Search

Sandhoff disease (SD) is a lysosomal storage disease, caused by loss of β-hexosaminidase (HEX) activity resulting in the accumulation of ganglioside GM2. There are shared features between SD and Parkinson’s disease (PD). α-synuclein (aSYN) inclusions, the diagnostic hallmark sign of PD, are frequently found in the brain in SD patients and HEX knockout mice, and HEX activity is reduced in the substantia nigra in PD. In this study, we biochemically demonstrate that HEX deficiency in mice causes formation of high-molecular weight (HMW) aSYN and ubiquitin in the brain. As expected from HEX enzymatic function requirements, overexpression in vivo of HEXA and B combined, but not either of the subunits expressed alone, increased HEX activity as evidenced by histochemical assays. Biochemically, such HEX gene expression resulted in increased conversion of GM2 to its breakdown product GM3. In a neurodegenerative model of overexpression of aSYN in rats, increasing HEX activity by AAV6 gene transfer in the substantia nigra reduced aSYN embedding in lipid compartments and rescued dopaminergic neurons from degeneration. Overall, these data are consistent with a paradigm shift where lipid abnormalities are central to or preceding protein changes typically associated with PD.

The catabolism of ganglioside GM2 is dependent on three gene products. Mutations in any of these genes result in a different type of GM2 gangliosidosis (Tay-Sachs disease, Sandhoff disease, and the B1 and AB variants of GM2 gangliosidosis), with GM2 as the major lysosomal storage compound. GM2 is also a secondary storage compound in lysosomal storage diseases such as Niemann-Pick disease types A–C, with primary storage of SM in type A and cholesterol in types B and C, respectively. The reconstitution of GM2 catabolism at liposomal surfaces carrying GM2 revealed that incorporating lipids into the GM2-carrying membrane such as cholesterol, SM, sphingosine, and sphinganine inhibits GM2 hydrolysis by α-hexosaminidase A assisted by GM2 activator protein, while anionic lipids, ceramide, fatty acids, lysophosphatidylcholine, and diacylglycerol stimulate GM2 catabolism. In contrast, the hydrolysis of the synthetic, water-soluble substrate 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-α-d-glucopyranoside was neither significantly affected by membrane lipids such as ceramide or SM nor stimulated by anionic lipids such as bis(monoacylglycero)phosphate added as liposomes, detergent micelles, or lipid aggregates. Moreover, hydrolysis-inhibiting lipids also had an inhibiting effect on the solubilization and mobilization of membrane-bound lipids by the GM2 activator protein, while the stimulating lipids enhanced lipid mobilization.—

Background Immune checkpoint inhibitors (ICI) have greatly improved the treatment options for patients with metastatic melanoma. Yet, a large percentage of melanoma patients do not respond to ICIs, there is a need for biomarkers that can predict patients‘ clinical benefit thereby identifying the patient population most likely to respond. Here, we apply unbiased discovery proteomics to deeply characterize global tumor proteomes and associate proteins and pathways at baseline with clinical response to anti-PD-1 immunotherapy. Methods Unbiased, data-independent acquisition (DIA) mass spectrometry was used to analyze Formalin Fixed Paraffin Embedded (FFPE) tumor tissue from subjects with stage IIIc-IV melanoma which were resected prior to initiation of first-line anti-PD-1 therapy. The selected samples represent two distinct clinical subgroups; those who received clinical benefit (CR or PR by RECIST criteria or OS >1 year with SD by RECIST criteria, n = 13), and those with no clinical benefit (PD by RECIST criteria or OS Results 8’548 proteins were quantified across all samples, with 7’416 quantified on average per sample. Univariate statistical testing between groups identified 285 proteins that were significantly regulated in subjects who received clinical benefit. Through partial least squares discriminant analysis (PLS-DA) a set of 25 proteins was identified that describe the variance between the two sample groups. Ganglioside GM2 activator (GM2A) and other members of its interaction network such as HEXB, HRNR and CPPED1 were identified to be upregulated in the non-responder group. Conclusions Global profiling of the baseline tumor proteome provides a unique characterization of melanoma tumor biology. A signature of 25 protein markers was identified as a driver of separation between responder and non-responder patients to PD-1 blockade. Among the protein markers, GM2A and its interactors, were previously shown to perturb T cell function, which might explain their enrichment in the non-responder group and provide an attractive target for improving patient response to immunotherapy.

β-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.

Approximately 70 lysosomal storage diseases are currently known, resulting from mutations in genes encoding lysosomal enzymes and membrane proteins. Defects in lysosomal enzymes that hydrolyze sphingolipids have been relatively well studied. Gaucher disease is caused by the loss of activity of glucocerebrosidase, leading to accumulation of glucosylceramide. Gaucher disease exhibits a number of subtypes, with types 2 and 3 showing significant neuropathology. Sandhoff disease results from the defective activity of β-hexosaminidase, leading to accumulation of ganglioside GM2. Niemann-Pick type C disease is primarily caused by the loss of activity of the lysosomal membrane protein, NPC1, leading to storage of cholesterol and sphingosine. All three disorders display significant neuropathology, accompanied by neuroinflammation. It is commonly assumed that neuroinflammation is the result of infiltration of monocyte-derived macrophages into the brain; for instance, cells resembling lipid-engorged macrophages ('Gaucher cells') have been observed in the brain of Gaucher disease patients. We now review the evidence that inflammatory macrophages are recruited into the brain in these diseases and then go on to provide some experimental data that, at least in the three mouse models tested, monocyte-derived macrophages do not appear to infiltrate the brain. Resident microglia, which are phenotypically distinct from infiltrating macrophages, are the only myeloid population present in significant numbers within the brain parenchyma in these authentic mouse models, even during the late symptomatic stages of disease when there is substantial neuroinflammation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. This article is part of the Special Issue "Lysosomal Storage Disorders".

