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.