Formation of a ternary complex between GM2 activator protein, GM2 ganglioside and hexosaminidase A.

The GM2 activator is a 17 kDa protein required for the hydrolysis of GM2 ganglioside by the lysosomal enzyme hexosaminidase A (HexA). The activator behaves as a substrate binding protein, solubilizing GM2 ganglioside monomers from micelles (in vitro) or membranes (in vivo). However, the activator also shows a high order of specificity for activation of lysosomal hydrolases and has been predicted to form a ternary complex with the heterodimeric enzyme (alphabeta) Hex A and GM2 ganglioside. We demonstrated a transient interaction between HexA and the GM2 activator. A chimeric protein containing the FLAG epitope sequence upstream of the GM2 activator was expressed in Escherichia coli and purified using the M1 immunoaffinity (anti-FLAG) column. Binding of the FLAG-GM2 activator (FLAG-AP) fusion protein to the M1 column led to the specific retardation of Hex A applied to the column. Other proteins were not retarded by the column nor did they compete with Hex A for binding to FLAG-AP. Hex A and GM2 ganglioside could be simultaneously bound to the column, but the binding of each ligand was independent of the other. The homodimeric (beta beta) isozyme Hex B did not bind to the immobilized activator. The alpha alpha homodimer, HexS, bound weakly, confirming that a hexosaminidase alpha subunit is required for interaction of enzyme and activator.

A chronic GM2 gangliosidosis variant with a HEXA splicing defect: quantitation of HEXA mRNAs in normal and mutant fibroblasts.

Over 72 mutations have been identified in the HEXA gene of which only four (T538C, A590C, G805A, and C1495T) are believed to cause a chronic form of Tay-Sachs disease (TSD). We identified a novel HEXA mutation (IVS7, -7 G-->A) leading to chronic TSD in a Canadian patient of English ancestry. The second allele in this patient was the exon 11 4-bp insertion mutation (/1277TATC), which is the most frequent TSD allele in Ashkenazi Jews. The IVS7, -7 G-->A mutation introduces a new 3' splice acceptor site 5 bp upstream of the normal intron 7 splice acceptor site. The mutation leads to reduction of steady-state levels of HEXA mRNA by more than 80%. Two mRNA species are produced by the IVS7, -7 G-->A allele; a normal nRNA species and an mRNA lacking exon 8. No mRNA species that was spliced at the upstream 3' splice acceptor site was detected. We used competitive PCR to quantitate mRNA species in fibroblasts obtained from this patient. We compared the amounts of three identified mRNA species to HEXA mRNA levels in cells from normal individuals and from individuals heterozygous for /1277TATC. The steady-state level of HEXA mRNA in cells from a normal individual was 17.3 pg/microg RNA. An individual heterozygous for /1277TATC produced 8.7 pg of normal HEXA mRNA/microg RNA. The HEXA mRNA species with the insertion mutation was present in patient cells at 4.8% of the level of normal HEXA nRNA in homozygous normal cells. In fibroblasts from the patient carrying the IVS7, -7 G-->A mutation, the steady-state level of exon 8-deleted HEXA mRNA was 5.9% the level of that produced by homozygous normal cells. The level of normal HEXA nRNA in this patient's cells was 10.4%.

A Pst+ polymorphism in the HEXA gene with an unusual geographic distribution.

A polymorphic variant in the human HEXA gene is described. This gene encodes the alpha-subunit of hexosaminidase A, the enzyme which is deficient in Tay-Sachs disease (TSD). In individuals carrying the polymorphism there is a T-->C transition at position -6 in intron 13. The substitution creates a site for the restriction endonuclease Pst1. This variant has an unusual ethnogeographic distribution. It occurs on 1.4% of non-TSD carrier chromosomes in Ashkenazi Jews. All individuals ascertained carrying the Pst+ allele have ancestry in Lithuania, Belarus and Ukraine. By contrast, no individuals carrying the Pst+ allele have been detected among non-Jewish Lithuanians, Jews of Sephardic origin or in several other ethnic groups. Two unrelated non-Jewish families have been identified in which the Pst+ variant occurs. In both cases the variant occurs on a chromosome carrying a novel TSD mutation (G772C) association with the B1 phenotype. The Pst+ G772C chromosomes are of Scots-Irish descent.

