. In this review, we will discuss the possible applications of plant lectins as anti-coronaviruses including SARS-CoV-2, antinociceptive, anti-inflammatory, and antiulcer agents with the proposed mechanism of their actions.
... Nettle has been used medicinally probably since the Middle Ages, particularly for menstrual problems. It remains a popular remedy for bladder disorders (Shibuya , 1996). ...
... UDA, derived from stinging nettle, is annotated as a GlcNAc binding lectin [33]. ...
Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving, however the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well-defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use glycan microarray analysis of 116 commercially available lectins, including different preparations of the same lectin, to extract the specific glycan features required for lectin binding. Data was obtained using the Consortium for Functional Glycomics microarray (CFG v5.0) containing 611 glycans. We use a combination of machine learning algorithms to define lectin specificity, mapping inputs (glycan sequences) to outputs (lectin-glycan binding) for a large-scale evaluation of lectin-glycan binding behaviours. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono-and disaccharides-and linkages. Using a combination of machine learning and manual annotation of the data, we created a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.
... GalNAc GalNAc, GalNAcα3Gal [34] SNA Sambucus nigra Sia Siaα2-6Gal/GalNAc [36] SSA Sambucus sieboldiana Sia Siaα2-6Gal/GalNAc [49] STL Solanum tuberosum GlcNAc (GlcNAcβ) n , (GlcNAcβ4MurNAc) n (peptidoglycan backbone) [50] TJAÀ I Tanthes japonica Sia Siaα2-6Gal/GalNAc [51] UDA Urtica dioica GlcNAc GlcNAcβ4GlcNAc, Man5~Man9 [52] UEAÀ I Ulex europaeus Fuc Fucα2Galβ4GlcNAc (H-type 2) [53] VVA Vicia villosa GalNAc α GalNAc, GalNAcα3Gal [54] WFA Wisteria floribunda GalNAc GalNAcβ4GlcNAc, Galβ3(-6) GalNAc [34] a) Fuc, fucose; Gal, galactose; GlcNAc, N-acetyl-D-glucosamine; Man, mannose; GalNAc, N-Acetyl-D-galactosamine; Sia, sialic acid ...
Many proteins in living organisms are glycosylated. As their glycan patterns exhibit protein‐, cell‐, and tissue‐specific heterogeneity, changes in the glycosylation levels could serve as useful indicators of various pathological and physiological states. Thus, the identification of glycoprotein biomarkers from specific changes in the glycan profiles of glycoproteins is a trending field. Lectin microarrays provide a new glycan analysis platform, which enables rapid and sensitive analysis of complex glycans without requiring the release of glycans from the protein. Recent developments in lectin microarray technology enable high‐throughput analysis of glycans in complex biological samples. In this review, we will discuss the basic concepts and recent progress in lectin microarray technology, the application of lectin microarrays in biomarker discovery, and the challenges and future development of this technology. Given the tremendous technical advancements that have been made, lectin microarrays will become an indispensable tool for the discovery of glycoprotein biomarkers. A powerful tool to find novel biomarkers! Lectin microarray technology is a fast, high‐throughput, and inexpensive tool for the discovery of glycol‐biomarkers in complex biological samples. This review summarizes the recent progress and application of lectin microarrays in biomarker discovery under different physiopathological states.
... 4.4.1. Urtica dioica agglutinin (UDA) Urtica dioica Agglutinin (UDA) was isolated from stinging nettle (Urtica dioica) and is monomeric with a molecular weight of 8.5 kDa (Shibuya et al., 1986;Wagner et al., 1989). The lectin has two internal repeats with 46% sequence identity with R1 including residues 1e46 and R2 including residues 47e90. ...
Many natural lectins have been reported to have antiviral activity. As some of these have been put forward as potential development candidates for preventing or treating viral infections, we have set out in this review to survey the literature on antiviral lectins. The review groups lectins by structural class and class of source organism we also detail their carbohydrate specificity and their reported antiviral activities. The review concludes with a brief discussion of several of the pertinent hurdles that heterologous proteins must clear to be useful clinical candidates and cites examples where such studies have been reported for antiviral lectins. Though the clearest path currently being followed is the use of antiviral lectins as anti-HIV microbicides via topical mucosal administration, some investigators have also found systemic efficacy against acute infections following subcutaneous administration.
... stigmasterol, campesterol, stigmast-4-en-3-on, hecogenin. [24,41] Lignans: neo-olivil, secoisolariciresinol, dehydrodiconiferyl alcohol, isolariciresinol, pinoresinol, and 3,4divanillyltetrahydrofuran. [27,39] Coumarins: Scopoletin [42,43] Fruit (seeds) [39,43] Fixed oil: saturated and unsaturated fatty acids. ...
Urtica dioica L. is a herbaceous plant belonging to the family of Urticaceae that has been used for centuries against a variety of diseases. Thanks to its high content of nutriments and bioactive compounds like poly phenols, vitamins and minerals, nettle possesses a great nutritional value and a large number of pharmacological effects, including anti-proliferative, anti-inflammatory, antioxidant, analgesic, immunostimulatory, anti-infectious, hypotensive, antiulcer activities and cardiovascular disease prevention. Stinging nettle is considered safe and has been shown to be side effects free, when taken by mouth of up to 18 gram per day. The most common stinging nettle preparations usually include the crude dried powder, dry extract, infusion (herbal tea), decoction or fresh juice. Stinging nettle root is mainly used for mictional disorders related to benign prostatic hyperplasia while the leaves are used for arthritis, rheumatism and allergic rhinitis. This up to date review highlights the current knowledge and scientific advances concerning Urtica dioica. © 2015, International Journal of Pharmacy and Pharmaceutical Science. All rights reserved.
... As a consequence, available information about lectins in medicinal plants that are listed in national pharmacopeias is rather limited [11]. In particular, a PubMed-based search (December 2012) on lectins (also using the synonymous term "agglutinin") in 43 herbs from Dietary Supplements Official Monographs of the 2012 US Pharmacopeia has revealed that information about lectins in medicinal plants is available only for ~ 21 % of the listed herbs (Table 3.1) [12][13][14][15][16][17][18][19][20][21][22][23]. ...
Lectins represent a family of glycan-binding proteins that are differentially expressed in various plant tissues and organs. As a component of traditional herbs, some purified plant lectins are known to possess immunomodulatory, cytotoxic, and anticancer activities with a potential biomedical application. In this chapter, we summarize our procedures for lectin isolation from medicinal plants and methods for lectin screening and biotesting based on a variety of cellular responses (cell aggregation, generation of hydrogen peroxide, and secretion of vascular endothelial growth factor C). The importance of detecting and characterizing lectins in herbal preparations is discussed in the context of safety and efficacy of lectin-based phytotherapeutical approaches.
... Urtica dioica agglutinin (UDA) is a monomeric GlcNAc-specific protein derived from the stinging nettle rhizomes. It has a total molecular weight of 8.5 kDa and contains two carbohydrate-binding sites with different affinities [75,76]. Structural modeling indicated that UDA efficiently interacts with the GlcNAc2Man1 residue, which is part of the core unit ( Figure 1A) of glycans on gp120 [34]. ...
The glycoproteins on the surfaces of enveloped viruses, such as HIV, can be considered as a unique target for antiviral therapy. Different carbohydrate-binding agents (CBAs) target specific glycans present on viral glycoproteins of enveloped viruses. It has been shown that long-term CBA pressure in vitro can result in mutant HIV-1 isolates with several N-linked glycan deletions on gp120. These studies demonstrated that mainly high-mannose type glycans are deleted. However, interestingly, N241, N262 and N356 on gp120 have never been found to be affected after prolonged CBA exposure. Here, we review the mutation and (cross)-resistance profiles of eleven specific generated CBA-resistant HIV-1 strains. We observed that the broad-neutralizing anti-carbohydrate binding mAb 2G12 became completely inactive against all the generated CBA-resistant HIV-1 clade B isolates. In addition, all of the CBAs discussed in this review, with the exception of NICTABA, interfered with the binding of 2G12 mAb to gp120 expressed on HIV-1-infected T cells. The cross-resistance profiles of mutant HIV-1 strains are varying from increased susceptibility to very high resistance levels, even among different classes of CBAs with dissimilar sugar specificities or binding moieties [e.g., α(1,3), α(1,2), α(1,6)]. Recent studies demonstrated promising results in non-topical formulations (e.g., intranasally or subcutaneously), highlighting their potential for prevention (microbicides) and antiviral therapy.
... Èçó÷åíî âçàèìîäåéñòâèå óãëåâîäîðîäíîé ñâÿçè â UDA ñ ïîìîùüþ êîëè÷åñòâåííîãî îñàaeäåíèÿ, èíãèáèðîâàíèÿ ãàïòåíîì, ðàâíîâåñíîãî äèàëèçà è ÓÔ-ñïåêòðîñêîïèè. Ïîëîaeåíèå óãëåâîäîðîäíîé ñâÿçè â UDA áûëî îïðåäåëåíî äîïîëíèòåëüíî ñ N,N¢, N²-òðèàöåòèëõèòîòðèîçîé. Óñòàíîâëåíî, ÷òî â UDA 2 óãëåâîäîðîäíûå ñâÿçè ðàñïîëîaeåíû â ìîëåêóëå, ñîñòîÿùåé èç 2 ïîëèïåïòèäíûõ öåïî÷åê [189,190]. ...
