©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of an Alternatively Spliced G Activator Protein, G Protein
AN ACTIVATOR PROTEIN WHICH STIMULATES THE ENZYMATIC HYDROLYSIS OF N-ACETYLNEURAMINIC ACID, BUT NOT N-ACETYLGALACTOSAMINE, FROM G(*)

(Received for publication, December 29, 1995; and in revised form, February 1, 1996)

Yan Yun Wu Sandro Sonnino (1) Yu-Teh Li Su-Chen Li (§)

From the Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112 Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Milan 20133, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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


INTRODUCTION

It has been well established that the catabolism of ganglioside G(^1)requires the assistance of a protein cofactor called G activator protein(1, 2) . The physiological importance of G activator protein has been shown by the presence of an autosomal recessive genetic disease, the AB variant of Tay-Sachs disease, which is caused by the deficiency or the defect of G activator protein(3, 4, 5) . In addition to G activator protein, four other activator proteins for the catabolism of glycosphingolipids have been reported. These four activator proteins are derived from the proteolytic processing of a single precursor protein, prosaposin(6, 7, 8) , and have been named saposin A, B, C, and D according to their placements from the amino terminus of the prosaposin(9) . Saposin B is also known as a nonspecific activator protein and has been found to have a detergent-like activity which stimulates the hydrolyses of various glycolipids by different glycosidases(10) . Among the five activator proteins, only G activator protein is derived from a separate gene(11) . We have isolated two distinct cDNA clones for human G activator protein(12) . One of them, G activator cDNA, which has also been isolated by others(13, 14) , encodes almost the entire amino acid sequence of the native G activator protein isolated from human kidney (15) . The other clone, G cDNA, which was reported only by us, has an identical 5`-terminal sequence as that of G activator cDNA from nucleotides 1 to 302, but different for the next 346 nucleotides toward the 3` end. Klima et al.(16) isolated the genomic DNA which covered 94% of G activator cDNA, and identified the presence of three introns and four exons. The last exon, exon 4, spanned the segment coding for the carboxyl terminus of G activator protein and the entire 3`-untranslated region of the G activator cDNA. Comparing the sequence of G cDNA with this genomic DNA, we found that the last 346 nucleotides of G cDNA were identical to the sequence of 5` end of intron 3 (the exons and the introns are defined based on G activator mRNA). Thus, G mRNA is an alternative splicing product of G activator RNA in which the potential 5` splicing site between exon 3 and intron 3 is not subjected to the splicing process. As shown in Fig. 1, the coding region of G activator cDNA contains the end portion of exon 1, all of exons 2 and 3, and the front portion of exon 4. While the coding region of G cDNA contains the identical exon 1, 2, and 3 as in G activator cDNA, and a stretch of 9 nucleotides encoding a tripeptide, VST, at the COOH terminus which is derived from intron 3. This 9-nucleotide sequence is immediately followed by a stop codon.


Figure 1: Gene structure of G activator protein and G protein. E1, E2, E3, and E4 represent exon 1, exon 2, exon 3, and exon 4, respectively. I1, I2, and I3 represent intron 1, intron 2, and intron 3, respectively. The word ``stop'' means stop codon. VST is the last three amino acids encoded by intron 3. The sizes of the gene fragments are not in the exact proportion.



It has been postulated that the function of G activator protein is to extract a single G molecule from the micelles and to present the substrate-activator complex to beta-hexosaminidase A(17) , or to lift G from biological membranes where the sugar chain of G molecules may be shielded by other complex lipids with larger headgroups(18, 2) . In contrast, we have shown that the action of G activator protein in stimulating the hydrolysis of G by beta-hexosaminidase A may be due to its ability to recognize and interact with the branched trisaccharide of G, the G epitope(19) . This view is further supported by the finding that G activator protein also stimulates the hydrolysis of NeuAc from G by clostridial sialidase(19) . Based on the selective hydrolysis of only the terminal NeuAc residue in GalNAc-GD1a, we postulated that G activator protein can specifically recognize the G epitope in this ganglioside (20) .

Since the amino acid sequence encoded by G cDNA consists of the segment of G activator protein from amino acid residues 1 to 109 and an additional tripeptide sequence VST at the COOH terminus, the protein encoded by G cDNA (G protein) can be regarded as a truncated form of G activator protein. It is, therefore, important to compare the specificity of G protein with that of G activator protein. We have produced the recombinant G protein and found that this activator protein possesses only one of the two known activities of G activator protein.


