Molecular genetics of GM2-gangliosidosis AB variant: a novel mutation and expression in BHK cells

The GM2 activator is a hexosaminidase A-specific glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. Genetic deficiency of GM2 activator leads to a neurological disorder, an atypical form of Tay-Sachs disease (GM2 gangliosidosis variant AB). Here, we describe a G⁵⁰⁶ to C transversion (Arg¹⁶⁹ to Pro) in the mRNA of an infantile patient suffering from GM2-gangliosidosis variant AB. Using the polymerase chain reaction amplification and direct-sequencing technique, we found the patient to be homozygous for the mutation, whereas the parents were, as expected, heterozygous. BHK cells transfected with a construct of mutant cDNA gave no GM2 activator protein detectable by the Western blotting technique, whereas those transfected by a wild-type cDNA construct showed a significant level of human GM2 activator protein. The substitution of proline for the normal Arg¹⁶⁹ therefore appears to result in premature degradation of the mutant GM2 activator, either during the post-translational processing steps or after reaching the lysosome. The basis for the phenotype of GM2 gangliosidosis variant AB may therefore be either inactivation of the physiological activator function by the point mutation or instability of the mutant protein.     

In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits,Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.     

A mutation in the gene of a glycolipid-binding protein (GM2 activator) that causes GM2-gangliosidosis variant AB

GM2-gangliosidoses are neurological disorders caused by a genetic deficiency of either the β-hexosaminidase A or the GM2 activator, a glycolipid binding protein. In a patient with an immunologically proven GM2 activator protein deficiency, A T⁴¹² → C transition (counted from A of the initiation codon) was found in the coding sequence, which results in the substitution of Arg for the normal Cys¹⁰⁷ in the mature GM2 activator protein. The remainder of the coding sequence remained entirely normal.     

Isolation and expression of a full-length cDNA encoding the human GM2 activator protein

We report the construction of a cDNA clone encoding a functional GM2-activator protein. The sequence of the complete 5' end of the coding region was determined by direct nucleotide sequencing of a fragment generated by multiple RACE PCR procedures from Hela cell cDNA. Specific oligonucleotides were synthesized from these data which allowed us to produce a PCR fragment that contained the complete coding sequence of the protein. This was then cloned into a mammalian expression vector. The ability of purified hexosaminidase A (beta-N-acetylhexosaminidase, EC 3.2.1.52) to hydrolyse labeled GM2 ganglioside was enhanced 10-fold more by the addition in the assay mix of lysate from transfected COS-1 cells than by the addition of identical amounts of lysate from mock transfected cells. Direct sequencing of PCR fragments from two sources also identified three polymorphisms.     

Biochemical consequences of mutations causing the GM2 gangliosidoses

The hydrolysis of GM2-ganglioside is unusual in its requirements for the correct synthesis, processing, and ultimate combination of three gene products. Whereas two of these proteins are the alpha- (HEXA gene) and beta- (HEXB) subunits of beta-hexosaminidase A, the third is a small glycolipid transport protein, the GM2 activator protein (GM2A), which acts as a substrate specific co-factor for the enzyme. A deficiency of any one of these proteins leads to storage of the ganglioside, primarily in the lysosomes of neuronal cells, and one of the three forms of GM2-gangliosidosis, Tay-Sachs disease, Sandhoff disease or the AB-variant form. Studies of the biochemical impact of naturally occurring mutations associated with the GM2 gangliosidoses on mRNA splicing and stability, and on the intracellular transport and stability of the affected protein have provided some general insights into these complex cellular mechanisms. However, such studies have revealed little in the way of structure-function information on the proteins. It appears that the detrimental effect of most mutations is not specifically on functional elements of the protein, but rather on the proteins' overall folding and/or intracellular transport. The few exceptions to this generalization are missense mutations at two codons in HEXA, causing the unique biochemical phenotype known as the B1-variant, and one codon in both the HEXB and GM2A genes. Biochemical characterization of these mutations has led to the localization of functional residues and/or domains within each of the encoded proteins.     

