GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies.

GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the ß-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay-Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood-brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.

Glycoside hydrolase family 18 and 20 enzymes are novel targets of the traditional medicine berberine.

Berberine is a traditional medicine that has multiple medicinal and agricultural applications. However, little is known about whether berberine can be a bioactive molecule toward carbohydrate-active enzymes, which play numerous vital roles in the life process. In this study, berberine and its analogs were discovered to be competitive inhibitors of glycoside hydrolase family 20 ß-N-acetyl-d-hexosaminidase (GH20 Hex) and GH18 chitinase from both humans and the insect pest Ostrinia furnacalis Berberine and its analog SYSU-1 inhibit insect GH20 Hex from O. furnacalis (OfHex1), with Ki values of 12 and 8.5 µm, respectively. Co-crystallization of berberine and its analog SYSU-1 in complex with OfHex1 revealed that the positively charged conjugate plane of berberine forms π-π stacking interactions with Trp490, which are vital to its inhibitory activity. Moreover, the 1,3-dioxole group of berberine binds an unexplored pocket formed by Trp322, Trp483, and Val484, which also contributes to its inhibitory activity. Berberine was also found to be an inhibitor of human GH20 Hex (HsHexB), human GH18 chitinase (HsCht and acidic mammalian chitinase), and insect GH18 chitinase (OfChtI). Besides GH18 and GH20 enzymes, berberine was shown to weakly inhibit human GH84 O-GlcNAcase (HsOGA) and Saccharomyces cerevisiae GH63 α-glucosidase I (ScGluI). By analyzing the published crystal structures, berberine was revealed to bind with its targets in an identical mechanism, namely via π-π stacking and electrostatic interactions with the aromatic and acidic residues in the binding pockets. This paper reports new molecular targets of berberine and may provide a berberine-based scaffold for developing multitarget drugs.

Cirsium maritimum Makino Inhibits the Antigen/Immunoglobulin-E-Mediated Allergic Response In Vitro and In Vivo.

We investigated whether Cirsium maritimum Makino can inhibit immunoglobulin-E-mediated allergic response in rat basophilic leukemia (RBL-2H3) cells and passive cutaneous anaphylaxis (PCA) in BALB/c mice. In vitro, the ethyl acetate extract of C. maritimum Makino (ECMM) significantly inhibited ß-hexosaminidase release and decreased intracellular Ca2+ levels in RBL-2H3 cells. Moreover, ECMM leaves more strongly suppressed the release of ß-hexosaminidase than ECMM flowers. ECMM leaves also significantly suppressed the PCA reaction in the murine model. High-performance liquid chromatography and 1H and 13C nuclear magnetic resonance indicated that cirsimaritin, a flavonoid, was concentrated in active fractions of the extract. Our findings suggest that ECMM leaves have a potential regulatory effect on allergic reactions that may be mediated by mast cells. Furthermore, cirsimaritin may be the active anti-allergic component in C. maritimum Makino.

The anti-allergic and anti-inflammatory effects of Benjakul extract (a Thai traditional medicine), its constituent plants and its some pure constituents using in vitro experiments.

Benjakul (BJK), a Thai traditional medicine preparation, has long been used for balanced health, controlled abnormal of element in the body, carminative, and relief of flatulence. It is composed of five plants: Piper interruptum Opiz., Piper longum L., Piper sarmentosum Roxb., Plumbago indica L., and Zingiber officinale Roscoe. The ethanolic extracts of BJK, its five individual plants, and pure constituents of BJK were investigated for their anti-allergic activity using immunoglobulin E (IgE)-sensitized ß-hexosaminidase in the rat basophilic leukemia-2H3 (RBL-2H3) cells and anti-inflammatory activity using lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) in the murine macrophage (RAW 264.7) cells. The ethanolic extracts of BJK showed anti-allergic activity (IC50=12.69µg/ml) and exhibited potent NO inhibitory effect (IC50=16.60µg/ml), but inactive on TNF-α release. Moreover, 6-shogaol and plumbagin, two pure compounds from BJK, showed higher anti-allergic activity than the ethanolic BJK extract with IC50 values of 0.28 and 4.03µg/ml, respectively. These compounds were significantly higher than chlorpheniramine (CPM), standard drug, with IC50 value of 17.98µg/ml. Determination of the anti-inflammatory activity by measuring the inhibition of NO production presented that plumbagin and 6-shogaol exhibited higher than crude BJK extract with IC50 values of 0.002 and 0.92µg/ml, respectively. In particular, plumbagin also showed higher anti-inflammatory than prednisolone, positive control, with IC50 value of 0.59µg/ml. 6-Shogaol also showed inhibitory effect on TNF-α release (IC50=9.16µg/ml). These preliminary results may provide some scientific support for the use of BJK for the anti-allergic treatment and inflammatory disorders through the inhibition of NO production.