EXPERIMENTAL PROCEDURES

Materials

G from Tay-Sachs brain (21) and the radioactive G(22, 23) were prepared as previously reported. The recombinant G activator protein and the recombinant saposin B were produced in Escherichia coli as described previously(19, 20) . beta-Hexosaminidase A (specific activity, 33.3 units/mg) (24) was isolated from human liver. GalNAc-G was isolated from the total ganglioside mixture of bovine brain(25) . The following reagents of the highest grade were obtained from commercial sources: clostridial sialidase Type X, isopropyl thio-beta-galactoside, ampicillin, and glutathione, Sigma; yeast extract and tryptone, Difco; restriction endonucleases and T4 DNA ligase, Life Technologies, Inc.; Taq DNA polymerase, Promega; T7 sequencing kit version 2.0, U. S. Biochemical Corp.; E. coli strain BL-21(DE3), Novagen; Universol (a scintillation mixture), ICN Biochemicals; the precoated Silica Gel-60 HPTLC plates, Merck (Darmstadt, Germany); Cellex D anion exchange cellulose and the prestained protein standard markers, Bio-Rad. A 23-mer peptide, PFKEGTYSLPKSEFVVPDLELPS-amide, which is identical to amino acids 106-128 of the mature G activator protein, was synthesized using a solid phase peptide synthesizer (Milligen 9050) by the Core Laboratories of Louisiana State University Medical Center.

Methods

Expression of G Protein

The fragment of G cDNA, which encodes amino acids 1-109 of the human G activator protein plus the tripeptide VST at the COOH terminus, was obtained by polymerase chain reaction using G-KS Bluescript as template. The upstream primer was: 5`-CGC-TCT-AGA-CGG-ATC-CCA-TAT-GTT-TTC-CTG-GGA-TAA-CTG-TGA-T-3` and the downstream primer was 5`-TCA-TCT-AGA-GGA-TCC-AAG-CTT-AGC-CAC-AGG-GGT-AAC-GCT-CTC-3`. This cDNA fragment was subcloned into pT7-7 expression vector at BamHI and HindIII sites, and was verified for its sequence. The recombinant G protein was expressed and purified according to the method described previously(19) . The production of G protein was assessed by SDS-PAGE of Laemmli (26) using silver staining and by Western blot analysis using polyclonal anti-G activator antibodies(5) . The NH(2)-terminal amino acid sequence of the purified G protein was confirmed by a gas-phase peptide sequencer (Applied Biosystems Model 477A).

Enzymatic Hydrolysis of NeuAc from G and GalNAc-G

Each ganglioside substrate in micellar form was incubated with the appropriate enzyme and the designated activator protein in a final volume of 100 µl at 37 °C. For the conversion of G into G, 40 µM G was incubated with 10 units of clostridial sialidase in 10 mM acetate buffer, pH 5.5, in the presence of activator protein. For the hydrolysis of NeuAc from GalNAc-G, 20 µM GalNAc-G was used. For quantitative analysis of the hydrolysis of NeuAc from G, the incubation conditions were identical to that described above, except three different concentrations of [^3H]G were used.

Enzymatic Hydrolysis of GalNAc from G

For the hydrolysis of GalNAc from G, 20 µM G was incubated at 37 °C with 0.2 units of beta-hexosaminidase A in 10 mM acetate buffer, pH 4.6, in the presence of the indicated amount of an activator protein. The quantitative analysis of the conversion of G to G was carried out by using [^3H]G as substrate at the indicated concentrations and measuring the release of [^3H]GalNAc.

Analysis of the Reaction Products

For analyzing the reaction products by TLC, the reaction was stopped by heating the tube in a bath of boiling water for 3 min, followed by adsorption of gangliosides on C18 beads as described previously(27) . The beads were then extracted by 0.5 ml of methanol and followed by 0.5 ml of chloroform/methanol (2:1, v/v)(27) . The extracts were combined, dried, and analyzed by TLC using the following solvents: chloroform/methanol/water (60:35:8, v/v/v) for the separation of G and G; chloroform/methanol/water (65:25:4, v/v/v) for the separation of G and G; methyl acetate, 1-propanol, chloroform, methanol, 0.25% KCl (25:20:20:20:17) for the separation of GalNAc-G, GalNAc-G, GalNAc-G, and GalNAc-G. The plates were sprayed with diphenylamine reagent (28) and heated at 110 °C for 15-20 min to reveal the glycosphingolipids. When [^3H]G was used, the reaction mixtures were evaporated to dryness under vacuum, then redissolved in 1 ml of chloroform/methanol (2:7) and passed through a DEAE-cellulose column (0.5 times 4 cm) which had been equilibrated with the same solvent. The column was washed with 4 ml of chloroform/methanol (2:7) and then eluted with 5 ml of chloroform/methanol (2:7) containing 20 mM sodium acetate. The breakthrough and the eluted fractions were separately collected, evaporated to dryness, dissolved in 0.5 ml of water, and mixed with 5 ml of Universol. The radioactivity was measured by using a Packard 1600CA liquid scintillation counter.


