Tumor-specific protein human galectin-1 interacts with anticancer agents Sabato D’Auria, a Lidia Petrova, b Constance John, c George Russev, b Antonio Varriale a and Vanya Bogoeva* b Received 25th March 2009, Accepted 29th July 2009 First published as an Advance Article on the web 18th August 2009 DOI: 10.1039/b905921k 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 mM 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. 1. Introduction Lectins are carbohydrate-binding proteins, known for their ability to interact with specific oligosaccharides. 1 Recently, in addition to their carbohydrate-binding capacity, it has been discovered that some plant lectins have hydrophobic sites for adenine and physiological compounds. 2–8 The affinity towards these physiological non-carbohydrate ligands was higher than their affinity for carbohydrates indicating that the interaction with hydrophobic ligands may be biologically relevant. Similarly, some animal lectins have non-carbohydrate-binding domains, 9–11 and it is shown that they bind non-carbohydrate ligands through hydrophobic interactions. 12–14 Therefore, we postulated that human lectins, in particular galectin-1, could possibly interact with non-carbohydrate ligands. Galectins are a group of mammalian beta-galactoside binding proteins with diverse functions that are not fully defined yet. It has been shown that galectins have intracellular regulatory roles in RNA splicing, act to inhibit or induce apoptosis, stimulate cell proliferation and differentiation, and regulate the cell cycle. One of the most well studied amongst the galectin family is hGal-1, which can induce apoptosis of activated T-cells and T-leukemia cell lines. 15 hGal-1 is a homodimeric protein, with a molecular mass of 14.5 kDa 16 , that acts extracellularly through its carbohydrate-binding ability to modulate immune and inflammatory responses, infection, transplantation, and cancer. Galectin-1 negatively regulates T-helper type 1 (T H 1) and interleukin-17 producing T helper (TH-17) cells that express cell surface glycans required for galectin-1-induced cell death, while T H 2 cells are protected through sialylation of cell surface glycoproteins. 17 Galectin-1 binds to several glycoconjugates that are implicated in cancer or are known to be cancer antigens such as the ovarian cancer antigen CA-125, 18 lysosome-associated membrane glycoprotein-1 (lamp-1), lamp-2, fibronectin, and carcinoembryonic antigen (CEA). 19 Recently, it was found that hGal-1 is involved not only in carbohydrate interactions, but is also engaged in protein– protein/lipid interactions. 20 Gemin 4 (implicated in pre-mRNA splicing) and oncogenic human Ras protein are found to bind hGal-1 via a hydrophobic binding site, 21–22 different from the carbohydrate-binding domain (CRD). In addition, a small-angle neutron scattering study of hGal-1 in solution indicated a conformational change upon ligand binding. 23 Drug–protein interactions are critical in the delivery of small molecules in biological systems. Understanding the molecular basis of drug–protein interactions is important in designing novel therapeutic agents with improved drug a Laboratory for Molecular Sensing, Institute of Protein Biochemistry, CNR, Via P. Castellino, 111, 80131 Naples, Italy b Department of Molecular Biology of Cell Cycle, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. ‘‘G. Bonchev’’ str., bl. 21, 1113 Sofia, Bulgaria. E-mail: vanya.bogoeva@gmail.com; Fax: +359-8723507; Tel: +359-9792638 c MandalMed, Inc., Suite 250, 665 3rd Street, San Francisco, CA 94107, USA This journal is  c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 1331–1336 | 1331 PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems