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PubMed2004

Congenital ocular motor apraxia associated with idiopathic generalized epilepsy in monozygotic twins.

Identical female twins (age 11 years) with congenital ocular motor apraxia and generalized idiopathic epilepsy are reported. Their presenting symptoms were a long history of abnormal head and eye movements. One twin developed partial sensory seizures. The patients underwent 16-channel EEG, electro-oculographic recordings, MRI of the brain, and genetic and metabolic investigations. EEG findings were consistent with idiopathic generalized epilepsy. Electrooculographic recordings of the saccades confirmed an inability to elicit horizontal saccades without preceding head movement; saccades to the left were better than saccades to the right. MR scans for one twin showed normal findings, however, for the twin who had meningitis they revealed asymmetry between the right and left temporal lobes but no specific abnormality. DNA analysis using a series of autosomal polymorphic markers confirmed the monozygocity of the twins. White blood cell enzyme analysis excluded Sandhoff disease, Tay-Sachs disease, GM1 gangliosidosis, metacromatic leucodystrophy, Gaucher disease, Niemann-Pick disease (A and B), and Krabbe leucodystrophy. Albumin and immunoglobulin (IgA, IgG, and IgM) levels were normal. It is concluded that autosomal recessive inheritance seems the most likely explanation here, as recent studies have found insertion and missense mutations of the aprataxin gene which have been related to an early onset form of ataxia with ocular motor apraxia and hypoalbuminaemia.

Apraxias↗

PubMed2000

Substrate deprivation: a new therapeutic approach for the glycosphingolipid lysosomal storage diseases.

The glycosphingolipid (GSL) lysosomal storage diseases are a family of human metabolic diseases that, in their severest forms, cause death in early infancy, as a result of progressive neurodegeneration. They are caused by mutations in the genes encoding the glycohydrolases or the activator proteins that catabolise GSLs within lysosomes. In these diseases the GSL substrate of the defective enzyme accumulates in the lysosome, where it is stored and leads to cellular dysfunction and disease. The therapeutic options for treating these diseases are relatively limited; in fact, there are currently no available therapies for most of these disorders. The problem is further compounded by difficulties in delivering therapeutic agents to the central nervous system, which is where the pathology is frequently manifested. To date, research effort has mainly focused on strategies for augmenting enzyme concentrations to compensate for the underlying defect. These strategies include bone-marrow transplantation, enzyme-replacement therapy and gene therapy. Our group has been exploring the alternative strategy of substrate deprivation. This approach aims to balance the rate of GSL synthesis with the impaired rate of GSL breakdown. Studies using an asymptomatic mouse model of Tay-Sachs disease have shown that substrate deprivation prevents GSL storage. In a severe neurodegenerative mouse model of Sandhoff disease, substrate deprivation delayed the onset of symptoms and disease progression, and significantly increased life expectancy. The implications of this research for human therapy have been discussed.

Journal Article↗

PubMed2001

Identification of the 6-sulfate binding site unique to alpha-subunit-containing isozymes of human beta-hexosaminidase.

In humans, beta-hexosaminidase A (alphabeta) is required to hydrolyze GM2 ganglioside. A deficiency of either the alpha- or beta-subunit leads to a severe neurological disease, Tay-Sachs or Sandhoff disease, respectively. In mammals beta-hexosaminidase B (betabeta) and S (alphaalpha) are other major and minor isozymes. The primary structures of the alpha- and beta-subunits are 60% identical, but only the alpha-containing isozymes can efficiently hydrolyze beta-linked GlcNAc-6-SO(4) from natural or artificial substrates. Hexosaminidase has been grouped with glycosidases in family 20. A molecular model of the active site of the human hexosaminidase has been generated from the crystal structure of a family 20 bacterial chitobiase. We now use the chitobiase structure to identify residues close to the carbon-6 oxygen of NAG-A, the nonreducing beta-GlcNAc residue of its bound substrate. The chitobiase side chains in the best interactive positions align with alpha-Asn(423)Arg(424) and beta-Asp(453)Leu(454). The change in charge from positive in alpha to negative in beta is consistent with the lower K(m) of hexosaminidase S, and the much higher K(m) and lower pH optimum of hexosaminidase B, toward sulfated versus unsulfated substrates. In vitro mutagenesis, CHO cell expression, and kinetic analyses of an alphaArg(424)Lys hexosaminidase S detected little change in V(max) but a 2-fold increase in K(m) for the sulfated substrate. Its K(m) for the nonsulfated substrate was unaffected. When alphaAsn(423) was converted to Asp, again only the K(m) for the sulfated substrate was changed, increasing by 6-fold. Neutralization of the charge on alphaArg(424) by substituting Gln produced a hexosaminidase S with a K(m) decrease of 3-fold and a V(max) increased by 6-fold for the unsulfated substrate, parameters nearly identical to those of hexosaminidase B at pH 4.2. As well, for the sulfated substrate at pH 4.2 its K(m) was increased 9-fold and its V(max) decreased 1.5-fold, values very similar to those of hexosaminidase B obtained at pH 3.0, where its betaAsp(453) becomes protonated.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed2000

Role of beta Arg211 in the active site of human beta-hexosaminidase B.

