Galectin-3 - A novel ligand of complement protein C1q.

In the present study we report a novel interaction of human C1q, a primary activator of the Complement system, with human Galectin-3 (Gal-3). We investigated the potential recognition between C1q and Gal-3 on a solid hydrophobic surface by ELISA, by fluorescence spectroscopy, molecular docking and molecular dynamics (MD). The data showed that C1q and Gal-3 had a pronounced affinity for protein-protein interaction and supramolecular binding, locating the binding sites within the globular domains of C1q (gC1q) and on the backside of the carbohydrate recognition domain (CRD) of Gal-3. Fluorescence spectroscopy gave quantitative assessment of the recognition with KD value of 0.04 µM. MD analysis showed that when the active AAs of the two proteins interacted, electrostatic attraction, aided by a large number of hydrogen bonds, was dominant for the stabilization of the complex. When the contact of C1q and Gal-3 was not limited to active residues, the complex between them was stabilized mainly by Van der Waals interactions and smaller in number but stronger hydrogen bonds. This is the first report analyzing the interaction of Gal-3 with C1q, which could open the way to new applications of this protein-protein complex.

Jacalin-Curcumin Complex Sensitizes the Breast Cancer MDA-MB-231 Cell Line.

Protein-drug interactions are crucial for understanding drug delivery and cell functions. Jacalin is a suitable molecule for such targeting, as it specifically recognizes the tumor-associated Thomsen-Friedenreich (TF) antigen that is expressed on the glycosylated proteins in cancer cells. The present paper describes the interaction of curcumin and jacalin, a possible carrier molecule for the delivery of antitumor drugs due to its ability to recognize tumor cells. Our results have shown that both steady-state fluorescence and fluorescent labelling of jacalin are two reliable methods to determine jacalin-curcumin interactions. The affinity of jacalin for curcumin is consistently within the micromolar range (using fluorescence and microscale thermophoresis) showing high-affinity binding of the complex. In vitro experiments on triple-negative breast cancer MDA-MB-231 cells indicated inhibition of cell growth after treating with the jacalin-curcumin complex for 48 h. The cell survival fraction was significantly reduced to 50% after combined treatment. In this paper, we report for the first time about the jacalin-curcumin interaction. We quantified this unique biomolecular interaction and gathered additional information on the binding event. We observed that the jacalin-curcumin complex inhibits the proliferation of the triple-negative breast cancer MDA-MB-231 cells.

Proteomic analysis reveals mechanisms underlying increased efficacy of bleomycin by photochemical internalization in bladder cancer cells.

Photochemical internalization (PCI) is a promising new technology for site-specific drug delivery, developed from photodynamic therapy (PDT). In PCI, light-induced activation of a photosensitizer trapped inside endosomes together with e.g. chemotherapeutics, nucleic acids or immunotoxins, allows cytosolic delivery and enhanced local therapeutic effect. Here we have evaluated the photosensitizer meso-tetraphenyl chlorine disulphonate (TPCS2a/fimaporfin) in a proteome analysis of AY-27 rat bladder cancer cells in combination with the chemotherapeutic drug bleomycin (BML). We find that BLMPCI attenuates oxidative stress responses induced by BLM alone, while concomitantly increasing transcriptional repression and DNA damage responses. BLMPCI also mediates downregulation of bleomycin hydrolase (Blmh), which is responsible for cellular degradation of BLM, as well as several factors known to be involved in fibrotic responses. PCI-mediated delivery might thus allow reduced dosage of BLM and alleviate unwanted side effects from treatment, including pulmonary fibrosis.

Anti-Idiotype scFv Localizes an Autoepitope in the Globular Domain of C1q.

