3Department of Molecular, Cellular, and Craniofacial Biology, School of Dentistry and Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, 501 S. Preston St., Louisville, KY 40292, USA, 4Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA, and 5James G. Brown Cancer Center, University of Louisville, Louisville, KY 40292, USA
Received on August 23, 2000; revised on October 2, 2000; accepted on October 4, 2000.
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Abstract |
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Key words: DXD motif/glycosyltransferase/GM2 synthase/ganglioside/Golgi
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Introduction |
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To date, little structural information is available concerning the catalytic site of GM2 synthase. Recently, however, an aspartateany residueaspartate (DXD) motif was found to be conserved in 13 families of glycosyltransferases, including GM2 synthase (Breton et al., 1998; Wiggins and Munro, 1998
), and in several of those families, this motif was found to be critical for enzymatic activity (Busch et al., 1998
; Wiggins and Munro, 1998
; Keusch et al., 2000a
,b; Moloney et al., 2000
; Munro and Freeman, 2000
). This motif is hhhhDxDxh, where h is a hydrophobic residue and x is any residue (Wiggins and Munro, 1998
), and in human GM2 synthase the motif is found in the sequence 352-VLWVDDDFV (Nagata et al., 1992
; Wiggins and Munro, 1998
). Mutation of the conserved aspartates of the motif in the large clostridial cytotoxins, which glucosylate Rho- and Ras-family GTPases, prevented photolabeling with an analog of UDP-glucose (Busch et al., 1998
). This finding led to the proposal that the DXD motif may participate in coordination of a divalent metal ion that is required for the binding of nucleotide sugar (Busch et al., 1998
). In striking contrast, mutations of the DXD motif in the Fringe signaling molecule eliminated biological activity (Moloney et al., 2000
; Munro and Freeman, 2000
) but did not alter photolabeling by a UDP analog (Munro and Freeman, 2000
). The conclusion drawn from this latter study that the DXD motif was not critical to UDP binding was supported by the crystal structure of the SpsA glycosyltransferase from Bacillus subtilis (Charnock and Davies, 1999
), which indicated that binding of nucleotide sugar could be mediated by residues outside the DXD motif and that the role of the DXD motif itself could be to assist leaving group departure during catalysis through coordination of a manganese ion. In summary, the differences reported on the cytotoxins as compared to Fringe raise the possibility that not all DXD motifs are functionally equivalent.
The purpose of the present study was to determine whether the DXD motif in GM2 synthase was similar to the motif described in the cytotoxins or to the motif in Fringe. We mutated residues within the DXD motif in GM2 synthase to determine its importance to GM2 synthase function and nucleotide binding. Our results indicate that the DXD motif in GM2 synthase is required for enzymatic activity but is not critical to the binding of UDP, findings similar to those described for Fringe (Munro and Freeman, 2000) and dissimilar to those for the large clostridial cytotoxins (Busch et al., 1998
).
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Results |
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As a second measure of proper folding, we assessed the ability of each mutated protein to form homodimers by analyzing anti-myc Western blots of cell extracts run under nonreducing conditions (Figure 2A). All of the mutants were able to produce some homodimers, but the ratio of dimer to monomer varied (Table I). Those closest to the wild-type ratio of 4.9 were the D356E, D356N, D357N, and D358N mutations (Table I). The DDD/NNN triple mutation as well as the D358A, V352A, and W354A mutations all had dimer to monomer ratios (Table I) significantly lower than wild-type enzyme indicating that these latter mutations experienced problems of folding that prevented proper dimer formation. The lowest ratio of dimer to monomer was seen for the D356A mutation and the double mutation DDD/ADA (Table I).