Purification and characterization of an activator protein for the degradation of glycolipids GM2 and GA2 by hexosaminidase A

The activator protein for the degradation of glycolipids GM2 and GA2 by hexosaminidase A was purified some 2 500-fold from normal human kidney. It has a molecular weight of approximately 25 000 is heat-stable up to 60 degrees C, possesses an isoelectric point of pH 4.8 and is digestible by proteases. Enzymic degradation of the lipid substrates in the presence of this activator proceeds optimally at pH 4.2. The mode of action of the activator was also studied: the protein most probably complexes lipid molecules and presents them to the enzyme which otherwise cannot attack the aggregates formed by the lipids in aqueous solution. The hydrolysis of water-soluble synthetic substrates is not affected by the activator protein. The activator is highly specific for hexosaminidase A: hydrolysis of glycolipids GA2 and GM2 by the hexosaminidase B isoenzyme is almost not enhanced by this protein. The isoenzymes' lipid substrate specificity measured in the presence of the activator is entirely different from that obtained with detergents and can satisfactorily account for the lipid storage pattern observed in patients with variant forms of infantile GM2- gangliosidosis.     

Ganglioside GM2 N-acetyl-beta-D-galactosaminidase activity in cultured fibroblasts of late-infantile and adult GM2 gangliosidosis patients and of healthy probands with low hexosaminidase level.

A sensitive assay was developed to assess the ability of extracts from cultured fibroblasts to catabolize ganglioside GM2, in the presence of the natural activator protein but without detergents. This method, which permitted the reliable determination of residual activities as low as 0.1% of normal controls, was then used to measure ganglioside GM2 hydrolase activities in fibroblasts from several hexosaminidase variants. The residual activities thus determined correlated well with the clinical status of the respective proband: infantile Tay-Sachs (0.1% of normal controls), late-infantile (0.5%), and adult GM2 gangliosidoses (2%-4%) and healthy probands with "low hexosaminidase" (11% and 20%). In contrast, beta-hexosaminidase A levels as measured with the synthetic substrate 4-MU-GlcNAc could not be relied on for diagnostic purposes (the late-infantile patient studied retained 80% of the activity of controls).     

Juvenile GM2 gangliosidosis (AMB variant): inability to activate hexosaminidase A by activator protein.

Two sibling from a consanguineous Puerto Rican marriage were found to have a juvenile-onset type of lipidosis first noted at age 2 1/2 by expressing difficulties with motor function and developmental delay. They continued to deteriorate, showing muscle atrophy, spasticity, and loss of speech, and death occurred at ages 7 and 8. Examination of the brains from these patients revealed that the concentration of GM2 ganglioside was about 56% of the total gangliosides. Hexosaminidase and percent hexosaminidase A (HEX A) and other lysosomal enzymes were normal in cultured skin fibroblasts, liver, and brain. The concentration of the activator protein required for the enzymatic hydrolysis of GM2 ganglioside was in high normal levels in the brain of the patient available. However, the HEX A from the patient's brain and liver as well as from skin fibroblast lysates could not be activated to hydrolyze GM2 ganglioside by the activator protein from a control or himself. The HEX A from a control could be activated by the activator protein from controls or this patient. These patients appear to have a defect in HEX A, which does not affect it heat stability, electrophoretic migration, and activity toward fluorogenic substrates, but may affect the binding of the activator protein required for GM2 ganglioside hydrolysis. We propose to call these patients the AMB variant of GM2 gangliosidosis to denote the mutation in HEX A but with normal levels of HEX A and B with synthetic substrates. This is to distinguish these patients from those missing the activator protein and normal HEX A and B levels.     

