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Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.

Myelination of axons by oligodendrocytes (OLGs) is essential for proper saltatory nerve conduction, i.e., rapid transmission of nerve impulses. Among others, extracellular matrix (ECM) molecules, neuronal signaling, and axonal adhesion regulate the biogenesis and maintenance of myelin membranes, driven by polarized transport of myelin-specific proteins and lipids. Galectin-4, a tandem-repeat-type lectin with affinity to sulfatide and nonsialylated termini of N-glycans, has the ability to regulate adhesion of cells to ECM components and is also involved in polarized membrane trafficking. We, therefore, anticipated that galectin-4 might play a role in myelination. Here, we show that in developing postnatal rat brains galectin-4 expression is downregulated just before the onset of myelination. Intriguingly, when immature OLGs were treated with galectin-4, OLG maturation was retarded, while a subset of the immature OLGs reverted to a morphologically less complex progenitor stage, displaying concomitantly an increase in proliferation. Similarly, myelination was inhibited when galectin-4 or anti-galectin-4 antibodies were added to co-cultures of dorsal root ganglion neurons and OLGs. Neurons and OLGs were identified as a possible source of galectin-4, both in vitro and in vivo. In culture, neurons but not OLGs released galectin-4. Interestingly, in co-cultures, a reduced release of endogenous galectin-4 correlated with the onset of myelination. Moreover, galectin-4-reactive sites are transiently expressed on processes of premyelinating primary OLGs, but not on neurons. Taken together, these results identify neuronal galectin-4 as a candidate for a soluble regulator of OLG differentiation and, hence, myelination. © 2012 Wiley Periodicals, Inc., (Copyright © 2012 Wiley Periodicals, Inc.)

The expression of ABO(H) blood group antigens causes deletion of cells that generate self-specific antibodies to these antigens but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against such pathogens, given these limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectin-4 (Gal-4) and Gal-8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing Escherichia coli while failing to alter the viability of other E. coli strains or other Gram-negative or Gram-positive organisms both in vitro and in vivo. The killing activity of both Gal-4 and Gal-8 is mediated by their C-terminal domains, occurs rapidly and independently of complement and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that express blood group-like antigens on their surface.

Our previous studies have found that expression of p27 in oligodendrocytes enhances myelin basic protein (MBP) gene expression through a mechanism that involves the transcription factor Sp1. In this study we show that this activation only requires the N-terminal 45 amino acids of p27 containing a functional cyclin-binding motif. In an effort to identify other cofactors that are involved in the p27-mediated activation of MBP gene expression, a yeast two-hybrid assay was performed using an N-terminal truncated p27 and a mouse embryo cDNA library. Galectin-4 was found to interact with p27 in the yeast two-hybrid assay. This novel interaction was also confirmed using a glutathione-S-transferase interaction assay and immunoprecipitation assays. Expression of galectin-4 in primary oligodendrocytes was confirmed by western blot. Additionally, the MBP promoter could be activated by expression of galectin-4 in CG4 oligodendrocytes, similar to the effects of increased p27 levels. We also show that Sp1 and galectin-4 interact in cells, while a complex of all three proteins could not be found. We conclude that galectin-4 is involved in the p27-mediated activation of the MBP gene, possibly through modulation of the glycosylation status of the transcription factor Sp1.

Cellular turnover is associated with exposure of surface phosphatidylserine (PS) in apoptotic cells, leading to their phagocytic recognition and removal. But recent studies indicate that surface PS exposure is not always associated with apoptosis. Here we show that several members of the human galectin family of glycan binding proteins (galectins-1, -2, and -4) induce PS exposure in a carbohydrate-dependent fashion in activated, but not resting, human neutrophils and in several leukocyte cell lines. PS exposure is not associated with apoptosis in activated neutrophils. The exposure of PS in cell lines treated with these galectins is sustained and does not affect cell viability. Unexpectedly, these galectins bind well to activated T lymphocytes, but do not induce either PS exposure or apoptosis, indicating that galectin's effects are cell specific. These results suggest novel immunoregulatory contribution of galectins in regulating leukocyte turnover independently of apoptosis.

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by "conventional" extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant "superrafts." These were enriched in galectin-4, a beta-galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.