The common ⌬F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe 508 deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and ⌬F508 in vivo. Incubation of transient and stable ⌬F508 tranfectants with 10% glycerol induced a significant accumulation of ⌬F508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued ⌬F508 polypeptides form functional plasma membrane CFTR channels. These effects were dose-and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) ⌬F508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.Cystic fibrosis (CF), 1 a lethal hereditary exocrinopathy affecting approximately one in two thousand live births among populations of Caucasian or northern European descent, is caused by the functional absence of a plasma membrane chloride channel, designated cystic fibrosis transmembrane conductance regulator (CFTR) (1). The vast majority of severe CF cases in these populations is linked to a single genetic lesion, deletion of a phenylalanine codon (⌬F508) (2, 3), which interferes with the folding of newly synthesized CFTR polypeptides. Nascent ⌬F508 molecules fail to traffic to the plasma membrane (4) but rather are retained by the ER quality control mechanism that prevents unfolded or misfolded proteins and unassociated subunits from exiting the ER. Instead, these retained immature ⌬F508 molecules are rapidly degraded (5, 6) in a pre-Golgi compartment by a process that appears to require covalent modification by ubiquitin (7). Moreover, plasma membrane CFTR-like Cl Ϫ channel activity can be detected when ⌬F508 is overexpressed (8) or synthesized at reduced temperature (9), suggesting that Phe 508 does not play an essential role in CFTR function and raising the possibility of therapeutic intervention in CF by increasing the efficiency of ⌬F508 folding.Glycerol and other polyols are known to stabilize protein conformation (10), increase the rate of in vitro protein refolding (11)...

Recruitment of neutrophils from blood vessels to sites of infection represents one of the most important elements of innate immunity. Movement of neutrophils across blood vessel walls to the site of infection first requires that the migrating cells firmly attach to the endothelial wall. Generally, neutrophil extravasation is mediated at least in part by two classes of adhesion molecules, β2 integrins and selectins. However, in the case of streptococcal pneumonia, recent studies have revealed that a significant proportion of neutrophil diapedesis is not mediated by the β2 integrin/selectin paradigm. Galectin-3 is a β-galactoside-binding lectin implicated in inflammatory responses as well as in cell adhesion. Using an in vivo streptococcal pneumonia mouse model, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs correlates closely with the onset of neutrophil extravasation. Furthermore, immunohistological analysis of infected lung tissue revealed the presence of galectin-3 in the lung tissue areas composed of epithelial and endothelial cell layers as well as of interstitial spaces. In vitro, galectin-3 was able to promote neutrophil adhesion to endothelial cells. Promotion of neutrophil adhesion by galectin-3 appeared to result from direct cross-linking of neutrophils to the endothelium and was dependent on galectin-3 oligomerization. Together, these results suggest that galectin-3 acts as an adhesion molecule that can mediate neutrophil adhesion to endothelial cells. However, accumulation of galectin-3 in lung was not observed during neutrophil emigration into alveoli induced by Escherichia coli infection, where the majority of neutrophil emigration is known to be β2 integrin dependent. Thus, based on our results, we propose that galectin-3 plays a role in β2 integrin-independent neutrophil extravasation, which occurs during alveolar infection with Streptococcus pneumoniae.

Growing evidence suggests that galectin-3 is involved in fine tuning of the inflammatory responses at the periphery, however, its role in injured brain is far less clear. Our previous work demonstrated upregulation and coexpression of galectin-3 and IGF-1 in a subset of activated/proliferating microglial cells after stroke. Here, we tested the hypothesis that galectin-3 plays a pivotal role in mediating injury-induced microglial activation and proliferation. By using a galectin-3 knock-out mouse (Gal-3KO), we demonstrated that targeted disruption of the galectin-3 gene significantly alters microglia activation and induces ϳ4-fold decrease in microglia proliferation. Defective microglia activation/proliferation was further associated with significant increase in the size of ischemic lesion, ϳ2-fold increase in the number of apoptotic neurons, and a marked deregulation of the IGF-1 levels. Next, our results revealed that contrary to WT cells, the Gal3-KO microglia failed to proliferate in response to IGF-1. Moreover, the IGF-1-mediated mitogenic microglia response was reduced by N-glycosylation inhibitor tunicamycine while coimmunoprecipitation experiments revealed galectin-3 binding to IGFreceptor 1 (R1), thus suggesting that interaction of galectin-3 with the N-linked glycans of receptors for growth factors is involved in IGF-R1 signaling. While the canonical IGF-1 signaling pathways were not affected, we observed an overexpression of IL-6 and SOCS3, suggesting an overactivation of JAK/STAT3, a shared signaling pathway for IGF-1/IL-6. Together, our findings suggest that galectin-3 is required for resident microglia activation and proliferation in response to ischemic injury.