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Effect of mutations on enzyme activity in whole cells
We first assessed by flow cytometry the ability of GM2 synthase mutants to produce GM2 ganglioside in whole cells. As we reported previously (Jaskiewicz et al., 1996b), clone C5 cells expressing a high level of wild-type GM2 synthase/myc displayed extremely strong staining with anti-GM2 (Figure 4). In striking contrast, none of the cells expressing mutations of the conserved aspartates had detectable GM2 on the cell surface (Figure 4). Only on the D356A expressing cells was the anti-GM2 staining slightly brighter than untransfected CHO cells, but on those D356A expressing cells, staining with the negative control anti-LeY antibody was essentially the same as anti-GM2 (data not shown). Thus, mutation of the highly conserved D356 and D358 as well as D357 to either Ala, Asn, or Glu reduced enzyme activity to background levels as defined by anti-GM2 flow cytometry. The only mutations that retained strong cell surface GM2 staining were V352A and W354A, both of which had cells that were even brighter than wild-type clone C5 cells (Figure 4, bottom row). The heterogeneity in GM2 display in W354A cells may reflect instability of GM2 synthase expression but is not the result of total loss of enzyme activity because all cells were cholera toxin positive (see below and Figure 5).
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The cells expressing the V352A mutated enzyme were strongly cholera toxin B subunit positive (Figure 5), consistent with the strong GM2 positive nature of these cells (Figure 4). Surprisingly, the cells expressing the W354A mutated enzyme were all positive for cholera toxin B subunit, and the majority were strongly positive (Figure 5) even though the majority of these cells were negative or very weak for GM2 cell surface display (Figure 4). Therefore, the GM2-negative cells still expressed GM2 synthase activity, but all of the GM2 that was produced was utilized as metabolic intermediate and was converted to GM1 product. In summary, flow cytometry demonstrated that mutation of either D356 or D358 to Ala or Asn produced an enzyme with no detectable activity, but the D356E mutant exhibited weak activity, indicating the importance of maintaining the negative charge at that conserved position.
Effect of mutations on enzyme activity in vitro
Examples have been reported of glycosphingolipids being present on the cell surface but being inaccessible or cryptic to probes such as antibodies or galactose oxidase (Urdal and Hakomori, 1983; Hamilton et al., 1994; Tatewaki et al., 1997
). Therefore, we also tested for the activity of GM2 synthase by in vitro assay of cell extracts (Table I). None of the mutations of the highly conserved D356 or D358 or the intervening D357 had activity above that of the background level of untransfected CHO cells (Table I). This included the D357N and D356E mutations which exhibited weak activity as detected by cholera toxin B subunit flow cytometry (Figure 5). Thus, the GM2 synthase in vitro activity of these mutated enzymes was more than 1800-fold reduced compared to wild-type enzyme in clone C5 cells. The only mutations showing detectable activity of the cell extracts were V352A and W354A (Table I), in agreement with these two mutations being the only ones that were positive by anti-GM2 flow cytometry (Figure 4).
The levels of in vitro enzyme activities of cell extracts are dependent on several factors, such as expression level in the case of a clonal cell population, stability of expression, or percent of the cell population that is expressing the enzyme in the case of a mixed cell population. In contrast, activity values for released enzyme provide a valid measure of enzyme-specific activity when those values are normalized based on the amount of enzyme present. Therefore, we determined the activity of the released mutant enzymes by in vitro assay and then normalized the data based on the intensities of anti-myc positive bands in the culture supernatants by Western blotting. No activity above background was detected for the released mutant enzymes D358A, D356N, D358N, DDD/NNN, or D356E (Table I), even though D356E exhibited activity by cholera toxin B subunit flow cytometry (Figure 5). However, the D357N mutation released enzyme did possess activity above background (Table I), in agreement with cholera toxin display being two- to threefold brighter on these cells than on D356E cells (Figure 5). The specific activity of the V352A mutated enzyme was actually higher than wild-type, and the specific activity of the W354A mutated enzyme was approximately fourfold less than wild-type (Table I). In summary, we could not detect any activity in vitro of cell extracts or of soluble enzyme in the culture supernatant when the invariant Asp 356 or 358 were mutated, indicating the critical importance of these residues for catalysis. Among the other residues within the DXD motif, D357 is also of great importance, whereas V352 and W354 influence GM2 synthase activity to a lesser extent.