Literature on the application, chemical composition, and standardization of nettle (Urtica) raw material and related medicinal plant preparations is reviewed.
... For example, WGA possesses both high-and low-affinity sites. Similarly, the specificity and affinity toward GlcNAc-oligomers of the two binding sites of UDA are different (Shibuya et al., 1986). A final remark concerns some peculiarities concerning the specificity of the Gramineae lectins. ...
Many plants contain carbohydrate-binding proteins that are commonly designated as lectins, agglutinins, or hemagglutinins. Due to the obvious differences in molecular structure, biochemical properties, and carbohydrate-binding specificity, plant lectins are usually considered a complex and heterogeneous group of proteins. Recent advances in the structural analysis of lectins and molecular cloning of lectin genes enable subdividision of plant lectins in a limited number of subgroups of structurally and evolutionary related proteins. Four major lectin families, namely, the legume lectins, the chitin-binding lectins composed of hevein domains, the type 2 ribosome-inactivating proteins, and the monocot mannose-binding lectins comprise the majority of all currently known plant lectins. In addition to these four large families the jacalin-related lectins, the amaranthin family, and the Cucurbitaceae phloem lectins are now recognized as separate subgroups. Each of the above-mentioned lectin families is discussed in detail. The description of the individual lectin families includes (1) a brief historical note, (2) an overview of the occurrence, molecular structure, and primary structure of the lectins, (3) a detailed discussion of the structure of the gene(s) and the biosynthesis and posttranslational processing of the primary translation products, (4) a summary of carbohydrate-binding specificity, (5) if relevant a note on the occurrence of lectin-related proteins, (6) a description of the three-dimensional structure of the lectins and the protomers, (7) a detailed discussion of the molecular evolution, and (8) a critical assessment of the physiological role of each group of lectins. Lectins that cannot be classified into one of the seven groups are discussed separately. General conclusions about the structure, evolution, and function of plant lectins are summarized in the concluding remarks.
... Binding studies revealed that the CBAs act during the adsorption phase of the virus to the host cell. HHA and GNA have been shown to interact with mannose-residues [121,122], and UDA can recognize specifically Glc-NAc residues [123]. Mannose and Glc-NAc molecules are present in the backbone of the high-mannose type glycans on the viral envelope protein. ...
Dengue virus (DENV) infections are expanding worldwide and, because of the lack of a vaccine, the search for antiviral products is imperative. Four serotypes of DENV are described and they all cause a similar disease outcome. It would be interesting to develop an antiviral product that can interact with all four serotypes, prevent host cell infection and subsequent immune activation. DENV entry is thus an interesting target for antiviral therapy. DENV enters the host cell through receptor-mediated endocytosis. Several cellular receptors have been proposed, and DC-SIGN, present on dendritic cells, is considered as the most important DENV receptor until now. Because DENV entry is a target for antiviral therapy, various classes of compounds have been investigated to inhibit this process. In this paper, an overview is given of all the putative DENV receptors, and the most promising DENV entry inhibitors are discussed.
... A second example are the lectins with hevein domains. Numerous lectins of the hevein family (e.g., WGA, UDA, potato, and tomato lectins) have been reported to specifically bind to GlcNAc oligomers (Peumans et al., 1984;Shibuya et al., 1986). In the case of UDA, detailed studies of its carbohydrate binding properties were performed using quantitative precipitation assays and hapten inhibition. ...
Plant lectins are being studied for over a century. Until a decade ago, most information was obtained from biochemical, molecular, and structural studies of a reasonably high but still limited number of abundant lectins from seeds and vegetative storage organs. Though the results of these studies are still valid, the recent progress made in several areas of plant lectin research urge to profoundly revise the prevailing concepts.This contribution aims to give a comprehensive overview of the occurrence, taxonomic distribution, molecular evolution, and physiological role of plant lectins with the emphasis on relevant novel developments. The overview comprises a description of the 12 different carbohydrate binding domains and annex lectin families that hitherto have been identified in plants. For each lectin family, a fairly detailed summary is given of the biochemical properties, taxonomical distribution, and evolutionary origin. Novel insights in some specific aspects of plant lectins like their biosynthesis and topogenesis, carbohydrate binding specificity, and regulation of expression are discussed. The last major section deals with an updated critical discussion of the physiological role of plant lectins with an emphasis on specific functions within the plant cell. Finally, the concept of “lectinome” is introduced.
Urtica dioica agglutinin (UDA) is a carbohydrate-binding small monomeric protein isolated from stinging nettle rhizomes. It inhibits replication of a broad range of viruses, including coronaviruses, in multiple cell types, with appealing selectivity. In this work, we investigated the potential of UDA as a broad-spectrum antiviral agent against SARS-CoV-2. UDA potently blocks transduction of pseudotyped SARS-CoV-2 in A549.ACE2 ⁺ -TMPRSS2 cells, with IC 50 values ranging from 0.32 to 1.22 µM. Furthermore, UDA prevents viral replication of the early Wuhan-Hu-1 strain in Vero E6 cells (IC 50 = 225 nM), but also the replication of SARS-CoV-2 variants of concern, including Alpha, Beta and Gamma (IC 50 ranging from 115 to 171 nM). In addition, UDA exerts antiviral activity against the latest circulating Delta and Omicron variant in U87.ACE2 ⁺ cells (IC 50 values are 1.6 and 0.9 µM, respectively). Importantly, when tested in Air-Liquid Interface (ALI) primary lung epithelial cell cultures, UDA preserves antiviral activity against SARS-CoV-2 (20A.EU2 variant) in the nanomolar range. Surface plasmon resonance (SPR) studies demonstrated a concentration-dependent binding of UDA to the viral spike protein of SARS-CoV-2, suggesting interference of UDA with cell attachment or subsequent virus entry. Moreover, in additional mechanistic studies with cell-cell fusion assays, UDA inhibited SARS-CoV-2 spike protein-mediated membrane fusion. Finally, pseudotyped SARS-CoV-2 mutants with N-glycosylation deletions in the S2 subunit of the spike protein remained sensitive to the antiviral activity of UDA. In conclusion, our data establish UDA as a potent fusion inhibitor for the current variants of SARS-CoV-2.
Urtica dioica agglutinin (UDA) is a carbohydrate-binding small monomeric protein isolated from stinging nettle rhizomes. It inhibits replication of a broad range of viruses, including coronaviruses, in multiple cell types, with appealing selectivity. In this work, we investigated the potential of UDA as a broad-spectrum antiviral agent against SARS-CoV-2. UDA potently blocks entry of pseudotyped SARS-CoV-2 in A549.ACE2+-TMPRSS2 cells, with IC50 values ranging from 0.32 to 1.22 microM. Furthermore, UDA prevents viral replication of the early Wuhan-Hu-1 strain in Vero E6 cells (IC50 = 225 nM), but also the replication of SARS-CoV-2 variants of concern, including Alpha, Beta and Gamma (IC50 ranging from 115 to 171 nM). In addition, UDA exerts antiviral activity against the latest circulating Delta and Omicron variant in U87.ACE2+ cells (IC50 values are 1.6 and 0.9 microM, respectively). Importantly, when tested in Air-Liquid Interface (ALI) primary lung epithelial cell cultures, UDA preserves antiviral activity against SARS-CoV-2 (20A.EU2 variant) in the nanomolar range. Surface plasmon resonance (SPR) studies demonstrated a concentration-dependent binding of UDA to the viral spike protein of SARS-CoV-2, suggesting interference of UDA with cell attachment or subsequent virus entry. Moreover, in additional mechanistic studies with cell-cell fusion assays, UDA inhibited SARS-CoV-2 spike protein-mediated membrane fusion. Finally, pseudotyped SARS-CoV-2 mutants with N-glycosylation deletions in the S2 subunit of the spike protein remained sensitive to the antiviral activity of UDA. In conclusion, our data establish UDA as a potent and broad-spectrum fusion inhibitor for SARS-CoV-2.
L’ortie, Urtica dioica L., est une plante herbacée de la famille des urticacées, utilisée empiriquement depuis des millénaires dans de nombreuses pathologies. De par sa composition chimique riche en polyphénols, en vitamines et en minéraux, l’ortie affiche une haute valeur nutritionnelle et une multiplicité d’actions pharmacologiques : antiproliférative, anti-inflammatoire, anti-oxydante, analgésique, anti-ulcéreuse, immunostimulante, anti-infectieuse, hypotensive et protectrice vis-à-vis des maladies cardiovasculaires. L’ortie est inoffensive et des doses allant jusqu’à dix-huit grammes par jour par voie orale chez l’Homme n’ont montré aucun effet secondaire. Les modes de préparation les plus fréquemment employés en phytothérapie sont la poudre totale sèche, l’extrait sec, les infusions, les décoctions et les sucs frais. La racine d’ortie est utilisée essentiellement dans le traitement des troubles de miction liés à l’hypertrophie bénigne de la prostate, alors que ses feuilles sont indiquées dans les arthrites, les rhumatismes et les rhinites allergiques. Le présent travail met en exergue les connaissances et les avancées scientifiques d’Urtica dioica.
Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving; however, the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well-defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use a combination of machine learning algorithms and expert annotation to define lectin specificity for this important probe set. Our analysis uses comprehensive glycan microarray analysis of commercially available lectins we obtained using version 5.0 of the Consortium for Functional Glycomics glycan microarray (CFGv5). This data set was made public in 2011. We report the creation of this data set and its use in large-scale evaluation of lectin−glycan binding behaviors. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono-and disaccharides and linkages. Combining machine learning with manual annotation, we create a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.
Lectins or clusters of carbohydrate-binding proteins of non-immune origin are distributed chiefly in the Plantae. Lectins have potent anti-infectivity properties for several RNA viruses including SARS-CoV-2. The primary purpose of this review is to review the ability of lectins mediated potential biotherapeutic and bioprophylactic strategy against coronavirus causing COVID-19. Lectins have binding affinity to the glycans of SARS-COV-2 Spike glycoprotein that has N-glycosylation sites. Apart from this, the complement lectin pathway is a “first line host defense” against the viral infection that is activated by mannose-binding lectins. Mannose-binding lectins deficiency in serum influences innate immunity of the host and facilitates infectious diseases including COVID-19. Our accumulated evidence obtained from scientific databases particularly PubMed and Google Scholar databases indicate that mannose-specific/mannose-binding lectins (MBL) have potent efficacies like anti-infectivity, complement cascade induction, immunoadjuvants, DC-SIGN antagonists, or glycomimetic approach, which can prove useful in the strategy of COVID-19 combat along with the glycobiological aspects of SARS-CoV-2 infections and antiviral immunity. For example, plant-derived mannose-specific lectins BanLac, FRIL, Lentil, and GRFT from red algae can inhibit and neutralize SARS-CoV-2 infectivity, as confirmed with in-vitro, in-vivo, and in-silico assessments. Furthermore, Bangladesh has a noteworthy resource of antiviral medicinal plants as well as plant lectins. Intensifying research on the antiviral plant lectins, adopting a glyco-biotechnological approach, and with deeper insights into the “glycovirological” aspects may result in the designing of alternative and potent blueprints against the 21st century's biological pandemic of SARS-CoV-2 causing COVID-19
Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid “spacer” domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.
Here, we report on the anti-influenza virus activity of the mannose-binding agents Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA) and the (N-acetylglucosamine)
n
-specific Urtica dioica agglutinin (UDA). These carbohydrate-binding agents (CBA) strongly inhibited various influenza A(H1N1), A(H3N2), and B viruses in vitro, with 50% effective concentration values ranging from 0.016 to 83 nM, generating selectivity indexes up to 125,000. Somewhat less activity was observed against A/Puerto Rico/8/34 and an A(H1N1)pdm09 strain. In time-of-addition experiments, these CBA lost their inhibitory activity when added 30 min postinfection (p.i.). Interference with virus entry processes was also evident from strong inhibition of virus-induced hemolysis at low pH. However, a direct effect on acid-induced refolding of the viral hemagglutinin (HA) was excluded by the tryptic digestion assay. Instead, HHA treatment of HA-expressing cells led to a significant reduction of plasma membrane mobility. Crosslinking of membrane glycoproteins, through interaction with HA, could also explain the inhibitory effect on the release of newly formed virions when HHA was added at 6 h p.i. These CBA presumably interact with one or more N-glycans on the globular head of HA, since their absence led to reduced activity against mutant influenza B viruses and HHA-resistant A(H1N1) viruses. The latter condition emerged only after 33 cell culture passages in the continuous presence of HHA, and the A(H3N2) virus retained full sensitivity even after 50 passages. Thus, these CBA qualify as potent inhibitors of influenza A and B viruses in vitro with a pleiotropic mechanism of action and a high barrier for viral resistance.
Glycans play a vital role in a large number of cellular processes. Their complex and flexible nature hampers structure-function studies using experimental techniques. Molecular dynamics (MD) simulations can help in understanding dynamic aspects of glycans if the forcefield (FF) parameters used can reproduce key experimentally observed properties. Here, we present optimized coarse-grained (CG) Martini FF parameters for N-glycans, calibrated against experimentally derived binding affinities for lectins. The CG bonded parameters were obtained from atomistic (ATM) simulations for different glycan topologies including high mannose and complex glycans with various branching patterns. In the CG model, additional elastic networks are shown to improve maintenance of the overall conformational distribution. Solvation free energies and octanol-water partition coefficients were also calculated for various n-glycan disaccharide combinations. When using standard Martini non-bonded parameters, we observed that glycans spontaneously aggregated in the solution and required down-scaling of their interactions for reproduction of ATM model radial distribution functions. We also optimised the non-bonded interactions for glycans interacting with seven lectin candidates and show that scaling down the glycan-protein interactions can reproduce free energies obtained from experimental studies. These parameters should be of use in studying the role of glycans in various glycoproteins, carbohydrate binding proteins (CBPs) as well as their complexes, while benefiting from the efficiency of CG sampling.
To protect themselves from invasion by pathogenic microorganisms, plants produce a wide array of proteins that exert direct antimicrobial activity. Based on homology at the level of the amino acid sequence and/or three-dimensional folding pattern, these antimicrobial proteins (AMPs) can be classified into over 18 distinct protein families. Some information is emerging on how these proteins interfere with the growth of microorganisms, fungi in particular. Hydrolases such as PR-3-type chitinases and PR-2-type glucanases, and possibly also PR-4-type proteins, affect fungal growth by disturbing the structural integrity of their cell wall. Thionins, 2S albumins, lipid transfer proteins and puroindolines have been demonstrated to partially lyse artificial phospholipid vesicles and are therefore believed to interfere with the phospholipid bilayer of the microbial plasma membrane. Other AMPs, such as PR-5-type proteins and plant defensins are proposed to affect plasma membrane receptors, although the evidence is still circumstantial. For many different types of AMPs it has been shown that particular combinations by two result in synergistic antimicrobial effects, suggesting that maximal antimicrobial potency of AMPs is achieved when they act in concert. Most plant tissues express simultaneously a number of AMP genes. In uninfected vegetative tissues expression is predominant in gateways for microbial invasion such as epidermal cells, stomata, hydathodes and cells in vascular strands. Infection of vegetative tissues by viruses, bacteria or fungi results in the coordinate activation of sets of AMP genes via multiple signalling pathways. These genes are in some but not all cases the same as those that confer basal expression in microbial gateway cells. At the subcellular level, AMPs are usually either deposited in the extracellular space or stored in vacuoles, in which case they are released when host cell lysis occurs. Vacuolar and extracellular AMP isoforms are usually products of different genes. The contribution of AMP genes to the resistance of plants has been studied extensively by overexpression or antisense down-regulation of AMP genes in transgenic plants, or by using mutant or transgenic plants showing either up- or down-regulation of signalling pathways leading to coordinate AMP gene expression. Many of these studies prove the concept that particular AMPs contribute to resistance to particular groups of microbial pathogens.
LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides is underexplored. Binding of a Mycobacterium smegmatis protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration which demonstrate the presence of two binding sites of non-identical affinities per dimeric MSL-LysM molecule. Affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in MSL-LysM, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the present case two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of MSL-LysM for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.
Isırgan otunun (yaprağı ve kökü), antihistaminik, antialerjik, antiromatizmal, antiinflamatuvar etkileri nedeniyle dünyanın her yerinde olduğu gibi Türkiye'de de yaygın bir kullanımı vardır. Bu derlemede amacımız, ısırgan yaprağının kontakt temas ve oral yolla alımı sonrası ortaya çıkan etkilerin değerlendirilmesidir. Anahtar Kelimeler: Isırgan yaprağı; Alerji; Histamin; Ürtiker.
Datura stramonium seeds contain at least three chitin-binding isolectins [termed Datura stramonium agglutinin (DSA)] as homo- or heterodimers of A and B subunits. We isolated a cDNA encoding isolectin B (DSA-B) from an immature
fruit cDNA library; this contained an open reading frame encoding 279 deduced amino acids, which was confirmed by partial
sequencing of the native DSA-B peptide. The sequence consisted of: (i) a cysteine (Cys)-rich carbohydrate-binding domain composed
of four conserved chitin-binding domains and (ii) an extensin-like domain of 37 residues containing four SerPro4–6 motifs that was inserted between the second and third chitin-binding domains (CBDs). Although each chitin-binding domain
contained eight conserved Cys residues, only the second chitin-binding domain contained an extra Cys residue, which may participate
in dimerization through inter-disulfide bridge formation. Using matrix-assisted laser desorption/ionization-time-of-flight
mass spectrometry, the molecular mass of homodimeric lectin composed of two B-subunits was determined as 68,821 Da. The molecular
mass of the S-pyridilethylated B-subunit were found to be 37,748 Da and that of the de-glycosylated form was 26,491 Da, which correlated
with the molecular weight estimated from the deduced sequence. Transgenic Arabidopsis plants overexpressing the dsa-b demonstrated hemagglutinating activity. Recombinant DSA-B was produced as a homodimeric glycoprotein with a similar molecular
mass to that of the native form. Moreover, the N-terminus of the purified recombinant DSA-B protein was identical to that
of the native DSA-B, confirming that the cloned cDNA encoded DSA-B.
