CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

GM2 ganglioside accumulation causes neuroinflammation and behavioral alterations in a mouse model of early onset Tay-Sachs disease.

BACKGROUND: Tay-Sachs disease (TSD), a type of GM2-gangliosidosis, is a progressive neurodegenerative lysosomal storage disorder caused by mutations in the α subunit of the lysosomal ß-hexosaminidase enzyme. This disease is characterized by excessive accumulation of GM2 ganglioside, predominantly in the central nervous system. Although Tay-Sachs patients appear normal at birth, the progressive accumulation of undegraded GM2 gangliosides in neurons leads to death. Recently, an early onset Tay-Sachs disease mouse model, with genotype Hexa-/-Neu3-/-, was generated. Progressive accumulation of GM2 led to premature death of the double KO mice. Importantly, this double-deficient mouse model displays typical features of Tay-Sachs patients, such as cytoplasmic vacuolization of nerve cells, deterioration of Purkinje cells, neuronal death, deceleration in movement, ataxia, and tremors. GM2-gangliosidosis is characterized by acute neurodegeneration preceded by activated microglia expansion, macrophage, and astrocyte activation, along with the production of inflammatory mediators. However, the mechanism of disease progression in Hexa-/-Neu3-/- mice, relevant to neuroinflammation is poorly understood. METHOD: In this study, we investigated the onset and progression of neuroinflammatory changes in the cortex, cerebellum, and retina of Hexa-/-Neu3-/- mice and control littermates by using a combination of molecular genetics and immunochemical procedures. RESULTS: We found elevated levels of pro-inflammatory cytokine and chemokine transcripts, such as Ccl2, Ccl3, Ccl4, and Cxcl10 and also extensive microglial and astrocyte activation and proliferation, accompanied by peripheral blood mononuclear cell infiltration in the vicinity of neurons and oligodendrocytes. Behavioral tests demonstrated a high level of anxiety, and age-dependent loss in both spatial learning and fear memory in Hexa-/-Neu3-/- mice compared with that in the controls. CONCLUSION: Altogether, our data suggest that Hexa-/-Neu3-/- mice display a phenotype similar to Tay-Sachs patients suffering from chronic neuroinflammation triggered by GM2 accumulation. Furthermore, our work contributes to better understanding of the neuropathology in a mouse model of early onset Tay-Sachs disease.

Comparative analysis of brain pathology in heparan sulphate storing mucopolysaccharidoses.

The cause of neurodegeneration in MPS mouse models is the focus of much debate and what the underlying cause of disease pathology in MPS mice is. The timing of development of pathology and when this can be reversed or impacted is the key to developing suitable therapies in MPS. This study is the first of its kind to correlate the biochemical changes with the functional outcome as assessed using non-invasive behaviour testing across multiple mucopolysaccharidosis (MPS) mouse models. In the MPS brain, the primary lysosomal enzyme dysfunction leads to accumulation of primary glycosaminoglycans (GAGs) with gangliosides (GM2 and GM3) being the major secondary storage products. With a focus on the neuropathology, a time course experiment was conducted in MPS I, MPS IIIA, MPS VII (severe and attenuated models) in order to understand the relative timing and level of GAG and ganglioside accumulation and how this correlates to behaviour deficits. Time course analysis from 1 to 6 months of age was conducted on brain samples to assess primary GAG (uronic acid), ß-hexosaminidase enzyme activity and levels of GM2 and GM3 gangliosides. This was compared to a battery of non-invasive behaviour tests including open field, inverted grid, rotarod and water cross maze were assessed to determine effects on motor function, activity and learning ability. The results show that the GAG and ganglioside accumulation begins prior to the onset of detectable changes in learning ability and behaviour. Interestingly, the highest levels of GAG and ganglioside accumulation was observed in the MPS IIIA mouse despite having 3% residual enzyme activity. Deficits in motor function were clearly observed in the severe Gusmps/mps, which were significantly delayed in the attenuated Gustm(L175F)Sly model despite their minimal increase in detectable enzyme activity. This suggests that genotype and residual enzyme activity are not indicative of severity of disease pathology in MPS disease and there exists a window when there are considerable storage products without detectable functional deficits which may allow an alteration to occur with therapy.

Amylin and pramlintide modulate γ-secretase level and APP processing in lipid rafts.

A major characteristic of Alzheimer's disease (AD) is the accumulation of misfolded amyloid-ß (Aß) peptide. Several studies linked AD with type 2 diabetes due to similarities between Aß and human amylin. This study investigates the effect of amylin and pramlintide on Aß pathogenesis and the predisposing molecular mechanism(s) behind the observed effects in TgSwDI mouse, a cerebral amyloid angiopathy (CAA) and AD model. Our findings showed that thirty days of intraperitoneal injection with amylin or pramlintide increased Aß burden in mice brains. Mechanistic studies revealed both peptides altered the amyloidogenic pathway and increased Aß production by modulating amyloid precursor protein (APP) and γ-secretase levels in lipid rafts. In addition, both peptides increased levels of B4GALNT1 enzyme and GM1 ganglioside, and only pramlintide increased the level of GM2 ganglioside. Increased levels of GM1 and GM2 gangliosides play an important role in regulating amyloidogenic pathway proteins in lipid rafts. Increased brain Aß burden by amylin and pramlintide was associated with synaptic loss, apoptosis, and microglia activation. In conclusion, our findings showed amylin or pramlintide increase Aß levels and related pathology in TgSwDI mice brains, and suggest that increased amylin levels or the therapeutic use of pramlintide could increase the risk of AD.

Ganglioside GM2, highly expressed in the MIA PaCa-2 pancreatic ductal adenocarcinoma cell line, is correlated with growth, invasion, and advanced stage.

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-ß1 signaling in these CSC-like cells, presumably by inhibiting the interaction between GM2 and TGFß RII and suppressing invasion. Furthermore, suppression of GM2 expression by MAPK inhibition also reduced TGF-ß1 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.

Reversibility of neuropathology in Tay-Sachs-related diseases.

The GM2 gangliosidoses are progressive neurodegenerative disorders due to defects in the lysosomal ß-N-acetylhexosaminidase system. Accumulation of ß-hexosaminidases A and B substrates is presumed to cause this fatal condition. An authentic mouse model of Sandhoff disease (SD) with pathological characteristics resembling those noted in infantile GM2 gangliosidosis has been described. We have shown that expression of ß-hexosaminidase by intracranial delivery of recombinant adeno-associated viral vectors to young adult SD mice can prevent many features of the disease and extends lifespan. To investigate the nature of the neurological injury in GM2 gangliosidosis and the extent of its reversibility, we have examined the evolution of disease in the SD mouse; we have moreover explored the effects of gene transfer delivered at key times during the course of the illness. Here we report greatly increased survival only when the therapeutic genes are expressed either before the disease is apparent or during its early manifestations. However, irrespective of when treatment was administered, widespread and abundant expression of ß-hexosaminidase with consequent clearance of glycoconjugates, α-synuclein and ubiquitinated proteins, and abrogation of inflammatory responses and neuronal loss was observed. We also show that defects in myelination occur in early life and cannot be easily resolved when treatment is given to the adult brain. These results indicate that there is a limited temporal opportunity in which function and survival can be improved-but regardless of resolution of the cardinal pathological features of GM2 gangliosidosis, a point is reached when functional deterioration and death cannot be prevented.

The trigeminal retrograde transfer pathway in the treatment of neurodegeneration.

The trigeminal sensory system was evaluated for the retrograde transfer of gene therapy vectors into the CNS. The feline immunodeficiency viral vector, FIV(HEXB), encoding for the human HEXB gene, was injected intra-articularly in the temporomandibular joint of 12 week-old HexB(-/-) mice displaying clinical and histopathological signs of Sandhoff disease. This treatment regiment reduced GM(2) storage and ameliorated neuroinflammation in the brain of HexB(-/-) mice, as well as attenuated behavioral deficits. In conclusion, retrograde transfer along trigeminal sensory nerves may prove to be a valuable route of gene therapy administration for the treatment of lysosomal storage disorders and other neurodegenerative diseases.