Divalent metal ion specificity of GM2 synthase
Conserved acidic amino acids have been found in other proteins to participate in metal ion coordination (Wiggins and Munro, 1998). Although Mn2+ has been commonly used for GM2 synthase activity assays (Ruan and Lloyd, 1992
; Jaskiewicz et al., 1996b
), the metal ion specificity of this enzyme is unclear. Whereas the rank order for cation requirement for GM2 synthase purified from rat liver was Ni2+ >> Mn2+
Fe2+ > Cu2+ > Zn2+
Co2+, Mg2+, and Ca2+ (Yanagisawa et al., 1987
), the order for the enzyme purified from mouse liver was Mn2+ > Co2+ > Ca2+
Mg2+
Fe2+ > Ni2+ (Hashimoto et al., 1993
). To clarify this discrepancy, we determined the activity of the soluble form of wild-type GM2 synthase from clone GTm1 (Zhu et al., 1998
) to be the following when tested in the presence of 3.5 mM metal (expressed as a percent of the activity in the presence of Co2+): Co2+ 100%, Mn2+ 70.1%, Ni2+ 45.7%, Cd2+ 17.1%, Mg2+ 2.6%, Ca2+ 1.3%. We then tested whether the activity of mutant enzymes could be restored by replacing Mn2+ with other metals. However, none of the soluble forms of the D356N, D358A, D358N, and D356E mutants displayed detectable activity in the presence of 1 mM Co2+, Ni2+, or Cd2+ (data not shown).
Effect of mutations on binding to UDP-beads
Mutation of the conserved aspartate residues of the DXD motif of large clostridial cytotoxins prevented their labeling with the photoaffinity probe azido-UDP-glucose, thus indicating an important role for the motif in nucleotide sugar binding (Busch et al., 1998). However, mutation of the aspartates of the DXD motif in Fringe had minimal effect on labeling with a UDP analog (Munro and Freeman, 2000
). To determine the role of the DXD motif in nucleotide binding in GM2 synthase, we tested for the binding of soluble, wild-type, and mutant GM2 synthase to UDP-beads. As expected, wild-type enzyme bound to UDP-beads in the presence of Mn2+ (Figure 6, lane 1). Surprisingly, however, binding was nearly as great in the presence of 5 mM EDTA (Figure 6, lane 3) indicating that Mn2+ was not critical to nucleotide sugar binding in GM2 synthase. Binding of wild-type enzyme to UDP-beads in the presence of Mn2+ was inhibited more than 80% by the presence of 22 mM UDP (Figure 6, lane 2), which was a 1000-fold excess of UDP as compared to the number of UDP coupled to the beads. The fact that such a relatively high concentration of inhibitor was necessary suggests the possibility that each monomer of the GM2 synthase homodimer has a functional nucleotide sugar binding site, and, therefore, the interaction being inhibited is between a bivalent enzyme and the multivalent beads. Precedents in the literature for the greatly increased affinity or avidity of such interactions include the 3000-fold difference in dissociation constants of the cation-independent mannose 6-phosphate receptor for oligosaccharides having two mannose 6-phosphate termini instead of one (Tong et al., 1989
).
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The soluble form of the DDD/NNN mutant of GM2 synthase also bound to UDP-beads both in the presence of Mn2+ (Figure 6, lane 5) as well as in the presence of EDTA (Figure 6, lane 7). Furthermore, this binding was inhibited by 22 mM UDP (Figure 6, lanes 6 and 8) although the extent of inhibition was not as great as for wild-type enzyme (Figure 6, lanes 2 and 4). These findings demonstrated that the DXD motif of GM2 synthase was not critical for the binding of nucleotide. In summary, these binding data suggest that GM2 synthase may be similar to Fringe in which the DXD motif is not critical to labeling with a UDP analog (Munro and Freeman, 2000).
