In this study, we have shown that exposure of E. histolytica
to bona fide
Gal/GalNAc lectin ligands (e.g.,. hRBCs or collagen) was accompanied by enrichment of the Gal/GalNAc lectin subunits, specifically Hgl and Lgl, in lipid raft domains. Previously, it was shown that cholesterol loading induced colocalization of Gal/GalNAc lectin subunits in rafts and increased activity of the Gal/GalNAc lectin (53
). Here, we have provided evidence that another condition, namely, ligand binding, can also influence the submembrane localization of the Gal/GalNAc lectin subunits. We have also shown that binding to ligand was necessary, but not sufficient, to induce enrichment of Hgl and Lgl in lipid rafts after ligand binding. Our data also indicate that PIP2
and calcium participate in the enrichment of Gal/GalNAc lectin subunits in rafts.
Enrichment of Hgl and Lgl in high-buoyancy lipid raft domains after ligand binding is similar to the clustering and activation of mammalian integrins in lipid rafts. For example, in Jurkat T lymphocytes, attachment to collagen type IV or fibronectin induces lipid raft enrichment of α2β1 and α4β1 integrins, respectively (17
). Furthermore, activation of another integrin in Jurkat T lymphocytes, lymphocyte function-associated antigen 1 (LFA-1), is correlated with its enrichment in lipid rafts (24
). Although these signaling pathways are well understood in immune cells, the current study is an important first step toward the understanding of downstream signaling pathways that arise from lipid rafts in a parasite model.
The present study shows that attachment to ligand results in colocalization of the three lectin subunits in lipid raft fractions. Previously, it was shown by immunoprecipitation that Igl associates with Hgl (30
). Importantly, we have not shown that Hgl and Lgl physically interact with Igl in lipid rafts. However, it is conceivable that the enrichment of Hgl and Lgl in raft regions, which already contain Igl, facilitates the assembly of the lectin into a functional trimer. This, in turn, may serve to activate subsequent raft-based signaling pathways related to virulence.
Exposure to hRBCs or collagen was correlated with the enrichment of Hgl and Lgl subunits in lipid rafts. Interestingly, these raft populations differed slightly in their buoyant densities. For example, after binding to hRBCs, Hgl and Lgl associated with rafts that were more buoyant than the rafts harboring these same subunits after collagen binding. It is possible that there are multiple types of lipid rafts within the parasite membrane, and binding to collagen or hRBCs causes the lectin to localize to distinct and separate lipid raft domains. In other systems, there is evidence for distinct raft populations. For example, purification of rafts from Madin-Darby canine kidney cells, using a variety of detergents, resulted in the isolation of distinct lipid raft domains with different protein residents (41
). Immunogold labeling and electron microscopy have shown that all lipid raft markers do not colocalize. These data from other systems support the notion that multiple lipid raft domains exist within the plasma membrane (55
). Our data suggest that the same is true in E. histolytica
Differences in the buoyant density of rafts containing the lectin may be due to the association of the lectin with a different set of signaling proteins or cytoskeletal proteins in a ligand-specific manner. In neutrophils, heavier detergent-resistant membranes were found to contain more cytoskeletal proteins (34
). Adhesion plaques, which contain actin, myosin I and II, α-actinin, vinculin, and tropomyosin (49
), have been observed in E. histolytica
upon attachment to ECM components but have not been observed upon attachment to hRBCs. Thus, the formation of a Gal/GalNAc lectin-containing adhesion plaque after exposure to collagen may explain why the lipid rafts harboring the lectin after collagen exposure are less buoyant than those harboring the lectin after hRBC exposure.
We showed that ligand binding was not correlated with the enrichment of Hgl and Lgl in rafts in a transgenic cell line with reduced levels of PIP2. We also showed that addition of exogenous PIP2 to this cell line partially rescued the phenotype. Together, these data provide strong genetic evidence for a role for PIP2 in regulating the submembrane distribution of the lectin subunits in E. histolytica. To our knowledge, this is the first study, in any system, to use a PIP2-deficient mutant to illustrate the role of PIP2 in protein-lipid raft interactions.
In the current study, intracellular calcium levels were increased upon exposure to collagen but not fibronectin. Others have shown that calcium levels increase when trophozoites are exposed to fibronectin (5
). One explanation for this difference is that we exposed cells to fibronectin-coated coverslips instead of fibronectin in solution (5
); adhesion to the solid ECM surface may initiate different signaling pathways. It is currently unknown whether the increased intracellular calcium levels are directly related to PIP2
hydrolysis in the cell or are attributable to other mechanisms related to calcium influx. In mammalian cells, the physical interaction between αII
integrin, sodium-proton exchangers, and sodium-calcium exchangers occurs simultaneously with integrin binding to ligand and results in increased intracellular calcium levels (56
). Additionally, in phagocytes, extracellular calcium influx was shown to be essential for movement of an integrin bound to adenylate cyclase toxin from Bordetella
into lipid rafts (3
). Similarly, in the current study, the increase in calcium levels was shown to be necessary for ligand-induced enrichment of Hgl and Lgl in lipid raft domains.
Other studies, with mammalian cells as well as with E. histolytica
, have supported the connection between calcium, PIP2
, regulation of actin cytoskeleton, regulation of transcription, and virulence. For example, in B cells, calcium signaling has been shown to activate transcriptional regulators, such as NF-κB and NFAT (13
). Likewise, attachment to collagen by trophozoites induces an increase in the binding of transcriptional regulators AP-1, STAT1, and STAT3 to DNA (7
) and an increase in the expression of several important virulence factors, including amoebapore and cysteine proteases (10
). In E. histolytica
, actin remodeling occurs during attachment to collagen (32
) and hRBCs (1
), and calcium mobilization can affect actin organization (5
). In mammalian cells, calpain, a calcium-dependent protease, has been shown to cleave the cytoskeletal elements talin, filamin, and α-actinin, thereby releasing integrins from the actin cytoskeleton (45
). It has been proposed previously that this cleavage of talin may be responsible for freeing proteins to allow their recruitment to lipid raft domains (3
also contributes to actin cytoskeletal reorganization by guiding and activating actin binding proteins (20
plays an important role in mammalian cells by binding to talin, thereby targeting it to focal adhesions, where it can interact with and activate integrins (25
). Together with our data, these findings suggest an intriguing link between parasite-host interactions, raft association of the Gal/GalNAc lectin, calcium mobilization, the cytoskeleton, and changes in gene expression.
The data presented here provide insights into signaling pathways in E. histolytica and, importantly, add to a developing model of the regulation of Gal/GalNAc lectin function. In the absence of ligand, GPI-anchored Igl subunits reside predominantly in raft-like domains, whereas Hgl-Lgl dimers are localized primarily to a different submembrane compartment. Binding to at least two biologically relevant ligands, hRBCs and collagen, brings all three subunits to the same raft fractions. Interestingly, our data are the first to show a correlation between the submembrane position of the lectin subunits and phosphoinositide-based signaling in this pathogen. In the future, it will be important to identify effectors that act downstream and in parallel with the Gal/GalNAc lectin after ligand binding and enrichment in lipid rafts. Fully understanding the behavior of this receptor after contact with extracellular ligands during invasion is necessary to fully appreciate virulence functions in E. histolytica.