More than 100 years have passed since the first lectin ricin was discovered. Since then, a wide variety of lectins (lect means "select" in Latin) have been isolated from plants, animals, fungi, bacteria, as well as viruses, and their structures and properties have been characterized. At present, as many as 48 protein scaffolds have been identified as functional lectins from the viewpoint of three-dimensional structures as described in this chapter. In this chapter, representative 53 lectins are selected, and their major properties that include hemagglutinating activity, mitogen activity, blood group specificity, molecular weight, metal requirement, and sugar specificities are summarized as a comprehensive table. The list will provide a practically useful, comprehensive list for not only experienced lectin users but also many other non-expert researchers, who are not familiar to lectins and, therefore, have no access to advanced lectin biotechnologies described in other chapters.
The lectins, that can be used as tools to study glycobiological systems are defined as applied lectins (1–14).They are easily obtained, stable and have their own binding specificity extending beyond the monosaccharide(1–4,15–19).Their biochemical application has been reviewed extensively by Goldstein and Poretz (19), Sharon and Lis (8) and Goldsteinet al. (11) Hundreds of lectins are used as applied lectins. Thus, organizing and grouping binding properties of these lectins should
facilitate the selection of lectins as structural probes for studying glycans as well as the interpretation, distribution
and properties of the carbohydrate chains on the cell surface. From the information provided by inhibition assays and binding
properties, the carbohydrate specificities of applied lectins are classified into six groups according to their specificities
to monosaccharides. The subgroups are based on lectin affinities to GalNAca1-0 to Ser(Thr) of the peptide chain, disaccharides,
trisaccharides and, the number and the location of LFucal-’linked to oligosaccharides; all of these structures are found mainly
in soluble glycoproteins and as cell surface glycoconjugates in mammals (1–4,18). Reviews concerning coding and classification of DGa1, GaINAc and Galf31-3/4G1cNAc specificities of applied lectins are
given in Secion 1–4 of this proceeding (18) together with references 1–4. A scheme of the classification is shown as follows.
Terpene diols and terpene diol glucosides were detected in methanolic root extracts of Urtica dioica. The structures of five new monoterpenoid components were elucidated by spectrometry. The preparation of trimethylsilyl derivatives of the monoterpene diols and the glucosides enhanced volatility and thermal stability. The mass spectra of the derivatives provided more information compared to those of the free compounds.
Previous studies have shown that lectins with specificity for GlcNAc residues, when fed in the diet of the cowpea weevil,Callosobruchus maculatus, cause delays in its development. We have begun anin vivo structure activity analysis to determine what molecular features lead to maximum lectin toxicity. Although rice lectin has a four-fold greater agglutinating activity toward mammalian erythrocytes than wheat germ agglutinin (WGA), its biological activity when fed toC. maculatus is similar to WGA. Stinging nettle lectin, a poor agglutinin, isca two to four times less effective than WGA.
This article provides extensive and exhaustive mathematical description of titration curves related to acid-base systems with mixtures of mono- and polyprotic acids and their salts and bases involved. The related curves are presented in compact forms facilitating further operations made for particular needs. Some derivative properties of the curves, such as buffer capacity and inflection points, are also discussed. The “windowed” (BV) buffer capacity is interrelated with “dynamic” (βV) buffer capacity, introduced for dynamic (titration) systems. The equations useful for searching the inflection points on titration curves are derived. A kind of “homogenization” of complex acid-base systems, with polyprotic acids with defined and/or undefined (e.g., fulvic acids) composition, with use of an approach based on Simms constants principle, has been considered in context with buffer capacity and alkalinity. The new concepts of total alkalinity (TAL) and total acidity (TAC), unlike ones considered hitherto, has been introduced. The TAL is determined according to curve–fitting method with use of iterative computer program, applied to nonlinear regression equation involving Simms or Hill constants. Searching the best fit of the related function is involved with addition of consecutive hyperbolic terms.
The lectin microarray is a novel platform for glycan analysis, having emerged only in recent years. Unlike other conventional methods, e.g., liquid chromatography and mass spectrometry, it enables rapid and high-sensitivity profiling of complex glycan features without the need for liberation of glycans. Target samples include an extensive range of glycoconjugates involved in cells, tissues, body fluids, as well as synthetic glycans and their mimics. Various procedures for rapid differential glycan profiling have been developed for glycan-related biomarkers. Such glycoproteomics targeting allows precise diagnosis of chronic diseases potentially related to cancer. Application of this method to evaluation of various types of stem cells resulted in the discovery of a new pluripotent cell-specific glycan marker. To explore this technology a more fundamental and extensive understanding of lectins is necessary in relation to the structural uniqueness of glycans. In this chapter, the essence of the lectin microarray is described with some focus on an evanescent-field-activated fluorescence detection principle as a system to achieve in situ (i.e., washing free) aqueous-phase observation under equilibrium conditions. The developed lectin microarray system allows even researchers with poor experience in glycan profiling to perform extensive high-throughput analysis targeting various forms of glycans and even cells.
The structure of an antibacterial and antitumor antibiotic setomimycin, produced by Streptomyces pseudovenezuelae AM-2947, was determined to be a unique substituted 9,9′-bianthryl 1 by means of various new 13C NMR techniques including 13C {1H} selective decoupling, 13C {1H} selective population transfer, and 13C {1H} NOE experiments. The biosynthetic studies were also carried out by labeling with [1-13C]- and [1,2-13C2]sodium acetates. The labeling pattern was determined by the 13C-13C coupling constants with the aid of 13C {13C} homonuclear decoupling experiments, which allowed for the elucidation that 1 is derived from two nonaketide metabolites via decarboxylation at the terminals.
The lectin from stinging nettle rhizomes, Urtica dioica agglutinin (UDA), did not affect the evolution of wet and dry weight, protein, nucleic acid, ATP, cAMP and glycerol content during early germination of Phycomyces blakesleeanus spores. However, earlier investigations established a strongly reduced mycelial growth of several phytopathogenic fungi by this small plant lectin. Total uptake and incorporation of radioactive precursors showed no differences between UDA or control hyphae, but UDA significantly altered the distribution patterns of [14C]-glucose incorporated into the walls of Phycomyces blakesleeanus (more label was recovered in the chitin fraction). Moreover, a small but significant stimulation of chitin synthase and a similar inhibition of chitin deacetylase was found in cell wall preparations. These observations could lead to a better understanding of plant-pathogen interrelationships and to a further elucidation of cell wall structure in fungi.
Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA- isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA- isolectins. The deduced amino acid sequences share 78.9–98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.
The nomenclature, structure, mode of action and applications of lectins are reviewed. This group of proteinaceous macromolecules has the property of interacting with carbohydrates through binding sites to create complexes. Such complex formation is dependent upon the particular lectin and its specificity for certain carbohydrate structures.The review deals with the vast array of these known lectins with emphasis on recent literature, and covers such additional aspects as occurrence, purification, specificity, and records the recent advances that have been made in the field since the original discovery of erythrocyte agglutination by plant extracts, and the original extensive work on the original lectin concanavalin A.
A novel lectin, called VisalbCBA, was isolated from European mistletoe (Viscum album). This lectin differs completely from the classical galactose/N-acetylgalactosamine-binding mistletoe lectins MLI, MLII and MLIII. Biochemical analyses indicated that VisalbCBA is a dimeric protein composed of two identical subunits of approx. 10 kDa. VisalbCBA exhibits specificity towards oligomers of N-acetylglucosamine and shows sequence homology to the previously isolated chitin-binding plant proteins. Although VisalbCBA is less toxic than the other mistletoe lectins, it definitely exhibits cytotoxic properties. The possible involvement of VisalbCBA in the biological and therapeutic effects of mistletoe is discussed.