Direct Intracranial Injection of AAVrh8 Encoding Monkey ß-N-Acetylhexosaminidase Causes Neurotoxicity in the Primate Brain.

GM2 gangliosidoses, including Tay-Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in ß-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or ß-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and ß-subunits. Three doses (3.2 × 1012 vg [n = 3]; 3.2 × 1011 vg [n = 2]; or 1.1 × 1011 vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/ß developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/ß, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/ß intracranial injection among different species, despite encoding for self-proteins.

Removal of O-GlcNAcylation is important for pig preimplantation development.

Glucose has been recognized as an energy source for a long time, but it has recently been suggested that the hexosamine biosynthesis pathway (HBP) and downstream protein O-GlcNAcylation have important functions in mouse preimplantation development. Thus, whether or not O-GlcNAcylation was present and what functions O-GlcNAcylation has in pig preimplantation development were investigated in the present study. The expressions of mRNA of glutaminefructose-6-phosphate aminotransferase (Gfpt), O-GlcNAc transferase (Ogt) and O-GlcNAcase (Oga), which are involved in the HBP and O-GlcNAc cycling, were examined in pig parthenogenetic diploids at each preimplantation developmental stage. Gfpt and Ogt were detected in diploids at all stages. Though Oga was detected at all stages except the 4-cell stage, OGA proteins were detected in diploids from the 2-cell to blastocyst stage. Furthermore, O-GlcNAcylated proteins in MII oocytes and diploids were also detected by immunofluorescence at every stage. Inhibition of OGT by 4.0 mM BADGP did not affect development up to the blastocyst stage, while inhibition of OGA by 300 µM PUGNAc decreased the proportion of diploids beyond the 4-cell stage. Four-cell diploids cultured with PUGNAc until 48 h developed to the blastocyst stage after culture in a PUGNAc-free medium until 144 h after electrostimulation. RNA polymerase II (Pol II) phosphorylation, which indicates the onset of mRNA transcription, was detected in nuclei of diploids in the control group at 48 h but not in the PUGNAc-treated group. These results indicate that HBP and O-GlcNAcylation have important functions in pig preimplantation development and that inhibition of OGA is fatal for development. It is also suggested that OGA inhibition disrupts normal Pol II regulation and may cause a zygotic gene activation error.

Cinnamaldehyde is the main mediator of cinnamon extract in mast cell inhibition.

PURPOSE: In terms of their involvement in allergic and inflammatory conditions, mast cells (MC) can be promising targets for medical agents in therapy. Because of their good compliance and effectiveness, phytochemicals are of great interest as new therapeutic tools in form of nutraceuticals. We found recently that cinnamon extract (CE) inhibits mast cell activation. Here, we analysed the effects of a major compound of CE, cinnamaldehyde (CA), on mast cell activation. METHODS: Release of prestored and de novo synthesised mediators as well as expression of pro-inflammatory cytokines and mast cell-specific proteases were analysed in RBL-2H3 cells or in human mast cells isolated from intestinal tissue (hiMC) treated with CA prior to stimulation by FcεRI crosslinking or IONO/PMA. The results were compared with the corresponding effects of CE. RESULTS: Following treatment with CA, release of ß-hexosaminidase in IgE-dependent or IgE-independent activated RBL-2H3 cells was down-regulated in a dose-dependent manner to about 10%. In hiMC, release of ß-hexosaminidase was also significantly reduced, and release of LTC4 and CXCL8 was almost completely inhibited by CA. Moreover, IgE-mediated expression of CXCL8, CCL2, CCL3 and CCL4 in hiMC was significantly down-regulated by CA. With the exception of the expression of the mast cell proteases tryptase and chymase, the inhibitory effects of CA were very similar to the effects shown for CE treatment. The reducing effect of CA on mast cell mediators-seen for long- and for short-term incubations-could be related to particular signalling pathways as CA caused a down-regulation in ERK as well as PLCγ1 phosphorylation. CONCLUSIONS: CA decreases release and expression of pro-inflammatory mast cell mediators. This inhibitory action is similar to the effects observed for CE indicating CA as the main active compound in CE leading to its anti-allergic properties.