Tay-Sachs disease screening and diagnosis: evolving technologies.

Tay-Sachs disease (TSD) is an autosomal recessive, progressive, and fatal neurodegenerative disorder. Within the last 25 years, the discovery of the enzymatic basis of the disease, the deficiency of the enzyme hexosaminidase A, has made possible both enzymatic diagnosis of TSD and heterozygote identification. TSD is the first genetic condition for which a community-based heterozygote screening program was attempted with the intention of reducing the incidence of a genetic disease. In this article we review the clinical, biochemical, and molecular features of TSD as well as the development of laboratory technology that has been deployed in community genetic screening programs. We describe the assay procedures used and some of the limitations in their accuracy. We consider the impact of DNA-based technology on the process of identification of individuals carrying mutant genes associated with TSD and we discuss the social context within which genetic screening occurs.

Synthesis of 4-methylumbelliferyl-beta-D-N-acetylglucosamine-6-sulfate and its use in classification of GM2 gangliosidosis genotypes.

Measurement of hexosaminidase A (Hex A) is an important clinical chemical procedure in the classification of GM2 gangliosidosis genotypes. We have synthesized a new substrate which may be useful in both the biochemical diagnosis of GM2 gangliosidosis and the detection of heterozygotes for the Tay-Sachs disease (TSD) allele. 4-Methylumbelliferyl-beta-D-N-acetylglucosamine-6-sulfate (4MUGS) was synthesized by sulfation of 4MU-beta-D-N-acetylglucosamine (4MUG) with chlorosulfonic acid and purified through gel filtration and ion-exchange chromatography. The structure of 4MUGS was verified by elemental analysis and NMR. Hex A is approximately 100 times more active toward 4MUGS than Hex B. The advantage of this increased specificity is that Hex A can be determined in a one-step procedure which allows separation of normal control serum values from those of obligate heterozygotes. Alternatively, assay values obtained using both substrates can be transformed by application of an empirical equation that allows the calculation of both Hex A and Hex B without the requirement of thermal fractionation. Lower values for % Hex A in serum have been obtained for Tay-Sachs homozygotes using the 4MUGS assay procedure. The results of Hex A assays on fibroblast cell strains obtained from Tay-Sachs homozygotes, variant forms of GM2 gangliosidosis and normal controls are also discussed.

Variable onset of metachromatic leukodystrophy in a Vietnamese family.

We report two siblings with metachromatic leukodystrophy, one who presented at 7 years of age (juvenile onset) and his sister who presented at 22 years of age (adult onset). They are compound heterozygotes for two novel mutations in the arylsufatase A gene (ARSA). The responsible mutations in this Vietnamese family consist of a missense mutation with 5% enzyme activity (R143G) and a nonsense mutation (W318ter), from which no enzyme activity would be expected. These mutations in the ARSA gene have not been previously reported and may be useful when diagnosing metachromatic leukodystrophy in other affected Vietnamese individuals. The variability in presentation suggests that the genotype alone is not sufficient to determine the onset and course of the disease and modifying genetic and perhaps nongenetic factors likely contribute.

Tay-Sachs disease: B1 variant.

This first child of non-Jewish parents had nystagmus at 4 months of age, bilateral cherry-red macular spots at 7 months of age, and hyperacusis at 8 months of age; the patient has deteriorated progressively following a clinical course typical of Tay-Sachs disease B variant. Total beta-N-acetylhexosaminidase assayed with 4-methylumbelliferyl-beta-glucosamine (4 MU GlcNAc) as substrate was within the normal range in plasma and cultured dermal fibroblasts and 2/3 the normal mean in leukocytes. The hexosaminidase A activity, assayed with the same substrate in plasma and cultured fibroblasts, approximated Tay-Sachs disease heterozygote levels; however, the activity of hexosaminidase A assayed with 4 MU Glc NAc-6-sulfate in the plasma, leukocytes, and cultured fibroblasts was less than 8, 2, and 1%, respectively of the control mean. This female infant with the B1 variant of Tay-Sachs disease demonstrated an earlier onset and more rapidly progressive course than was observed in 4 of the 5 previously reported patients with this Tay-Sachs disease variant.