Grouping of lectin-binding properties, based on determinant structure rather than monosaccharide-inhibition pattern, should facilitate the selection of lectins as structural probes for glycans, as well as for the interpretation of the distribution and the properties of the carbohydrate chains on the cell surface. Based on the binding specificities studied with glycan by precipitin-inhibition, competitive-binding, and hemagglutinin-inhibition assays, twenty d-galactose-or N-acetyl-d-galactosamine-(or both)-specific lectins have been divided into six classes according to their specificity for a disaccharide unit, as all or part of the determinants, and the α-d-GalpNAc-(1→3)-Ser(Thr) unit of the glycopeptide chain. A scheme of classification is shown as follows: (a) F-specific lectins [α-dGalpNAc-(1→3)-d-GalNAc, Forssman specific disaccharide]: Dolichos biflorus (DBL), Helix pomatia (HPL), hog peanut (ABL, Amphicarpaea bracteata), and Wistaria floribunda (WFL) lectins. (b) A-specific lectins [α-d-GalpNAc-(1→3)-d-Gal blood group A-specific disaccharide]: Griffonia (Bondeiraea) simplicifolia-A4 (GSI-A4), lima bean (LBL), soy bean(SBL), Vicia villosa (VVL), Wistaria floribunda (WFL), Dolichos biflorus (DBL), and Helix pomatia (HPL) lectins. (c) Tn-specific lectins [α-d-GalpNAc-(1→3)-Ser(Thr) of the protein core]: Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Machura pomifera (MPL), Bauhinia purpurea alba (BPL), HPL, and WFL, lectins. (d) T-specific lectins [β-d-Galp-(1→3)-d-GalNAc, the mucin-type sugar sequences on human erythrocyte membrane and T antigen, or the terminal, nonreducing disaccharide end-groups of the gangliosides]: Peanut (PNA), Bauhinia purpurea alba (BPL), Machura pomifera (MPL), Sophora japonica (SJL), Artocarpus integrifolia (Jacalin, AIL), and Artocarpus lakoocha (Artocarpin) lectins. (e) Type I and II specific lectins [β-d-Galp-(1→3 or 4)-d-GlCNAc, the disaccharide residues at the nonreducing end of the carbohydrate chains derived from either N- or O-glycans]: Ricinus communis agglutinin (RCAl), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL), lectins. (f) B-specific lectin [α-d-Galp-(1→3)-β-d-Galp, human blood group B-specific disaccharide]: Griffonia (Bandeiraea) simplicifolia B4 (GSI-B4) lectin. Many other GalNAc- or Gal-(or both)-specific lectins that can be used as tools are also described.
A legume-type lectin (L-Lectin) gene of the red algae Gracilaria fisheri (GFL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of GFL was 1714 bp and contained a 1542 bp open reading frame encoding 513 amino acids with a predicted molecular mass of 56.5 kDa. Analysis of the putative amino acid sequence with NCBI-BLAST revealed a high homology (30-68%) with legume-type lectins (L-lectin) from Griffithsia japonica, Clavispora lusitaniae, Acyrthosiphon pisum, Tetraodon nigroviridis and Xenopus tropicalis. Phylogenetic relationship analysis showed the highest sequence identity to a glycoprotein of the red algae Griffithsia japonica (68%) (GenBank number AAM93989). Conserved Domain Database analysis detected an N-terminal carbohydrate recognition domain (CRD), the characteristic of L-lectins, which contained two sugar binding sites and a metal binding site. The secondary structure prediction of GFL showed a beta-sheet structure, connected with turn and coil. The most abundant structural element of GFL was the random coil, while the alpha-helixes were distributed at the N- and C-termini, and 21 beta-sheets were distributed in the CRD. Computer analysis of three-dimensional structure showed a common feature of L-lectins of GFL, which included an overall globular shape that was composed of a beta-sandwich of two anti-parallel beta-sheets, monosaccharide binding sites, were on the top of the structure and in proximity with a metal binding site. Northern blot analysis using a DIG-labelled probe derived from a partial GFL sequence revealed a hybridization signal of (approx.) 1.7 kb consistent with the length of the full-length GFL cDNA identified by RACE. No detectable band was observed from control total RNA extracted from filamentous green algae.
In recent years, evidence has been accumulating that protein–carbohydrate interactions play an important role in host–pathogen
interaction(s), development, cell–cell communication, and cell signaling. To study the protein–carbohydrate recognition phenomena
that take place within or at the surface of a cell, it is requisite to have the appropriate tools for dissecting this type
of interaction. During the past decade, microarray technology has successfully been introduced into the field of glycobiology.
These carbohydrate or glycan microarrays allow the rapid and comprehensive screening of carbohydrate-binding proteins for
interaction with a large set of carbohydrate structures and characterization of their carbohydrate-binding properties.
The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydrate-binding sites. AH strongly interacts with gp120 derived from different X4 and R5 human immunodeficiency virus (HIV) strains, simian immunodeficiency virus (SIV) gp130, and HIV type 1 (HIV-1) gp41 with affinity constants (KD) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (alpha1,2-mannose)3 oligosaccharide but not by alpha1,3/alpha1,6-mannose- or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of alpha1,2-mannose-specific monoclonal antibody 2G12, and AH covers a broader epitope on gp120 than 2G12. Prolonged exposure of HIV-1-infected CEM T-cell cultures with escalating AH concentrations selects for mutant virus strains containing N-glycosylation site deletions (predominantly affecting high-mannose-type glycans) in gp120. In contrast to 2G12, AH has a high genetic barrier, since several concomitant N-glycosylation site deletions in gp120 are required to afford significant phenotypic drug resistance. AH is endowed with broadly neutralizing activity against laboratory-adapted HIV strains and a variety of X4 and/or R5 HIV-1 clinical clade isolates and blocks viral entry within a narrow concentration window of variation (approximately 5-fold). In contrast, the neutralizing activity of 2G12 varied up to 1,000-fold, depending on the virus strain. Since AH efficiently prevents syncytium formation in cocultures of persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T lymphocytes, inhibits dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated capture of HIV-1 and subsequent virus transmission to CD4+ T lymphocytes, does not upregulate cellular activation markers, lacks mitogenic activity, and does not induce cytokines/chemokines in peripheral blood mononuclear cell cultures, it should be considered a potential candidate drug for microbicidal use.
N-Glycans are major components of many glycoproteins. These sugar moieties are frequently involved in important physiological and disease processes via their interactions with a variety of glycan-binding proteins (GBP). Clustering effect is an important feature in many glycan-lectin interactions. We describe in this paper a chemoenzymatic synthesis of novel N-glycan clusters using a tandem endoglycosidase-catalyzed transglycosylation. It was found that the internal beta-1,2-linked GlcNAc moieties in the N-glycan core, once exposed in the nonreducing terminus, was able to serve as acceptors for transglycosylation catalyzed by Endo-A and EndoM-N175A. This efficient chemoenzymatic method allows a quick extension of the sugar chains to form a class of glycan clusters in which sugar residues are all connected by native glycosidic linkages found in natural N-glycans. In addition, a discriminative enzymatic reaction at the two GlcNAc residues could be fulfilled to afford novel hybrid clusters. Lectin microarray studies revealed unusual properties in glyco-epitope expression by this panel of structurally well-defined synthetic N-glycans. These new compounds are likely valuable for functional glycomics studies to unveil new functions of both glycans and carbohydrate-binding proteins.
Given the continuing expansion of the HIV pandemic, more emphasis on new methods of treatment and prevention of the HIV infection is urgently needed.
Several carbohydrate-binding agents, basically monomeric, dimeric or tetrameric proteins (lectins), are endowed with a pronounced anti-HIV activity in cell culture. They inhibit the viral entry (fusion) process and can block HIV infections by cell-free virions, as well as virus cell-cell transmission. Prolonged exposure to carbohydrate-binding agents slowly and progressively selects for deletions of glycans at N-glycosylation sites in HIV gp120. Such mutant virus strains retain full sensitivity to other anti-HIV drugs. Moreover, virus strains that contain deleted glycans in their gp120 envelope may trigger the immune system to produce neutralizing antibodies against the uncovered immunogenic epitopes on HIV gp120, contributing further to the elimination of the mutant virus particles from the bloodstream. Efficient expression of carbohydrate-binding agents by commensal bacteria has been achieved and may be an interesting novel approach to protect the vaginal mucosa against HIV infection/transmission.
Carbohydrate-binding proteins are a unique group of natural products that may qualify as efficient microbicides and as a potential tool to prevent mother-to-infant virus transmission.
Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.
Concanavalin A, a globulin found in the jack bean, reacts specifically to form a precipitate with a restricted group of branched polysaccharides. The various parameters optimal for this interaction were investigated by the quantitative precipitin method with a dextran as the precipitating polysaccharide. In this manner, an assay for concanavalin A activity was established. The analogy of this interaction with the antibody-antigen system is striking. A summary of findings follows.
1. Complete precipitation is achieved in 24 hours at 25°.
2. The pH range optimum for the reaction lies between 6.1 and 7.2.
3. The concentration of sodium chloride has no effect on the reaction when the system is buffered at pH 7.0 with phosphate. KI and KCNS, however, are inhibitory.
4. The presence of foreign proteins does not affect the total amount of concanavalin A precipitated.
5. The precipitate formed between concanavalin A and dextran is slightly soluble (1.5 µg of nitrogen per ml) at 25°.
6. More nitrogen is precipitated at 25° than at 0°, the relative amount depending upon the region of the equivalence curve examined.
