Targeting Glycosylation Aberrations to Improve the Efficiency of Cancer Phototherapy.

The use of photodynamic therapy in cancer still remains limited, partly because of the lack of photosensitizer (PS) specificity for the cancerous tissues. Various molecular tools are available to increase PS efficiency by targeting the cancer cell molecular alterations. Most strategies use the protein-protein interactions, e.g. monoclonal antibodies directed toward tumor antigens, such as HER2 or EGFR. An alternative could be the targeting of the tumor glycosylation aberrations, e.g. T/Tn antigens that are truncated O-glycans over-expressed in numerous tumors. Thus, to achieve an effective targeting, PS can be conjugated to molecules that specifically recognize the Oglycosylation aberrations at the cancer cell surface.

Juvenile hormone affects the splicing of Culex quinquefasciatus early trypsin messenger RNA.

The full length of Culex quiquefasciatus early trypsin has been cloned and sequenced and a three-dimensional (3D) model of the enzyme was built showing that the enzyme has the canonical trypsin's active pocket containing H78, D123, S129, and D128. The biosynthesis of juvenile hormone (JH) III by the corpora allata (CA) in female Cx. quiquefasciatus is sugar-dependent. Females that were maintained on water after emergence synthesize very little JH III, JH III bisepoxide, and methyl farnesoate (MF) (3.8, 1.1, and 0.8 fmol/4 hr/CA, respectively). One hour after sugar feeding, the synthesis of JH III and JH III bisepoxide reached a maximum (11.3 and 5.9 fmol/4 hr/CA, respectively) whereas MF biosynthesis reached a maximum at 24 hr (5.2 fmol/4 hr/CA). The early trypsin is transcribed with a short intron (51 nt) is spliced when JH III biosynthesis is high in sugar fed and at 1 hr after the blood meal (22 and 15 fmol/4 hr/CA, respectively). We investigated the transcriptional and posttranscriptional regulation of the early trypsin gene showing that JH III concentrations influence splicing. In the absence JH III the unspliced transcript is linked by a phosphoamide bond at the 5'-end to RNA ribonuleoprotein (RNP). The biosynthesis of the early trypsin was followed in ligated abdomens (without CA) of newly emerged females that fed blood by enema. Our results show that the early trypsin biosynthesis depends on sugar and blood feeding, whereas the late trypsin biosynthesis does not depend on sugar feeding, or JH III biosynthesis. Downregulating the early trypsin transcript does not affect the late trypsin.

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy.

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors.

Differences in the mannose oligomer specificities of the closely related lectins from Galanthus nivalis and Zea mays strongly determine their eventual anti-HIV activity.

BACKGROUND: In a recent report, the carbohydrate-binding specificities of the plant lectins Galanthus nivalis (GNA) and the closely related lectin from Zea mays (GNAmaize) were determined by glycan array analysis and indicated that GNAmaize recognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity. RESULTS: GNAmaize appeared to be ~20- to 100-fold less inhibitory than GNA against HIV infection, syncytia formation between persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T-lymphocyte SupT1 cells, HIV-1 capture by DC-SIGN and subsequent transmission of DC-SIGN-captured virions to uninfected CD4+ T-lymphocyte cells. In contrast to GNA, which preferentially selects for virus strains with deleted high-mannose-type glycans on gp120, prolonged exposure of HIV-1 to dose-escalating concentrations of GNAmaize selected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR) analysis revealed that GNA and GNAmaize interact with HIV IIIB gp120 with affinity constants (KD) of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNAmaize selectively binds complex-type GlcNAcß1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNAmaize to gp120, whereas GNAmaize cannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcß1,2man specific) or SNA (NeuAcα2,6X specific). CONCLUSION: The markedly reduced anti-HIV activity of GNAmaize compared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNAmaize that have high affinity for mannose oligomers. These findings underscore the need for mannose oligomer recognition of therapeutics to be endowed with anti-HIV activity and that mannose, but not complex-type glycan binding of chemotherapeutics to gp120, may result in a pronounced neutralizing activity against the virus.

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket.

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.

IgE-binding epitopic peptide mapping on a three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum).

The three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum) consists of three protomers exhibiting the cupin motif, arranged in a homotrimer around a three-fold symmetry axis. Using the SPOT technique, 11 continuous IgE-binding epitopic peptides were characterized on the molecular surface of the 13S globulin allergen of buckwheat. Except for one of them, they all correspond to well exposed regions containing electropositiveley and/or electronegatively charged residues, which cover up to 40% of the molecular surface of the allergen. Some of these epitopes come in close contact to probably create more extended discontinuous epitopes, especially those located on the edge of the 13S globulin homotrimer. Half of the identified epitope peptides remain unaltered in a core structure protected against hydrolysis by digestive proteases and are thus assumed to promote the allergenicity of the 13S globulin. In addition, a few of these epitopes coincide with sequential IgE-binding epitopes previously characterized in soybean 11S globulins, that could account for the IgE-binding cross-reactions observed between soybean and buckwheat in Western blot experiments.

Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of multispecificity through domain duplication and divergent evolution.

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.

A novel family of lectins evolutionarily related to class V chitinases: an example of neofunctionalization in legumes.