Photoaffinity labeling
As a separate test for the role of the DXD motif in nucleotide sugar binding, we compared the ability of the soluble forms of wild-type and mutant GM2 synthase to be labeled with the photoaffinity probe AAUTP (Rancour and Menon, 1998). In initial experiments we determined that AAUTP was a potent inhibitor of GM2 synthase activity. The IC50 of AAUTP was 23 µM, lower than the Km for UDP-GalNAc, which we measured to be 76 µM [similar to the 71 µM Km value determined for UDP-GalNAc previously (Ruan and Lloyd, 1992
)]. The ability of AAUTP to inhibit at such a low concentration relative to the Km for UDP-GalNAc suggested strong but possibly complex interactions between AAUTP and GM2 synthase. As suggested in other work with AAUTP (Kostova et al., 2000), it is possible that binding of the photoprobe to the sugar nucleotide binding pocket of the enzyme is reinforced by hydrophobic interactions between the arylazide moiety of the photoprobe and a hydrophobic region of the enzyme, such as the GM3 binding pocket. To analyze this possibility further and to test whether the photoprobe was differentially recognized by wild-type and mutant GM2 synthase, we carried out photolabeling studies with AAUTP[
-32P]. To quantitate our results, we normalized the autoradiogram signal resulting from photolabeling to an immunoblot signal of the same material obtained using anti-myc antibodies.
Labeling of wild-type GM2 synthase with AAUTP[-32P] was UV-irradiation dependent (compare Figure 7, lanes 1 and 2), and could be partially competed by unlabeled UDP, but not GDP, at concentrations near the Km for nucleotide sugar substrate (Figure 7, lanes 3 [UDP] and 4 [GDP], compared to lane 2 [no competitor]). These data indicate that photolabeling is nucleotide specific.
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To address directly the role of the DXD motif in binding of nucleotide sugar, we labeled the soluble form of the DDD/NNN mutant. Because this mutant protein was secreted at a lower level than wild-type enzyme, the SP-Sepharose purified mutant protein was only about 10% pure as compared to the wild-type, which was 8090% pure. Therefore, control experiments were performed that determined that the presence of background proteins did not significantly alter the amount of AAUTP cross-linked to GM2 synthase (data not shown). Hence, the ability to directly compare the photoaffinity labeling of the more highly purified wild-type enyzme to the DDD/NNN protein-containing mixture was concluded to be feasible.
Figure 9 shows that the DDD/NNN mutant form of GM2 synthase was photolabeled with AAUTP[-32P] and that the extent of labeling was equal to or slightly greater than that seen for the wild-type enzyme. This result indicates that AAUTP binds to the mutant as well as or better than to wild-type enzyme. In apparent contrast, the DDD/NNN mutant was less susceptible than wild type to inhibition by free UDP for its binding to the affinity matrix (Figure 6) which would indicate that the mutant binds UDP less avidly than the wild-type enzyme. This apparent disparity is due in part to the fact that these methods are based on different assay formats, involving different substrate presentation. Despite this difference, overall the photolabeling results reinforce the conclusion drawn from the binding experiments with UDP-beads and suggest that GM2 synthase is capable of recognizing nucleotides (and, by implication, its nucleotide sugar substrate) even when the DDD motif is compromised.
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Discussion |
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The effect of mutation of the residue between the highly conserved Asp residues differed, however. In the case of MNN1, mutation of Ile431 to Ala only reduced activity by 30%, whereas for GM2 synthase mutation of Asp357 to Asn destroyed detectable activity of cell extracts, reduced the specific activity of the soluble form of the enzyme approximately 125-fold (Table I), and reduced the in vivo activity such that only a small amount of GM2 was produced which was detectable as cell surface GM1 (Figures 4 and 5). Thus, the additional Asp at 357 of GM2 synthase may participate along with the invariant Asp at 356 and 358 to facilitate catalysis. Among the glycosyltransferases in which the effect of the DXD motif on enzyme activity has been studied, the only other enzyme possessing an Asp as the middle amino acid between the two invariant Asp is Fringe (Moloney et al., 2000; Munro and Freeman, 2000
). To date, the middle Asp of Fringe has not been separately mutated to assess its role in enzyme activity.
