Human GM2-activator Protein Research Articles

725. Assessment of CSF Route for Gene Delivery in Sandhoff Mice Using AAV9 Expressing an Hexosaminidase Isoenzyme

GM2 gangliosidoses are a group of neurodegenerative disorders, characterized by the malfunctioning Hexosaminidase A (HexA) enzyme. HexA is formed by heterodimerization of two subunits, α and β. Hex A, in interaction with GM2 activator protein (GM2AP), is the main isoenzyme able to hydrolyze GM2 gangliosides. HexA deficiencies result in Tay-Sachs (α-subunit deficiency) or Sandhoff disease (SD, β-subunit deficiency). In the recent work by Tropak et al. (Mol Ther Methods Clin Dev, in press), a hybrid human α-subunit, named “µ” and coded by HEXM, was created (patent pending) by incorporating the dimer stabilization and GM2AP binding sites of the β-subunit while maintaining the catalytic properties of the α-subunit. The µ-subunit is able to homodimerize to form a stable and functional enzyme, named HexM, which can interact with the human GM2AP and effectively hydrolyse GM2 gangliosides. Another advantage of this subunit (~1.6 kb) is that it can be packaged in a self-complementary adeno-associated virus (scAAV/HEXM). An intravenous route of scAAV administration has been shown to be successful, but brings with it translational issues including large scale viral preparation and relatively high vector uptake by liver. We tested the cerebrospinal fluid (CSF) route, as an alternative, for delivering scAAV9/HEXM. We injected 2.5E+11 vector genomes per mouse of scAAV9/HEXM via the cisterna magna at 6 weeks of age (n=13). Our controls include treatment with scAAV9/GFP (n=3) and vehicle (n=8). One additional cohort received an IV injection of 25% mannitol (2g/kg) post-AAV9 injection (n=10). We sacrificed part of the cohorts (n=4 each group) at 16 weeks of age (humane end-point of untreated SD mice) for tissue analysis. The remainder are being monitored for long-term survival. The parameters for analyses are survival benefit, locomotor behaviour, Hexosaminidase activity, GM2 ganglioside accumulation, and vector genome biodistribution. The preliminary results from this ongoing study show a significant survival advantage to the humane endpoint in HexM treated (average 28 weeks to date) as compared to negative controls (~16 weeks). The behaviour tests showed improved locomotor activity in HexM-treated mice as compared to negative controls. Results were similar when mannitol was administered in conjunction with scAAV9/HEXM. These preliminary results indicate that the intra-CSF route of administration of AAV9/HEXM is a tractable translatable approach for SD worth further exploration. Additional studies are focused on increased dosage and methods to improve distribution.

Chemical Synthesis of Biologically Active Monoglycosylated GM2‐Activator Protein Analogue Using N‐Sulfanylethylanilide Peptide

Going to SEA(lide): Total chemical synthesis of a 162-residue glycoprotein analogue of the monoglycosylated human GM2-activator protein (GM2AP) was achieved. Key steps were the use of N-sulfanylethylanilide (SEAlide) peptides in the kinetic chemical ligation synthesis of a large peptide fragment, and a convergent native chemical ligation for final fragment assembly.

Human Urinary GM2-activator Protein as a Potential Biomarker for Lung Cancer

Human urine can be obtained from most patients by noninvasive ways of collection. It is a rich source of biomarkers for diagnostic tests, therapeutic guidance and prognostic information for patients and clinicians. Recently, the protein expression pattern of urine samples from lung cancer patients and healthy controls were compared using two-dimensional gel electrophoresis (2-DE). GM2 activator protein (GM2AP) in lung cancer patients with 2.5-4.0 folds higher than that found in healthy controls was the protein of interest, and this protein was focused because it is related to tumor-associated gangliosides found in cancer progression. The aim of this study was to investigate the urinary biomarkers associated with lung cancer. First, we applied proteomic approach to confirm the increase level of GM2AP in urine samples from lung cancer patients and healthy controls. Then, the expression level of urinary GM2AP was quantified using Western blotting and Enzyme-Linked Immunosorbent Assay (ELISA). The results show that the expression level of GM2AP significantly increased in each subtype of lung cancer patients (P<0.05). In addition, nano liquid chromatography coupled online with tandem mass spectrometry (NanoLC-MS/ MS) provided evidence of an increase of GM2AP with the glycan structure identification from doublely charge at m/z 1202.1025 corresponding to the structure of (Hex)3(HexNAc)2(Fuc)1 linked with peptide (PIIVOGNVTLSVVG). Overall data suggested that the GM2AP may be associated with lung cancer and may be useful as a marker to monitor lung cancer prognosis.