We addressed the issue of C1q autoantigenicity by studying the structural features of the autoepitopes recognized by the polyclonal anti-C1q antibodies present in Lupus Nephritis (LN) sera. We used six fractions of anti-C1q as antigens and selected anti-idiotypic scFv antibodies from the phage library "Griffin.1". The monoclonal scFv A1 was the most potent inhibitor of the recognition of C1q and its fragments ghA, ghB and ghC, comprising the globular domain gC1q, by the lupus autoantibodies. It was sequenced and in silico folded by molecular dynamics into a 3D structure. The generated 3D model of A1 elucidated CDR similarity to the apical region of gC1q, thus mapping indirectly for the first time a globular autoepitope of C1q. The VH CDR2 of A1 mimicked the ghA sequence GSEAD suggested as a cross-epitope between anti-DNA and anti-C1q antibodies. Other potential inhibitors of the recognition of C1q by the LN autoantibodies among the selected recombinant antibodies were the monoclonal scFv F6, F9 and A12.

Binding of Gold(III) Porphyrin by the Pro-metastatic Regulatory Protein Human Galectin-3.

Gold(III) porphyrin presents an attractive alternative to the use of, for example, cisplatin in chemotherapy. However, approaches that allow to selectively target cancer cells are highly sought. Many plant and mammalian lectins have been shown to bind oligosaccharide sequences of the aberrant glycosylation pattern found on cancerous tumors. For example human galectin-3, of the galectin family specific for ß-galactoside, is overexpressed in the extracellular matrix of tumorigenous and metastatic tissues. We searched for non-carbohydrate ligands for galectin-3 that can guide a cytotoxic drug to the cancer cells by maintaining its affinity for tumor associated carbohydrate antigens. Previous findings showed that zinc tetrasulfonatophenylporphyrin can bind galectin-3 with sub-micromolar affinity without disturbing lactose binding. Gold(III) porphyrin is not only cytotoxic to cancer cells, it knows also a potential application as photosensitiser in photodynamic therapy. We investigated the binding of gold(III) porphyrin to galectin-3 using different biophysical interaction techniques and demonstrated a low micromolar affinity of human galectin-3 for the cytotoxic compound. Co-crystallization attempts in order to understand the binding mode of gold porphyrin to galectin-3 failed, but molecular docking emphasized a highly populated secondary binding site that does not hinder lactose or Thomsen Friendenreich disaccharide binding. This suggests that gold(III) porphyrin might significantly enhance its concentration and delivery to cancer cells by binding to human galectin-3 that keeps its orientation towards tumor associated carbohydrate antigens.

Ruthenium porphyrin-induced photodamage in bladder cancer cells.

Photodynamic therapy (PDT) is a noninvasive treatment for solid malignant and flat tumors. Light activated sensitizers catalyze photochemical reactions that produce reactive oxygen species which can cause cancer cell death. In this work we investigated the photophysical properties of the photosensitizer ruthenium(II) porphyrin (RuP), along with its PDT efficiency onto rat bladder cancer cells (AY27). Optical spectroscopy verified that RuP is capable to activate singlet oxygen via blue and red absorption bands and inter system crossing (ISC) to the triplet state. In vitro experiments on AY27 indicated increased photo-toxicity of RuP (20µM, 18h incubation) after cell illumination (at 435nm), as a function of blue light exposure. Cell survival fraction was significantly reduced to 14% after illumination of 20µM RuP with 15.6J/cm(2), whereas the "dark toxicity" of 20µM RuP was 17%. Structural and morphological changes of cells were observed, due to RuP accumulation, as well as light-dependent cell death was recorded by confocal microscopy. Flow cytometry verified that PDT-RuP (50µM) triggered significant photo-induced cellular destruction with a photoxicity of (93%±0.9%). Interestingly, the present investigation of RuP-PDT showed that the dominating mode of cell death is necrosis. RuP "dark toxicity" compared to the conventional chemotherapeutic drug cisplatin was higher, both evaluated by the MTT assay (24h). In conclusion, the present investigation shows that RuP with or without photoactivation induces cell death of bladder cancer cells.

A critical evaluation of Amicon Ultra centrifugal filters for separating proteins, drugs and nanoparticles in biosamples.

Amicon(®) Ultra centrifugal filters were critically evaluated for various sample preparations, namely (a) proteome fractionation, (b) sample cleanup prior to liquid chromatography mass spectrometry (LC-MS) measurement of small molecules in cell lysate, and (c) separating drug-loaded nanoparticles and released drugs for accurate release profiling in biological samples. (a) Filters of supposedly differing molar mass (MM) selectivity (10, 30, 50 and 100K) were combined to attempt fractionation of samples of various complexity and concentration. However, the products had surprisingly similar MM retentate/filtrate profiles, and the filters were unsuited for proteome fractionation. (b) Centrifugal filtration was the only clean-up procedure in a FDA-guideline validated LC-MS method for determining anti-tuberculosis agents rifampicin and thioridazine in macrophage cell lysate. An additional organic solvent washing step (drug/protein-binding disruption) was required for satisfactory recovery. (c) The centrifugation filters are well suited for separating drugs and nanoparticles in simple aqueous solutions, but significantly less so for biological samples, as common drug-protein binding disruptors can dissolve NPs or be incompatible with LC-MS instrumentation.

Thioridazine in PLGA nanoparticles reduces toxicity and improves rifampicin therapy against mycobacterial infection in zebrafish.

Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.

Autoantigenicity of human C1q is associated with increased hydrophobicity due to conformational transitions in the globular heads.

We analyzed the structural features of C1q that underlie its autoantigenicity by means of a model system using the amphiphilic polyzwitterion (PZ), poly(ethylene oxide-b-N,N-dimethyl(methacryloyloxyethyl) ammonium propanesulfonate) in the process of C1q immobilization. The source of anti-C1q autoantibodies was human sera from patients with Lupus Nephritis (LN). Both analyzed concentrations of PZ, 25 mM and 50 mM, were found to be applicable for inducing conformational transitions which resulted in increased recognition of C1q and the globular domain of its B polypeptide chain, designated ghB, by the LN autoantibodies. The registered conformational transitions displayed a hydrophobic enhancement of the protein microenvironment due to the presence of hydrophobic binding sites in ghB which consequently affected the autoantigenicity of the whole C1q molecule.

Photophysical characterisation and studies of the effect of palladium(II) 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin on isometric contraction of isolated human mesenteric artery: good news for photodynamic therapy.

BACKGROUND: Considering the important roles of porphyrins in biological systems and their promising use in photodynamic therapy (PDT), the present work investigated the photophysical properties of palladium(II) 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin (PdTSPP) and the effects of non-activated by light form of this porphyrin on contractile behaviour of isolated healthy endothelium-denuded human mesenteric arteries. METHODS: The photophysical characterisation of PdTSPP: the formation of the triplet states and the singlet oxygen were studied using laser flash photolysis. The effect of PdTSPP on the isometric contraction of artery segments from human mesentery was assessed utilising the precise method of artery isometric tension recording using Mulvany-Halpern wire myograph. RESULTS: We found that PdTSPP had a high lifetime of the triplet states τT=270µs. The calculated Stern Volmer rate constant kq=1.7×10(9)M(-1)s(-1) showed an efficient quenching by oxygen that indicated formation of singlet oxygen, O2((1)Δg). The photophysical parameters of PdTSPP, in particular its ability to generate O2((1)Δg) has defined it as an exceptionally interesting molecule for PDT. The results of the contraction study showed that PdTSPP applied in increasing concentrations (1-100µM) had no effect on the basal tone of human mesenteric artery under isometric condition. Furthermore, PdTSPP failed to potentiate or to attenuate the isometric contraction of the artery preparations precontracted with high extracellular potassium (42mM KCl) or with 1nM endothelin-1. CONCLUSIONS: The excellent photophysical properties of PdTSPP as well as the lack of an effect on the contractility of human vasculature in vitro characterise PdTSPP as a suitable compound for potential medical applications.

New Activity of a Protein from Canavalia ensiformis.