Activating proteins for ganglioside GM2 degradation by beta-hexosaminidase isoenzymes in tissue extracts from different species

The existence of activator proteins that stimulate hydrolysis of ganglioside GM2 by beta-hexosaminidase was demonstrated in kidney extracts from four species (rat, mouse, cattle and pig). The extent to which these preparations, as well as their human counterpart, promote ganglioside GM2 catabolism by autologous and heterologous hexosaminidase isoenzymes was compared. It was found that these activators can replace each other functionally, although the animal activator proteins do not cross-react immunochemically with an antiserum against the human protein. All preparations examined catalysed the transfer of ganglioside GM2 between liposomal membranes, indicating that the animal activator proteins act by a mechanism similar to the human GM2 activator.     

Isolation of a cDNA encoding the human GM2 activator protein

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.     

Complexing of glycolipids and their transfer between membranes by the activator protein for degradation of lysosomal ganglioside GM2

The lysosomal degradation of ganglioside GM2 by hexosaminidase A depends on the presence of the specific activator protein which mediates the interaction between micellar or membrane-bound ganglioside and water-soluble hydrolase. The mechanism and the glycolipid specificity of this activator were studied in more detail. 1. It could be shown with three different techniques (isoelectric focusing, centrifugation and electrophoresis) that the activator protein extracts glycolipid monomers from micelles or liposomes to give water-soluble complexes with a stoichiometry of 1 mol of glycolipid/mol of activator protein. Liposome-bound ganglioside GM2 is considerably more stable against extraction and degradation than micellar ganglioside. 2. In the absence of enzyme the activator acts in vitro as glycolipid transfer protein, transporting glycolipids from donor to acceptor membranes. 3. The activator protein is rather specific for ganglioside GM2. Other glycolipids (GM3 GM1, GD1a and GA2) form less stable complexes with the activator and are transferred at a slower rate (except for ganglioside GM1) than ganglioside GM2.     

Catabolism of asialo-GM2 in man and mouse. Specificity of human/mouse chimeric GM2 activator proteins.

Tay-Sachs disease is an inborn lysosomal disease characterized by excessive cerebral accumulation of GM2. The catabolism of GM2 to GM3 in man requires beta-hexosaminidase A (HexA) and a protein cofactor, the GM2 activator. Thus, Tay-Sachs disease can be caused by the deficiency of either HexA or the GM2 activator. The same cofactor found in mouse shares 74.1% amino acid identity (67% nucleotide identity) with the human counterpart. Between the two activators, the mouse GM2 activator can effectively stimulate the hydrolysis of both GM2 and asialo-GM2 (GA2) by HexA and, to a lesser extent, also stimulate HexB to hydrolyze GA2, whereas the human activator is ineffective in stimulating the hydrolysis of GA2 (Yuziuk, J. A., Bertoni, C., Beccari, T., Orlacchio, A., Wu, Y.-Y., Li, S.-C., and Li, Y.-T. (1998) J. Biol. Chem. 273, 66-72). To understand the role of these two activators in stimulating the hydrolyses of GM2 and GA2, we have constructed human/mouse chimeric GM2 activators and studied their specificities. We have identified a narrow region (Asn(106)-Tyr(114)) in the mouse cDNA sequence that might be responsible for stimulating the hydrolysis of GA2. Replacement of the corresponding site in the human sequence with the specific mouse sequence converted the ineffective human activator into an effective chimeric protein for stimulating the hydrolysis of GA2. This chimeric activator protein, like the mouse protein, is also able to stimulate the hydrolysis of GA2 by HexB. The mouse model of human type B Tay-Sachs disease recently engineered by the targeted disruption of the Hexa gene showed less severe clinical manifestation than found in human patients. This has been considered to be the result of the catabolism of GM2 via converting it to GA2 and further hydrolysis of GA2 to lactosylceramide by HexB with the assistance of mouse GM2 activator protein. The chimeric activator protein that bears the characteristics of the mouse GM2 activator may therefore be able to induce an alternative catabolic pathway for GM2 in human type B Tay-Sachs patients.     