Tay-Sachs or Sandhoff disease results from a deficiency of either the alpha- or the beta-subunits of beta-hexosaminidase A, respectively. These evolutionarily related subunits have been grouped with the "Family 20" glycosidases. Molecular modeling of human hexosaminidase has been carried out on the basis of the three-dimensional structure of a bacterial member of Family 20, Serratia marcescens chitobiase. The primary sequence identity between the two enzymes is only 26% and restricted to their active site regions; therefore, the validity of this model must be determined experimentally. Because human hexosaminidase cannot be functionally expressed in bacteria, characterization of mutagenized hexosaminidase must be carried out using eukaryotic cell expression systems that all produce endogenous hexosaminidase activity. Even small amounts of endogenous enzyme can interfere with accurate K(m) or V(max) determinations. We report the expression, purification, and characterization of a C-terminal His(6)-tag precursor form of hexosaminidase B that is 99.99% free of endogenous enzyme from the host cells. Control experiments are reported confirming that the kinetic parameters of the His(6)-tag precursor are the same as the untagged precursor, which in turn are identical to the mature isoenzyme. Using highly purified wild-type and Arg(211)Lys-substituted hexosaminidase B, we reexamine the role of Arg(211) in the active site. As we previously reported, this very conservative substitution nevertheless reduces k(cat) by 500-fold. However, the removal of all endogenous activity has now allowed us to detect a 10-fold increase in K(m) that was not apparent in our previous study. That this increase in K(m) reflects a decrease in the strength of substrate binding was confirmed by the inability of the mutant isozyme to efficiently bind an immobilized substrate analogue, i.e., a hexosaminidase affinity column. Thus, Arg(211) is involved in substrate binding, as predicted by the chitobiase model, as well as catalysis.

Animals↗

PubMed2006

Altered gene expression in cells from patients with lysosomal storage disorders suggests impairment of the ubiquitin pathway.

By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.

Animals↗

PubMed2001

Degradation of G(M1) and G(M2) by mammalian sialidases.

In mammalian tissues, the pathway known for the catabolism of G(M1) [Galbeta3GalNAcbeta4(Neu5Acalpha3)Galbeta4GlcCer; where Cer is ceramide] is the conversion of this ganglioside into G(M2) [GalNAcbeta4(Neu5Acalpha3)Galbeta4GlcbetaCer] by beta-galactosidase followed by the conversion of G(M2) into G(M3) (Neu5Acalpha3Galbeta4GlcbetaCer) by beta-N-acetylhexosaminidase A (Hex A). However, the question of whether or not G(M1) and G(M2) can also be respectively converted into asialo-G(M1) (Galbeta3GalNAcbeta4Galbeta4GlcCer; G(A1)) and asialo-G(M2) (GalNAcbeta4Galbeta4GlcbetaCer, G(A2)) by mammalian sialidases has not been resolved. This is due to the fact that sialidases purified from mammalian tissues always contained detergents that interfered with the in vitro hydrolysis of G(M1) and G(M2) in the presence of an activator protein. The mouse model of human type B Tay-Sachs disease created by the disruption of the Hexa gene showed no neurological abnormalities, with milder clinical symptoms than the human counterpart, and the accumulation of G(M2) in the brains of affected mice was only limited to certain regions [Sango, Yamanaka, Hoffmann, Okuda, Grinberg, Westphal, McDonald, Crawley, Sandhoff, Suzuki and Proia (1995) Nat. Genet. 11, 170-176]. These results suggest the possible presence of an alternative catabolic pathway (the G(A2) pathway) in mouse to convert G(M2) into G(A2) by sialidase. To show the existence of this pathway, we have used recombinant mammalian cytosolic sialidase and membrane-associated sialidase to study the desialylation of G(M1) and G(M2). We found that the mouse membrane-bound sialidase was able to convert G(M1) and G(M2) into their respective asialo-derivatives in the presence of human or mouse G(M2) activator protein. The cytosolic sialidase did not exhibit this activity. Our results suggest that, in vivo, the stable NeuAc of G(M1) and G(M2) may be removed by the mammalian membrane-associated sialidase in the presence of G(M2) activator protein. They also support the presence of the G(A2) pathway for the catabolism of G(M2) in mouse.

