Carbohydrate Recognition Mechanism of the Mushroom Galectin ACG
25 Jan 2018-Trends in Glycoscience and Glycotechnology (FCCA(Forum: Carbohydrates Coming of Age))-Vol. 30, Iss: 172
About: This article is published in Trends in Glycoscience and Glycotechnology.The article was published on 2018-01-25 and is currently open access. It has received 9 citations till now. The article focuses on the topics: Cis trans isomerization & Galectin.
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TL;DR: A primer to the concept of the sugar code by glycan-(ga)lectin recognition is presented, followed by a survey on galectin-3 (considering common and distinct features within this family of multifunctional proteins expressed at various cellular sites and cell types).
Abstract: Introduction: The discoveries that sugars are a highly versatile platform to generate biochemical messages and that glycan-specific receptors (lectins) are a link between these signals and their bioactivity explain the interest in endogenous lectins such as galectins. Their analysis is a highly dynamic field. It is often referred to as being promising for innovative drug design. Area covered: We present a primer to the concept of the sugar code by glycan-(ga)lectin recognition, followed by a survey on galectin-3 (considering common and distinct features within this family of multifunctional proteins expressed at various cellular sites and cell types). Finally, we discuss strategies capable of blocking (ga)lectin activity, with an eye on current challenges and inherent obstacles. Expert opinion: The emerging broad profile of homeostatic and pathophysiological bioactivities stimulates further efforts to explore galectin (Gal-3) functionality, alone and then in mixtures. Like thoroughly assessing the pros and cons of blocking approaches for a multifunctional protein active at different sites, identifying a clinical situation, in which the galectin is essential in the disease process, will be critical.
25 citations
TL;DR: The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ.
Abstract: The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure–activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms.
18 citations
Cites background from "Carbohydrate Recognition Mechanism ..."
...2008; Iwaki and Hirabayashi 2018)....
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...With focus on the adhesion/growth-regulatory galectins, these multifunctional effectors are separated into three groups in vertebrates according to this criterion (Kasai and Hirabayashi 1996; Barondes 1997; Hirabayashi 1997, 2018; Cooper 2002; Kaltner et al....
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...Since structural aspects of the CRDs of many galectins are already well characterized (Iwaki and Hirabayashi 2018; Kamitori 2018; Romero and Gabius 2019), these endogenous lectins are highly suited for our work towards the aim to relate protein design to activity....
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TL;DR: Findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch forGal-7.
Abstract: Human galectin-7 (Gal-7; also termed p53-induced gene 1 product) is a multifunctional effector by productive pairing with distinct glycoconjugates and protein counter-receptors in the cytoplasm and nucleus, as well as on the cell surface. Its structural analysis by NMR spectroscopy detected doubling of a set of particular resonances, an indicator of Gal-7 existing in two conformational states in slow exchange on the chemical shift time scale. Structural positioning of this set of amino acids around the P4 residue and loss of this phenomenon in the bioactive P4L mutant indicated cis-trans isomerization at this site. Respective resonance assignments confirmed our proposal of two Gal-7 conformers. Mapping hydrogen bonds and considering van der Waals interactions in molecular dynamics simulations revealed a structural difference for the N-terminal peptide, with the trans-state being more exposed to solvent and more mobile than the cis-state. Affinity for lactose or glycan-inhibitable neuroblastoma cell surface contact formation was not affected, because both conformers associated with an overall increase in order parameters (S2). At low µM concentrations, homodimer dissociation is more favored for the cis-state of the protein than its trans-state. These findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch for Gal-7.
8 citations
TL;DR: This study broadens the scope for galectin targeting by calixarene‐based compounds and opens the perspective for selective Gal‐7 blocking by removing the N‐dimethyl alkyl chain amide groups.
Abstract: Calix[4]arene PTX008 is an angiostatic agent that inhibits tumor growth in mice by binding to galectin-1, a β-galactoside-binding lectin. To assess the affinity profile of PTX008 for galectins, we used 15 N,1 H HSQC NMR spectroscopy to show that PTX008 also binds to galectin-3 (Gal-3), albeit more weakly. We identified the contact site for PTX008 on the F-face of the Gal-3 carbohydrate recognition domain. STD NMR revealed that the hydrophobic phenyl ring crown of the calixarene is the binding epitope. With this information, we performed molecular modeling of the complex to assist in improving the rather low affinity of PTX008 for Gal-3. By removing the N-dimethyl alkyl chain amide groups, we produced PTX013 whose reduced alkyl chain length and polar character led to an approximately eightfold stronger binding than PTX008. PTX013 also binds Gal-1 more strongly than PTX008, whereas neither interacts strongly, if at all, with Gal-7. In addition, PTX013, like PTX008, is an allosteric inhibitor of galectin binding to the canonical ligand lactose. This study broadens the scope for galectin targeting by calixarene-based compounds and opens the perspective for selective galectin blocking.
7 citations
References
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TL;DR: Protein engineering and peptide synthesis have been used in order to understand the basis of these pathogenic mechanisms as well as to produce hypoallergenic proteins with potential diagnostic and immunotherapeutic applications.