Characterization of an activating factor required for hydrolysis of Gm2 ganglioside catalyzed by hexosaminidase A.

Separation of the hexosaminidase A (EC 3.2.1.52) and B isozymes of human liver by ion-exchange chromatography results in recovery of greater than 80% of the activity in crude extracts when synthetic substrates are used to monitor enzyme activity. Only 15% of hexosaminidase activity toward the N-acetylgalactosaminyl (N-acetylneuraminyl) galactosyl glucosylceramide (Gm2 ganglioside) substrate is recovered and all of this activity is associated with the hexosaminidase A Fraction. The low level of Gm2 ganglioside hydrolase activity in the hexosaminidase A fraction could be enhanced by coincubation with column fractions which contain hexosaminidase B. The activating factor, which has been partially purified by gel filtration, is a heat-stable protein with a molecular weight of 36 000 and is without enzyme activity toward hexosaminidase substrates. Highly purified hexosaminidase A or crude hexosaminidase A recovered after gel filtration on Sephadex G-100 has no Gm2 ganglioside hydrolase activity. The Gm2 ganglioside hydrolase activity of these hexosaminidase. A preparations can be completely restored by addition of activating factor. The activating factor does not affect the rate of hydrolysis of synthetic substrate or asialo Gm2 ganglioside catalyzed by hexosaminidase A.

Apparent hexosaminidase B deficiency in two healthy members of a pedigree.

A family is described in which all members have decreased serum and leukocyte hexosaminidase activity. Two individuals, the mother and the younger daughter, have a normal ratio of hexosaminidase B (HEX B) to total hexosaminidase, but their serum enzymes display respectively partial or complete lability to heat. It is proposed that the proband is a double heterozygote for the Sandhoff allele and for an allele producing thermolabile beta subunits.

Genetic control of ganglioside biosynthesis in mice.

The enzymatic basis for the differences in hepatic ganglioside patterns in the mouse strains C57Bl/6 and Swiss White (SW) was investigated. SW has a "Swiss-type" ganglioside profile, expressing GM1- and GD1a- in addition to GM2- as major hepatic gangliosides, whereas C57Bl/6 shows a "GM2-type" profile, expressing only GM2- as the major hepatic ganglioside. The enzyme UDP-galactose:GM2 ganglioside galactosyltransferase (GM2-GalT), which catalyzes the synthesis of GM1 ganglioside, showed a four- to fivefold elevation in intact and solubilized liver Golgi membrane fractions of the SW strain compared to C57Bl/6. Crosses between C57Bl/6 and SW produced an F1 generation with a hepatic ganglioside and enzymatic phenotype intermediate between those of the two parental strains. All three genotypic groups show two forms of the Golgi apparatus enzyme with isoelectric points of 6.5-6.8 and 8.3-9.0. The simplest mode of action of genes which control the enzymatic phenotype that would be consistent with these findings are one or two structural genes or one or two cis-regulatory genes affecting the rate of enzyme synthesis.

Prolidase deficiency in cultured human fibroblasts: biochemical pathology and iminodipeptide-enhanced growth.