Effect of DXD mutations on binding of nucleotide
Mutation of either or both of the invariant Asp of the DXD motif of the large clostridial cytotoxins prevented labeling with azido-UDP-glucose (Busch et al., 1998). These findings led to a model in which the two invariant Asp residues of the motif might coordinate Mn2+, which in turn would be important for UDP-glucose binding by interaction with the two phosphate moieties of UDP. For two reasons our present results with binding to UDP-beads and labeling by AAUTP indicate that this model does not apply to GM2 synthase. First, binding of wild-type GM2 synthase to UDP-beads was not dependent on the presence of Mn2+ but instead occurred to nearly the same extent in the presence of EDTA as in the presence of Mn2+ (Figure 6). Therefore, although it is still possible that Mn2+ coordination of the phosphates of UDP may contribute to binding of UDP-GalNAc in GM2 synthase, that contribution is not critical to binding. Second, the soluble form of the DDD/NNN mutant bound to UDP-beads (Figure 6) and was labeled by AAUTP (Figure 7), indicating that binding of nucleotide was not dependent on the invariant Asp residues of the motif.
The Notch signaling regulator Fringe is a ß1,3 GlcNAc transferase that adds GlcNAc to the O-linked fucose residues on the EGF repeats of Notch (Moloney et al., 2000). In contrast to the results reported for the cytotoxins, mutation of the conserved Asp of the DXD motif in Fringe destroyed biological activity (Moloney et al., 2000
; Munro and Freeman, 2000
) but had no effect on labeling by a UDP analog (Munro and Freeman, 2000
). Thus, our results with GM2 synthase are compatible with the findings for Fringe and dissimilar to those for the cytotoxins. In summary, the DXD motif is not critical for UDP binding in Fringe or GM2 synthase.
Nearly all Golgi glycosyltransferases have the same domain organization and are type II membrane proteins. However, little overall structural homology is apparent for these enzymes, even for those sharing similar acceptor and donor substrates. With the goal of being able to use sequence similarities to predict folding similarities, glycosyltransferases were classified into 26 families based on sequence similarities (Campbell et al., 1997). GM2 synthase was placed in family 12, which consists only of GM2 synthase and the closely related GALGT2 enzyme (Smith and Lowe, 1994
). Therefore, family 12 may have a novel fold. The recently available crystal structures of family 2 member SpsA (Charnock and Davies, 1999
) and family 7 member galactosyltransferase (ß4GalT-1; Gastinel et al., 1999
) will be of greatest use in predicting the structures of other members of those families. In fact, however, all inverting glycosyltransferases are expected to share structural features relevant to common functions, such as the binding of nucleotide. Therefore, our results of mutation of the DXD motif are discussed below in the context of those two crystal structures.
The probable mechanism of catalysis for GM2 synthase, as for many inverting glycosyltransferases, is an SN2 reaction (Charnock and Davies, 1999). In this reaction a general base would activate the nucleophilic acceptor species by deprotonation, followed by facilitation of the departure of the UDP leaving group by Mn2+. The crystal structures of ß4GalT-1 (Gastinel et al., 1999
) and SpsA (Charnock and Davies, 1999
) reveal similar means for binding the uracyl ring of UDP; in ß4GalT-1 uracil is bound by a stacking interaction between Phe 226 and Arg 191 and by hydrogen bonding with Arg 189, whereas in SpsA the stacking interaction is with Tyr 11, plus there is hydrogen bonding with Arg 71 and Asp 39. In both crystals the ribose moiety is bound by several H-bonds. Although Mn2+ was not visible in ß4GalT-1, in the SpsA crystal Mn2+ interacted with Asp 99 of the DXD motif and also was located between the two phosphates of UDP. In summary, there are multiple residues responsible for binding of nucleotide for both ß4GalT-1 and SpsA. Therefore, Charnock and Davies speculated that the role for Asp 99 of the DXD motif of SpsA was in leaving group departure, which would be facilitated by Mn2+ (Charnock and Davies, 1999
).
If the catalytic site of GM2 synthase shares elements with that of SpsA, then our results with binding to UDP-beads and labeling by AAUTP can be explained using SpsA as a model. In the case of the large clostridial cytotoxins the contribution of the DXD motif to binding of UDP-glucose was great enough so that after mutation of the motif the azido-UDP-glucose probe no longer bound (Busch et al., 1998). In contrast, in the case of GM2 synthase the DXD motif may be only one of several regions of the enzyme that are responsible for binding of UDP-GalNAc as is the case for SpsA. Interaction of the DXD motif with the phosphates of the UDP moiety may be mediated by Mn2+, but because the contribution of the motif to overall binding of UDP is relatively small, binding to UDP-beads occurred in the absence of Mn2+. Similarly, binding to UDP-beads and labeling by AAUTP of the DDD/NNN mutant of GM2 synthase occurred for the same reason, because the contribution of the other residues to binding of the uracil and ribose moieties was sufficient to overcome mutation of the motif.
GM2 synthase is a homodimer in which the monomers are linked by disulfide bonds in the lumenal domain (Jaskiewicz et al., 1996a; Zhu et al., 1997
). In a separate study, we recently determined that the pattern of these disulfide bonds results in an antiparallel orientation of the lumenal domains (Li et al., 2000
). To date we have not been able to generate catalytically active monomers, raising the possibility that the monomers must cooperate in the dimer to produce active enzyme. With regard to the number of nucleotide-sugar binding sites per dimer, a high concentration of free UDP was required to prevent binding of GM2 synthase to UDP-beads suggesting that each dimer might bear two binding sites (Figure 6). In contrast, however, binding of AAUTP saturated at close to 50% of the available binding sites (Figure 8), thus suggesting only one UDP binding site per dimer. Future structural studies will be required to define the stoichiometry of UDP binding and the functional relationship of the monomers of GM2 synthase.
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Materials and methods |
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Immunofluorescence and flow cytometry
Staining of transfected CHO cells with monoclonal IgM anti-GM2 10-11 and anti-myc 9E10 was performed as described previously (Zhu et al., 1998). Briefly, for flow cytometry cells were removed from monolayer culture with trypsin-EDTA and incubated for 30 min at 4°C with the primary antibody in PBS-BSA. The cells were then washed in cold PBS-BSA and incubated for 30 min at 4°C in PBS-BSA containing fluorescein-conjugated goat anti-mouse Ig. For staining with the FITC-labeled B subunit of cholera toxin, transfected CHO cells were trypsinized and incubated for 30 min on ice with 10 µg/ml FITC-cholera toxin B subunit (Sigma, St. Louis, MO) diluted in PBS-BSA. After washing, the cells were resuspended in PBS-BSA and analyzed on an Epics Elite flow cytometer (Coulter, Hialeah, FL).
Western blotting, subcellular fractionation, and GM2 synthase activity assay
Western blotting with anti-myc was described previously (Jaskiewicz et al., 1996b) and quantitated with a Personal Densitometer SI and Image Quant version 3.3 software (Molecular Dynamics, Sunnyvale, CA). For subcellular fractionation, cells were homogenized, and Golgi and ER fractions were separated on sucrose gradients as described previously (Jaskiewicz et al., 1996a
). The in vitro assay for GM2 synthase activity was performed as described elsewhere (Jaskiewicz et al., 1996b
) except that the assay mixture contained 0.1 mM UDP-[3H] GalNAc and 10 mM CDP-choline, and the product GM2 was isolated by butanol-water partitioning. Large-scale cell culture was achieved in roller bottles using the serum-free medium CHO-S-SFM II (Life Technologies) according to Kolhekar et al. (1997)
and in bioreactors at the National Cell Culture Center (Minneapolis, MN). The soluble form of GM2 synthase was purified from culture supernatants by SP-Sepharose chromatography using a 00.25 M NaCl gradient in 50 mM HEPES pH 7.6, 5 mM MnCl2.