GM2 Gangliosidoses are a group of autosomal recessive genetic disorders caused by intra-lysosomal deposition of ganglioside GM2 mainly in the neuronal cells. GM2-Activator protein deficiency is an extremely rare type of GM2 gangliosidosis (AB variant) caused by the mutation of GM2A.We report a case of a female child who presented with clinical features similar to classical Tay-Sachs disease, but with normal beta hexosaminidase enzyme levels. Molecular study revealed a novel homozygous intronic mutation which confirmed the diagnosis of GM2 Activator protein deficiency. GM2 Activator protein deficiency is a mimic of Classical Tay-Sachs disease and should be a differential diagnosis in children who present with neuroregression, cherry red spots without hepatosplenomegaly and with normal beta hexosaminidase enzyme levels.

Gangliosides, a group of glycosphingolipids, are known to be cell surface markers and functional factors in several cancers. However, the association between gangliosides and pancreatic ductal adenocarcinoma (PDAC) has not been well elucidated. In this study, we examined the expression and roles of ganglioside GM2 in PDAC. GM2+ cells showed a higher growth rate than GM2− cells in the adherent condition. When GM2– and GM2+ cells were cultured three-dimensionally, almost all cells in the spheres expressed GM2, including cancer stem cell (CSC)-like cells. A glycolipid synthesis inhibitor reduced GM2 expression and TGF-β1 signaling in these CSC-like cells, presumably by inhibiting the interaction between GM2 and TGFβ RII and suppressing invasion. Furthermore, suppression of GM2 expression by MAPK inhibition also reduced TGF-β1 signaling and suppressed invasion. GM2+ cells formed larger subcutaneous tumors at a high incidence in nude mice than did GM2– cells. In PDAC cases, GM2 expression was significantly associated with younger age, larger tumor size, advanced stage and higher histological grade. These findings suggest that GM2 could be used as a novel diagnostic and therapeutic target for PDAC.

Sandhoff disease (SD) is a genetic disorder caused by a mutation of HEXB, which is the β-subunit gene of β-hexosaminidase A and B (HexA and HexB) in humans. HEXB mutation reduces HexA and HexB enzymatic activities, and results in the massive accumulation of ganglioside GM2 in the nervous system. Severe phenotypes of SD show progressive neurodegeneration in human infants, and lysosomal dysfunction that may affect the early development of the nervous system. In a previous study, neural stem cells (NSCs) and induced pluripotent stem cells derived from SD model mice, which are Hexb-deficient (Hexb-/-), demonstrated impaired neuronal differentiation. This study investigated early neurodevelopment in vivo using Hexb-/- mice. The structure of adult cerebral cortices of Hexb-/- mice was normal. However, the expression of Sox2, an NSC-related gene, was reduced in the embryonic cerebral cortices of Hexb-/- mice. Moreover, a reduction of early neuronal migration and differentiation was observed in the embryonic cerebral cortices of Hexb-/- mice. In addition, we showed that the production of layer-specific neurons was delayed in somatosensory cerebral cortices of Hexb-/- mice. These findings suggest that the alterations observed in embryonic Hexb-/- mice may contribute to deficits in neurodevelopment of SD.

The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor.

Objective: A rare case of possible hereditary predisposition to autoimmune neuropathy Background: This case report is of a patient who presented two episodes of Facial paresis in her live without ascertained apparent causes. Bell’s palsy is commonly known as peripheral idiopathic facial nerve palsy, because in the most cases the triggers remain unknown (1). Case Report: I want to present a case of a 34 year old woman who we will call A.B. and who came to us to evaluate the severity of the sequelae of a Bell´s palsy and any eventual need for cosmetic measure. But she had another Bell´s Palsy 9 years ago, so I started to investigate the most common and treatable causes of Facial paresis. I found a significate increase of anti-bodies against ganglioside GM2. When I explained to her that these anti-bodies is commonly related with several neurological diseases, she started to investigate her family history and she found that his father’s brother died of GuillainBarré syndrome about 40 years ago.

(a) Purpose: Gangliosides are molecules composed of glycosphingolipids linked with one or more sialic acids, and are known to be key components of lipid rafts, which act as platforms for signal transduction. Changes in ganglioside levels affect the expression of raft-associated proteins on the cell surface and lead to reduced membrane fluidity, thus, resulting in cellular dysfunction, such as impaired signal transduction. The ganglioside GM2 is one of the major series of gangliosides that has several biological functions, such as in cell adhesion and signal transduction. GM2 is highly expressed in several types of human malignant tumors, such as melanomas, gliomas, and neuroblastomas, but is absent or weakly expressed in normal tissues. However, the association between gangliosides and pancreatic ductal adenocarcinoma (PDAC) has not been elucidated yet. Thus, in this study, we aimed to clarify the expression and role of ganglioside GM2 in PDAC. (b) Experimental procedures: GM2 expression in eight human PDAC cell lines was examined by fluorescence-activated cell sorter (FACS) analysis. The morphology, growth, and stemness of GM2- and GM2+ sorted-cells were compared by transmission electron microscopy, growth assays, real-time PCR analysis of the markers of stemness, and sphere forming assays. Cell motility was evaluated by invasion assay. For comparing tumorigenicity between the GM2- and GM2+ sorted-cells, heterotopic implantations were performed. The expression levels and clinico-pathological roles of GM2 in patients with PDAC were examined by immunohistochemical analysis of 117 pancreatic tissue samples. (c) Results: The fraction of GM2+ cells was the highest (21.4%) in MIA PaCa-2 from among the eight cell lines, as revealed by FACS analysis. GM2-expressing MIA PaCa-2 cells had higher growth rates under adherent growth conditions. In 3D-culture, which enriches cancer stem cells (CSCs), most MIA PaCa-2 cells expressed GM2 and the cells responded to TGF-β1 treatment to promote invasion. Transplantation of the GM2-expressing cells into nude mice resulted in development of larger tumors as compared to that for control cells that did not express GM2. In the PDAC cases, GM2 expression was significantly associated with younger age, larger tumor size, advanced stage, and higher histological grade. (d) Conclusions: We show that a PDAC cell line overexpressing GM2 exhibits high growth rate and high tumor initiation in 2D-culture. Further, pancreatic CSC-like cells expressing GM2 exhibit responsiveness against TGF-β1, resulting in enhanced invasion in 3D-culture. Furthermore, GM2 expression is associated with the growth and advanced stage of human PDAC. Further studies will be required for the development of an early detection method for GM2-overexpressing pancreatic cancers and to develop novel therapeutic strategies targeting GM2. Citation Format: Norihiko Sasaki, Kenichi Hirabayashi, Masaki Michishita, Kimimasa Takahashi, Fumio Hasegawa, Fujiya Gomi, Yoko Itakura, Naoya Nakamura, Masashi Toyoda, Toshiyuki Ishiwata. Increased expression of ganglioside GM2 correlates with aggressiveness of human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 135.

The catabolism of ganglioside GM2 is dependent on the lysosomal enzyme β-hexosaminidase A and a supporting lipid transfer protein, the GM2 activator protein. A genetically based disturbance of GM2 catabolism, leads to several subtypes of the GM2 gangliosidosis: Tay-Sachs disease, Sandhoff disease, the AB-variant and the B1-variant, all of them having GM2 as major lysosomal storage compound. Further on it is known that the gangliosides GM2 and GM3 accumulate as secondary storage compounds in mucopolysaccharidoses, especially in Hunter disease, Hurler disease, Sanfilippo disease and Sly syndrome, with chondroitin sulfate as primary storage compound. The exact mechanism of ganglioside accumulation in mucopolysaccaridoses is still a matter of debate. Here, we show that chondroitin sulfate strongly inhibits the catabolism of membrane-bound GM2 by β-hexosaminidase A in presence of GM2 activator protein in vitro already at low micromolar concentrations. In contrast, hyaluronan, the major storage compound in mucopolysaccharidosis IX, a milder disease without secondary ganglioside accumulation, is a less effective inhibitor. On the other hand, hydrolysis of micellar-bound GM2 by β-hexosaminidase A without the assistance of GM2AP was not impeded by chondroitin sulfate implicating that the inhibition of GM2 hydrolysis by chondroitin sulfate is most likely based on an interaction with GM2AP, the GM2AP-GM2 complex or the GM2-carrying membranes. We also studied the influence of some cationic amphiphilic drugs (desipramine, chlorpromazine, imipramine and chloroquine), provoking drug induced phospholipidosis and found that all of them inhibited the hydrolysis of GM2 massively.

Gangliosidoses are caused by monogenic defects of a specific hydrolase or an ancillary sphingolipid activator protein essential for a specific step in the catabolism of gangliosides. Such defects in lysosomal function cause a primary accumulation of multiple undegradable gangliosides and glycosphingolipids. In reality, however, predominantly small gangliosides also accumulate in many lysosomal diseases as secondary storage material without any known defect in their catabolic pathway. In recent reconstitution experiments, we identified primary storage materials like sphingomyelin, cholesterol, lysosphingolipids, and chondroitin sulfate as strong inhibitors of sphingolipid activator proteins (like GM2 activator protein, saposin A and B), essential for the catabolism of many gangliosides and glycosphingolipids, as well as inhibitors of specific catabolic steps in lysosomal ganglioside catabolism and cholesterol turnover. In particular, they trigger a secondary accumulation of ganglioside GM2, glucosylceramide and cholesterol in Niemann–Pick disease type A and B, and of GM2 and glucosylceramide in Niemann–Pick disease type C. Chondroitin sulfate effectively inhibits GM2 catabolism in mucopolysaccharidoses like Hurler, Hunter, Sanfilippo, and Sly syndrome and causes a secondary neuronal ganglioside GM2 accumulation, triggering neurodegeneration. Secondary ganglioside and lipid accumulation is furthermore known in many more lysosomal storage diseases, so far without known molecular basis.

The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qβ. Although the copper-catalyzed azide–alkyne cyclo-addition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.

Malignant pleural mesothelioma (MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action. Ganglioside GM2 is a glycolipid that has been shown to be overexpressed in various types of cancer. However, there are no published reports regarding the use of GM2 as a potential therapeutic target in cases of MPM. In this study, we evaluated the efficacy of the anti-GM2 antibody BIW-8962 as an anti-MPM therapeutic using in vitro and in vivo assays. Consequently, the GM2 expression in the MPM cell lines was confirmed using flow cytometry. In addition, eight of 11 cell lines were GM2-positive (73%), although the GM2 expression was variable. BIW-8962 showed a significant antibody-dependent cellular cytotoxicity activity against the GM2-expressing MPM cell line MSTO-211H, the effect of which depended on the antibody concentration and effector/target ratio. In an in vivo orthotropic mouse model using MSTO-211H cells, BIW-8962 significantly decreased the incidence and size of tumors. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. Fifty-eight percent of the MPM tumors were positive for GM2, with individual variation in the intensity and frequency of staining. These data suggest that anti-GM2 antibodies may become a therapeutic option for MPM patients. In this study, the anti-GM2 antibody BIW-8962 first showed a significant ADCC activity against malignant pleural mesothelioma (MPM) cell line and therapeutic activity in an in vivo orthotropic mouse model using the cell line. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. These data suggest that anti-GM2 antibodies may become a therapeutic option for MPM patients. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Zika virus (ZIKV) infection has recently emerged as a major concern worldwide due to its strong association with nervous system malformation (microcephaly) of fetuses in pregnant women infected by the virus. Signs and symptoms of ZIKV infection are often mistaken with other common viral infections. Since transmission may occur through biological fluids exchange and coitus, in addition to mosquito bite, this condition is an important infectious disease. Thus, understanding the mechanism of viral infection has become an important research focus, as well as providing potential targets for assertive clinical diagnosis and quality screening for hemoderivatives. Within this context, the present work analyzed blood plasma from 79 subjects, divided as a control group and a ZIKV-infected group. Samples underwent direct-infusion mass spectrometry and statistical analysis, where eight markers related to the pathophysiological process of ZIKV infection were elected and characterized. Among these, Angiotensin (1-7) and Angiotensin I were upregulated under infection, showing an attempt to induce autophagy of the infected cells. However, this finding is concerning about hypertensive individuals under treatment with inhibitors of the Renin-Angiotensin System (RAS), which could reduce this response against the virus and exacerbate the symptoms of the infection. Moreover, one of the most abundant glycosphingolipids in the nervous tissue, Ganglioside GM2, was also elected in the present study as an infection biomarker. Considered an important pathogen receptor at membrane's outer layer, this finding represents the importance of gangliosides for ZIKV infection and its association with brain tropism. Furthermore, a series of phosphatidylinositols were also identified as biomarkers, implying a significant role of the PI3K-AKT-mTOR Pathway in this mechanism. Finally, these pathways may also be understood as potential targets to be considered in pharmacological intervention studies on ZIKV infection management.

Explosive detonations generate atmospheric pressure changes that produce nonpenetrating blast induced "mild" traumatic brain injury (bTBI). The structural basis for mild bTBI has been extremely controversial. The present study applies matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to track the distribution of gangliosides in mouse brain tissue that were exposed to very low level of explosive detonations (2.5-5.5 psi peak overpressure). We observed major increases of the ganglioside GM2 in the hippocampus, thalamus, and hypothalamus after a single blast exposure. Moreover, these changes were accompanied by depletion of ceramides. No neurological or brain structural signs of injury could be inferred using standard light microscopic techniques. The first source of variability is generated by the Latency between blast and tissue sampling (peak intensity of the blast wave). These findings suggest that subtle molecular changes in intracellular membranes and plasmalemma compartments may be biomarkers for biological responses to mild bTBI. This is also the first report of a GM2 increase in the brains of mature mice from a nongenetic etiology.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (MALDI-MSI) allows us to investigate the distribution of lipid molecules within tissues. We used MALDI-MSI to identify prognostic gangliosides in tissue sections of rat intracranial allografts of rat glioma and mouse intracranial xenografts of human medulloblastoma. In the healthy adult rodent brain, GM1 and GD1 were the main types of glycolipids. Both gangliosides were absent in both intracranial transplants. The ganglioside GM3 was not present in the healthy adult brain but was highly expressed in rat glioma allografts. In combination with tandem mass spectrometry GM3 (d18:1/C24:0) was identified as the most abundant ganglioside species in the glioma allotransplant. By contrast, mouse xenografts of human medulloblastoma were characterized by prominent expression of the ganglioside GM2 (d18:0/C18:0). Together, these data demonstrate that tissue-based MALDI-MSI of gangliosides is able to discriminate between different brain tumors and may be a useful clinical tool for their classification and grading.

Sandhoff Disease (SD) involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the β-subunit gene of β-hexosaminidase A and B (Hexb gene). Substrate reduction therapy, utilizing imino sugar N-butyldeoxygalactonojirimycin (NB-DGJ), reduces ganglioside biosynthesis and levels of stored GM2 in SD mice. Intracranial transplantation of Neural Stem Cells (NSCs) can provide enzymatic cross correction, to help reduce ganglioside storage and extend life. Here we tested the effect of NSCs and NB-DGJ, alone and together, on brain β-hexosaminidase activity, GM2, and GA2 content in juvenile SD mice. The SD mice received either cerebral NSC transplantation at post-natal day 0 (p-0), intraperitoneal injection of NB-DGJ (500 mg/kg/day) from p-9 to p-15, or received dual treatments. The brains were analyzed at p-15. β-galactosidase staining confirmed engraftment of lacZ-expressing NSCs in the cerebral cortex. Compared to untreated and sham-treated SD controls, NSC treatment alone provided a slight increase in Hex activity and significantly decreased GA2 content. However, NSCs had no effect on GM2 content when analyzed at p-15. NB-DGJ alone had no effect on Hex activity, but significantly reduced GM2 and GA2 content. Hex activity was slightly elevated in the NSC + drug-treated mice. GM2 and GA2 content in the dual treated mice were similar to that of the NB-DGJ treated mice. These data indicate that NB-DGJ alone was more effective in targeting storage in juvenile SD mice than were NSCs alone. No additive or synergistic effect between NSC and drug was found in these juvenile SD mice.

Monolithic columns containing ganglioside GM2 and GM3 mimics were prepared for selective removal of serum anti-ganglioside antibodies from patients with acute and chronic immune-mediated neuropathies. ELISA results demonstrated that anti-GM2 IgM antibodies in human sera and a mouse monoclonal anti-GM2 antibody were specifically and selectively adsorbed by monolithic GM2 mimic columns and not by blank monolithic columns or monolithic GM3 mimic columns. In control studies, serum antibodies against the ganglioside GQ1b from another neuropathy patient were not depleted by monolithic GM2 mimic columns. Fluorescence microscopy with FITC-conjugated anti-human immunoglobulin antibodies showed that the immobilized ganglioside mimics were evenly distributed along the column. The columns were able to capture similar to 95% of the anti-GM2 antibodies of patients after only 2 min of incubation. A monolithic column of 4.4 mu L can deplete 28.2 mu L of undiluted serum. These columns are potential diagnostic and therapeutic tools for neuropathies related to anti-ganglioside antibodies.

GOTO cells, a neuroblastoma cell line retaining the ability to differentiate into neuronal or Schwann cells, were found to be rich in membrane rafts containing ganglioside GM2 and hypersensitive to lipid raft-disrupting methyl-beta-cyclodextrin (MbetaCD); the GM2-rich rafts and sensitivity to MbetaCD were markedly diminished upon their differentiation into Schwann cells. We first raised a monoclonal antibody that specifically binds to GOTO cells but not to differentiated Schwann cells and determined its target antigen as ganglioside GM2, which was shown to be highly concentrated in lipid rafts by its colocalization with flotillin, a marker protein of rafts. Disturbance of normal structure of the lipid raft by depleting its major constituent, cholesterol, with MbetaCD resulted in acute apoptotic cell death of GOTO cells, but little effects were seen on differentiated Schwann cells. Until this study, GM2-rich rafts are poorly characterized and MbetaCD hypersensitivity, which may have clinical implications, has not been reported.

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes the beta-subunit of N-acetyl-beta-hexosaminidase A and B, resulting in the accumulation of the ganglioside GM2. We isolated astrocytes from the neonatal brain of Sandhoff disease model mice in which the N-acetyl-beta-hexosaminidase beta-subunit gene is genetically disrupted (ASD). Glycolipid profiles revealed that GM2/GA2 accumulated in the lysosomes and not on the cell surface of ASD astrocytes. In addition, GM3 was increased on the cell surface. We found remarkable differences in the cell proliferation of ASD astrocytes when compared with cells isolated from wild-type mice, with a faster growth rate of ASD cells. In addition, we observed increased extracellular, signal-regulated kinase (ERK) phosphorylation in ASD cells, but Akt phosphorylation was decreased. Furthermore, the phosphorylation of ERK in ASD cells was not dependent upon extracellular growth factors. Treatment of ASD astrocytes with recombinant N-acetyl-beta-hexosaminidase A resulted in a decrease of their growth rate and ERK phosphorylation. These results indicated that the up-regulation of ERK phosphorylation and the increase in proliferation of ASD astrocytes were dependent upon GM2/GA2 accumulation. These findings may represent a mechanism in linking the nerve cell death and reactive gliosis observed in Sandhoff disease.

Sandhoff's disease is a lysosomal storage disease in which the ganglioside GM2 accumulates in lysosomes. It has been reported that MRI cannot detect abnormalities in spin echo images in clinically presymptomatic Sandhoff's disease patients. Because one of the results of GM2 accumulation is cell swelling and lysosomal distension, our goal was to determine if changes in the diffusion of water is perturbed. We utilized the MRI imaging modality diffusion-weighted imaging to measure the apparent diffusion coefficient in a mouse models of Sandhoff's disease, the hexb−/− mouse, and determined if diffusion-weighted imaging could be utilized to detect early changes prior to behavioral or overt disease symptom onset. Here we report for the first time a comprehensive behavioral characterization of the hexb−/− mouse in conjunction with the apparent diffusion coefficient measurement. Our data indicate that the apparent diffusion coefficient decreases in the hexb−/− mouse in many but not all brain regions prior to disease symptoms (

Ganglioside GM2 and its asialo-derivative, GA2 were radiolabeled in their N-acetyl-D-galactosaminyl moieties by oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Specific activities of 6 X 10(4) dpm/nmol (GM2) and 1.8 X 10(6) dpm/nmol (GA2) were achieved. About 98% of the label was in N-acetyl-D-galactosamine. Using these substrates, an assay was developed for GM2-N-acetyl-beta-D-galactosaminidase (E.C.3.2.1.30) and GA2-N-acetyl-beta-D-galactosaminidase (E.C.3.2.1.30) activities in human cultured skin fibroblasts. The products of the GM2 cleaving reaction were identified as N-acetylgalactosamine and ganglioside GM3. Both GM2 and GA2 cleaving activities were stimulated about 5-fold by purified sodium taurocholate, and this stimulation was inhibited by neutral detergents, lipids and albumin at low concentrations. Addition of various salts, reducing agents and a protein activator factor from human liver of Li et al. (1973) did not stimulate GM2-N-acetyl-beta-D-galactosaminidase activity beyond that found with sodium taurocholate. Under optimal conditions, control fibroblast supernates cleaved ganglioside GM2 at a rate of 3.7 nmol/mg protein/h compared to 1100 for GA2-N-acetyl-beta-D-galactosaminidase and 4700 for 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminidase. Supernates from two patients with Tay-Sachs disease had markedly reduced activity levels for GM2-N-acetyl-beta-D-galactosaminidase but not for the other two substrates. Supernates from two patients with Sandhoff's disease had reduced activities for all three substrates. A supernate from one patient with juvenile GM2 gangliosidosis cleaved GM2 at a somewhat faster rate than those from Tay-Sachs or Sandhoff's patients. Two healthy adult women with markedly reduced hexosaminidase A activities using 4MU-N-acetyl-beta-D-glucosaminide as substrate had approximately half-normal activities using GM2 as substrate. A patient with the Tay-Sachs phenotype but with a partial deficiency of hexosaminidase A using the 4-MU substrate had a profound deficiency using GM2 as substrate. In such unusual hexosaminidase mutants, assays using GM2 as substrate are better indicators of phenotype than those using synthetic substrates.

The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.

Summary Background Sandhoff disease is an autosomal recessive disorder caused by β-hexosaminidase deficiency in which the ganglioside GM2 and other glycolipids accumulate intracellularly within lysosomes. This process results in progressive motor neuron manifestations, death from respiratory failure and infections in infantiles. Case Report This report presents a 22-month-old girl with infantile type Sandhoff disease that was hospitalized for generalized seizures and psychomotor retardation. She was diagnosed with a genetically proven novel mutation and by demonstrating it’s specific imaging findings. Conclusions Determination of spesific changes in neuroimaging which are initial findings for GM2 gangliosidosis is important from the point of diagnosis and follow-up in infants suspected of having a neurodegenerative disease.

An outbreak of Guillain-Barré syndrome (GBS), subsequent to Campylobacter jejuni enteritis, occurred in China in 2007. Serum anti-ganglioside antibodies were measured in GBS patients and controls. Genome sequencing was used to determine the phylogenetic relationship among three C. jejuni strains from a patient with GBS (ICDCCJ07001), a patient with gastroenteritis (ICDCCJ07002) and a healthy carrier (ICDCCJ07004), which were all associated with the outbreak. The ganglioside-like structures of the lipo-oligosaccharides of these strains were determined by mass spectrometry. Seventeen (53%) of the GBS patients had anti-GT1a IgG antibodies. GT1a mimicry was found in the lipo-oligosaccharides of strain ICDCCJ07002 and ICDCCJ07004; but a combination of GM3/GD3 mimics was observed in ICDCCJ07001, although this patient had anti-GT1a IgG antibodies. A single-base deletion in a glycosyltransferase gene caused the absence of GT1a mimicry in ICDCCJ07001. The phylogenetic tree showed that ICDCCJ07002 and ICDCCJ07004 were genetically closer to each other than to ICDCCJ07001. C. jejuni, bearing a GT1a-like lipo-oligosaccharide, might have caused the GBS outbreak and the loss of GT1a mimicry may have helped ICDCCJ07001 to survive in the host.

Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the β-subunit of β-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients.

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes for the β-subunit of β-hexosaminidase A and B, resulting in ganglioside GM2 accumulation in the brain. We now demonstrate that phospholipid metabolism is altered in both cultured neurons and in brain tissue from a mouse model of Sandhoff disease, the Hexb–/– mouse. Metabolic labelling using [methyl-14C]choline and l-[3-3H]serine demonstrated reduced incorporation of [methyl-14C]choline into phospholipids in brain tissue but not in liver or spleen. Phospholipid mass was also reduced in brain. The activities of CTP : phosphocholine cytidylyltransferase (CCT) and phosphatidylserine synthase were also reduced in brain tissue from Hexb–/– mice, probably because of post-translational modification as no changes were observed in levels of enzyme expression. The relevance of these findings to Sandhoff disease in human patients is strengthened by observations made over 30 years ago on autopsy tissue of Tay Sachs and Sandhoff disease patients, in which reduced phospholipid levels were observed. We suggest that changes in phospholipid metabolism are not simply because of loss of neuronal tissue as a result of degeneration but rather may cause degeneration, and we discuss the possible effects that changes in phospholipid metabolism could play in the neuropathophysiology of Sandhoff disease.

The GM2-activator protein (GM2AP) is an essential cofactor for the degradation of ganglioside GM2 by lysosomal beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-bound substrate at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. To elucidate the mode of action of this glycoprotein cofactor, we synthesized the two photoaffinity labels [14C]C3-TPD-GM2 and [14C]C7-TPD-GM2. Incubation of GM2AP with these substrate analogues and subsequent irradiation led to covalent labelling of the protein. After separation of tryptic peptides by reverse-phase HPLC, the labelled peptide fractions were analysed by MALDI-TOF and sequenced by ESI-Q-TOF mass spectrometry. Both labels were found to be specifically photoincorporated into a part of the surface loop comprising residues V153-L163, a stretch of amino acids that was previously identified as the most flexible region in the crystal structure of the activator. Our results provide strong evidence that this loop constitutes the part of the activator protein that directly interacts with the ganglioside substrate, suggesting that the hydrophobicity and the great structural mobility of this element are crucial for the extraction of the membrane-embedded glycolipid, its stabilization inside the spacious cavity and its guidance to the enzyme's active site. This study demonstrates that the approach of photoaffinity labelling in conjunction with accurate mass measurements can provide insight into substrate binding interactions that complements structural information. D-53121 Bonn, Germany.

Tay-Sachs disease (TSD) is a classical glycosphingolipid (GSL) storage disease. Although the genetic and biochemical bases for a massive cerebral accumulation of ganglioside GM2 in TSD have been well established, the mechanism for the neural dysfunction in TSD remains elusive. Upon analysis of GSLs from a variant B TS brain, we have detected a novel GSL that has not been previously revealed. We have isolated this GSL in pure form. Using NMR spectroscopy, mass spectrometry, and chemical synthesis, the structure of this unusual GSL was established to be a taurine-conjugated GM2 (tauro-GM2) in which the carboxyl group of N-acetylneuraminic acid was amidated by taurine. Using a rabbit anti-tauro-GM2 serum, we also detected the presence of tauro-GM2 in three other small brain samples from one variant B and two variant O TSD patients. On the other hand, tauro-GM2 was not found in three normal human brain samples. The presence of tauro-GM2 in TS brains, but not in normal brains, indicates the possible association of this unusual GM2 derivative with the pathogenesis of TSD. Our findings point to taurine conjugation as a heretofore unelucidated mechanism for TS brain to cope with water-insoluble GM2.

Gangliosides are found at high levels in neuronal tissues where they play a variety of important functions. In the gangliosidoses, gangliosides accumulate because of defective activity of the lysosomal proteins responsible for their degradation, usually resulting in a rapidly progressive neurodegenerative disease. However, the molecular mechanism(s) leading from ganglioside accumulation to neurodegeneration is not known. We now examine the effect of ganglioside GM2 accumulation in a mouse model of Sandhoff disease (one of the GM2 gangliosidoses), the Hexb-/- mouse. Microsomes from Hexb-/- mouse brain showed a significant reduction in the rate of Ca2+-uptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which was prevented by feeding Hexb-/- mice with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glycolipid synthesis that reduces GM2 storage. Changes in SERCA activity were not due to transcriptional regulation but rather because of a decrease in Vmax. Moreover, exogenously added GM2 had a similar effect on SERCA activity. The functional significance of these findings was established by the enhanced sensitivity of neurons cultured from embryonic Hexb-/- mice to cell death induced by thapsigargin, a specific SERCA inhibitor, and by the enhanced sensitivity of Hexb-/- microsomes to calcium-induced calcium release. This study suggests a mechanistic link among GM2 accumulation, reduced SERCA activity, and neuronal cell death, which may be of significance for delineating the neuropathophysiology of Sandhoff disease.

OBJECTIVES The GM2 gangliosidoses are a group of genetic disorders caused by the accumulation of ganglioside GM2 in neuronal cells. We examined the alpha- and beta-subunits of beta-hexosaminidases by a non-radioisotopes detecting system to evaluate whether it was a useful method for understanding of the pathophysiologies of GM2 gangliosidoses. MATERIALS AND METHODS We investigated the alpha- and beta-subunits of beta-hexosaminidases in cultured fibroblasts from cases of various forms of GM2 gangliosidosis by means of Western blotting and a chemiluminescence detection system. RESULTS In a patient with infantile Tay-Sachs disease [HEXA genotype, Int5-SA(g-1-->t)/Int5-SA(g-1-->t)], the mature alpha-subunit was undetectable. In a patient with infantile Sandhoff disease (HEXB genotype, C534Y/C534Y), the mature beta-subunit was deficient. However, a small amount of the mature beta-subunit was detected in a patient with adult Sandhoff disease (HEXB genotype, R505Q(+I207V)/R505Q(+I207V)), which may have resulted in the residual enzyme activity and mild clinical course. Normal amounts of alpha- and beta-subunits were detected in a patient with GM2 activator deficiency. CONCLUSION This method is easy and sensitive for detecting target proteins, and is useful for clarification of the pathophysiologies of GM2 gangliosidoses.

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexameric enzyme that catalyzes the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. Mutations in the α/β subunit precursor gene cause the severe lysosomal storage disorder mucolipidosis II (ML II) or the more moderate mucolipidosis III alpha/beta (ML III α/β), while mutations in the γ subunit gene cause the mildest disorder, mucolipidosis III gamma (ML III γ). Here we report neurologic consequences of mouse models of ML II and ML III γ. The ML II mice have a total loss of acid hydrolase phosphorylation, which results in depletion of acid hydrolases in mesenchymal-derived cells. The ML III γ mice retain partial phosphorylation. However, in both cases, total brain extracts have normal or near normal activity of many acid hydrolases reflecting mannose 6-phosphate-independent lysosomal targeting pathways. While behavioral deficits occur in both models, the onset of these changes occurs sooner and the severity is greater in the ML II mice. The ML II mice undergo progressive neurodegeneration with neuronal loss, astrocytosis, microgliosis and Purkinje cell depletion which was evident at 4 months whereas ML III γ mice have only mild to moderate astrocytosis and microgliosis at 12 months. Both models accumulate the ganglioside GM2, but only ML II mice accumulate fucosylated glycans. We conclude that in spite of active mannose 6-phosphate-independent targeting pathways in the brain, there are cell types that require at least partial phosphorylation function to avoid lysosomal dysfunction and the associated neurodegeneration and behavioral impairments.

This work describes the in vivo expression and distribution of glioma-associated gangliosides (GD3, GM2, 3′-isoLM1) in a novel human brain tumour nude rat xenograft model. In this model, the tumours, which are established directly from human glioblastoma biopsies, show extensive infiltrative growth within the rat brain. This model therefore provides an opportunity to study ganglioside expression not only within the macroscopic tumour, but also in brain areas with tumour cell infiltration. The ganglioside expression was studied by confocal microscopy of immunostained brain sections using antiganglioside monoclonal antibodies. Xenografts from four human glioblastoma multiformes were established in rats and the brains removed after 3–4 months. Ganglioside GD3 was expressed in the tumour parenchyma while ganglioside 3′-isoLM1 was more abundantly expressed in the periphery of the tumour associated with areas of tumour cell invasion. GM2 expression was only seen in one tumour, where it was located within the main tumour mass. Double staining with a pan antihuman monoclonal antibody (3B4) and the antiganglioside monoclonal antibodies confirmed that the ganglioside expression was associated with tumour cells. This work supports the concept of different biological roles for individual gangliosides and indicates that antibodies or ligands directed against GD3 and 3′-isoLM1 might be complementary when applied in the treatment of human glioblastomas.

According to a recent hypothesis, glycosphingolipids originating from the plasma membrane are degraded in the acidic compartments of the cell as components of intraendosomal and intralysosomal vesicles and structures. Since most previous in vitro investigations used micellar ganglioside GM2 as substrate, we studied the degradation of membrane-bound ganglioside GM2 by water-soluble β-hexosaminidase A in the presence of the GM2 activator protein in a detergent-free, liposomal assay system. Our results show that anionic lipids such as the lysosomal components bis(monoacylglycero)phosphate or phosphatidylinositol stimulate the degradation of GM2 by β-hexosaminidase A up to 180-fold in the presence of GM2 activator protein. In contrast, the degradation rate of GM2 incorporated into liposomes composed of neutral lysosomal lipids such as dolichol, cholesterol, or phosphatidylcholine was significantly lower than in negatively charged liposomes. This demonstrates that both, the GM2 activator protein and anionic lysosomal phospholipids, are needed to achieve a significant degradation of membrane-bound GM2 under physiological conditions. The interaction of GM2 activator protein with immobilized membranes was studied with surface plasmon resonance spectroscopy at an acidic pH value as it occurs in the lysosomes. Increasing the concentration of bis(monoacylglycero)phosphate in immobilized liposomes led to a significant drop of the resonance signal in the presence of GM2 activator protein. This suggests that in the presence of bis(monoacylglycero)phosphate, which has been shown to occur in inner membranes of the acidic compartment, GM2 activator protein is able to solubilize lipids from the surface of immobilized membrane structures.

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts. The human skin fibroblast cell populations were metabolically pulse labeled with (14)C-galactose (48 h). The amounts of (14)C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by beta-scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns. The incorporation of (14)C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.

According to our hypothesis (Furst, W., and Sandhoff, K. (1992) Biochim. Biophys. Acta 1126, 1–16) glycosphingolipids of the plasma membrane are digested after endocytosis as components of intraendosomal and intralysosomal vesicles and membrane structures. The lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acid exohydrolases requires small, non-enzymatic cofactors, called sphingolipid activator proteins (SAPs). A total of five activator proteins have been identified as follows: namely the saposins SAP-A, -B, -C, and -D, which are derived from the single chain SAP-precursor protein (prosaposin), and the GM2 activator protein. A deficiency of prosaposin results in the storage of ceramide and sphingolipids with short oligosaccharide head groups. The loss of the GM2 activator protein blocks the degradation of the ganglioside GM2. The enzymatic hydrolysis of the ganglioside GM1 is catalyzed by β-galactosidase, a water-soluble acid exohydrolase. The lack of ganglioside GM1 accumulation in patients suffering from either prosaposin or GM2 activator protein deficiency has led to the hypothesis that SAPs are not needed for the hydrolysis of the ganglioside GM1 in vivo. In this study we demonstrate that an activator protein is required for the enzymatic degradation of membrane-bound ganglioside GM1 and that both SAP-B and the GM2 activator protein significantly enhance the degradation of the ganglioside GM1 by acid β-galactosidase in a liposomal, detergent-free assay system. These findings offer a possible explanation for the observation that no storage of the ganglioside GM1 has been observed in patients with either isolated prosaposin or isolated GM2 activator deficiency. We also demonstrate that anionic phospholipids such as bis(monoacylglycero)phosphate and phosphatidylinositol, which specifically occur in inner membranes of endosomes and in lysosomes, are essential for the activator-stimulated hydrolysis of the ganglioside GM1. Assays utilizing surface plasmon resonance spectroscopy showed that bis(monoacylglycero)phosphate increases the binding of both β-galactosidase and activator proteins to substrate-carrying membranes.

Gangliosides on tumor cells have been suggested as potential target antigens for specific immunotherapy in various types of cancer including small cell lung cancer (SCLC). In this study we have compared the expression of three gangliosides that have been described as tumor-associated antigens, FucGM1, GM2 and GD3 in SCLC tissue specimens collected at autopsy, using a double-layer immunofluorescence staining method and specific monoclonal antibodies (Mabs) directed against these ganliosides. We found expression of FucGM1, GD3 and GM2 in (70% ( n =20), 60% ( n =15) and 40% ( n =20) of SCLC tissue specimens, respectively. Lymph node metastases appeared to express less antigen than SCLC lesions at other sites (lung, brain and liver). Reactivity of Mab with >75% of tumor cells in individual lesions was seen in 55% of specimens for anti-FucGM1, 20% for anti-GD3, and in no specimen with anti-GM2. Only Mab F12 reacted with >75% of the tumor cells in all lesions from the same patient (five of eight cases). Our results indicate that FucGM1 is a relevant ganglioside antigen in SCLC, and suggest that specific immunotherapy involving more than one ganglioside antigen in SCLC should at least include FucGM1 and GD3.

GM2 ganglioside is a potential peripheral nerve antigen for neuropathy-associated autoantibodies. However little data are available on their pathogenic effects, if any. In this study we have screened both neuropathy-associated and control sera for anti-GM2 antibodies and subsequently used high titre sera for immunohistological and complement mediated cytotoxicity studies. We identified abnormally elevated anti-GM2 antisera in the normal population, as well as in patients with peripheral neuropathies and other neurological diseases. GM2 antibodies were either mono-reactive, cross-reactive with GM1a, or cross-reactive with GalNAc-GM1b and/or GalNAc-GD1a. All GM2 antisera from neuropathy subjects and normal controls bound to, and were capable of complement-mediated lysis of the NSC-34 cell line which expresses high levels of membrane-associated GM2. However, in immunohistological studies on human and rodent peripheral nervous system tissues, no specific binding was seen with GM2 antisera, either cross-reactive with GalNAc-GM1b and GalNAc-GDla, or with GM1a. These data indicate that although GM2 antisera can lyse neural membranes containing GM2, this antigen(s) is not detectable by standard immunohistological techniques in human or rodent peripheral nerve. This raises doubts about their pathophysiological significance in human autoimmune neuropathy.

Ganglioside GM2 is expressed on the surface of neuroblastoma and glioblastoma cells, and may also be detected on lung cancer cells. We reported previously that anti-ganglioside GM2 antibody exhibited strong in vitro anti-tumor activity against adriamycin-resistant cancer cells, which overexpressed ganglioside GM2. In the present study, we examined the in vivo anti-tumor effect of the chimeric anti-ganglioside GM2 antibody, KM966, against human lung and breast carcinoma cells, SBC-3 and MCF-7, and respective adriamycinresistant clones, SBC-3/ADM and AdrR MCF-7 in BALB/c nu/nu mice. Ratios of tumor volume (T/C) between KM966treated group and control group were 0.01 for SBC-3, 0.00 for SBC-3/ADM, 0.85 for MCF-7 and 0.34 for AdrR MCF-7 cells, respectively. Nude mice, which were pretreated with antiasialo GM1 antibody to remove natural killer cells, were transplanted with 4 3 10 7 of SBC-3 and SBC-3/ADM subcutaneously. Seven days later, when tumors had grown to a diameter of over 8 mm, mice began to receive intravenous treatment of 120 mg/mouse KM966 daily. Fourteen daily treatments induced regression to less than 4-mm diameter in 4/5 SBC-3 tumors and 5/5 of SBC-3/ADM tumors. All SBC-3/ ADM tumors disappeared completely, suggesting that KM966 exerts a strong in vivo anti-tumor effect on ganglioside GM2-expressing cancer cells. In KM966-treated mice, the surface of the tumor cells stained positive with anti-human IgG. In addition, numerous leukocytes had infiltrated into the tumor mass. Antibody-dependent cell-mediated cytotoxicity (ADCC) of KM966 against tumor cells was examined in vitro by 51 Cr-release assay and revealed that KM966 induces ADCC activity against ganglioside GM2-expressing tumors. Our results suggest that immunotherapy using KM966 may be useful for the treatment of ganglioside GM2-expressing solid tumors. Int. J. Cancer 82:759‐764, 1999. r 1999 Wiley-Liss, Inc.