GM1 and GM2 gangliosides: recent developments

Introduction

GM1 and GM2 are monosialic gangliosides, a class of glycosphingolipids that are found predominantly in the outer leaflet of the plasma membrane and, to a lesser extent, in intracellular membranes. Gangliosides are amphipathic molecules with a negatively charged head group modified with carbohydrate groups that vary among the more than 200 identified species of ganglioside. In the case of GM1 and GM2, the head group consists of a single sialic acid. Two hydrocarbon chains of the ceramide moiety anchor gangliosides in the plasma membrane. Gangliosides are found in the plasma membrane of vertebrate cells and are particularly enriched in the nervous system.

Gangliosides are constantly synthesized and catabolized by highly regulated pathways. Ceramide, which forms the hydrophobic backbone of gangliosides, is synthesized in the endoplasmic reticulum, and glycosylated and sialylated in the Golgi apparatus. Glycoslytransferases add the final complement of carbohydrate groups to form individual gangliosides before vesicles of the secretory pathway transport the gangliosides to the cell surface.

Gangliosides undergo catabolism via the lysosomal recycling pathway. Endocytosis and endosomal sorting pathways chaperone gangliosides to endosomes and eventually lysosomes, where they are sequentially degraded by a series of hydrolytic enzymes assisted by lipid-binding proteins. Defects in the catabolic pathway lead to an accumulation of ganglioside species and result in lysosomal storage disorders and gangliosidoses, including Tay-Sachs disease (1).

In this paper, we survey the latest methods and developments in our understanding of the gangliosides GM1 and GM2, from their basic biochemistry and cellular function to the interactions with amyloid β (Aβ) peptide in Alzheimer’s disease. We also focus on the influence of GM1 and GM2 in other diseases including cancer, viral infection, and diabetes.

New approaches to study the cell biology and membrane biochemistry of gangliosides

The gangliosides GM1 and, to a lesser extent, GM2 are primarily recognized as essential components of membrane rafts. Membrane rafts, or lipid rafts, are heterogeneous clusters of cholesterol, sphingolipids, gangliosides, and membrane-associated proteins, including G protein-coupled receptors (GPCRs). These clusters form stabilized, liquid-ordered nanostructures important in a variety of physiological processes including signal transduction, protein and lipid sorting, and trafficking. Here, we mention the most innovative methods that have been recently used to gain new knowledge on the complex biology of gangliosides (Table 1).

The segregation of plasma membrane lipids into membrane microdomains has been reviewed extensively (2). A new study demonstrates the colocalization of cholesterol and GM1 in membrane microdomains with unprecedented precision using secondary ion mass spectrometry (SIMS) of individual isotope-labeled lipid components in model membranes (3). The results suggest several regimes of membrane domains classified by size: broad micrometer-scale domains and nanometer-scale domains that can be found either within the larger domains or outside of them. The micrometer-scale domains contained cholesterol and GM1 to the exclusion of other species. Nanometer-scale domains within larger domains contained increased palmitoyl sphingomyelin, whereas the nanometer-scale domains outside the larger domains have increased GM1. The detailed molecular information made visible by the SIMS technique represents a new step into the conceptual overview of membrane raft distribution.

Liquid-ordered (L_o) domains in model membranes have also been shown recently to respond to acidic gradients in a GM1-dependent manner (4). GM1-containing domains (but not GM1-free or asialo-GM1, which lacks the acidic head group) reorganized into larger clusters facing away from the source of the gradient. The authors speculate that this could constitute a potential mechanism by which membranes segregate into polarized regions during cell polarization.

GM1 in membrane raft domains has been shown to influence the functions of lipid raft-associated proteins. Mahmood et al. have recently demonstrated a direct relationship between the lipid composition in model bilayers and the distinct conformational states of a GPCR, the β₂ adrenergic receptor (5). The addition of GM1 to these synthetic membranes increased the stability of both the unbound and the ligand-bound β₂ adrenergic receptor. Their report suggests that beyond the role that lipid rafts play in recruiting membrane receptors and cofactors into close proximity, they may also play a direct role in the activation of membrane receptors. At the same time, another group has described a new method for identifying interactions between gangliosides, including GM1 and GM2, and membrane-associated proteins (6). Gangliosides extracted from a cell pellet are purified and passed through a column of immobilized beads functionalized with a target protein. Mass spectrometry allows the identification of minute amounts of an array of ganglioside species. The protocol improves upon earlier methods in its sensitivity to low concentrations of ganglioside species and has the potential to identify other GPCRs with activity-modulating interactions with GM1 or GM2.