A lectin has been identified in black locust (Robinia pseudoacacia) bark that shares approximately 50% sequence identity with plant class V chitinases but is essentially devoid of chitinase activity. Specificity studies indicated that the black locust chitinase-related agglutinin (RobpsCRA) preferentially binds to high-mannose N-glycans comprising the proximal pentasaccharide core structure. Closely related orthologs of RobpsCRA could be identified in the legumes Glycine max, Medicago truncatula, and Lotus japonicus but in no other plant species, suggesting that this novel lectin family most probably evolved in an ancient legume species or possibly an earlier ancestor. This identification of RobpsCRA not only illustrates neofunctionalization in plants, but also provides firm evidence that plants are capable of developing a sugar-binding domain from an existing structural scaffold with a different activity and accordingly sheds new light on the molecular evolution of plant lectins.

Potato lectin: an updated model of a unique chimeric plant protein.

A complete cDNA encoding a potato tuber lectin has been identified and sequenced. Based on the deduced amino acid sequence, the still enigmatic molecular structure of the classical chimeric potato lectin could eventually be determined. Basically, the potato lectin consists of two nearly identical chitin-binding modules, built up of two in-tandem arrayed hevein domains that are interconnected by an extensin-like domain of approximately 60 amino acid residues. Although this structure confirms the 'canonical' chimeric nature of the Solanaceae lectins, it differs fundamentally from all previously proposed models. The new insights in the structure are also discussed in view of the physiological role of the Solanaceae lectins.

Comparative membrane interaction study of viscotoxins A3, A2 and B from mistletoe (Viscum album) and connections with their structures.

Viscotoxins A2 (VA2) and B (VB) are, together with viscotoxin A3 (VA3), among the most abundant viscotoxin isoforms that occur in mistletoe-derived medicines used in anti-cancer therapy. Although these isoforms have a high degree of amino-acid-sequence similarity, they are strikingly different from each other in their in vitro cytotoxic potency towards tumour cells. First, as VA3 is the only viscotoxin whose three-dimensional (3D) structure has been solved to date, we report the NMR determination of the 3D structures of VA2 and VB. Secondly, to account for the in vitro cytotoxicity discrepancy, we carried out a comparative study of the interaction of the three viscotoxins with model membranes. Although the overall 3D structure is highly conserved among the three isoforms, some discrete structural features and associated surface properties readily account for the different affinity and perturbation of model membranes. VA3 and VA2 interact in a similar way, but the weaker hydrophobic character of VA2 is thought to be mainly responsible for the apparent different affinity towards membranes. VB is much less active than the other two viscotoxins and does not insert into model membranes. This could be related to the occurrence of a single residue (Arg25) protruding outside the hydrophobic plane formed by the two amphipathic alpha-helices, through which viscotoxins are supposed to interact with plasma membranes.

Homology modeling of the cellulose-binding domain of a pollen allergen from rye grass: structural basis for the cellulose recognition and associated allergenic properties.

A three-dimensional model of the cellulose-binding domain of the rye-grass pollen allergen Lol pI built by homology modeling is proposed as a structural scaffold for expansins and other expansin-related proteins. A groove and an extended strip of aromatic and polar residues presumably account for the cellulose-binding properties of the protein domain. Two of the four predicted T-cell epitopes readily exposed on the surface of the cellulose-binding domain match with previously reported IgE-binding regions. A close structural relationship occurs between the cellulose-binding and allergenic properties.

Biochemical, molecular and structural analysis of multiple thaumatin-like proteins from the elderberry tree (Sambucus nigra L.).

Thaumatin-like proteins (TLPs) were isolated and characterized from fruits and leaves of elderberry (Sambucus nigra) and their corresponding genes cloned. In addition, the developmental regulation and induction of the different TLPs was followed in some detail. Ripening berries accumulated a fruit-specific TLP during the final stages of maturation. This fruit-specific TLP had no antifungal activity and was devoid of beta-glucanase activity. Leaves constitutively expressed a TLP that closely resembled the fruit-specific homologue. Treatment with jasmonate methyl ester induced two additional TLPs in leaves but did not induce or enhance the expression of TLPs in immature berries. In contrast to jasmonate methyl ester, both ethephon and garlic extract induced the expression of a TLP in unripe berries that normally do not express any TLP. Sequence analysis and molecular modeling indicated that all elderberry thaumatin-like proteins share a high sequence similarity with group-5 pathogenesis-related proteins. However, the proteins encoded by the different sequences differed from each other in isoelectric point and the distribution of the charges on the surface of the molecule.

Modes of membrane interaction of a natural cysteine-rich peptide: viscotoxin A3.

Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from other members because it is cytotoxic against tumoral cells but weakly hemolytic. We studied the interactions between the most active of these toxins, viscotoxin A3 (VA3), and model membranes made of phosphatidylcholine and phosphatidylserine (PS), the major zwitterionic and acidic phospholipids found in eukaryotic cells. Monolayer studies showed that electrostatic forces are essential for the interaction and are mainly involved in modulating the embedding of the toxin in the PS head group region. This in turn induces membrane stiffening, as shown by fluorescence polarization assays with 1,6-diphenyl-1,3,5-hexatriene and its derivatives. Moreover, vesicle permeabilization analyses showed that there are two modes of interaction, which are directly related to the stiffening effect and depend on the amount of VA3 bound to the surface of the vesicles. We propose an interaction model in which the embedding of VA3 in the membrane induces membrane defects leading to the gradual release of encapsulated dye. When the surfaces of the vesicles are saturated with the viscotoxin, complete vesicle destabilization is induced which leads to bilayer disruption, all-or-none encapsulated dye release and rearrangement of the vesicles.

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similiar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConsA looked very similar. However, doking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, reveled conformational changes in side chains of the animo acid residues invlved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConsA requires conformational chances of this monosaccharide-binding site.