Prolidase deficiency is a rare autosomal recessive disorder characterized by iminodipeptiduria, severe skin ulcers, recurrent infections, and mental retardation. The enzyme prolidase hydrolyzes dipeptides containing C-terminal proline or hydroxyproline. We investigated the metabolic abnormality caused by prolidase deficiency in human cultured skin fibroblasts. These studies were undertaken to test biochemical hypotheses regarding the metabolic origins of the skin lesion occurring in this disease. Our results indicate that prolidase plays a major role in the recycling of dipeptide-bound proline. Control fibroblasts were able to use iminodipeptides in lieu of proline to sustain normal growth, whereas cells homozygous for the prolidase deficiency mutation were not. Proline derived from iminodipeptides diluted incorporation of radiolabeled extracellular proline into cellular protein in normal cells but not in mutant cells. Substitution of a prolidase-free medium for FCS did not affect the growth rate of control cell lines but increased the doubling time of prolidase-deficient cells by 19% (28% in the presence of iminodipeptides). Iminodipeptides added to control and mutant cells maintained in serum-free medium showed no adverse effects on protein synthesis. These results are consistent with a mechanism of biochemical pathology in which proline deprivation caused by the enzyme deficit is a primary cause of damage to skin cells. Prolidase regulation by product and substrate was studied. A 44% decrease in activity was observed in fibroblasts grown for 3 wk in proline-containing medium relative to proline-free medium. However, cells grown in medium in which iminodipeptides replaced proline showed no significant difference in prolidase activity.

Purification and properties of the hexosaminidase A-activating protein from human liver.

Human liver extracts contain an activating protein which is required for hexosaminidase A-catalysed hydrolysis of the N-acetylgalactosaminyl linkage of G(M2) ganglioside [N-acetylgalactosaminyl-(N-acetylneuraminyl) galactosylglucosylceramide]. A partially purified preparation of human liver hexosaminidase A that is substantially free of G(M2) ganglioside hydrolase activity is used to assay the activating protein. The proceudres of heat and alcohol denaturation, ion-exchange chromatography and gel filtration were used to purify the activating protein over 100-fold from crude human liver extracts. When the purified activating protein is analysed by polyacrylamide-gel disc electrophoresis, two closely migrating protein bands are seen. When purified activating protein is used to reconstitute the G(M2) ganglioside hydrolase activity, the rate of reaction is proportional to the amount of hexosaminidase A used. The activation is specific for G(M2) ganglioside and and hexosaminidase A. The activating protein did not stimulate hydrolysis of asialo-G(M2) ganglioside by either hexosaminidase A or B. Hexosaminidase B did not catalyse hydrolysis of G(M2) ganglioside with or without the activator. Kinetic experiments suggest the presence of an enzyme-activator complex. The dissociation constant of this complex is decreased when higher concentrations of substrate are used, suggesting the formation of a ternary complex between enzyme, activator and substrate. Determination of the molecular weight of the activating protein by gel-filtration and sedimentation-velocity methods gave values of 36000 and 39000 respectively.

Purification and properties of two enzymes catalyzing galactose transfer to GM2 ganglioside from rat liver Golgi.

An enzyme activity which catalyzed the transfer of galactose from UDP-galactose to GM2 ganglioside was demonstrated in rat liver homogenate and enriched 38-fold in specific activity by preparation of Golgi membranes. This activity could be solubilized from Golgi membranes by sonication and extraction with 1% Triton X-100. The solubilized activity catalyzed the formation of GM1 ganglioside and was completely dependent upon the addition of acceptor. The rate of galactose incorporation was constant for up to 5 h at 30 degrees C. This enzyme activity was further purified by gel filtration on Sepharose CL-6B and ion exchange chromatography on DEAE-Sepharose. The elution position on gel filtration corresponded to a molecular weight for the enzyme of 38,000 which was in good agreement with that obtained by sedimentation velocity studies. Ion exchange chromatography resolved GM2 ganglioside galactosyltransferase into two species. The more basic enzyme (I) comprising 28% of the recovered activity was not retarded by the column, whereas enzyme II was eluted from the resin following the application of a salt gradient. Net purification was 120- to 140-fold for each enzyme with a total recovery of 42%. Unlike the activity in the Golgi extract, the purified enzymes I and II were labile to freezing and could be stored at -20 degrees C only in the presence of 50% glycerol. Both enzymes I and II had similar molecular weights and Michaelis constants and both had a strict requirement for Mn2+. Properties which distinguish the two enzymes included pH optima (enzyme I 7.0, enzyme II 6.0) and surfactant requirements. Neither enzyme was active following removal of Triton X-100 from the preparation. Among a series of glycolipids tested for ability to serve as substrates for galactose transfer only GM2 and asialo-GM2 ganglioside served as acceptors.

