Visualization of Cytosolic Galectin Accumulation Around Damaged Vesicles and Organelles.

Galectins are animal lectins that recognize ß-galactoside and bind glycans. Recent studies have indicated that cytosolic galectins recognize cytosolically exposed glycans and accumulate around endocytic vesicles or organelles damaged by various disruptive substances. Accumulated galectins engage other cytosolic proteins toward damaged vesicles, leading to cellular responses, such as autophagy. Disruptive substances include bacteria, viruses, particulate matters, and protein aggregates; thus, this process is implicated in the pathogenesis of various diseases. In this chapter, we describe methods for studying three disruptive substances: photosensitizers, Listeria monocytogenes, and Helicobacter pylori. We summarize the tools used for the detection of cytosolic galectin accumulation around damaged vesicles.

Intracellular galectins sense cytosolically exposed glycans as danger and mediate cellular responses.

Galectins are animal lectins that recognize carbohydrates and play important roles in maintaining cellular homeostasis. Recent studies have indicated that under a variety of challenges, intracellular galectins bind to host glycans displayed on damaged endocytic vesicles and accumulate around these damaged organelles. Accumulated galectins then engage cellular proteins and subsequently control cellular responses, such as autophagy. In this review, we have summarized the stimuli that lead to the accumulation of galectins, the molecular mechanisms of galectin accumulation, and galectin-mediated cellular responses, and elaborate on the differential regulatory effects among galectins.

Galectins in Host Defense Against Microbial Infections.

Galectins are differentially expressed in a variety of cell types, including immune cells, and characterized by the affinity for ß-galactoside-containing glycans. There are fifteen galectin members in mammals. Galectins are primarily located intracellularly, but can be secreted outside the cells. They exhibit pivotal roles during microbial infection, such as pathogen recognition and innate and adaptive immunity, and this review aims to discuss the functions of endogenous galectins during infection by four main types of microbes (bacteria, fungi, viruses, and parasites). Extracellular galectins are known to exert a bacteriostatic effect on some bacteria via association with bacterial glycans, whereas cytosolic galectins are recognized to control antibacterial autophagy by binding to luminal host glycans of ruptured endo-lysosomes. With regard to fungal infections, most studies deal with galectin-3. Galectin-3 modulates fungal burdens, the adaptive immune responses, and mortality in fungi-infected mice, which has been shown to be associated with its ability to manipulate fungicidal functions in neutrophils and cytokine expression in dendritic cells. Some viral infections, such as human immunodeficiency virus (HIV) and influenza virus infections, can be regulated by galectin-1 and -3, and they affect various aspects of viral infections, including viral binding, replication, budding, transmission, and infection-associated inflammation. Functions of galectins during a number of different parasitic infections have been identified in studies using galectin-knockout mice. Different parasitic infections have consistently demonstrated a role of galectins in tuning T helper immune responses in infected hosts.

Helicobacter pylori induces intracellular galectin-8 aggregation around damaged lysosomes within gastric epithelial cells in a host O-glycan-dependent manner.

Galectin-8, a beta-galactoside-binding lectin, is upregulated in the gastric tissues of rhesus macaques infected with Helicobacter pylori. In this study, we found that H. pylori infection triggers intracellular galectin-8 aggregation in human-derived AGS gastric epithelial cells, and that these aggregates colocalize with lysosomes. Notably, this aggregation is markedly reduced following the attenuation of host O-glycan processing. This indicates that H. pylori infection induces lysosomal damage, which in turn results in the accumulation of cytosolic galectin-8 around damaged lysosomes through the recognition of exposed vacuolar host O-glycans. H. pylori-induced galectin-8 aggregates also colocalize with autophagosomes, and galectin-8 ablation reduces the activation of autophagy by H. pylori. This suggests that galectin-8 aggregates may enhance autophagy activity in infected cells. We also observed that both autophagy and NDP52, an autophagy adapter, contribute to the augmentation of galectin-8 aggregation by H. pylori. Additionally, vacuolating cytotoxin A, a secreted H. pylori cytotoxin, may contribute to the increased galectin-8 aggregation and elevated autophagy response in infected cells. Collectively, these results suggest that H. pylori promotes intracellular galectin-8 aggregation, and that galectin-8 aggregation and autophagy may reciprocally regulate each other during infection.

Breast tumor kinase phosphorylates p190RhoGAP to regulate rho and ras and promote breast carcinoma growth, migration, and invasion.

Breast tumor kinase (Brk), an Src-like nonreceptor tyrosine kinase, is overexpressed in breast cancer and several other cancer types. Our previous study indicates that Brk promotes cell migration and tumor invasion by phosphorylating the focal adhesion protein paxillin. Here, we report the identification of p190RhoGAP-A (p190) as a Brk substrate. Brk phosphorylates p190 at the Y(1105) residue both in vitro and in vivo, thereby promoting the association of p190 with p120RasGAP (p120). As a consequence, Brk stimulates p190 and attenuates p120 functions, leading to RhoA inactivation and Ras activation, respectively. In carcinoma cells expressing high levels of Brk, endogenous Brk functions as a key contributor to epidermal growth factor-induced p190 tyrosine phosphorylation. We present evidence showing that p190 phosphorylation plays essential roles in both migratory and proliferative effects of Brk. Furthermore, disruption of p190 phosphorylation-induced p190/p120 complex in breast cancer cells abolishes not only the abilities of Brk to regulate RhoA and Ras but also the stimulatory effects of Brk on proliferation, migration, invasion, transformation, and tumorigenicity. Together, our findings reveal a previously unknown function of Brk in regulating both RhoA and Ras by phosphorylating p190 and provide evidence for the crucial roles of this Brk-elicited signaling pathway in promoting breast malignancy.