Anti-inflammatory constituents from Tabebuia avellanedae.

Five novel compounds were isolated from the water extract of Tabebuia avellanedae, and their structures were established by analysis of NMR spectroscopy and mass spectrometry. Compounds 1-5 at 25µM showed strong inhibitory activity on the inflammatory cytokine, tumor-necrosis factor-α and interleukin-1ß production in cultured human myeloma THP-1 cells co-stimulated with lipopolysaccharide without any significant cytotoxicity, and their anti-allergic and antioxidant activities were evaluated.

Inflammatory and degranulation effect of yellow sand on RBL-2H3 cells in relation to chemical and biological constituents.

Recent studie pointed out that allergic diseases have increased during the Asian dust storm event (ADSE) in Japan. Daily observations and the atmospheric concentrations of yellow sand (YS) aerosol have been increasing. In this study, YS samples collected from three sites of Japan during ADSE in 2009-2010 were used. The particles were analyzed by X-ray photoelectron spectroscopy (XPS) and X-ray fluorescence-energy dispersive spectrometer (XRF-EDS). We investigate ability of YS extract on enhancing the chemical mediator release and cytokine production from rat basophilic leukemia (RBL-2H3) cells. The dust particles at Fukuoka and Tsukuba were abundant in aluminum (Al), iron (Fe), potassium (K) and titan (Ti) than those at Naha. Concentration of the trace endotoxin and Cryptomeria japonica pollen allergen (Cry j 1) were measured in YS extract. After exposure of RBL-2H3 cells to YS extract, the ß-hexosaminidase (ß-hex) release, tumor necrosis factor-alpha (TNF-α) production were enhanced in RBL-2H3 cells. This process depends on endotoxin, Cry j 1 and other allergen present in the YS extract. YS water extract also show a strong cytotoxic effect on the cells. This data suggest that low levels of endotoxin and Cry j 1 in YS may cause allergy during the ADSE.

Therapeutic effects of stem cells and substrate reduction in juvenile Sandhoff mice.

Sandhoff Disease (SD) involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the ß-subunit gene of ß-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 ß-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. ß-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.

Restricted ketogenic diet enhances the therapeutic action of N-butyldeoxynojirimycin towards brain GM2 accumulation in adult Sandhoff disease mice.

Sandhoff disease is an autosomal recessive, neurodegenerative disease involving the storage of brain ganglioside GM2 and asialo-GM2. Previous studies showed that caloric restriction, which augments longevity, and N-butyldeoxynojirimycin (NB-DNJ, Miglustat), an imino sugar that hinders the glucosyltransferase catalyzing the first step in glycosphingolipid biosynthesis, both increase longevity and improve motor behavior in the beta-hexosaminidase (Hexb) knockout (-/-) murine model of Sandhoff disease. In this study, we used a restricted ketogenic diet (KD-R) and NB-DNJ to combat ganglioside accumulation. Adult Hexb-/- mice were placed into one of the following groups: (i) a standard diet (SD), (ii) a SD with NB-DNJ (SD + NB-DNJ), (iii) a KD-R, and (iv) a KD-R with NB-DNJ (KD-R + NB-DNJ). Forebrain GM2 content (mug sialic acid/100 mg dry wt) in the four groups was 375 +/- 15, 312 +/- 8, 340 +/- 28, and 279 +/- 26, respectively, indicating an additive interaction between NB-DNJ and the KD-R. Most interestingly, brain NB-DNJ content was 3.5-fold greater in the KD-R + NB-DNJ mice than in the SD + NB-DNJ mice. These data suggest that the KD-R and NB-DNJ may be a potential combinatorial therapy for Sandhoff disease by enhancing NB-DNJ delivery to the brain and may allow lower dosing to achieve the same degree of efficacy as high dose monotherapy.