GM1 has also been shown to regulate the formation of cell-cell junctions. Cell-cell junctions promote cell adhesion in tissues and play important roles in collective cell migration and the invasion of cancer cells in metastasis. New results indicate that flotillin mediates the interaction of cadherin with GM1 during the formation of cell-cell junctions (7). Another group demonstrated that GM1 localizes rapidly (within 10 s) to newly formed focal adhesions induced by a fibronectin-coated nanoelectrode (8). They further show that after recruitment of GM1, focal adhesions act as mechanosensors, transmitting signals from mechanical perturbations across the cell length.

GM1 gangliosides have also been shown to be central in cell polarization and motility. GM1 accumulates at the leading edge of migrating cells, and we have recently shown that this polarization is dependent on activation of phosphatidylinositide 3-kinases (PI3K), but not mitogen-activated protein kinase (MEK)/extracellular-signal-regulated kinases (ERK) (9). GM1 polarization was also demonstrated to be mechanistically and biochemically independent of the polarization of the Golgi apparatus, suggesting that de novo synthesis and transport are not responsible for the polarized localization. Other recently published results suggest that actin filaments form nucleation sites at the plasma membrane that enrich GM1-containing microdomains (10). Stabilized actin filaments with attachments to the plasma membrane at the leading edge are a common feature of polarized cell architecture, so these results indicate a plausible mechanism for enrichment of GM1 at the leading edge.

A general protocol for GM1 extraction that can potentially be applied to the analysis of disease-associated changes to GM1-enriched intracellular membranes has been recently proposed by Waugh (11).

Pathological involvement of GM1 and GM2

GM1 and GM2 in infections, cancer, and insulin metabolism

Gangliosides have long been known to play a central role in cholera infection, but recently, new reports suggest apparent roles in diseases as diverse as cancer (47–49), serotype 1 reovirus infection (50), and insulin secretion and regulation (51). GM2 appears to play an important role in adhesion, motility, and proliferation in tumor cells through interactions with growth factor receptors and focal adhesion proteins, including integrins, and caveolins [for a review, see (47)]. GM2 plays a protective role, and when its expression is decreased, characteristics of greater cancer malignancy are observed. More recently, analysis of glycosphingolipid profiles, including GM1 and GM2, in cancer stem cells revealed that GM2 and GM1 are upregulated in breast cancer stem cells compared to cancer non-stem cells and may provide a useful target against the development of cancer stem cell phenotypes (49). Interestingly, hexosaminidase subunit β (HEXB), which is involved in the catabolism of GM2, was found to be upregulated, presumably along with a downregulation of GM2 in blood vessels associated with invasive human breast cancer (48). Depletion of HEXB via siRNA was found to decrease the angiogenic potential. Another study using GM2 and GM3 synthase double knockout oncogene-transformed fibroblasts in a cancer model in mice found that downregulation of GM1 induces strikingly slower tumor growth (52). Although the precise role of GM2 in cancer progression is still not completely understood, it may prove to be an interesting therapeutic target.

GM2 was also identified recently to be a specific receptor for reovirus strain type 1 Lang (T1L) (50). Given the uneven distribution of GM2 in different tissue types, with a prominent expression in neural tissues, this finding could open the door to understanding other viral tissue infection patterns. It is also particularly interesting as reoviruses are currently being developed as delivery systems for cancer therapy, and given that GM2 expression is altered in many cancer types, understanding the interaction between reoviruses and GM2 could prove essential to these cancer therapies.

Finally, the GM2 activator protein (GM2AP), which acts as a cofactor to HexA in the catabolism of GM2, has been recently identified to be an adipokine that increases insulin secretion while at the same time impairing insulin and nerve growth factor signal transduction. Insulin receptors are localized in lipid rafts, and diabetes and insulin resistance have been hypothesized to be related to membrane microdomain function (51). GM2AP now appears to play a significant role in insulin metabolism, although a direct connection to GM2 has not yet been observed.

Considerations on the role of gangliosides in disease

Gangliosides are involved in important aspects of homeostasis, and levels of GM1 and GM2 gangliosides must be tightly and accurately regulated. Misregulation of GM1 or GM2 is implicated in a diverse array of diseases mostly associated with the nervous system (Figure 1). An accumulation of GM1 in neurons can lead to the deleterious effects observed in gangliosidoses. By contrast, GM1 depletion is associated to Parkinson’s and Huntington’s diseases, and administration of exogenous GM1 seems to reduce the pathological symptoms (in humans and mice) in both cases. Moreover, interaction of virus components and amyloid species with GM1 can trigger pathological pathways in cholera infection and Alzheimer’s disease, respectively. Finally, high levels of anti-ganglioside antibodies are frequently found in the serum of patients affected by autoimmune neuropathy, where they are suggested to play a pathogenic role. The importance of gangliosides, especially GM1, is evident from these types of diseases, showing that perturbations in one sense or in the other can result in dramatic consequences for the organism.

Figure 1

Pathological involvement of GM1 and GM2.