Animals↗

PubMed1974

A low-molecular-weight protein cross-reacting with human liver N-acetyl-beta-D-glucosaminidase.

Antisera were raised to preparations of hexosaminidase isoenzymes A and B purified from human liver. Protein that cross-reacted with the liver hexosaminidase was detected by an antibody-consumption method. A cross-reacting protein with a low molecular weight (20000) was partially characterized and purified from control human liver. This protein is also present in the liver of patients with Tay-Sachs disease or with Sandhoff's disease. Hexosaminidases A and B gave an immunological reaction of partial identity with the low-molecular-weight protein. The possible identity of the low-molecular-weight cross-reacting protein as a subunit of hexosaminidase is discussed.

Acetamides↗

PubMed1976

Studies on human N-acetyl-Beta-d-hexosaminidase C separated from neonatal brain.

Human brain hexosaminidase C was separated from isoenzymes A and B by Sephadex G-200 gel filtration. Properties of the enzyme were studied, particularly its isoelectric-focusing profile, pI4.80. These findings indicate that hexosaminidase C is identical with the major residual component of Sandhoff fibroblasts with respect to substrate specificity, pI and activity pH optimum.

Brain↗

PubMed1977

Purification and properties of human kidney-cortex hexosaminidases A and B.

Hexosaminidases (EC 3.2.1.30) A and B from human kidney cortex were purified to homogeneity by using concanavalin A affinity chromatography, ion-exchange chromatography and gel filtration. The yield of homogeneous isoenzymes improved approx. 20-fold, giving preparations of hexosaminidases A and B with specific activities of about 200 and 325 units/mg of protein respectively. The kinetic and structural properties of kidney hexosaminidase isoenzymes were studied and compared with the hexosaminidase isoenzymes from human placenta. The amino acid composition of hexosaminidase A was significantly different from that of hexosaminidase B. In the event of success in developing enzyme-replacement therapy for Tay-Sachs and Sandhoff's diseases, this modified procedure can furnish larger amounts of homogeneous isoenzymes.

Amino Acids↗

PubMed1981

Degradation of keratan sulphate by beta-N-acetylhexosaminidases A and B.

Enzymic cleavage of beta-N-acetylglucosamine residues of keratan sulphate was studied in vitro by using substrate a [3H]glucosamine-labelled desulphated keratan sulphate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal beta-N-acetylhexosaminidases A and B are proposed to participate in the degradation of keratan sulphate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay--Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of beta-N-acetylhexosaminidase from human liver the peaks of keratan sulphate-degrading activity coincided with the activity towards p-nitrophenyl beta-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulphate-degrading activity. Both isoenzymes had the same apparent Km of 4mM, but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH--activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the p-nitrophenyl derivative.

Cells, Cultured↗

PubMed1990

An enzyme with properties similar to those of beta-N-acetylhexosaminidase S is expressed in the promyelocytic cell line HL-60.

Extracts of the human promyelocytic cell line HL-60 contain a form of beta-N-acetylhexosaminidase that is not retained on columns of benzeneboronate-agarose ('phenylboronate-agarose') and has a pI value lower than that of beta-N-acetylhexosaminidase A. It is clearly distinct from beta-N-acetylhexosaminidase A in its behaviour on DEAE-cellulose columns, and it requires a higher concentration of salt for its elution. This 'extra' form has a higher ratio of activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate and 4-methylumbelliferyl beta-N-acetylglucosaminide than has beta-N-acetylhexosaminidase A and is less stable when heated at 50 degrees C. It has a pH optimum of 4.5 and is therefore not beta-N-acetylglucosaminidase C. Anti-(human beta-N-acetylhexosaminidase alpha-subunit) serum precipitated both beta-N-acetylhexosaminidase A and the 'extra' form, whereas an anti-(beta-subunit) serum precipitated beta-N-acetylhexosaminidase A but not the 'extra' form. Western blotting and immunodetection of polypeptides derived from the 'extra' form revealed a band corresponding in size to mature alpha-subunits. On the basis of this and of its behaviour on isoelectric focusing, chromatofocusing and its kinetic properties, we conclude that the 'extra' form is composed of alpha-subunits and resembles beta-N-acetylhexosaminidase S, the residual form in Sandhoff's disease.