RESULTS AND DISCUSSION

Expression and Purification of G Protein

As in the case of the recombinant G activator protein(19) , the recombinant G protein produced in E. coli was also found to accumulate in the inclusion bodies. The same scheme used for the extraction, refolding, and purification of the recombinant G activator protein (19) was used for the preparation of the recombinant G protein. As shown in the SDS-PAGE profile (Fig. 2), the purified recombinant G protein (lane 2) moved at the position corresponding to 13.5 kDa, the calculated molecular size of G protein. It is considerably smaller than the recombinant G activator protein (Fig. 2, lane 3). The identity of G protein was also verified by the microsequencing of the NH(2)-terminal amino acid sequence. In Western blot analysis, G protein was also recognized by the polyclonal antibodies against G activator protein(5) .


Figure 2: SDS-PAGE analysis of the purified recombinant G protein and G activator proteins. A high density gel of Phamacia Phast system was used. Lane 1, the prestained protein standards: from the top, phosphorylase b, 112 kDa; bovine serum albumin, 84 kDa; ovalbumin, 53.2 kDa; carbonic anhydrase, 34.9 kDa; soybean trypsin inhibitor, 28.7 kDa; and lysozyme, 20.5 kDa. Lane 2, the recombinant G protein (0.2 µg). Lane 3, the recombinant G activator protein (0.2 µg). The gel was stained with silver staining.



Hydrolysis of NeuAc from G by Clostridial Sialidase in the Presence of G Activator Protein or G Protein

Our previous results indicate that G activator protein can stimulate the hydrolysis of both GalNAc and NeuAc from G by beta-hexosaminidase A and clostridial sialidase, respectively, and that it may be able to recognize the branched trisaccharide structure GalNAcbeta14(NeuAcalpha23)Gal-, the G epitope(19) . Since G protein contains only the NH(2)-terminal 109 amino acids of G activator protein without the COOH terminus 110-160 amino acids which were derived from exon 4, it would be important to examine whether or not this short version of the activator protein possesses the two known biological activities expressed by G activator protein. The TLC analysis of the conversion of G to G (Fig. 3A) showed that, as in the case of G activator protein (lane 4), G protein (lane 5) also stimulated the hydrolysis of NeuAc from G by clostridial sialidase. However, G protein was found to be slightly less effective than G activator protein. In order to compare the stimulatory potency of these two activator proteins, quantitative analysis was performed using [^3H]G at three different substrate concentrations instead of triplicates of one concentration. As shown in Fig. 3B, at all three substrate concentrations tested, the conversion of G to G was greatly enhanced by the presence of 2.5 µM of either G activator protein or G protein. G protein was about 20% less effective than G activator protein.


Figure 3: Effect of G activator protein and G protein on the hydrolysis of G by clostridial sialidase. A, each tube contained 40 µM G, 10 units of clostridial sialidase, and 2.5 µM of the indicated activator protein in a final volume of 100 µl as described under ``Experimental Procedures.'' The incubations were carried out at 37 °C for 16 h. M2, G; E, clostridial sialidase; P2, G activator protein; P2A, G protein. B, the incubation conditions were identical to that of A except using [^3H]G at three different concentrations as indicated. The mixtures were incubated at 37 °C for 3 h. The black bars represent the analyses performed in the absence of an activator protein, the white bars, in the presence of 2.5 µM G protein, and the hatched bars, in the presence of 2.5 µM G activator protein.



Hydrolysis of NeuAc from GalNAc-G by Clostridial Sialidase in the Presence of Activator Proteins