Obesity causes a shift in metabolic flow of gangliosides in adipose tissues.

Obesity is associated with insulin resistance and a mild chronic inflammation in adipose tissues. Recent studies suggested that GM3 ganglioside mediates dysfunction in insulin signaling. However, it has not been determined the ganglioside profiling in adipose tissues of obese animals. Here, we for the first time examined semi-quantitative ganglioside profiles in the adipose tissues of high fat- and high sucrose-induced obese, diabetic C57BL/6J mice by TLC and HPLC/mass spectrometry. In control adipose tissues GM3 dominated with traces of GM1 and GD1a; obesity led to a dramatic increase in GM2, GM1, and GD1a with the GM3 content unchanged. Similar results were obtained in KK and KKAy mice. Adipocytes separated from stromal vascular cells including macrophages contained more of those gangliosides in KKAy mice than in KK mice. These results underscore those gangliosides in the pathophysiology of obesity-related diseases.

Glycosphingolipids are not pivotal receptors for Subtilase cytotoxin in vivo: sensitivity analysis with glycosylation-defective mutant mice.

Certain glycosphingolipids play important roles as cellular receptor for bacterial toxins with high specificity and strong affinity. In particular AB(5) toxins exhibit typical modes of cell attachment with B5 and invasion and biological effects in cells with A subunit. Subtilase cytotoxin (SubAB) is the prototype of a recently discovered AB(5) cytotoxin family produced by certain strains of Shiga toxigenic Escherichia coli, and shows highly specific serine protease activity toward endoplasmic reticulum chaperone Bip. Since this toxin bound to a mimic of ganglioside GM2, GM2 has been considered to be possible receptor for SubAB. Using six kinds of glycosylation-defective knockout mice lacking certain group of glycosphingolipids, sensitivity to SubAB in vivo was analyzed. Consequently, all mutant mice died at around 70h after intraperitoneal injection of 10 microg (or 7.5 microg) of SubAB as well as wild type mice. These results indicated none of glycolipids are not pivotal receptor for SubAB in the body.

N-butyldeoxygalactonojirimycin reduces brain ganglioside and GM2 content in neonatal Sandhoff disease mice.

Sandhoff disease involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the beta-subunit gene of beta-hexosaminidase A and B (Hexb gene). Accumulation of these glycosphingolipids (GSLs) produces progressive neurodegeneration, ultimately leading to death. Substrate reduction therapy (SRT) aims to decrease the rate of glycosphingolipid (GSL) biosynthesis to compensate for the impaired rate of catabolism. The imino sugar, N-butyldeoxygalactonojirimycin (NB-DGJ) inhibits the first committed step in GSL biosynthesis. NB-DGJ treatment, administered from postnatal day 2 (p-2) to p-5 (600 mg/kg/day)), significantly reduced total brain ganglioside and GM2 content in the Sandhoff disease (Hexb(-/-)) mice, but did not reduce the content of GA2. We also found that NB-DGJ treatment caused a slight, but significant elevation in brain sialidase activity. The drug had no adverse effects on viability, body weight, brain weight, or brain water content in the mice. No significant alterations in neutral lipids or acidic phospholipids were observed in the NB-DGJ-treated Hexb(-/-) mice. Our results show that NB-DGJ is effective in reducing total brain ganglioside and GM2 content at early neonatal ages.

TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

Gangliosides, Ab1 and Ab2 antibodies III. The idiotype of anti-ganglioside mAb P3 is immunogenic in a T cell-dependent manner.

P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA. We used small immune proteins (SIP) consisting on the idiotype in the scFv format, covalently linked to gamma1CH3, the self-dimerizing domain of murine IgG1. SIPs were previously shown to be appropriate to induce specific anti-idiotypic responses. By gene gun immunization, a polyspecific response was occasionally generated, particularly with the P3 idiotype. A single shot of DNA was sufficient to induce a strong and long-lasting anti-P3 idiotype response. In addition, by delivery of the same DNA construct with a recombinant adeno-associated virus the unique immunogenicity of the P3 idiotype was demonstrated. The requirement of T cells in the anti-P3 idiotype response was indicated by the lack of P3-specific anti-idiotypic antibodies following immunization of both, allogeneic C57BL/6 and athymic BALB/c mice.

Presence of an unusual GM2 derivative, taurine-conjugated GM2, in Tay-Sachs brain.

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.

Retrovirus-mediated transfer and expression of beta-hexosaminidase alpha-chain cDNA in human fibroblasts from G(M2)-gangliosidosis B1 variant.

Mutations in the alpha-chain of lysosomal hexosaminidase (EC 3.2.1.52) underlie two distinct biochemical phenotypes known as variant B and variant B1 of G(M2) gangliosidosis. This paper shows that the transduction of human B1-type fibroblasts (producing catalytically inactive alpha-chains) with a retroviral vector encoding the human hexosaminidase alpha-chain leads to a complete correction of HexA (alpha beta dimer) activity with both synthetic and natural substrates. The alpha-subunit overexpression leads to a partial HexB (beta beta dimer) depletion corresponding to about 10% of control HexB activity. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. The high levels of recombinant enzyme correctly produced the metabolic defect, enabling the cells efficiently to degrade the accumulated storage product present in lysosomes. The transduced fibroblasts are also able to secrete HexA efficiently into the culture medium. Moreover, transfer of the human transgene product to B1-type deficient fibroblasts lead to an increase of activity against 4MUGS, the alpha-chain specific synthetic substrate, up to 30% of the control mean activity level. This level of activity might be sufficient to restore the normal ganglioside G(M2) metabolism in recipient cells. The data obtained demonstrate that B1-type phenotype can be efficiently corrected by retrovirus-mediated gene transfer.

Promoter characterization and expression of the gene coding for the human GM2 activator protein.

Genomic clones of the human GM2 activator protein have been isolated and analyzed. The 5' region of the gene demonstrated promoter activity as ascertained by its ability to drive luciferase gene expression in transfected COS cells. This sequence contains GC rich region and several putative promoter elements were present, including Sp1, AP2, cAMP-responsive element, and B-cell-specific activating protein. Analysis of tissue distribution of the GM2 activator protein gene revealed tissue-specific variations in transcript levels. Placenta, bone marrow, mammary gland, bladder, lymph node, and spleen had the highest mRNA levels.

Gangliosides as modulators of dendritogenesis in normal and storage disease-affected pyramidal neurons.

Pyramidal cells initiate the formation of dendritic arbors in a prolific burst of neurite outgrowth during early cortical development. Although morphologically mature pyramidal neurons do not normally sprout additional primary dendrites, the discovery of ectopic dendritogenesis in neuronal storage diseases has revealed that these cells do retain this ability under appropriate stimulation. The capacity for renewal of dendritogenesis has been found to exhibit a species gradient with human > cat, dog, sheep > mouse. A consistent metabolic feature of ectopic dendrite-bearing pyramidal neurons is a heightened intracellular expression of GM2 ganglioside. Elevated expression of this same glycosphingolipid has also been found to correlate with normal dendritogenesis. Immature neurons in developing cat and ferret cortex exhibit high levels of GM2 ganglioside immunoreactivity coincident with normal dendritic sprouting and a similar relationship has now been shown for human cortical development. Ultrastructural studies of all three species revealed GM2 localized to vesicles in a manner consistent with Golgi synthesis and exocytic trafficking to the somatic-dendritic plasmalemma. We propose that GM2 ganglioside functions in glycosphingolipid-enriched microdomains (lipid rafts) in the plasmalemma to promote dendritic initiation through modulation of specific membrane proteins and/or their associated second messenger cascades.

Distribution of enzyme-bearing cells in GM2 gangliosidosis mice: regionally specific pattern of cellular infiltration following bone marrow transplantation.

Tissue distribution of beta-hexosaminidase was investigated using 5-bromo-4-chloro-3-indolyl N-acetyl beta-D-glucosaminide (X-Hex) as substrate in wild-type mice, four GM2 gangliosidosis model mice (Hexa-/-, Hexb-/-, Gm2a-/- and Hexa-/-Hexb-/-) and Hexb-/- mice that received bone marrow transplantation (BMT). In wild-type mice histochemical localization of beta-hexosaminidase was detected in the perikarya of the majority of neurons, small process-bearing microglial cells, perivascular macrophages, and macrophages in the choroid plexus and leptomeninges. X-Hex positivity was also noted in the renal tubular epithelium and macrophages in the liver and spleen. The staining pattern in the Gm2a-/- and Hexa-/- mice was generally similar to those of wild type, but in these mice, X-Hex stain was also noted in some storage neurons with swollen perikarya. No X-Hex-positive cells were detected in Hexb-/- or Hexa-/-Hexb-/- (DKO) mice. In Hexb-/- mice that received wild-type BMT (Hexb-/- +WBMT), many X-Hex-positive cells were detected in the spleen, and to a far lesser extent, in liver and kidney. In the CNS of these mice, X-Hex-positive cells were largely detected in the leptomeninges and choroid plexus. Some positive cells were also detected, mostly in the perivascular regions of the cerebrum, in particular in the regions of the posterior thalamus, brain stem and spinal cord. Some of X-Hex-positive cells were immunoreactive with Mac-1 and F4/80 antibodies and, thus, were cells of microglia/macrophage lineage. X-Hex-positive staining was not detected in neurons in these mice despite clinical improvement following BMT. This is the first time, as far as we know, that the regional distribution of the donor cells in the CNS has been investigated in a model of neuronal storage disease. Our study indicated that donor-derived cells of microglia/macrophage lineage infiltrated the CNS in a regionally specific manner following the BMT.

Immunological circumvention of multiple organ metastases of multidrug resistant human small cell lung cancer cells by mouse-human chimeric anti-ganglioside GM2 antibody KM966.

serum against SBC-3/DOX cells to a similar extent compared with parental SBC-3 cells. Pretreatment of human effector cells with various cytokines induced further enhancement of the KM966-dependent ADCC against SBC-3/DOX cells. Intravenous injection of SBC-3 or SBC-3/DOX cells into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice developed metastases in multiple organs (liver, kidneys and lymph nodes). Interestingly, SBC-3/DOX cells produced metastases more rapidly than SBC-3 cells, suggesting more aggressive phenotype of SBC-3/DOX cells than their parental cells in vivo. Systemic treatment with KM966, given on days 2 and 7, drastically inhibited the formation of multiple-organ metastases produced by both SBC-3 and SBC-3/DOX cells, indicating that KM966 can eradicate metastasis by SCLC cells irrespective of MDR phenotype. These findings suggest that the mouse-human chimeric KM966 targets the GM2 antigen, and might be useful for the immunological circumvention of multiple-organ metastases of refractory SCLC.

Ganglioside GM2-activator protein and vesicular transport in collecting duct intercalated cells.

This study describes the molecular characterization of an antigen defined by an autoantibody from a woman with habitual abortion as GM2-activator protein. The patient showed no disorder of renal function. Accidentally with routine serum screening for autoantibodies, an immunoreactivity was found in kidney collecting duct intercalated cells. Three distinct patterns of immunostaining of intercalated cells were observed: staining of the apical pole, basolateral pole, and diffuse cytoplasmic labeling. Ultrastructurally, the immunoreactivity was associated with "studs," which represent the cytoplasmic domain of the vacuolar proton pump in intercalated cells. This pump is subjected to a shuttling mechanism from cytoplasmic stores to the cell membrane, which exclusively occurs in intercalated cells. Peptide sequences of a 23-kD protein purified from rat kidney cortex showed complete identity with corresponding sequences of GM2-activator protein. In the brain, GM2-activator protein is required for hexosaminidase A to split a sugar from ganglioside GM2. Because neither ganglioside GM2 nor GM1 (its precursor) is present in significant amounts in the kidney, the previous finding that this tissue contains the highest level of activator protein in the body was confusing. In this study, a novel role for GM2-activator protein in intercalated cells is proposed, and possible roles in the shuttling mechanism are discussed.