A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

Functional characterisation of a nematode secreted GM2-activator protein

We have identified a GM2-activator protein (GM2AP) with highly unusual properties secreted by the nematode parasite Trichinella spiralis. Expression in Pichia pastoris resulted in a hyperglycosylated protein of 28 kDa, but the 18 kDa native protein was not glycosylated. The parasite GM2AP does not facilitate degradation of GM2 ganglioside by N-acetyl-β-hexosaminidase A, although it does inhibit phospholipase D activity. Lack of the former activity might be explained by the absence of a domain implicated in binding to hexosaminidase. In addition, and contrary to data on the human GM2AP, the nematode homologue does not inhibit platelet activating factor-induced calcium mobilisation in neutrophils, but actually enhances mediator-induced chemotaxis.

Oxidized Low-density Lipoprotein Upregulates GM2 Activator Protein Gene Expression

Oxidized low-density lipoprotein (LDL) has been shown to be a powerful regulator of gene expression in monocyte-derived macrophage. To determine the effects of oxidized LDL on macrophage gene expression, macrophages incubated with native or oxidized LDL were analyzed by differential display technique. The differentially expressed cDNA (387 bp) fragment by oxidized LDL showed 100% homology to the part of 3’-untranslated region of the gene of human GM2 activator protein. Quantitative RT-PCR data using specific TaqMan probe showed the increased GM2 activator protein mRNA expression in macrophages incubated with oxidized LDL by about twice (215±4 31.6 %) of that in macrophages incubated with native LDL. Our results shows that ox-LDL upregulates GM2 activator protein gene expression, indicating that GM2 activator protein may be associated with the pathogenesis of atherosclerosis.

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 β-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 1 L 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) 2Fuc(Man) 3.

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.

Degradation of GM1 and GM2 by mammalian sialidases

In mammalian tissues, the pathway known for the catabolism ofGM1[Galβ3GalNAcβ4(Neu5Acα3)Galβ4GlcCer;where Cer is ceramide] is the conversion of this ganglioside into GM2 [GalNAcβ4(Neu5Acα3)Galβ4GlcβCer] by β-galactosidase followed by the conversion of GM2 into GM3 (Neu5Acα3Galβ4GlcβCer) by β-N-acetylhexosaminidase A (Hex A). However, the question of whether or not GM1 and GM2 can also be respectively converted into asialo-GM1 (Galβ3GalNAcβ4Galβ4GlcCer; GA1) and asialo-GM2 (GalNAcβ4Galβ4GlcβCer, GA2) 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 GM1 and GM2 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 GM2 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 GA2 pathway) in mouse to convert GM2 into GA2 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 GM1 and GM2. We found that the mouse membrane-bound sialidase was able to convert GM1 and GM2 into their respective asialo-derivatives in the presence of human or mouse GM2 activator protein. The cytosolic sialidase did not exhibit this activity. Our results suggest that, in vivo, the stable NeuAc of GM1 and GM2 may be removed by the mammalian membrane-associated sialidase in the presence of GM2 activator protein. They also support the presence of the GA2 pathway for the catabolism of GM2 in mouse.

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

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

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Crystal structure of human GM2-activator protein with a novel β-cup topology

GM2 activator protein (GM2-AP) belongs to a small group of non- enzymatic lysosomal proteins that act as cofactors in the sequential degradation of gangliosides. It has been postulated that GM2-AP extracts single GM2 molecules from membranes and presents them in soluble form to β-hexosaminidase A for cleavage of N-acetyl- d-galactosamine and conversion to GM3. The high affinity of GM2-AP for GM2 is based on specfic recognition of the oligosaccharide moiety as well as the ceramide lipid tail. Genetic defects in GM2-AP result in an atypical form of Tay-Sachs disease known as variant AB GM2 gangliosidosis. The 2.0 Å resolution crystal structure of GM2-AP reported here reveals a previously unobserved fold whose main feature is an eight-stranded cup-shaped anti-parallel β-pleated sheet. The striking feature of the GM2-AP structure is that it possesses an accessible central hydrophobic cavity rather than a buried hydrophobic core. The dimensions of this cavity (12 Å × 14 Å × 22 Å) are suitable for binding 18-carbon lipid acyl chains. Flexible surface loops and a short α-helix decorate the mouth of the β-cup and may control lipid entry to the cavity.