Previously we have shown that G activator protein specifically recognized and stimulated the hydrolysis of the NeuAc residue in the G epitope of GalNAc-G by clostridial sialidase(20) . We, therefore, examined the possible recognition of the same NeuAc residue in GalNAc-G by G protein. As shown in Fig. 4, G protein did preferentially stimulate the hydrolysis of the external NeuAc in GalNAc-G. In the presence of 2.5 µM G protein, the clostridial sialidase produced GalNAc-G as the major product and GalNAc-G as the minor product from GalNAc-G (Fig. 4A, lane 6). The same products were produced from GalNAc-G by the clostridial sialidase in the presence of 2.5 µM G activator protein as seen in Fig. 4A, lane 5. The products GalNAc-G, GalNAc-G, and GalNAc-G were analyzed by secondary ion mass spectrometry as described previously(20) . The strict specificity of G activator protein and G protein toward the hydrolysis of the terminal NeuAc from GalNAc-G was further demonstrated by the comparison of this result with that of the parallel experiments carried out in the presence of 10 or 20 µM saposin B. The concentrations of saposin B were chosen according to our previous experience(20) . Our results clearly showed that in addition to GalNAc-G and GalNAc-G, GalNAc-G was also produced from GalNAc-G in the presence of saposin B (Fig. 4A, 20 µM in lane 7 and 10 µM in lane 8). This indicates that saposin B can stimulate the hydrolyses of both NeuAc residues from GalNAc-G. Since the concentrations of saposin B used in Fig. 4A were much higher than that of the two other activator proteins, we repeated the experiment using 20 µM of each activator protein. As shown in Fig. 4B, in the presence of G protein (lane 2`) or G activator protein (lane 3`), only GalNAc-G and GalNAc-G were produced. However, in the presence of saposin B (lane 1`), GalNAc-G was also produced in addition to GalNAc-G and GalNAc-G. These results suggest that G activator protein and G protein have the same specificity in recognizing the external NeuAc residue of GalNAc-G. It is of interest to note that in the presence of 10 µM saposin B (Fig. 4A, lane 8), slightly more GalNAc-G was produced than GalNAc-G, while in the presence of 20 µM saposin B (Fig. 4A, lane 7), the reverse was observed. Thus, the concentration of saposin B appeared to influence the ratio of GalNAc-G to GalNAc-G produced from GalNAc-G. In contrast, the production of only GalNAc-G in the presence of G activator protein or G protein was not affected by the activator protein concentration. In spite of being different in their molecular sizes (18.5 kDa for G activator protein and 13.5 kDa for G protein), both G activator protein and G protein were able to stimulate the hydrolysis of the same NeuAc from GalNAc-G.


Figure 4: Hydrolysis of GalNAc-G by clostridial sialidase in the presence of G protein, G activator protein, or saposin B. A, each tube contained 20 µM GalNAc-G, 10 units of clostridial sialidase, and the activator protein as indicated in a final volume of 100 µl as described under ``Experimental Procedures.'' The incubations were carried out at 37 °C for 16 h. ND1a, GalNAc-G; D1a, G; E, clostridial sialidase; P2, G activator protein, 2.5 µM; P2A, G protein, 2.5 µM; P1, saposin B, 20 µM in lane 7 and 10 µM in lane 8. B, the incubation conditions were identical to A except each incubation mixture contained 20 µM of each activator protein.



The above results strongly suggest that G protein is also able to recognize the NeuAc residue in the G epitope and its mode of action in stimulating the liberation of NeuAc from G by clostridial sialidase is identical to that of G activator protein. In view of the facts that the amino acid sequence of G protein (except VST) is identical to the sequence of G activator protein from 1 to 109 and that both proteins have the same stimulatory activity for the hydrolyses of the NeuAc from G and GalNAc-G, it is logical to assign the NeuAc recognition domain of G activator protein to be within amino acids 1-109.