Ym1/2 promotes Th2 cytokine expression by inhibiting 12/15(S)-lipoxygenase: identification of a novel pathway for regulating allergic inflammation.

The Ym1/2 lectin is expressed abundantly in the allergic mouse lung in an IL-13-dependent manner. However, the role of Ym1/2 in the development of allergic airways disease is largely unknown. In this investigation, we show that treatment of mice with anti-Ym1/2 Ab during induction of allergic airways disease attenuated mediastinal lymph node production of IL-5 and IL-13. Ym1/2 was found to be expressed by dendritic cells (DCs) in an IL-13-dependent manner and supplementation of DC/CD4(+) T cell cocultures with Ym1/2 enhanced the ability of IL-13(-/-) DCs to stimulate the secretion of IL-5 and IL-13. Affinity chromatography identified 12/15(S)-lipoxygenase (12/15-LOX) as a Ym1/2-interacting protein and functional studies suggested that Ym1/2 promoted the ability of DCs to stimulate cytokine production by inhibiting 12/15-LOX-mediated catalysis of 12-hydroxyeicosatetraenoic acid (12(S)-HETE). Treatment of DC/CD4(+) T cell cultures with the 12/15-LOX inhibitor baicalein enhanced, whereas 12(S)-HETE inhibited the production of Th2 cytokines. Notably, delivery of 12(S)-HETE to the airways of mice significantly attenuated the development of allergic airways inflammation and the production of IL-5 and IL-13. In summary, our results suggest that production of Ym1/2 in response to IL-13 promotes Th2 cytokine production and allergic airways inflammation by inhibiting the production of 12(S)-HETE by 12/15-LOX.

Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease.

Sandhoff disease is a severe neurodegenerative glycosphingolipid (GSL) lysosomal storage disorder, currently without treatment options. One therapeutic approach under investigation is substrate reduction therapy (SRT). By partially inhibiting GSL biosynthesis, the impaired rate of GSL catabolism is balanced by a slower rate of influx of GSLs into the lysosome. In a previous study, we reported the beneficial effects of treating Sandhoff disease mice with the glucose analogue N-butyldeoxynojirimycin (NB-DNJ), a compound that inhibits the first step of GSL biosynthesis catalysed by the ceramide specific glucosyltransferase. NB-DNJ, however, exhibits adverse effects at high doses such as weight loss and GI tract distress (due to glucosidase inhibition). This might limit the therapeutic potential of NB-DNJ for treating diseases affecting the CNS where high dose therapy may be required to achieve therapeutic levels of the drug in the brain. In the present study, a more selective compound, the galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ), was evaluated in the Sandhoff disease mouse model. Treatment with NB-DGJ showed greater therapeutic efficacy than NB-DNJ with no detectable side effects. The ability to escalate the dose of NB-DGJ, leading to extended life expectancy and increased delay in symptom onset, demonstrates the greater therapeutic potential of NB-DGJ for the treatment of the human gangliosidoses.

Origin and spread of the 1278insTATC mutation causing Tay-Sachs disease in Ashkenazi Jews: genetic drift as a robust and parsimonious hypothesis.