Characterization of an Alternatively Spliced GM2 Activator Protein, GM2A Protein: AN ACTIVATOR PROTEIN WHICH STIMULATES THE ENZYMATIC HYDROLYSIS OF N-ACETYLNEURAMINIC ACID, BUT NOT N-ACETYLGALACTOSAMINE, FROM GM2

GM2 activator protein is a protein cofactor which stimulates the enzymatic hydrolysis of both GalNAc and NeuAc from GM2. We have previously isolated two cDNA clones, GM2 activator cDNA and GM2A cDNA, for human GM2 activator protein (Nagarajan, S., Chen, H.-C., Li, S.-C., Li, Y.-T., and Lockyer, J. M. (1992) Biochem. J. 282, 807-813). GM2A mRNA is an RNA alternative splicing product that contains exons 1, 2, 3, and intron 3 of the genomic DNA sequence of GM2 activator protein (Klima, H., Tanaka, A., Schnabel, D., Nakano, T., Schröder, M., Suzuki, K., and Sandhoff, K. (1991) FEBS Lett. 289, 260-264). GM2A cDNA encodes a protein (GM2A protein) containing 1-109 of the 160 amino acids of human GM2 activator protein, plus a tripeptide (VST) encoded by intron 3 at the COOH terminus. Thus, GM2A protein can be regarded as a form (truncated version) of GM2 activator protein. We have expressed GM2A cDNA in Escherichia coli using pT7-7 as the vector. The recombinant GM2A protein was purified to an electrophoretically homogeneous form and was found to stimulate the hydrolysis of NeuAc from GM2 by clostridial sialidase, but not the hydrolysis of GalNAc from GM2 by beta-hexosaminidase A. Like GM2 activator protein, GM2A protein also specifically recognized the terminal GM2 epitope in GalNAc-GD1a and stimulated the hydrolysis of only the external NeuAc from this ganglioside by clostridial sialidase. These results enabled us to discern the enzymatic hydrolyses of GalNAc and NeuAc from the GM2 epitope and established that the NeuAc recognition domain of GM2 activator protein is located within amino acids 1-109. The presence of GM2A mRNA in human tissues and the selective stimulation of NeuAc hydrolysis by GM2A protein indicate that this activator protein may be involved in the catabolism of GM2 through the asialo-GM2 pathway.

Specific Recognition of N-Acetylneuraminic Acid in the GM2 Epitope by Human GM2 Activator Protein

GM2 Activator is a low molecular weight protein cofactor that stimulates the enzymatic conversion of GM2 into GM3 by human beta-hexosaminidase A and also the conversion of GM2 into GA2 by clostridial sialidase (Wu, Y.-Y., Lockyer, J.M., Sugiyama, E., Pavlova, N.V., Li, Y.-T., and Li, S.-C. (1994) J. Biol. Chem. 269, 16276-16283). Among the five known activator proteins for the enzymatic hydrolysis of glycosphingolipids, only GM2 activator is effective in stimulating the hydrolysis of GM2. However, the mechanism of action of GM2 activator is still not well understood. Using a unique disialosylganglioside, GalNAc-GD1a, as the substrate, we were able to show that in the presence of GM2 activator, GalNAc-GD1a was specifically converted into GalNAc-GM1a by clostridial sialidase, while in the presence of saposin B, a nonspecific activator protein, GalNAc-GD1a was converted into both GalNAc-GM1a and GalNAc-GM1b. Individual products generated from GalNAc-GD1a by clostridial sialidase were identified by thin layer chromatography, negative secondary ion mass spectrometry, and immunostaining with a monoclonal IgM that recognizes the GM2 epitope. Our results clearly show that GM2 activator recognizes the GM2 epitope in GalNAc-GD1a. Thus, GM2 activator may interact with the trisaccharide structure of the GM2 epitope and render the GalNAc and NeuAc residues accessible to beta-hexosaminidase A and sialidase, respectively.

Identification of a lysosomal protein causing lipid transfer, using a fluorescence assay designed to monitor membrane fusion between rat liver endosomes and lysosomes.

In the present and previous studies [Mullock, Perez, Kuwana, Gray and Luzio (1994) J. Cell Biol. 126, 1173-1182], we have attempted to investigate endosome-lysosome fusion using an assay based on the dilution of the self-quenching fluorescent lipid probe octadecylrhodamine. Although some characteristics of fluorescence dequenching were consistent with those observed in other cell-free assays, we have now demonstrated that increased fluorescence was due to leakage of an intralysosomal lipid-transfer protein. This protein was purified and found to be a 22 kDa molecule with sequence, immunological and functional characteristics strongly suggesting that it is the rat homologue of human GM2-activator protein. Both the 22 kDa protein and recombinant human GM2-activator protein caused fluorescence dequenching either when mixed with octadecylrhodamine-loaded endosomes and lysosomal membranes or in a liposome system. The data were consistent with GM2-activator protein acting as an octadecylrhodamine-transfer protein. Antibodies to the 22 kDa protein added to cell-free endosome-lysosome content-mixing assays had no effect, although they could inhibit fluorescence dequenching caused by the protein. Thus this protein is not required in any fusion event involved in delivery of ligands from endosomes to lysosomes. The existence within an intracellular organelle of a protein capable of acting as an octadecylrhodamine-transfer protein suggests the need for caution in the interpretation of fluorescence-dequenching assays using mammalian subcellular fractions.