Concanavalin A is a legume lectin which preferentially agglutinates transformed cells and shows antitumor effects on human breast carcinoma cells in vitro and in vivo. It is considered as a new potential antineoplastic agent targeting apoptosis, autophagy, and anti-angiogenesis in preclinical or clinical trials for cancer therapeutics, which has recently become the object of intensive study. In the present investigation, we show the capacity of the lectin to bind manganese, gold, iron, and zinc porphyrins: all potential anticancer agents. The interaction of the legume lectin with the studied compounds has been investigated by tryptophan fluorescence, showing conformational changes within the quaternary and tertiary structures of the protein. The binding of Con A with manganese, gold, and iron porphyrins, as well as adenine, was studied by fluorescence quenching. In contrast, the interaction of Con A with zinc porphyrin caused an increase in Trp fluorescence and a red shift of 10 nm of the emission maximum position. However, the binding of Con A to iron porphyrin was accompanied by a 5 nm blue shift of the emission maximum, and a kD of 0.95 ± 0.13 µM was calculated, respectively. The sigmoidal shape of the curve showed cooperative interactions, which indicated the presence of more than one class of binding site within the Con A molecule for iron porphyrin, confirmed by the Hill slope (h = 1.89±0.46). We have found that the legume lectin interacts with porphyrins and adenine with an affinity (0.14-1.89 µM) similar to that of the non-legume lectin, wheat germ agglutinin. In conclusion, the protein Con A shows new binding activity towards porphyrins with anticancer activities and could find prospective application as a drug delivery molecule that specifically targets cancer cells.

Characterization of metalloanticancer capacity of an agglutinin from wheat.

Many anticancer drugs cannot recognize selectively tumor tissues, and cause destruction to normal ones. Although it is very toxic, cisplatin is still one of the most applied chemotherapeutics used for treatment of sarcomas, carcinomas, etc. It causes severe side effects as a result of the lack of selectivity of the drug to tumor tissue and acquired or intrinsic resistance occurs. Wheat germ agglutinin (WGA) is a lectin that specifically recognizes transformed cells: prostate cancer cells, pancreatic cells etc., and is uptaken into the tumor cells for which it appears to be a suitable target for anticancer agents. A fluorescence spectroscopy method was used to study the interaction of WGA with four metal-based anticancer drugs: cisplatin, Pt porphyrin and two gold porphyrins. The affinity constant (k(D)) for binding of cisplatin with WGA was k(D) = 6.67 ± 2.5 µM. The hyperbolic curve indicated the presence of a single cisplatin binding site. The affinity of Au and Pt porphyrin to WGA (k(D) = 0.08-0.49 µM) is almost two orders of magnitude higher than that for cisplatin. We found that Pt porphyrin could displace fluorescent dye ANS showing an increase in the fluorescence intensity with a concomitant blue shift of the emission maximum suggesting that the compounds accommodate the same binding site. Current research characterizes the metalloanticancer binding capacity of WGA. Our results indicate that four metal-based anticancer agents have high affinity for WGA. Since WGA recognizes transformed cells, the obtained data show that this protein might have putative usage as a drug delivery molecule in cancer.

Human galectin-3 interacts with two anticancer drugs.

Human galectin-3 (hGal-3) is a mammalian lectin involved in regulation of RNA splicing, apoptosis, cell differentiation, and proliferation. Multimerized extracellular hGal-3 is thought to crosslink cells by binding to glycoproteins and glycosylated cancer antigens on the cell surface or extracellular matrix. Fluorescence spectroscopy and circular dichroism were used to study the interaction of hGal-3 with two anticancer agents: bohemine and Zn porphyrin (ZnTPPS(4)). The dissociation constant (k(D)) for binding of bohemine with hGal-3 was k(D) 0.23+/-0.05 microM. The hyperbolic titration curve indicated the presence of a single bohemine binding site. The binding of ZnTPPS(4) to hGal-3 (with and without lactose) is of high affinity having k(D)=0.18-0.20 microM and is not inhibited by lactose, indicating that ZnTPPS(4) and carbohydrate bind different sites. Circular dichroism spectra of the hGal-3 complexes suggested that the binding of the hydrophobic compounds changed the hGal-3 secondary structure. In summary, we show that two compounds with anticancer activity, bohemine and ZnTPPS(4), have high affinity for hGal-3 at a site that is distinct from its carbohydrate site. Since hGal-3 binds to several carbohydrate cancer antigens, the results suggest that it may have utility in the targeted delivery of drugs for cancer.

Tumor-specific protein human galectin-1 interacts with anticancer agents.

The present work shows a novel binding activity of the tumor specific lectin--recombinant human galectin-1 (hGal-1)--to three porphyrin compounds: (1) Zn-porphyrin (ZnTPPS); (2) Mn-porphyrin and (3) Au-porphyrin. These compounds are widely applied in the photodynamic therapy of cancer (PDT). Our data indicate that hGal-1, similar to some plant lectins, a bacterial lectin from Pseudomonas aeruginosa and an animal lectin from Helix pomatia, possesses dual functions binding to both carbohydrate and non-carbohydrate ligands. The interaction of ZnTPPS with hGal-1 was studied by the specific fluorescence emission of the porphyrin. The protein binding properties to Mn/Au-porphyrins and adenine were measured by intrinsic protein fluorescence quenching. The values determined for the apparent dissociation constants (K(D)) of 0.6-1.5 microM are similar to the K(D) for complexes of concanavalin A and porphyrin, and are indicative of the high affinity of hGal-1 for these porphyrins. In addition, the analysis of the hyperbolic binding curves obtained suggests the presence of one hGal-1 binding site for porphyrins or adenine. Additionally, we found that hGal-1 interacts with the fluorescent probe 2-(p-toluidinyl)naphthalene sulfonic acid (TNS), that was used to identify the hydrophobic regions within hGal-1. Homodimeric hGal-1 has more than one class of binding site for TNS as revealed by the sigmoidal shape of the fluorescence titration curve. hGal-1 can be characterized as a porphyrin-binding protein based on its interactions with the Zn/Mn- and Au-porphyrins, and this indicates that hGal-1 may have potential as a delivery molecule to target systems (e.g., tumor cells) with possible application in photodynamic therapy.

Fluorescence study of steroid hormone binding activity of Helix pomatia agglutinin.

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin, found in the reproductive gland of a Roman snail. The present study has shown that HPA, in addition to its carbohydrate binding capacity possesses a hydrophobic binding activity. This protein binds with high affinity (k(D)=1.9-2.4 microM) steroid hormones: testosterone and progesterone, identified as putative ligands for the animal lectin HPA. Additionally, we have found that this lectin also interacts with adenine (k(D)=5.4+/-0.5 microM) and arylaminonaphthalene sulfonate TNS (k(D)=12+/-0.3 microM). Binding of HPA to hormones and adenine was accompanied by a significant increase of the intrinsic Trp fluorescence (up to 50%), characterizing the conformational changes in the lectin molecule. The hyperbolic shape of the binding curves indicated one high affinity site for the two steroid hormones and adenine, and more than one hydrophobic site for TNS, showed by the sigmoidal curve fit and Hill coefficient of (n(H)=1.5+/-0.2). Hormones and adenine compete for an identical binding site, suggested to occupy the central hydrophobic cavity of the HPA hexamer. Fluorescence resonance energy transfer (FRET) was applied to calculate the intramolecular distance between TNS and Trp chromophores.

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones.

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).

Fluorescence analysis of hormone binding activities of wheat germ agglutinin.

Wheat germ agglutinin (WGA) from embryos of the monocotyledonous plant Triticum vulgaris (Graminaceae) is a carbohydrate binding protein characterized by high specificity to N-acetyl-d-glucosamine and N-acetyl-d-neuraminic acid. In this study we show that parallel to its carbohydrate binding activities, WGA binds with several orders of magnitude higher affinity adenine, adenine-related cytokinins: kinetin, zeatin and isopentenyl-adenine as well as abscisic and gibberellic acids (K(d) 0.43-0.65 microM). Its interactions with these ligands cause conformational rearrangements in the protein molecules and significant enhancement of the protein tryptophan fluorescence (up to 60%) allowing characterization of the protein-hormone complexes. Dimeric WGA molecules possess two different classes of binding sites for the fluorescent hydrophobic probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) as suggested by the sigmoid shape of the fluorescence titration curve and the value of the Hill coefficient (n(H) 1.6+/-0.3). The plant hormones displace part of the bound TNS probe and share the higher affinity TNS binding sites. These results characterize WGA as a hormone-binding protein.