Blotting, Western↗

PubMed1992

Additional biochemical findings in a patient and fetal sibling with a genetic defect in the sphingolipid activator protein (SAP) precursor, prosaposin. Evidence for a deficiency in SAP-1 and for a normal lysosomal neuraminidase.

It has been shown that sphingolipid activator proteins (SAPs) 1 and 2 are encoded on the same gene along with two other putative activator proteins [Fürst, Machleidt & Sandhoff (1988) Biol. Chem. Hoppe-Seyler 369, 317-328 and O'Brien, Kretz, Dewji, Wenger, Esch & Fluharty (1988) Science 241, 1098-1101]. We have undertaken further biochemical investigations on a patient and fetal sibling, who were previously shown to have a unique sphingolipid storage disorder associated with an SAP-2 deficiency [Harzer, Paton, Poulos, Kustermann-Kuhn, Roggendorf, Grisar & Popp (1989) Eur. J. Pediatr. 149, 31-39]. The severity of their disorder suggested that other products of the SAP precursor or prosaposin gene may also be deficient. The turnover of cerebroside sulphate and globotriaosylceramide were investigated and were both impaired in fibroblasts from the patient and fetus. However, the activities of cerebroside sulphate sulphatase and globotriaosylceramide alpha-galactosidase in vitro were normal in cells from the fetus and patient respectively. In addition, there was an increase in cerebroside sulphate concentration in the kidney of the affected fetus. These results indicate that, in addition to the SAP-2 deficiency, there was a defect in SAP-1 function in this disorder. Additional increases in the concentration of monohexosyl- and dihexosyl-ceramide in the fetal kidney probably reflect the deficiency of SAP-2 in the case of monohexosylceramides, and the combined activator deficiency in the case of dihexosylceramides. Lactosylceramide-loading studies confirmed that there was a defect in the turnover of this lipid in fibroblasts from the affected patient and fetus but not from a patient with an isolated SAP-1 deficiency, or from patients with Krabbe disease, GM1 gangliosidosis or galactosialidosis. It has been suggested [Potier, Lamontagne, Michaud & Tranchemontagne (1990) Biochem. Biophys. Res. Commun. 173, 449-456] that the prosaposin gene also codes for lysosomal neuroaminidase. However, we found normal neuraminidase activity in fibroblasts from our patient, using assay conditions which are diagnostic for sialidosis patients. The role of prosaposin gene products in sphingolipid metabolism is discussed in view of our biochemical findings in this genetic disorder.

Cells, Cultured↗

PubMed2000

Metabolic cardiomyopathies.

The energy needed by cardiac muscle to maintain proper function is supplied by adenosine Ariphosphate primarily (ATP) production through breakdown of fatty acids. Metabolic cardiomyopathies can be caused by disturbances in metabolism, for example diabetes mellitus, hypertrophy and heart failure or alcoholic cardiomyopathy. Deficiency in enzymes of the mitochondrial beta-oxidation show a varying degree of cardiac manifestation. Aberrations of mitochondrial DNA lead to a wide variety of cardiac disorders, without any obvious correlation between genotype and phenotype. A completely different pathogenetic model comprises cardiac manifestation of systemic metabolic diseases caused by deficiencies of various enzymes in a variety of metabolic pathways. Examples of these disorders are glycogen storage diseases (e.g. glycogenosis type II and III), lysosomal storage diseases (e.g. Niemann-Pick disease, Gaucher disease, I-cell disease, various types of mucopolysaccharidoses, GM1 gangliosidosis, galactosialidosis, carbohydrate-deficient glycoprotein syndromes and Sandhoff's disease). There are some systemic diseases which can also affect the heart, for example triosephosphate isomerase deficiency, hereditary haemochromatosis, CD 36 defect or propionic acidaemia.

Adult↗

PubMed1975

Absence of hexosaminidase A and B in a normal adult.

In the course of screening for heterozygotes for beta-hexosaminidase deficiency, the serum and white cells of a clinically normal father of deficient children were found to have an apparent deficiency for both hexosaminidases A and B, assayed with an artificial substrate, 4-methylumbelliferyl-beta-glucosaminide. No inhibitor was present. Assayed with a natural substrate, n-acetylgalactosaminyl beta 1-4 galactosyl beta 1-4 glucosyl ceramide, which had been isolated from the brain of a patient with Tay--Sachs disease and labeled in the terminal n-acetyl-galactosamine, a value in the heterozygote range was found. It was concluded that the proband is probably a double heterozygote for two mutations; one is the classic Sandhoff type (lack of hexosaminidases A and B), giving rise to deficient offspring when combined with the same mutation borne by the wife. The other obscures any activity with the artificial substrate but allows an action on natural substrates, explaining the normal life of its carrier.