The 1278insTATC is the most prevalent beta-hexosaminidase A ( HEXA) gene mutation causing Tay-Sachs disease (TSD), one of the four lysosomal storage diseases (LSDs) occurring at elevated frequencies among Ashkenazi Jews (AJs). To investigate the genetic history of this mutation in the AJ population, a conserved haplotype (D15S981:175-D15S131:240-D15S1050:284-D15S197:144-D15S188:418) was identified in 1278insTATC chromosomes from 55 unrelated AJ individuals (15 homozygotes and 40 heterozygotes for the TSD mutation), suggesting the occurrence of a common founder. When two methods were used for analysis of linkage disequilibrium (LD) between flanking polymorphic markers and the disease locus and for the study of the decay of LD over time, the estimated age of the insertion was found to be 40+/-12 generations (95% confidence interval: 30-50 generations), so that the most recent common ancestor of the mutation-bearing chromosomes would date to the 8th-9th century. This corresponds with the demographic expansion of AJs in central Europe, following the founding of the Ashkenaz settlement in the early Middle Ages. The results are consistent with the geographic distribution of the main TSD mutation, 1278insTATC being more common in central Europe, and with the coalescent times of mutations causing two other LSDs, Gaucher disease and mucolipidosis type IV. Evidence for the absence of a determinant positive selection (heterozygote advantage) over the mutation is provided by a comparison between the estimated age of 1278insTATC and the probability of the current AJ frequency of the mutant allele as a function of its age, calculated by use of a branching-process model. Therefore, the founder effect in a rapidly expanding population arising from a bottleneck provides a robust parsimonious hypothesis explaining the spread of 1278insTATC-linked TSD in AJ individuals.

Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin.

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the beta-subunit of beta-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.

Biology and potential strategies for the treatment of GM2 gangliosidoses.

The GM2 gangliosidoses are a group of heritable neurodegenerative disorders caused by excessive accumulation of the ganglioside GM2 owing to deficiency in beta-hexosaminidase activity. Tay-Sachs and Sandhoff diseases have similar clinical phenotypes resulting from a deficiency in human hexosaminidase alpha and beta subunits, respectively. The lack of treatment for GM2 gangliosidoses stimulated interest in developing animal models to understand the molecular mechanisms underlying the various forms of this disease and to test new potential therapies. In this review, we discuss the molecular biology of GM2 gangliosidoses and the different strategies that have been tested in animal models for the treatment of this genetic disorder, including gene transfer and cell engraftment of neural stem cells engineered to express the hexosaminidase isoenzymes.

Identification of an active acidic residue in the catalytic site of beta-hexosaminidase.

Human beta-hexosaminidases A and B (EC 3.2.1.52) are dimeric lysosomal glycosidases composed of evolutionarily related alpha and/or beta subunits. Both isozymes hydrolyze terminal beta-linked GalNAc or GlcNAc residues from numerous artificial and natural substrates; however, in vivo GM2 ganglioside is a substrate for only the heterodimeric A isozyme. Thus, mutations in either gene encoding its alpha or beta subunits can result in GM2 ganglioside storage and Tay-Sachs or Sandhoff disease, respectively. All glycosyl hydrolases ae believed to have one or more acidic residues in their catalytic site. We demonstrate that incubation of hexosaminidase with a chemical modifier specific for carboxyl side chains produces a time-dependent loss of activity, and that this effect can be blocked by the inclusion of a strong competitive inhibitor in the reaction mix. We hypothesized that the catalytic acid residue(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human alpha and beta subunits, as well as hexosaminidases from other species, including bacteria. Such a region is encoded by exons 5-6 of the HEXA and HEXB genes. This region includes beta Arg211 (invariant in 15 sequences), which we have previously shown to be an active residue. This region also contains two invariant and one conserved acidic residues. A fourth acidic residue, Asp alpha 258, beta 290, in exon 7 was also investigated because of its association with the B1 variant of Tay-Sachs disease. Conservative substitutions were made at each candidate residue by in vitro mutagenesis of a beta cDNA, followed by cellular expression. Of these, only the beta Asp196Asn substitution decreased the kcat (350-910-fold) without any noticeable effect on the K(m). Mutagenesis of either beta Asp240 or beta Asp290 to Asn decreased kcat by 10- or 1.4-fold but also raised the K(m) of the enzyme 11- of 3-fold, respectively. The above results strongly suggest that beta Asp196 is a catalytic acid residue in beta-hexosaminidase.