The glycocalyx is a glycan layer found on the surfaces of host cells as well as microorganisms and enveloped virus. Its thickness may easily exceed 50 nm. The glycocalyx does not only serve as a physical protective barrier but also contains various structurally different glycans, which provide cell- or microorganism-specific 'glycoinformation'. This information is decoded by host glycan-binding proteins, lectins. The roles of lectins in innate immunity are well established, as exemplified by collectins, dectin-1, and dendritic cell (DC)-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). These mammalian lectins are synthesized in the secretory pathway and presented on the cell surface to bind to specific glycan 'epitopes'. As they recognize non-self glycans presented by microorganisms, they can be considered as receptors for pathogen-associated molecular patterns (PAMPs), i.e. pattern recognition receptors (PRRs). One notable exception is the galectin family. Galectins are synthesized and stored in the cytoplasm, but upon infection-initiated tissue damage and/or following prolonged infection, cytosolic galectins are either passively released by dying cells or actively secreted by inflammatory activated cells through a non-classical pathway, the 'leaderless' secretory pathway. Once exported, galectins act as PRR, as well as immunomodulators (or cytokine-like modulators) in the innate response to some infectious diseases. As galectins are dominantly found in the lesions where pathogen-initiated tissue damage signals appear, this lectin family is also considered as potential damage-associated molecular pattern (DAMP) candidates that orchestrate innate immune responses alongside the PAMP system.

Galectin-3, a member of the galectin family of carbohydrate binding proteins, is widely expressed, particularly in cells involved in the immune response. Galectin-3 has also been indicated to play a role in various biological activities ranging from cell repression to cell activation and adhesion and has, thus, been recognized as an immunomodulator. Whereas those activities are likely to be associated with ligand cross-linking by this lectin, galectin-3, unlike other members of the galectin family, exists as a monomer. It has consequently been proposed that oligomerization of the N-terminal domains of galectin-3 molecules, after ligand binding by the C-terminal domain, is responsible for this cross-linking. The oligomerization status of galectin-3 could, thus, control the majority of its extracellular activities. However, little is known about the actual mode of action through which galectin-3 exerts its function. In this report we present data suggesting that oligomerization of galectin-3 molecules occurs on cell surfaces with physiological concentrations of the lectin. Using galectin-3 labeled at the C terminus with Alexa 488 or Alexa 555, the oligomerization between galectin-3 molecules on cell surfaces was detected using fluorescence resonance energy transfer. We observed this fluorescence resonance energy transfer signal in different biological settings representing the different modes of action of galectin-3 that we previously proposed; that is, ligand crosslinking leading to cell activation, cell-cell interaction/adhesion, and lattice formation. Furthermore, our data suggest that galectin-3 lattices are robust and could, thus, be involved, as previously proposed, in the restriction of receptor clustering.Galectin-3 is a member of the family of soluble host carbohydrate-binding proteins called galectins (1-3). Members of this lectin family are characterized by conserved peptide sequences in their carbohydrate recognition domains (CRDs), 2 which have an affinity for ␤-galactoside containing glycoconjugates (1-3). Galectin-3 has been implicated in various biological functions such as cell activation and cell adhesion (for a review, see Refs. 4 -7). It has been reported that galectin-3 can induce mast cell degranulation (8), oxidative burst and interleukin-1 production in monocytes (9, 10), and migration of monocytes/macrophages (11) as well as L-selectin shedding and interleukin-8 production in neutrophils (12). Galectin-3 is also involved in cell adhesion of different cell types, such as neutrophils, to extracellular matrix proteins and to the endothelium (13,14).Most galectins are multivalent, being intrinsically composed of two CRDs or existing in a dimerized form (1-3). This multivalency of galectins enables their involvement in cell-cell and cell-pathogen interactions along with signal transduction events and lattice formation upon ligand cross-linking (for a review, see Refs. 4 -7 and 15-19). Interestingly, galectin-3 does not comprise two CRDs nor does it form dimers in solution (20,21). Rather, galec...