Aminoglycosides↗

PubMed1982

Synthesis of beta-hexosaminidase in cell-free translation and in intact fibroblasts: an insoluble precursor alpha chain in a rare form of Tay-Sachs disease.

RNA was isolated from human term placenta or cultured fibroblasts and translated in a rabbit reticulocyte system in the presence of [35S]methionine; the translation products were immunoprecipitated with antisera made against beta-hexosaminidase or its isolated alpha and beta chains and analyzed by polyacrylamide gel electrophoresis. The largest translated alpha and beta chain polypeptides had Mrs of 65,000 and 59,000, respectively. These are approximately equal to 2,000 greater than the Mrs of precursor chains synthesized by intact fibroblasts and deglycosylated with endo-beta-N-acetylglucosaminidase H suggesting the presence of a signal sequence. RNA of fibroblast cultures from two patients with Sandhoff disease did not direct the translation of immunoprecipitable beta chain; RNA of fibroblast cultures from four patients with Tay-Sachs disease (three of Ashkenazi Jewish descent and one of non-Jewish descent) did not direct the translation of immunoprecipitable alpha chain. In contrast, a normal amount of alpha chain was made in the presence of RNA from the fibroblast culture of another non-Jewish Tay-Sachs patient (GM 1110). Intact fibroblasts from this patient also synthesized the alpha chain as shown by labeling with [3H]leucine; however, strong detergent was required for extraction. The alpha chain could be labeled with [3H]mannose but not with [32P]phosphate; it was neither secreted nor accumulated in the proteolytically processed form, and it disappeared within a day of synthesis. A plausible though not unique explanation is that the insoluble alpha chain is not transported from the endoplasmic reticulum (the site of glycosylation) to the Golgi apparatus (the site of phosphorylation) nor to further points of destination--lysosomes and the exterior of the cell.

Cells, Cultured↗

PubMed1990

Distribution of saposin proteins (sphingolipid activator proteins) in lysosomal storage and other diseases.

Saposins (A, B, C, and D) are small glycoproteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Concentrations of these saposins in brain, liver, and spleen from normal humans as well as patients with lysosomal storage disease were determined. A quantitative HPLC method was used for saposin A, C, and D and a stimulation assay was used for saposin B. In normal tissues, saposin D was the most abundant of the four saposins. Massive accumulations of saposins, especially saposin A (about 80-fold increase over normal), were found in brain of patients with Tay-Sachs disease or infantile Sandhoff disease. In spleen of adult patients with Gaucher disease, saposin A and D accumulations (60- and 17-fold, respectively, over normal) were higher than that of saposin C (about 16-fold over normal). Similar massive accumulations of saposins A and D were found in liver of patients with fucosidosis (about 70- and 20-fold, respectively, over normal). Saposin D was the primary saposin stored in the liver of a patient with Niemann-Pick disease (about 30-fold over normal). Moderate increases of saposins B and D were found in a patient with GM1 gangliosidosis. Normal or near normal levels of all saposins were found in patients with Krabbe disease, metachromatic leukodystrophy, Fabry disease, adrenoleukodystrophy, I-cell disease, mucopolysaccharidosis types 2 and 3B, or Jansky-Bielschowsky disease. The implications of the storage of saposins in these diseases are discussed.

Adult↗

PubMed1992

Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.

Development of a strategy for efficient delivery of exogenous enzyme to neuronal lysosomes is essential to achieve enzyme replacement in neurodegenerative lysosomal storage diseases. We tested whether effective lysosomal targeting of the human enzyme beta-N-acetylhexosaminidase A (Hex A; beta-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) can be obtained by coupling it via disulfide linkage to the atoxic fragment C of tetanus toxin (TTC) that is bound avidly by neuronal membrane. TTC-Hex A conjugation resulted in neuronal surface binding and enhanced endocytosis of enzyme as observed in immunofluorescence studies with rat brain cultures. In immunoelectrophoretic quantitative uptake studies, rat neuronal cell cultures contained 16- and 40-fold greater amounts of enzyme after incubation with TTC-Hex A than with nonderivatized Hex A. In cerebral cortex cell cultures from a feline model of human GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases), binding and uptake patterns of the enzymes were similar to those in the rat brain cell cultures. After exposure to extracellular concentrations of enzyme attainable in vivo, lysosomal storage of immunodetectable GM2 ganglioside was virtually eliminated in neurons exposed to TTC-Hex A, whereas a minimal effect was observed with Hex A. These findings demonstrate the usefulness of TTC adducts for effective neuronal lysosomal enzyme replacement.

Animals↗