Interaction of activating protein and surfactants with human liver hexosaminidase A and GM2 ganglioside.

The effects of surfactants on the human liver hexosaminidase A-catalysed hydrolysis of G(m2) ganglioside were assessed. Some non-ionic surfactants, including Triton X-100 and Cutscum, and some anionic surfactants, including sodium taurocholate, sodium dodecyl sulphate, phosphatidylinositol and N-dodecylsarcosinate, were able to replace the hexosaminidase A-activator protein [Hechtman (1977) Can. J. Biochem.55, 315-324; Hechtman & Leblanc (1977) Biochem. J.167, 693-701) and also stimulated the enzymic hydrolysis of substrate in the presence of saturating concentrations of activator. Other non-ionic surfactants, such as Tween 80, Brij 35 and Nonidet P40, and anionic surfactants, such as phosphatidylethanolamine, did not enhance enzymic hydrolysis of G(m2) ganglioside and inhibited hydrolysis in the presence of activator. The concentration of surfactants at which micelles form was determined by measurements of the minimum surface-tension values of reaction mixtures containing a series of concentrations of surfactant. In the case of Triton X-100, Cutscum, sodium taurocholate, N-dodecylsarcosinate and other surfactants the concentration range at which stimulation of enzymic activity occurs correlates well with the critical micellar concentration. None of the surfactants tested affected the rate of hexosaminidase A-catalysed hydrolysis of 4-methylumbelliferyl N-acetyl-beta-d-glucopyranoside. Both activator and surfactants that stimulate hydrolysis of G(m2) ganglioside decrease the K(m) for G(m2) ganglioside. Inhibitory surfactants are competitive with the activator protein. Evidence for a direct interaction between surfactants and G(m2) ganglioside was obtained by comparing gel-filtration profiles of (3)H-labelled G(M2) ganglioside in the presence and absence of surfactants. The results are discussed in terms of a model wherein a mixed micelle of surfactant or activator and G(M2) ganglioside is the preferred substrate for enzymic hydrolysis.

High-capacity method for purification of human liver hexosaminidase B using hydrophobic chromatography.

Hydrophobic chromatography was investigated as a purification procedure for human liver hexosaminidases. Both phenyl-Sepharose and valine-Sepharose have a high binding capacity of hexosaminidases. A degree of resolution between the A and B ioszymes is achieved with phenyl-Sepharose. Both hydrophobic supports must be used close to their capacity in order to recover the applied enzyme. Two purification procedures for human liver hexosaminidase B were employed, which resulted in recoveries of approximately 48 and 24% with final specific activities of 33,400 and 4840 nmole/min.mg, respectively.

Rat liver Golgi galactosyltransferases. Distinct enzymes for glycolipid and glycoprotein acceptor substrates.