The establishment of HIV type 1 (HIV-1) infection is initiated by the stable attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between virus-encoded gp120 and cell surface CD4, a number of distinct interactions influence binding of HIV-1 to host cells. In this study, we report that galectin-1, a dimeric β-galactoside-binding protein, promotes infection with R5, X4, and R5X4 variants. Galectin-1 acts as a soluble adhesion molecule by facilitating attachment of HIV-1 to the cell surface. This postulate is based on experiments where galectin-1 rendered HIV-1 particles more refractory to various agents that block HIV-1 adsorption and coreceptor binding (i.e., a blocking anti-CD4, soluble CD4, human anti-HIV-1 polyclonal Abs; stromal cell-derived factor-1α; RANTES). Experiments performed with the fusion inhibitor T-20 confirmed that galectin-1 is primarily affecting HIV-1 attachment. The relevance of the present findings for the pathogenesis of HIV-1 infection is provided by the fact that galectin-1 is abundantly expressed in the thymus and lymph nodes, organs that represent major reservoirs for HIV-1. Moreover, galectin-1 is secreted by activated CD8+ T lymphocytes, which are found in high numbers in HIV-1-positive patients. Therefore, it is proposed that galectin-1, which is released in an exocrine fashion at HIV-1 replication sites, can cross-link HIV-1 and target cells and promote a firmer adhesion of the virus to the cell surface, thereby augmenting the efficiency of the infection process. Overall, our findings suggest that galectin-1 might affect the pathogenesis of HIV-1 infection.

The neutrophil is the first line of defense against infection. As a part of the innate immune response, neutrophils start to emigrate from blood to an affected site and their state is altered from passively circulating naïve to primed, and then to fully activated. The extent of neutrophil activation and their subsequent response varies depending on the stimuli and environment that neutrophils encounter. Because neutrophils can also induce deleterious effects on host tissues, tight regulation of recruitment and functions of neutrophils is required for efficient recovery. Galectin-3, a soluble beta-galactoside binding protein, of which expression is up-regulated during inflammation/infection, is suggested to be involved in various inflammatory responses. However, the precise roles of this lectin in innate immunity remain unknown, while it has been demonstrated that galectin-3 binds to naïve and primed neutrophils. Here we report that galectin-3 can induce L-selectin shedding and interleukin-8 production in naïve and primed neutrophils. These activities were shown to be dependent on the presence of the C-terminal lectin domain and the N-terminal nonlectin domain of galectin-3, which is involved in oligomerization of this lectin. We also found that, after galectin-3 binds to neutrophils, primed but not naïve neutrophils can cleave galectin-3, mainly through elastase, which results in the formation of truncated galectin-3 lacking the N-terminal domain. Together, these results suggest that galectin-3 activates naïve and primed neutrophils, and galectin-3-activated primed neutrophils have an ability to inactivate galectin-3.

Pneumonia can be caused by a variety of pathogens, among which Streptococcus pneumoniae causes one of the most common forms of community-acquired pneumonia. Depending on the invading pathogen, the elements of the immune response triggered will vary. For most pathogens, such as Escherichia coli, neutrophil recruitment involves a well-described family of adhesion molecules, β2-integrins. In the case of streptococcal pneumonia, however, neutrophil recruitment occurs mainly through a β2-integrin-independent pathway. Despite decades of research on this issue, the adhesion molecules involved in neutrophil recruitment during lung infection by S. pneumoniae have not been identified. We have previously shown that galectin-3, a soluble mammalian lectin, can be found in lungs infected by S. pneumoniae, but not by E. coli, and can mediate the adhesion of neutrophils on the endothelial cell layer, implying its role in the recruitment of neutrophils to lungs infected with S. pneumoniae. In this study, using galectin-3 null mice, we report further evidence of the involvement of this soluble lectin in the recruitment of neutrophils to S. pneumonia-infected lungs. Indeed, in the absence of galectin-3, lower numbers of leukocytes, mainly neutrophils, were recruited to the infected lungs during infection by S. pneumoniae. In the case of β2-integrin-dependent recruitment induced by lung infection with E. coli, the number of recruited neutrophils was not reduced. Thus, taken together, our data suggest that galectin-3 plays a role as a soluble adhesion molecule in the recruitment of neutrophils to lungs infected by S. pneumoniae, which induces β2-integrin-independent migration.