Abstract: RNase T1 is the best known representative of a large family of ribonucleolytic
proteins secreted by fungi, mostly Aspergillus and Penicillium species. Ribotoxins
stand out among them by their cytotoxic character. They exert their toxic action by
first entering the cells and then cleaving a single phosphodiester bond located
within a universally conserved sequence of the large rRNA gene, known as the
sarcin–ricin loop. This cleavage leads to inhibition of protein biosynthesis,
followed by cellular death by apoptosis. Although no protein receptor has been
found for ribotoxins, they preferentially kill cells showing altered membrane
permeability, such as those that are infected with virus or transformed. Many steps
of the cytotoxic process have been elucidated at the molecular level by means of a
variety of methodological approaches and the construction and purification of
different mutant versions of these ribotoxins. Ribotoxins have been used for the
construction of immunotoxins, because of their cytotoxicity. Besides this activity,
Aspf1, a ribotoxin produced by Aspergillus fumigatus, has been shown to be one of
the major allergens involved in allergic aspergillosis-related pathologies. Protein
engineering and peptide synthesis have been used in order to understand the basis
of these pathogenic mechanisms as well as to produce hypoallergenic proteins with
potential diagnostic and immunotherapeutic applications.
139 citations
TL;DR: Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content—arabinogalactan inhibited hemagglutination significantly.
Abstract: Pectic polysaccharides from dietary sources such as Decalepis hamiltonii--swallow root (SRPP), Hemidesmus indicus (HPP), Nigella sativa--black cumin (BCPP), Andrographis serpyllifolia-(APP), Zingiber officinale--ginger (GRPP) and, citrus pectin (CPP) were examined for galectin inhibitory activity. Inhibition of (a) galectin-3 of MDA-MB-231 cells induced hemagglutination of red blood cells; (b) galectin-3 mediated interaction between normal/metastatic human buccal cells (NBC)/(MBC) and; (c) invasion of MDA-MB-231 and MBC in the invasive chamber was assessed. Results indicated that SRPP inhibited hemagglutination at Minimum Inhibitory Concentration (MIC) of 1.86 microg ml(-1) equivalent of carbohydrate as apposed to those of BCPP (130 microg ml(-1)), APP (40 microg ml(-1)), HPP (40 microg ml(-1)) and CPP (25 microg ml(-1)). GRPP even at concentration >1-6 mg ml(-1) did not inhibit agglutination. Also SRPP showed approximately 15 and 2 fold potent anti hemagglutination activity relative to that of galectin-3 specific sugars-galactose (MIC-27.1 microg ml(-1)) and lactose (MIC-4.16 microg ml(-1)) respectively. Further, SRPP at 10 microg ml(-1) inhibited agglutination of NBC by galectin-3 of MDA-MB-231 cells. Modified swallow root pectic polysaccharide (MSRPP) of 50 kDa retained anti hemagglutination activity (MIC of 1.03 microg ml(-1)) and inhibited MDA-MB-231 and MBC invasion by 73 and 50% with an IC(50) of 136 and 200 microg ml(-1) respectively. Both SRPP and MSRPP induced apoptosis up to 80% at 100 microg ml(-1) concentration by activating approximately 2 and 8 folds of Caspase-3 activity. Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content-arabinogalactan inhibited hemagglutination significantly.
110 citations
TL;DR: It is proposed that filamentous fungi possess an inducible resistance against predators and parasites mediated by lectins that are specific for glycans of these antagonists.
Abstract: Fruiting body lectins are ubiquitous in higher fungi and characterized by being synthesized in the cytoplasm and up-regulated during sexual development. The function of these lectins is unclear. A lack of phenotype in sexual development upon inactivation of the respective genes argues against a function in this process. We tested a series of characterized fruiting body lectins from different fungi for toxicity towards the nematode Caenorhabditis elegans, the mosquito Aedes aegypti and the amoeba Acanthamoeba castellanii. Most of the fungal lectins were found to be toxic towards at least one of the three target organisms. By altering either the fungal lectin or the glycans of the target organisms, or by including soluble carbohydrate ligands as competitors, we demonstrate that the observed toxicity is dependent on the interaction between the fungal lectins and specific glycans in the target organisms. The toxicity was found to be dose-dependent such that low levels of lectin were no longer toxic but still led to food avoidance by C. elegans. Finally, we show, in an ecologically more relevant scenario, that challenging the vegetative mycelium of Coprinopsis cinerea with the fungal-feeding nematode Aphelenchus avenae induces the expression of the nematotoxic fruiting body lectins CGL1 and CGL2. Based on these findings, we propose that filamentous fungi possess an inducible resistance against predators and parasites mediated by lectins that are specific for glycans of these antagonists.
91 citations
TL;DR: A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F and strongly interacted with glycoconjugates containing NeuAcα2,3Galβ1,3GlcNAc-/GalNAc sequences.
Abstract: A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein.
56 citations
TL;DR: Results indicate that the integrity of key amino acid residues responsible for recognition of lactose or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectin genes.
48 citations