Carlota Wolfenstein-Todel

GALECTINS: MATRICELLULAR GLYCAN-BINDING PROTEINS LINKING CELL ADHESION, MIGRATION, AND SURVIVAL

Abstract.Galectins are a taxonomically widespread family of glycan-binding proteins, defined by at least one conserved carbohydrate-recognition domain with a canonical amino acid sequence and affinity for β-galactosides. Because of their anti-adhesive as well as pro-adhesive extracellular functions, galectins appear to be a novel class of adhesion-modulating proteins collectively known as matricellular proteins (which include thrombospondin, SPARC, tenascin, hevin, and disintegrins). Accordingly, galectins can display de-adhesive effects when presented as soluble proteins to cells in a strong adhesive state. In this context, the de-adhesive properties of galectins should be considered as physiologically relevant as the proadhesive effects of these glycan-binding proteins. This article focuses on the roles of mammalian galectins in cell adhesion, spreading, and migration, and the crossregulation of these functions. Although careful attention should be paid when examining individual galectin functions due to overlapping distributions, these intriguing glycan-binding proteins offer promising possibilities for the treatment and intervention of a wide variety of pathological processes, including cancer, inflammation, and autoimmunity.

Modulation of endothelial cell migration and angiogenesis: a novel function for the “tandem-repeat” lectin galectin-8

Angiogenesis, the growth of new capillaries from preexisting blood vessels, is a complex process involving endothelial cell (EC) activation, disruption of vascular basement membranes, and migration and proliferation of ECs. Glycan‐mediated recognition has been proposed to play an instrumental role in mediating cell‐cell and cell‐matrix interactions. Ga‐lectins (Gal), a family of glycan‐binding proteins with affinity for β‐galactosides and a conserved sequence motif, can decipher glycan‐containing information and mediate cell‐cell communication. Galectin‐8 (Gal‐8), a member of this family, is a bivalent “tandem‐repeat”‐type galectin, which possesses 2 CRDs connected by a linker peptide. Here, we show that Gal‐8 is endowed with proangiogeneic properties. Functional assays revealed a critical role for this lectin in the regulation of capillary‐tube formation and EC migration. Moreover, Matrigel, either supplemented with Gal‐8 or vascular endothelial growth factor (VEGF), injected in mice resulted in induction of in vivo angiogenesis. Remarkably, Gal‐8 was expressed both in the cytoplasm and nucleus in ECs of normal and tumor vessels. Furthermore, CD166 [activated leukocyte cell adhesion molecule (ALCAM)] was identified as a specific Gal‐8‐binding partner in normal vascular ECs. Collectively, these data provide the first evidence demonstrating an essential role for Gal‐8 in the regulation of angiogenesis with critical implications in tumor biology.—CCárdenasrdenas Delgado, V. M., Nugnes, L. G., Colombo, L. L., Troncoso, M. F., Fernández, M. M., Malchiodi, E. L., Frahm, I., Croci, D. O., Compagno, D., Rabinovich, G. A., Wolfenstein‐Todel, C., Elola, M. T. Modulation of endothelial cell migration and angiogenesis: a novel function for the “tandem‐repeat” lectin galectin‐8. FASEB J. 25, 242–254 (2011). www.fasebj.org

Novel roles of galectin-1 in hepatocellular carcinoma cell adhesion, polarization, and in vivo tumor growth.

Galectin‐1 (Gal‐1), a widely expressed β‐galactoside–binding protein, exerts pleiotropic biological functions. Gal‐1 is up‐regulated in hepatocarcinoma cells, although its role in liver pathophysiology remains uncertain. We investigated the effects of Gal‐1 on HepG2 hepatocellular carcinoma (HCC) cell adhesion and polarization. Soluble and immobilized recombinant Gal‐1 (rGal‐1) promoted HepG2 cell adhesion to uncoated plates and also increased adhesion to laminin. Antibody‐mediated blockade experiments revealed the involvement of different integrins as critical mediators of these biological effects. In addition, exposure to rGal‐1 markedly accelerated the development of apical bile canaliculi as shown by TRITC‐phalloidin labeling and immunostaining for multidrug resistance associated‐protein 2 (MRP2). Notably, rGal‐1 did not interfere with multidrug resistance protein 1/P‐glycoprotein or MRP2 apical localization, neither with transfer nor secretion of 5‐chloromethylfluorescein diacetate through MRP2. Stimulation of cell adhesion and polarization by rGal‐1 was abrogated in the presence of thiodigalactoside, a galectin‐specific sugar, suggesting the involvement of protein–carbohydrate interactions in these effects. Additionally, Gal‐1 effects were abrogated in the presence of wortmmanin, PD98059 or H89, suggesting involvement of phosphoinositide 3‐kinase (PI3K), mitogen‐activated protein kinase and cyclic adenosine monophosphate–dependent protein kinase signaling pathways in these functions. Finally, expression levels of this endogenous lectin correlated with HCC cell adhesion and polarization and up‐regulation of Gal‐1–favored growth of hepatocarcinoma in vivo. Conclusion: Our results provide the first evidence of a role of Gal‐1 in modulating HCC cell adhesion, polarization, and in vivo tumor growth, with critical implications in liver pathophysiology. (HEPATOLOGY 2011;)

A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.