1. The purification of wheat-germ agglutinin from commercial wheat germ is described. By ion-exchange chromatography three active proteins (isolectins) were separated, one of which was examined in detail. 2. The amino acid composition is unusual, as 20% of residues are half-cystine and 21% are glycine. Unlike most lectins and contrary to previous reports, this protein is not a glycoprotein. 3. The efficiency of various saccharides as inhibitors of the agglutination reaction was investigated and from this the specificity of the binding site was inferred. Of monosaccharides, only derivatives of glucose with a 2-acetamido group and a free 3-hydroxyl group are effective inhibitors, and glycosides of either anomeric configuration are bound. Oligosaccharides are much more powerful inhibitors of agglutination than are monosaccharides. 4. It is proposed that the binding site consists of three or four subsites with differing specificities, in a cleft in the molecule resembling that proposed for hen's-egg-white lysozyme.
A lectin was isolated from elder (Sambucus nigra) bark by affinity chromatography on fetuin-agarose. It is a tetrameric molecule (Mr 140000) composed of two different subunits of Mr 34500 and 37500 respectively, held together by intramolecular disulphide bridges. The lectin is a glycoprotein and is especially rich in asparagine/aspartic acid, glutamine/glutamic acid, valine and leucine. It is also the first lectin isolated from a species belonging to the plant family Caprifoliaceae.
In the tomato (Lycopersicon esculentum) plant, the fruit juice was found to be the richest source of agglutinating activity. The lectin responsible could be inhibited by oligomers of N-acetylglucosamine, and this property was exploited to purify the lectin by affinity adsorption on trypsin-treated erythrocytes. The lectin is a glycoprotein that cross-reacts immunologically with the lectin from Datura stramonium (thorn-apple).
An N-acetylgalactosamine-specific lectin has been isolated from root stocks of Bryonia dioica by affinity chromatography on fetuin-agarose. It is a dimeric protein composed of two different subunits of relative molecular masses 32,000 and 30,000, held together by intermolecular disulphide bonds. Although most abundant in root stocks, the lectin occurs in all vegetative parts of the plant but not in seeds. Bryony lectin differs from other Cucurbitaceae lectins and from all known N-acetylgalactosamine-specific lectins.
A lectin has been isolated from rhizomes of ground elder (Aegopodium podagraria) using a combination of affinity chromatography on erythrocyte membrane proteins immobilized on cross-linked agarose and hydroxyapatite, and ion-exchange chromatography. The molecular structure of the lectin was determined by gelfiltration, sucrose density-gradient centrifugation and gel electrophoresis under denaturing conditions. It has an unusually high Mr (about 480000) and is most probably an octamer composed of two distinct types of subunits with slightly different Mr (about 60000). Hapten inhibition assays indicated that the Aegopodium lectin is preferentially inhibited by N-acetylgalactosamine. Nevertheless, it does not agglutinate preferentially blood-group-A erythrocytes. The ground-elder lectin is a typical non-seed lectin, which occurs virtually exclusively in the underground rhizomes. In this organ it is an abundant protein as it represents up to 5% of the total protein content. The lectin content of the rhizome tissue varies strongly according to its particular location along the organ. In addition, the lectin content changes dramatically as a function of the seasons. The ground-elder lectin differs from all other plant lectins by its unusually high molecular weight. In addition, it is the first lectin to be isolated from a species of the family Apiaceae.
An unusual lectin has been isolated from stinging nettle (Urtica dioica L.) rhizomes. It is a small (8.5 kDa) monomeric protein with high contents of glycine, cysteine and tryptophan. The U. dioica agglutinin (UDA) is not blood group-specific and is specifically inhibited by N-acetylglucosamine oligomers. As compared to other plant lectins, UDA has a very low specific agglutination activity. Nevertheless, it induces HulFN-γ in human lymphocytes at concentrations comparable to those of other inducers.
Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/23955/1/0000202.pdf
Wheat germ agglutinin (WGA) specifically binds N-acetylneuraminic acid and N-acetyl-D-galactosamine in addition to N-acetyl-D-glucosamine and its β-(1→4)-linked oligosaccharides. Both ovine submaxillary mucin (OSM) and asialo ovine submaxillary mucin, glycoconjugates lacking N-acetyl-D-glucosamine units, precipitate WGA. The precipitation of WGA by OSM is inhibited completely by D-GlcNAc as well as by the following saccharides (potency relative to GlcNAc (1.0) in parentheses): N,N′-diacetylchitobiose (32); N-acetylneuraminic acid (0.30); N-acetylneuraminic acid methyl ester (0.27); NeuNAc-7, 5-acetamido-3,5-dideoxy-Larabino-heptulosonic acid (0.80); N-acetyl-D-galactosamine (0.20). Selective conversion of the N-acetylneuraminic acid groups of fetuin and of OSM to NeuNAc-7 (two fewer carbon atoms) by periodate oxidation-borohydride reduction yielded analogue glycoproteins that reacted more strongly with WGA than the parent glycoproteins. Analogue fetuin, for example, precipitated 2.5 times more WGA from solution than native fetuin. A threefold higher concentration of N,N′-diacetylchitobiose also was required to inhibit the analogue fetuinWGA interaction than to inhibit equivalently the native fetuin-WGA interaction. Sialic acid groups appear to be immunodominant in fetuin-WGA interaction. Removal of sialic acid from fetuin or analogue fetuin by mild acid hydrolysis abolished the ability of the resulting substrates to precipitate WGA but had no effect on their interaction with the phyto-hemagglutinin from Phaseolus vulgaris. These results are interpreted in terms of the configurational similarity of NeuNAc and D-GalNAc with D-GlcNAc at positions C-2 (N-acetamido group) and C-3 (hydroxyl group) of the pyranose ring; these are the positions critical to productive contact with the WGA combining site.
Bandeiraea simplicifolia I plant seed isolectins comprise a family of tetrameric alpha-D-galactopyranosyl-binding glycoproteins composed of various combinations of teo different kinds of subunits designated A and B. Subtypes of the A (Aa, Ab, Ac, Ad, and Ae) and B (Ba, Bb, Bc, Bd, and Be) subunits were demotypes varies from seed to seed (e.g., some seeds contain only B subunits, others only A subunits), subtypes Ac and Bc predominate in a natural mixture of the isolectins. Two-dimensional agar gel diffusion studies indicate that, in addition to common structural features, each subunit contains its own distinct antigenic determinants. Although the A and B subunits have closely similar amino acid compositions, they differ markedly in one respect: the B subunit has one methionine residue whereas the A subunit contains no methionine. The neutral carbohydrate content of both subunits is identical. The ability of biopolymers and synthetic glycoproteins to precipitate A4 and B4, as well as the capacity of sugars and oligosaccharides to inhibit precipitate formation, was examined. On the basis of these studies, it is suggested that hydrogen bonding occurs between the hydrogen atoms of the C-3 and C-4 hydroxyl groups of alpha-D-GalNAcp and alpha-D-galp units and the A and B subunits, respectively.
Lectins play an important role in the development of immunology. Lectins also find application in serological laboratories for typing blood and determining secretor status, separating leucocytes from erythrocytes, and agglutinating cells from blood in the preparation of plasma. They serve as reagents for the detection, isolation, and characterization of carbohydrate-containing macromolecules, including blood-group antigens. In their interaction with saccharides, lectins serve as models for carbohydrate-specific antibodies, with the important advantage to purify lectins in gram quantities. Lectins are classified according to their carbohydrate-binding specificity that includes D-mannose(D-glucose)-binding lectins and 2-acetamido-2-deoxy-D-glucose-binding lectins. The chapter considers only those lectins that have been purified to homogeneity, and studied with regard to their biophysical, biochemical, and carbohydrate-binding specificity. The chapter also describes the cell-binding and biological properties of lectins. The chapter concludes with the description of several glycopeptide structures showing the carbohydrate-binding loci with which various lectins interact.
The lectin of black locust (Robinia pseudacacia) bark was isolated by specific adsorption on formaldehyde-fixed human erythrocytes and elution with a borate solution. The lectin is homogeneous on disc electrophoresis and ultracentrifugation (s20,w = 5.8 S) but yields three bands on isoelectric focusing. It has a molecular weight of approximately 110,000 and consists of two types of subunit (mol. wt 29,000 and 31,500). Its pI is approximately 5.9; it contains high amounts of aspartic acid, threonine and serine, no cysteine and very little methionine. Also 7.2% of covalently bound neutral sugar and 0.47% of glucosamine are present. The lectin is nonspecific in agglutination of human erythrocytes, it is inhibited by high concentrations of N-acetyl-D-galactosamine and is mitogenic in rabbit lymph node lymphocytes.
A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 microgram protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with Mr approximately 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-D-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.
The leaves and stems of the Dolichos biflorus plant contain a protein that cross reacts with antibodies to the seed lectin. This cross reactive material (CRM) has been isolated by (NH4)2SO4 precipitation and ion-exchange chromatography on DEAE-cellulose and CM-cellulose. The isolated CRM forms a single diffuse band in polyacrylamide gel electrophoresis at pH 9.7 and gives a reaction of partial identity to the seed lectin when tested in immunodiffusion against antibodies to the seed lectin. The CRM has an amino acid composition similar to that of the seed lectin and has a molecular weight of 68 000-70 000 as estimated by measurements of retardation of electrophoretic mobility with increasing gel concentrations. Discontinuous polyacrylamide gel electrophoresis of the CRM in 0.1% sodium dodecyl sulfate and 8.0 M urea shows the CRM has a subunit identical in mobility with that of subunit IA of the seed lectin and a second subunit of higher molecular weight than subunit IA. Sequence determination of the first 13 NH2-terminal residues of the CRM indicates that both subunits have the same NH2-terminal sequence; this sequence is identical with the sequences of the seed lectin subunits with the exception of an aspartic acid in place of an asparagine at the second residue. Unlike the seed lectin, the CRM does not agglutinate nor inhibit the agglutination of type A erythrocytes, nor does it bind to polyleucyl hog blood group A + H substance. The relationship of the CRM to the seed lectin is discussed, including the possibility that the CRM may be a precursor to the active lectin.
The interaction of ricin, one of the two lectins of Ricinus sanguineus, with its specific ligands galactose and lactose (4-O-beta-D-galactopyranosyl-D-glucopyranose) has been studied by means of equilibrium dialysis, analytical ultracentrifugation and fluorescence polarization. In the studied concentration range, only one molecule of galactose is bound per molecule of ricin with an association constant, Ka = 6900 m-1 at 4 degrees C. Scatchard plots of equilibrium dialysis data show that two molecules of lactose bind to one molecule of ricin, without modification of molecular weight of the lectin. Together with results of microcalorimetric experiments and agglutination of erythrocytes by ricin, equilibrium dialysis data indicate that the lectin contains two distinct saccharide binding sites. Regardless of the existence of extended sites, it is not possible to select between the two models: (a) two independent sites (Ka1 = 35 000 M-1, Ka2 = 2800 M-1 at 4 degrees C) or (b) two identical sites with negative cooperativity.
The ability of wheat germ agglutinin to form precipitates with a series of synthetic carbohydrate-protein conjugates and with carcinoembryonic antigen and its Smith degradation products was investigated. The precipitation reaction between wheat germ agglutinin and p-azophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside-bovine serum albumin was selected to examine the capacity of a large number of sugar haptens to inhibit this system. Our results indicate that the wheat germ agglutinin binding site is complementary to a sequence of three beta-(1 leads to 4)-linked N-acetyl-D-glucosamine units (N,N'N"-triacetyl chitotriose). The internal carbohydrate portion of carcinoembryonic antigen probably contains two such units and wheat germ agglutinin precipitates with untreated as well as sequentially Smith degraded carcinoembryonic antigen. Compared with other reports certain discrepancies in the relative binding affinities of per N-acetylated chitodextrins and N-acetyl-D-glucosamine were found. These differences are discussed in terms of the methods used and the proposed subsite hypothesis of Allen, A.K., Neuberger, A. and Sharon, N. (1973) Biochem. J. 131, 155-162.
1. Potato lectin has been purified and shown to be a glycoprotein containing about 50% of carbohydrate. Most of the sugar residues (92%) are arabinose; small amounts of galactose, glucose and glucosamine are also present. 2. The most abundant amino acid is hydroxyproline (16% of the residues), 11.5% of the residues are half-cystine and phenylalanine is absent. The lectin also contains about one residue/molecule of a basic amino acid, not usually found in proteins, which has been tentatively identified as ornithine. There is indirect evidence that the components of the glycoprotein are linked through hydroxyproline and arabinose. 3. By gel filtration in 6m-guanidine-HCl on Sepharose 4B, it was found that both the native glycoprotein and its S-carboxymethylated derivative had subunit molecular weights of 46000 (+/-5000). In a non-denaturing solution, two of these units appear to be associated. 4. The lectin is specifically inhibited in its agglutination reaction by oligosaccharides that contain N-acetylglucosamine. Its specificity is similar to, but not identical with, that of wheat-germ agglutinin.
A number of meta-alkylphenyl [beta]--glucopyranosides were synthesized and their ability to inhibit the concanavalin A-polysaccharide system was examined. The binding constants of these compounds as well as other substituted phenyl [amalgamation or coproduct]--glucopyranosides were related to the hydrophobic ([pi]) and electronic ([sigma]) nature of the substituents utilizing the equations devised by Hansch S and Hammett[paragraph sign] respectively.Regression analysis of these relationships revealed that: (1) no linear correlation between the binding constants and the electronic properties of the aromatic substituents was evident; (2) the molecular volume of mono-ortho-substituents does not significantly effect the binding of aromatic [beta]--glucopyranosides to concanavalin A; and (3) the hydrophobic nature ([pi]) of ortho- and meta- but not para-substituents is closely associated with the binding of aryl [beta]--glucosides to concanavalin A.It is proposed that apolar binding involving hydrophobic interactions associated with ortho and meta but not with the para positions of the aromatic nucleus are the predominant forces involved in the binding of the phenyl moiety of phenyl [beta]--glucosides to concanavalin A. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/33567/1/0000068.pdf
The interaction of concanavalin A, the phytohemagglutinin of the jack bean, with a variety of glycosides has been studied by the technique of ultraviolet difference spectroscopy. Whereas methyl α-d-gluco- and manno-pyranoside gave rise to relatively low intensity difference spectra, p-nitrophenyl α-d-mannopyranoside and α-d-glucopyranoside yielded large difference spectra upon interaction with concanavalin A. Using this technique as a measure of concanavalin A activity, it was demonstrated that the protein specifically binds low molecular weight carbohydrates at much lower pH values (e. g. pH 2.4) than previously believed. Although polysaccharides are also bound at these low pH values, they are not precipitated by concanavalin A. Molecular weight studies in acid media indicate that the protein does not dissociate and it is suggested that electrostatic repulsion of the protein molecules due to their high net positive charge prevents protein-polysaccharide lattice formation and hence failure of the complex to precipitate.
For the study of the location of tyrosyl and tryptophyl groups in proteins by the solvent perturbation technique, and the simultaneous assessment of the number or fraction of both of these chromophores exposed in proteins, carefully measured molar absorptivity difference values of the N-acetyl ethyl esters of tyrosine and tryptophan have been obtained with nine of the most commonly employed perturbants ranging in diameter from 2.0 to 9.4 Å. Molar absorptivity differences in the wavelength region from 350 to 240 mμ have been collected and tabulated at convenient wavelength intervals, using aqueous solutions of 90% deuterium oxide and 20% methanol, dimethyl sulfoxide, ethylene glycol, glycerol, erythritol, glucose, hexaethylene glycol (Carbowax 300), and sucrose as perturbants. For the study of urea-denatured proteins, model data have also been obtained in 8 M urea, with 20% dimethyl sulfoxide, ethylene glycol, glycerol, and sucrose as perturbants. The additivity of solvent perturbation difference spectra has been checked and found valid for both tyrosyl and tryptophyl model compounds and proteins, and the range of applicability in studies of proteins, rich in both tyrosine and tryptophan, has been defined and tested.
Concanavalin A, a globulin found in the jack bean, reacts specifically to form a precipitate with a restricted group of branched
polysaccharides. The various parameters optimal for this interaction were investigated by the quantitative precipitin method
with a dextran as the precipitating polysaccharide. In this manner, an assay for concanavalin A activity was established.
The analogy of this interaction with the antibody-antigen system is striking. A summary of findings follows.
1. Complete precipitation is achieved in 24 hours at 25°.
2. The pH range optimum for the reaction lies between 6.1 and 7.2.
3. The concentration of sodium chloride has no effect on the reaction when the system is buffered at pH 7.0 with phosphate.
KI and KCNS, however, are inhibitory.
4. The presence of foreign proteins does not affect the total amount of concanavalin A precipitated.
5. The precipitate formed between concanavalin A and dextran is slightly soluble (1.5 µg of nitrogen per ml) at 25°.
6. More nitrogen is precipitated at 25° than at 0°, the relative amount depending upon the region of the equivalence curve
examined.
The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (Mr = 86,000) possesses two carbohydrate-binding sites for N,N'N",N"'- tetraacetylchitotetritol /mol protein, with an apparent Ka = 8.7 X 10(3) M-1 at 4 degrees C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N'- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N',N"'- triacetylchitotriose was over 6-fold more potent than the disaccharide (N,N'- diacetylchitobiose ) which, in turn, was 90 times more reactive than N-acetyl-D-glucosamine. N-Acetyllactosamine [beta-D-Gal-(1----4)-D-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N'- diacetylchitobiose . The requirement for an N-acetyl-D-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.