Binding to UDP-beads
A 10-µl aliquot of UDP-beads (glycosyltransferase affinity gel-UDP, Calbiochem, San Diego, CA) that had been diluted 1:10 with nonderivatized beads (Fractogel EMD, EM Industries, Hawthorne, NY) was incubated at 4°C for 30 min with rotation with conditioned medium from GTm1 cells that contained 2.5 µg wild-type GM2 synthase or with 2.5 µg of the partially purified soluble form of the D356N/D357N/D358N (referred below as DDD/NNN) mutant of GM2 synthase in 0.5 ml in 50 mM HEPES, pH 7.6, containing either 5 mM MnCl2 or 5 mM EDTA in the presence or absence of 22 mM UDP. Following incubation, the beads were centrifuged and boiled in nonreducing sample buffer, then the samples electrophoresed on nonreducing SDSpolyacrylamide gels.
Photoaffinity labeling of GM2 synthase
[-32P]-Labeled P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP) was prepared as described previously (Rancour and Menon, 1998
). Equivalent GM2 synthase protein amounts (
0.26 µg) were used for each photolabeling reaction, and thus the total protein content per reaction varied due to the respective purity of the GM2 synthase fractions with wild-type enzyme having 0.26 µg and the DDD/NNN mutant having
2.6 µg total protein per reaction, respectively. Photolabeling was performed in a darkroom under a filtered safe-light. Reactions were carried out in 50 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM MnCl2 in a final volume of 10 µl in the presence or absence of 100 µM UDP or GDP. Samples were incubated 5 min in a room-temperature water bath followed by 1 min on ice and 2 min UV irradiation on ice. Photolysis was quenched by the addition of 10 mM ß-mercaptoethanol (1 mM final) and further incubation for 5 min. SDS (10% w/v stock) was added to a final concentration of 1% (w/v), and the samples were subsequently diluted to 50 µl with the addition of ß4GalT-1 as a carrier protein (5 µg), water, and NaCl (5 M stock; 500 mM final). The protein was organic solventprecipitated as described previously (Wessel and Flugge, 1984
). Dried protein pellets were dissolved in 25 µl 2x SDSPAGE sample buffer by heating at 100°C for 5 min. The entire sample was resolved by SDSPAGE followed by transfer to nitrocellulose and detection by Western blotting using anti-myc. Following immunoblot development, the protein containing-nitrocellulose was rinsed several times with TBS and allowed to dry overnight under normal lab lighting. The dry nitrocellulose was exposed to a PhosphorImager screen (Molecular Dynamics) to obtain an autoradiogram which was quantitated using ImageQuant software (Molecular Dynamics) associated with the PhosphorImager instrument. Quantification of the immunoblot signal was obtained by densitometer analysis (Molecular Dynamics) using ImageQuant software of multiple film exposures of the developed immunoblots to insure linearity of the film signal.
Cyanogen bromide fragmentation of photolabeled GM2 synthase
GM2 synthase (0.78 µg) was photoaffinity labeled with 1 µM AAUTP[-32P] in buffer (60 mM HEPES/NaOH pH 7.5, 4.5 mM MnCl2, 25 mM NaCl) in a total volume of 30 µl. Following photolysis and quenching of the reaction with 2.5 mM ß-mercaptoethanol, protein was organic-solvent precipitated (Wessel and Flugge, 1984
). The dried protein pellet was resuspended in 40 µl cleavage solution (20 mM citrate/HCl pH 4.5, 0.2 % w/v SDS). Cyanogen bromide crystals were added to one tube, followed by flushing of the reaction tubes with N2(g) and sealing of the tubes and incubation overnight in the dark. Cleavage reactions were diluted ten-fold with water, snap-frozen in liquid nitrogen, and solvent removed by sublimation in a centrifugal vacuum concentrator. The remaining residue was solubilized in 2x SDSPAGE sample buffer by heating at 100°C for 5 min and resolved by SDSPAGE (15% resolving gel). Gels were either directly fixed or silver-stained (Bloom et al., 1987), followed by drying and autoradiography on a PhosphorImager screen (Molecular Dynamics).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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Bloom, H., Beier, H., and Gros, H.S. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis., 8, 9399.[ISI]
Breton, C., Bettler, E., Joziasse, D.H., Geremia, R.A., and Imberty, A. (1998) Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases. J. Biochem. (Tokyo), 123, 10001009.[Abstract]
Busch, C., Hofmann, F., Selzer, J., Munro, S., Jeckel, D., and Aktories, K. (1998) A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J. Biol. Chem., 273, 1956619572.