Identification of an equilibrium intermediate in the unfolding process of galectin-1, which retains its carbohydrate-binding specificity

The unfolding process of galectin-1 (Gal-1) in the presence of a denaturing agent was examined using fluorescence and far-UV circular dichroism (CD) spectroscopy determinations, and was found to be completely reversible. The data showed that the transitions of guanidine hydrochloride (GdnHCl)-induced lectin unfolding, in the absence of ligand, were biphasic in nature, clearly showing the existence of at least one stable intermediate. On the other hand, the unfolding in the presence of disaccharide yielded data that could fit very well to a two-state model, indicating a stabilizing effect of the ligand. The folding intermediate was further characterized by size exclusion chromatography, near-UV CD and anilinonaphtalene sulfonate binding, and shown to belong to the molten globule type. Strikingly, this intermediate retained its carbohydrate-binding specificity, as evidenced by the tryptophan fluorescence changes detected upon its interaction with lactose.

The heparin-binding lectin from ovine placenta: Purification and identification as histone H4

The heparin-binding lectin complex from ovine placental cotyledons was purified by affinity chromatography on heparin-agarose column. It showed three protein bands, which had molecular weights of 13 000, 15 000 and 17 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of DNA by agarose gel electrophoresis. The protein components of the complex were separated by reverse-phase HPLC. The minimum inhibitory concentrations of glycosaminoglycans were significantly different for the lectin complex and the separated proteins, suggesting affinity changes upon DNA binding. The haemagglutinating activity specificity allowed the characterization of the fraction with a molecular weight of 13 000 as the heparin-binding lectin. This protein was identified as histone H4 by internal sequencing, thus showing that this is the histone responsible for the heparin-binding property of the complex. The accompanying proteins were tentatively identified as histones H2A and H2B.

Galectin-1 Controls the Proliferation and Migration of Liver Sinusoidal Endothelial Cells and Their Interaction With Hepatocarcinoma Cells.

Galectin‐1 (Gal1), a β‐galactoside‐binding protein elevated in hepatocellular carcinoma (HCC), promotes epithelial‐mesenchymal transition (EMT) and its expression correlates with HCC growth, invasiveness, and metastasis. During the early stages of HCC, transforming growth factor β1 (TGF‐β1) acts as a tumor suppressor; however in advanced stages, HCC cells lose their cytostatic response to TGF‐β1 and undergo EMT. Here, we investigated the role of Gal1 on liver endothelial cell biology, and the interplay between Gal1 and TGF‐β1 in HCC progression. By Western blot and immunofluorescence, we analyzed Gal1 expression, secretion and localization in HepG2 and HuH‐7 human HCC cells, and in SK‐HEP‐1 human liver sinusoidal endothelial cells (SECs). We used loss‐of‐function and gain‐of‐function experiments to down‐ or up‐regulate Gal1 expression, respectively, in HepG2 cells. We cultured SK‐HEP‐1 cells with conditioned media from HCC cells secreting different levels of Gal1, and demonstrated that Gal1 derived from tumor hepatocytes induced its own expression in SECs. Colorimetric and scratch‐wound assays revealed that secretion of Gal1 by HCC cells induced SEC proliferation and migration. Moreover, by fluorescence microscopy we demonstrated that Gal1 promoted glycan‐dependent heterotypic adhesion of HepG2 cells to SK‐HEP‐1 SECs. Furthermore, TGF‐β1 induced Gal1 expression and secretion by HCC cells, and promoted HepG2 cell adhesion to SK‐HEP‐1 SECs through a Gal1‐dependent mechanism. Finally, Gal1 modulated HepG2 cell proliferation and sensitivity to TGF‐β1‐induced growth inhibition. Our results suggest that Gal1 and TGF‐β1 might function coordinately within the HCC microenvironment to regulate tumor growth, invasion, metastasis, and angiogenesis. J. Cell. Physiol. 231: 1522–1533, 2016.