The two unique sugar binding sites in wheat germ agglutinin, located in the subunit/subunit interface of the dimer molecule and termed primary and secondary binding sites, are compared in the light of the newly obtained chemical amino acid sequence and a high-resolution electron density map (1.8 A). Homology was found in the three amino acid residues directly involved in sugar binding: Tyr73II, Ser62II, Glu115I in the primary site, and Tyr159I, Ser148I, Asp29II in the secondary site (subscripts refer to promoters I and II). Thirteen corresponding side-chain atoms of these three homologous residues in the two sites could be superimposed with a root-mean-square difference of 1.39 A. The three sugar binding residues are located in subsite 1 of each extended binding location and contribute to binding of the terminal, non-reducing N-acetyl-D-glucosamine and N-acetyl-D-neuraminic acid residues only, and they provide three hydrogen bonds for complex stabilization. Two hydrogen bonds are made with the carbonyl and amido portions of the N-acetyl group and the third with the C-3 OH group of the sugar ring. It is suggested that small differences in the sugar binding affinities at these two unique sites exist, due to the different numbers of van der Waals' interactions made at these sites, which contribute to stabilizing, for instance, the wheat germ agglutinin/N,N'-diacetyl-chitobiose complex. The single tryptophan residue is located at a distance of approximately 13 A from the primary site and is thought to have no affect on sugar binding. In addition, the disposition of the four saccharide binding sites of the dimer with respect to three local, pseudo 2-fold symmetry axes, relating domains of opposite protomers, is discussed.
Potato lectin (Solanum tuberosum agglutinin, STA) was found to contain fluorescent tryptophan residues highly exposed to solvent. The binding of chitin oligosaccharides to STA induced fluorescence quenching, a shift of the fluorescence maximum to shorter wavelength, a decrease in the quenching constant of iodide ion and a decrease of the number of tryptophan residues modifiable by N-bromosuccinimide. The results suggested that one tryptophan residues is located at or near a sugar binding site of STA, and that its environment is altered from hydrophilic to relatively more hydrophobic upon interaction with specific sugars. The binding constants of STA with chitin oligosaccharides were determined by measuring the peak-trough heights in the fluorescence difference spectra induced by various concentrations of sugars. The inhibition constants of chitin oligosaccharides for the hemagglutinating activity of STA were obtained by the method of Pitts and Yang [(1981) Biochem. J. 195, 435-439] and the results were in good agreement with those obtained by the fluorescence spectral method. Standard and unitary free energy changes (delta G0 and delta Gu) and standard enthalpy changes (delta H0) were also obtained. These values decreased with sugar chain length up to at least the tetramer. Thus, it was assumed that there are at least 4 subsites, A, B, C, and D, in the sugar binding site of STA. The contributions to the binding energy (delta Gu) were -17.0, -12.6, -7.3, and -4.4 kJ/mol at subsites A, B, C, and D, respectively, and the bindings of chitin monomer (GlcNAc), dimer, trimer, and tetramer were assumed to occur at subsite A, AB, ABC, and ABCD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Despite differences in their accessibility in the crystal and specificity toward the two types of acetylated sugars, the two unique binding locations exhibit very similar saccharide binding modes. The main contribution to binding of oligosaccharides comes from interactions of the acetamido and one hydroxyl group of the non-reducing terminal sugars with the protein at subsite 1, whereas little or no contribution is provided by subsites 2 and 3. Moreover, oligosaccharides binding at the primary binding location which possess terminal sialic acid, assume a distinctly different orientation from that of N-acetylglucosamine oligomers, although they share subsite 1. This is due to a difference in position of their glycosidic hydroxyl groups. Space limitations in crosslinked crystals prevent oligomers of N-acetylglucosamine larger than the dimer from binding either at the primary or secondary binding locations.
It was found that the addition of specific sugars to solutions of several lectins induced ultraviolet difference spectra. The difference spectra of lectins from Lens culinaris, Sophora japonica, Solanum tuberosum and wheat germ have two peaks at 292 nm and 284-287 nm which are characteristic of the tryptophanyl residue. The difference spectrum of Arachis hypogaea agglutinin has two peaks at 285 nm and 279 nm which seem to be characteristic of the tyrosyl residue. In addition to the identification of these amino acid residues as being in or near the sugar binding sites of the lectins, the binding constants of these lectins with the specific sugars can be easily determined from the intensities of the difference spectra at various concentrations of sugars. This method may be useful for the simple and direct determination of the binding constants of lectins with various naturally occurring sugars which have no chromogenic groups.
Leaves from mature Griffonia simplicifolia plants were examined for the presence of leaf lectins possessing sugar binding specificities similar to the four known seed lectins (GS-I, GS-II, GS-III, GS-IV). Three (GS-I, -II, -IV) of the four known G. simplicifolia seed lectins were present in the leaves. Leaf G. simplicifolia lectins I and IV were similar to the respective seed lectins. Leaf GS-II, however, was composed of two types of subunits (M(r) = 33,000 and 19,000), whereas the seed lectin consists of only one type of subunit (M(r) 32,500). Seed and leaf GS-II lectins also had different isoelectric points. All leaf and seed lectins were similar with respect to their hemagglutination and glycoconjugate precipitation properties and all subunits contained covalently bound carbohydrate. Leaf GS-IV appeared slightly under-glycosylated compared to seed GS-IV.The fate of GS-I and GS-II seed lectins in aging cotyledons was investigated. GS-I isolectins usually contain isolectin subtypes associated with each main isolectin. Upon inbibition and germination, these GS-I isolectin subtypes disappeared. Over time, GS-II lectin did not change its disc gel electrophoretic properties.
- F B Yanke
- W E And Brill
DAZZO, F. B., YANKE, W. E., AND BRILL, W. J. (1978)
Biochim.
Biophys.
Acta 539,276-286.
- V Haskovec
- C And Kocourek
HOREJSI, V., HASKOVEC,
C., AND KOCOUREK,
J.
(1978) Biochim.
Biophys. Acta 532,98-104.
- C F Talbot
- M E Etzler
TALBOT, C. F., AND ETZLER, M. E. (1978) Biochemistry 17, 1474-1479.
- B D Ebisu
- S Goldstein
- I J And Flashner
PETERS, B. D., EBISU, S., GOLDSTEIN,
I. J., AND
FLASHNER,
M. (1979) Biochemistry
18, 5505-
5511.
- R And
- E Van Driessche
R., AND VAN DRIESSCHE, E. (1984) Planta 160,
222-228.
- W J Nsimba-Lubaki
- M Peeters
- B And Broekaert
PEUMANS, W. J., NSIMBA-LUBAKI,
M., PEETERS, B.,
AND BROEKAERT, W. F. (1984) Planta
164, 75-
82.
- A K Neuberger
- A And Sharon
ALLEN, A. K., NEUBERGER,
A., AND SHARON, N.
(1973) Biochem.
J 131,155-162.
- S Peters
- B Roberts
- D D Gold-Stein
- I J And Liotta
SHIBATA, S., PETERS, B., ROBERTS, D. D., GOLD-
STEIN, I. J., AND LIOTTA, L. A. (1982) FEBS Lett.
142,194-198.
- R D And Goldstein
PORETZ, R. D., AND GOLDSTEIN, I. J. (1971) Biochem.
PharmacoL
20,2727-2739.
- A Seno
- N And Matsumoto
JIMBO, A., SENO, N., AND MATSUMOTO,
I. (1984) J.
- J F Goldstein
- I J Arnarp
- J And Lonngren
CROWLEY, J. F., GOLDSTEIN, I. J., ARNARP, J., AND
LONNGREN,
J. (1984) Arch. Biochem.
Biophya
231, 524-533.
- H S Herskovits
- T T And
- S M Sorensen
SIMMS, H. S. (1926) J. Amer. Chem. Sot. 48,1239-
1250.
22. HERSKOVITS,
T. T., AND SORENSEN, S. M. (1968)
Biochemistry
7,2523-2532.
- W J De Ley
- M And Broekaert
PEUMANS, W. J., DE LEY, M., AND BROEKAERT, W.
F. (1984) FEBS Lett. 177,999103.
- I Jimbo
- A Kitagaki
- H Go-Lovtchenko-Matsumoto
- A M And Seno
MATSUMOTO,
I., JIMBO, A., KITAGAKI,
H., Go-
LOVTCHENKO-MATSUMOTO,
A. M., AND SENO, N.
(1980) J. Biochem.
88,1093-1096.
- I J Goldstein
- C E Hayes
GOLDSTEIN,
I. J., AND HAYES, C. E. (1978) Adv.
Carbohydr.
Chem. Biochem.
35.127-340.
- J E Lamb
- S Shibata
- I J Goldstein
LAMB, J. E., SHIBATA, S., AND GOLDSTEIN,
I. J.
(1983) P&t
Physiol. 71, 879-887.
- A K Allen
- A Neuberger
ALLEN, A. K., AND NEUBERGER,
A. (1973) Biochemistry
135,307-314.
- W J Peljmans
- M Nsimba-Lubaki
- A R Carlier
- E Van Driessche
PELJMANS, W. J., NSIMBA-LUBAKI,
M., CARLIER, A.
R., AND VAN DRIESSCHE, E. (1984) Planta 160,
222-228.