Two enzymes that catalyse the transfer of galactose from UDP-galactose to GM2 ganglioside were partially purified from rat liver Golgi membranes. These preparations, designated enzyme I (basic) and enzyme II (acidic), utilized as acceptors GM2 ganglioside and asialo GM2 ganglioside as well as ovalbumin, desialodegalactofetuin, desialodegalacto-orosomucoid, desialo bovine submaxillary mucin and GM2 oligosaccharide. Enzyme II catalysed disaccharide synthesis in the presence of the monosaccharide acceptors N-acetylglucosamine and N-acetylgalactosamine. The affinity adsorbent alpha-lactalbumin-agarose, which did not retard GM2 ganglioside galactosyltransferase, was used to remove most or all of galactosyltransferase activity towards glycoprotein and monosaccharide acceptors from the extracted Golgi preparation. After treatment of the extracted Golgi preparation with alpha-lactalbumin-agarose, enzyme I and enzyme II GM2 ganglioside galactosyltransferase activities, prepared by using DEAE-Sepharose chromatography, were distinguishable from transferase activity towards GM2 oligosaccharide and glycoproteins by the criterion of thermolability. This residual galactosyltransferase activity towards glycoprotein substrates was also shown to be distinct from GM2 ganglioside galactosyltransferase in both enzyme preparations I and II by the absence of competition between the two acceptor substrates. The two types of transferase activities could be further distinguished by their response to the presence of the protein effector alpha-lactalbumin. GM2 ganglioside galactosyltransferase was stimulated in the presence of alpha-lactalbumin, whereas the transferase activity towards desialodegalactofetuin was inhibited in the presence of this protein. The results of purification studies, comparison of thermolability properties and competition analysis suggested the presence of a minimum of five galactosyltransferase species in the Golgi extract. Five peaks of galactosyltransferase activity were resolved by isoelectric focusing. Two of these peaks (pI 8.6 and 6.3) catalysed transfer of galactose to GM2 ganglioside, and three peaks (pI 8.1, 6.8 and 6.3) catalysed transfer to glycoprotein acceptors.

The GM2 gangliosidoses databases: allelic variation at the HEXA, HEXB, and GM2A gene loci.

The GM2 gangliosidoses are a group of recessive disorders characterized by accumulation of GM2 ganglioside in neuronal cells. The genes responsible for these disorders are HEXA (Tay-Sachs disease and variants), HEXB (Sandhoff disease and variants), and GM2A (AB variant of GM2 gangliosidosis). We report the establishment of three relational locus-specific databases recording allelic variation at the HEXA, HEXB, and GM2A genes and accessed at the GM2 gangliosidoses home page (http://data.mch.mcgill.ca/gm2-gangliosidoses). Submission forms are available for the addition of new mutations to the databases. The databases are available online for users to search and retrieve information about specific alleles by a number of fields describing mutations, phenotypes, or author(s).

Four novel PEPD alleles causing prolidase deficiency.

Mutations at the PEPD locus cause prolidase deficiency (McKusick 170100), a rare autosomal recessive disorder characterized by iminodipeptiduria, skin ulcers, mental retardation, and recurrent infections. Four PEPD mutations from five severely affected individuals were characterized by analysis of reverse-transcribed, PCR-amplified (RT-PCR) cDNA. We used SSCP analysis on four overlapping cDNA fragments covering the entire coding region of the PEPD gene and detected abnormal SSCP bands for the fragment spanning all or part of exons 13-15 in three of the probands. Direct sequencing of the mutant cDNAs showed a G-->A, 1342 substitution (G448R) in two patients and a 3-bp deletion (delta E452 or delta E453) in another. In the other two probands the amplified products were of reduced size. Direct sequencing of these mutant cDNAs revealed a deletion of exon 5 in one patient and of exon 7 in the other. Intronic sequences flanking exons 5 and 7 were identified using inverse PCR followed by direct sequencing. Conventional PCR and direct sequencing then established the intron-exon borders of the mutant genomic DNA revealing two splice acceptor mutations: a G-->C substitution at position -1 of intron 4 and an A-->G substitution at position -2 of intron 6. Our results indicate that the severe form of prolidase deficiency is caused by multiple PEPD alleles. In this report we attempt to begin the process of describing these alleles and cataloging their phenotypic expression.

Neutral amino acid transport in Pseudomonas fluorescens.

Membrane transport of beta-alanine, l-alanine, and l-proline was studied in a beta-alanine transaminaseless mutant (strain 67) of Pseudomonas fluorescens. In this mutant beta-alanine is metabolically inert, and it was therefore possible to demonstrate active transport of this substrate in the absence of intracellular catabolism. The permease which catalyzes the uptake of beta-alanine also transports l-proline and l-alanine. This common transport system was distinguished from permeases which transport only l-alanine and only l-proline by competition studies in strain 67 and by studies of transport specificity in a permeaseless mutant (strain 67/4MTR).