An alpha-subunit loop structure is required for GM2 activator protein binding by beta-hexosaminidase A

The alpha- and/or beta-subunits of human beta-hexosaminidase A (alphabeta) and B (betabeta) are approximately 60% identical. In vivo only beta-hexosaminidase A can utilize GM2 ganglioside as a substrate, but requires the GM2 activator protein to bind GM2 ganglioside and then interact with the enzyme, placing the terminal GalNAc residue in the active site of the alpha-subunit. A model for this interaction suggests that two loop structures, present only in the alpha-subunit, may be critical to this binding. Three amino acids in one of these loops are not encoded in the HEXB gene, while four from the other are removed posttranslationally from the pro-beta-subunit. Natural substrate assays with forms of hexosaminidase A containing mutant alpha-subunits demonstrate that only the site that is removed from the beta-subunit during its maturation is critical for the interaction. Our data suggest an unexpected biological role for such proteolytic processing events.     

The GM2 activator protein, a novel inhibitor of platelet-activating factor.

The GM2 activator protein is required as a substrate-specific cofactor for beta-hexosaminidase A to hydrolyze GM2 ganglioside. The GM2 activator protein reversibly binds and solubilizes individual GM2 ganglioside molecules, making them available as substrate. Although GM2 ganglioside is the strongest binding ligand for the activator protein, it can also bind and transport between membranes a series of other glycolipids, even at neutral pH. Biosynthetic studies have shown that a large portion of newly synthesized GM2 activator molecules are not targeted to the lysosome, but are secreted and can then be recaptured by other cells through a carbohydrate independent mechanism. Thus, the GM2 activator protein may have other in vivo functions. We found that the GM2 activator protein can inhibit, through specific binding, the ability of platelet activating factor (PAF) to stimulate the release of intracellular Ca2+ pools by human neutrophils. PAF is a biologically potent phosphoacylglycerol. Inhibitors for PAF's role in the pathogenesis of inflammatory bowel disease and asthma have been sought as potential therapeutic agents. The inherent stability and protease resistance of the small, monomeric GM2 activator protein, coupled with the ability to produce large quantities of the functional protein in transformed bacteria, suggest it may serve as such an agent.     

Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry

The GM2-activator protein (GM2AP) is a small non-enzymatic cofactor assisting the enzyme beta-hexosaminidase A in the lysosomal degradation of ganglioside GM2. Mutations in the gene encoding this glycoprotein lead to a fatal neurological disorder, the AB variant of GM2-gangliosidoses. In this paper, we describe the overexpression of GM2AP in Sf21 cells using both the baculovirus expression vector system (BEVS) and a non-lytic, plasmid-based insect cell expression system (InsectSelect). For the BEVS, the cDNA encoding human GM2AP-preproprotein was cloned in the expression vector pAcMP3. The recombinant virus generated by cotransfection with linearized baculovirus DNA was used to infect Sf21 cells. For the non-lytic expression system, the cDNA of GM2AP was inserted into the vector pIZ/V5-His, which was used for the constitutive expression in stably transformed Sf21 cells. As it was shown by immunoblot analysis of the cell culture supernatant, in both expression systems the GM2AP precursor protein was efficiently secreted into the medium. Following expression in the BEVS, the GM2AP was purified by sequential chromatography on Ni-NTA-agarose and Con A-Sepharose, resulting in a yield of up to 9 mg purified protein from 1L of cell culture supernatant. Following expression in stably transformed insect cells, the secreted protein was first concentrated by cation-exchange and purified by metal-ion affinity chromatography, with a yield of 0.1 mg/L cell culture supernatant. The biological activity of the recombinant protein was demonstrated by its ability to stimulate the hexosaminidase A-catalyzed degradation of ganglioside GM2, and the homogeneity and glycosylation were assessed by ESI-TOF mass spectrometry. While the protein expression in the BEVS led to partly glycosylated and partly non-glycosylated protein, the stably transformed cells produced only glycosylated protein. In both expression systems, the glycosylation was found to be identical and corresponded to the structure (GlcNAc)(2)Fuc(Man)(3).