Hydrolysis of GalNAc from G in the Presence of G Activator Protein or G Protein

As shown in Fig. 5A, while G activator protein was an effective activator for the enzymatic hydrolysis of GalNAc from G (0.5 µM in lane 3, and 2.5 µM in lane 4), G protein did not stimulate the hydrolysis of GalNAc from G by beta-hexosaminidase A (0.5 µM in lane 5 and 2.5 µM in lane 6). The inability of G protein to stimulate the conversion of G to G was further confirmed by examining the reaction at three different substrate concentrations (Fig. 5B). These results indicate that whatever enables G activator protein to exert the stimulatory activity toward the hydrolysis of GalNAc from G by beta-hexosaminidase A is absent in G protein. It is reasonable to conclude that, in G activator protein, the portion of the peptide sequence encoded by exon 4 may govern the recognition of the GalNAc residue in G epitope and/or contribute to the formation of the essential conformation required for the specific recognition of the GalNAc residue. Thus, the 51 amino acids at the COOH terminus of G activator protein must be crucial to make the GalNAc residue accessible to beta-hexosaminidase A. The functional importance of the COOH-terminal segment of G activator protein is supported by the fact that a case of type AB Tay-Sachs disease was found to be caused by a Arg Pro mutation at the exon 4 coding region(29) . The Arg Pro mutation may disrupt the tertiary structure of G activator protein which is vital for its activity and/or stability. Although our results suggest that amino acids 1-109 of G activator protein is sufficient for the stimulation of the cleavage of NeuAc from G by clostridial sialidase and the 51 amino acids at the COOH terminus is essential for the stimulation of GalNAc cleavage from G by beta-hexosaminidase A, it is not possible at this point to ascertain if G activator protein requires a simultaneous interaction with both GalNAc and NeuAc residues in the G epitope for the stimulation of hydrolysis of GalNAc from G. The fact that G activator protein is not as effective in stimulating the hydrolysis of G as that of G by beta-hexosaminidase A (19, 30) suggests that the binding of both the NeuAc and the GalNAc residues in the G epitope may be necessary for the action of G activator protein on the hydrolysis of GalNAc from G. We have synthesized a 23-mer peptide (see ``Experimental Procedures'') which covers amino acids 106 through 128 of the G activator protein. This segment of the peptide is encoded mostly by exon 4. The synthetic 23-mer peptide showed neither the stimulatory activity for the hydrolysis of GalNAc nor for the hydrolysis of NeuAc from G. When this peptide was mixed with G protein, it did not enable G protein to stimulate the hydrolysis of GalNAc from G. Taken together, our results indicate that the recognition of both the NeuAc and GalNAc residues in the G epitope is a unique function of G activator protein. As in the case of G activator protein, G protein also requires the hydrophobic lipid moiety of the substrate to express the stimulatory activity, since the oligosaccharide derived from G was not hydrolyzed by beta-hexosaminidase A or clostridial sialidase in the presence of G protein.


Figure 5: Effect of G activator protein and G protein on the hydrolysis of G by beta-hexosaminidase A. A, TLC analysis showing the conversion of G to G. The incubations were carried out at 37 °C for 3 h. M2, G; E, beta-hexosaminidase A; P2, G activator protein; P2A, G protein. The numbers in parentheses (0.5 and 2.5) showed concentration (µM) of activator proteins used. The detailed incubation conditions are described under ``Experimental Procedures.'' B, quantitative analysis of the liberation of GalNAc from G by beta-hexosaminidase A in the presence of activator proteins. The incubation mixtures were essentially the same as A except [^3H]G at three indicated concentrations were used. The black bars represent the experiments performed in the absence of an activator protein, the white bars, in the presence of 2.5 µM G protein, and the hatched bars, in the presence of 2.5 µM G activator protein.



As reported previously, G mRNA was found in both human placenta and fibroblasts, although in a much lower abundance than the mRNA of G activator protein(12) . This suggests that the existence of an alternative splicing for G activator RNA may be physiologically important. Nature may use the alternative splicing of G activator RNA to direct the catabolism of G. The production of G activator mRNA or G mRNA should lead to the production of G activator protein or G protein, respectively. In the presence of G activator protein, the catabolism of G may preferentially go through the cleavage of GalNAc residue, which is a well established pathway for the catabolism of G. This pathway explains the biochemical bases of Tay-Sachs diseases caused by the deficiency or the defect of beta-hexosaminidase A, or G activator protein. However, in the presence of G protein the catabolism of G might shift to a possible alternate pathway, GG, since G protein can only stimulate the hydrolysis of NeuAc from G by clostridial sialidase, but not the hydrolysis of GalNAc. This would mean that the control of the production of G activator mRNA and G mRNA could be the branching point to direct the G hydrolysis to GG or GG pathways. The possible in vivo pathway for the conversion of G to G has been proposed by Riboni et al.(31) in studies on the Neuro2a cell line. The authors suggested that the G pathway was carried out by a specific sialidase which could convert G to G. It has also been suggested by Fingerhut et al.(32) that the degradation of gangliosides by lysosomal sialidase also required an activator protein. The question of the physiological role of G protein will remain unanswered until the native G protein is isolated. By SDS-PAGE and Western blotting analysis, we have observed the presence of a protein band corresponding to 13 kDa which reacted with the antibody against G activator protein in the partially purified placental G activator protein preparation. At the present time, the isolation of G protein is hampered by the lack of an assay method which can distinguish G protein from G activator protein.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant NS 09626 and Grant 93.02246.PF39 from the target project ``ACRO'' from Consiglio Nazionale delle Ricerche (CNR), Rome, Italy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112. Tel.: 504-584-2459; Fax: 504-584-2739.

(^1)
The abbreviations used are: G, II^3NeuAcGgOse(3)Cer; G, II^3NeuAcLacCer; GalNAc-G, IV^4GalNAc, IV^3NeuAc,II^3NeuAcGgOse(4)Cer; G, GgOse(3)Cer; PAGE, polyacrylamide gel electrophoresis.


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