The Mouse Gene Encoding the GM2 Activator Protein (Gm2a): cDNA Sequence, Expression, and Chromosome Mapping

The GM2 activator protein forms a substrate-complex with GM2 ganglioside, which enables degradation of the ganglioside by beta-hexosaminidase A. Mutations in the human GM2 activator protein gene (GM2A) result in the GM2 gangliosidosis AB variant, a severe neurological disease. We have isolated and sequenced a mouse GM2 activator protein (Gm2a) cDNA with complete protein coding and 3' untranslated regions. Expression of the Gm2a transcript (approximately 2.3 kb) was apparent in all tissues examined and was most abundant in kidney and testis. The Gm2a gene was mapped to a region on mouse chromosome (Chr) 11 that is homologous with a segment of human chromosome 5 containing the orthologous human gene. In addition, a Gm2a-related sequence (Gm2a-rs1) was mapped to mouse Chr 5.

Expression and specificity of human GM2 activator protein.

The cDNA encoding GM2 activator was expressed in the Escherichia coli/pT7-7 system. The yield of the GM2 activator with greater than 99% purity was about 3 mg per liter culture. The recombinant GM2 activator was found to be as active as that isolated from human kidney. The availability of the recombinant GM2 activator enabled us to critically examine the specificity of this activator protein. Our results show that the specificity of GM2 activator is not as strict as that reported previously. Although GM2 activator stimulates most efficiently the degradation of GM2 carried out by beta-N-acetylhexosaminidase A (Hex A), this activator also stimulates the following reactions: (a) conversion of GM2 to GA2 by clostridial sialidase; (b) hydrolysis of GalNAc from dipalmitoylphosphatidylethanolamine-II3NeuAcGgOse3 by Hex A; and (c) liberation of Gal from GM1 by beta-galactosidase at a high activator concentration. Thus, this activator does not differentiate between GM2 and dipalmitoylphosphatidylethanolamine-II3NeuAcGgOse3 or between Hex A and clostridial sialidase. The micellar forms of GD2 and GalNAc-GD1a were found to be more readily hydrolyzed by Hex A than GM2 in the absence of GM2 activator. Our results also show that saposin B can enhance the stimulatory activity of GM2 activator, but it cannot promote the stimulatory activity of sodium taurodeoxycholate. Taken together, our results suggest that the mechanism of action of GM2 activator is different from saposin B, and the action of GM2 activator is more than to solubilize lipid substrates. The effectiveness of GM2 activator in stimulating the hydrolysis of GM2 may be due to its ability to recognize the specific trisaccharide structure of the GM2 epitope, GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal-, and to modify the GalNAc-NeuAc interaction in this structure.

Cloning and sequence analysis of a cDNA clone coding for the mouse GM2 activator protein.

A cDNA (1.1 kb) containing the complete coding sequence for the mouse GM2 activator protein was isolated from a mouse macrophage library using a cDNA for the human protein as a probe. There was a single ATG located 12 bp from the 5' end of the cDNA clone followed by an open reading frame of 579 bp. Northern blot analysis of mouse macrophage RNA showed that there was a single band with a mobility corresponding to a size of 2.3 kb. We deduce from this that the mouse mRNA, in common with the mRNA for the human GM2 activator protein, has a long 3' untranslated sequence of approx. 1.7 kb. Alignment of the mouse and human deduced amino acid sequences showed 68% identity overall and 75% identity for the sequence on the C-terminal side of the first 31 residues, which in the human GM2 activator protein contains the signal peptide. Hydropathicity plots showed great similarity between the mouse and human sequences even in regions of low sequence similarity. There is a single N-glycosylation site in the mouse GM2 activator protein sequence (Asn151-Phe-Thr) which differs in its location from the single site reported in the human GM2 activator protein sequence (Asn63-Val-Thr).

Over-expression of a functionally active human GM2-activator protein in Escherichia coli

The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17. The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus. The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded. The hexahistidine tail could be removed when desired by digestion with Factor Xa. In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator.

Evidence for two cDNA clones encoding human GM2-activator protein.

Two cDNAs encoding GM2 activator, pGM2A (648 bp) and GAP (1093 bp), were isolated from human placenta lambda gt11 libraries. The DNA sequence of pGM2A from 1 to 302 was almost identical with GAP, but diverged from 303-648. PCR was used to demonstrate the presence of both species of GM2 activator in placental RNA. Both cDNAs hybridized to mRNAs of approximately 2.3 kb and to identical single bands on genomic Southern blots.