Campbell, J.A., Davies, G.J., Bulone, V., and Henrissat, B. (1997) A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem. J., 326, 929939.[ISI][Medline]
Charnock, S.J. and Davies, G.J. (1999) Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry, 38, 63806385.[ISI][Medline]
Dunphy, W.G. and Rothman, J.E. (1983) Compartmentation of asparagine-linked oligosaccharide processing in the Golgi apparatus. J. Cell Biol., 97, 270275.[Abstract]
Gastinel, L.N., Cambillau, C., and Bourne, Y. (1999) Crystal structure of the bovine beta4 galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose. EMBO J., 18, 35463557.
Hakomori, S. (1996) Tumor malignancy defined by aberrant glycosylation and sphingo(glyco)lipid metabolism. Cancer Res,. 56, 53095318.[Abstract]
Hamilton, K.S., Briere, K., Jarrell, H.C., and Grant, C.W.M. (1994) Acyl chain length effects related to glycosphingolipid crypticity in phospholipid membranes: probed by 2H-NMR. Biochim. Biophys. Acta Bio-Membr., 1190, 367375.[ISI][Medline]
Hammond, C. and Helenius, A. (1995) Quality control in the secretory pathway. Curr. Opin. Cell Biol., 7, 523529.[ISI][Medline]
Hashimoto, Y., Sekine, M., Iwasaki, K., and Suzuki, A. (1993) Purification and characterization of UDP-N-acetylgalactosamine GM3/GD3 N-acetylgalactosaminyltransferase from mouse liver. J. Biol. Chem., 268, 2585725864.
Jaskiewicz, E., Zhu, G., Bassi, R., Darling, D.S., and Young, W.W. Jr. (1996a) Beta-1,4 N-acetylgalactosaminyltransferase (GM2 synthase) is released from Golgi membranes as a neuraminidase sensitive, disulfide-bonded dimer by a cathepsin D-like protease. J. Biol. Chem., 271, 2639526403.
Jaskiewicz, E., Zhu, G., Taatjes, D.J., Darling, D.S., Zwanzig, G.E. Jr., and Young, W.W. Jr. (1996b) Cloned beta1,4 N-Acetylgalactosaminyltransferase: Subcellular localization and formation of disulfide bonded species. Glycoconj. J., 13, 213223.[ISI][Medline]
Keusch, J., Manzella, S.M., Nyame, K.A., Cummings, R.D., and Baenziger, J.U. (2000a) Expression cloning of a new member of the ABO blood group glycosyltransferases, iGb3 synthase, that directs the synthesis of isogloboglycosphingolipids. J. Biol. Chem., 275, 2530825314.
Keusch, J., Manzella, S.M., Nyame, K.A., Cummings, R.D., and Baenziger, J.U. (2000b) Cloning of Gb3 synthase, the key enzyme in globo-series glycosphingolipid synthesis, predicts a family of alpha1,4 glycosyltransferases conserved in plants, insects, and mammals. J. Biol. Chem., 275, 2531525321.
Kolhekar, A.S., Keutmann, H.T., Mains, R.E., Quon, A.S.W., and Eipper, B.A. (1997) Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core. Biochemistry, 36, 1090110909.[ISI][Medline]
Kostova, Z., Rancour, D.M., Menon, A.K., and Orlean, P. (2000) Photoaffinity labeling with P3-(4-azidoanilido)uridine 5'-triphosphate identifies Gpi3p as the UDPGlcNAc-binding subunit of the enzyme that catalyzes formation of N-acetylglucosaminyl phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis. Biochem. J., 350, 815822.[ISI][Medline]
Leal, W.S., Nikovna, L., and Peng, G. (1999) Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori. FEBS Lett., 464, 8590.[ISI][Medline]
Li, J., Yen, T., Allende, M.L., Joshi, R.K., Cai, J., Pierce, W.M., Jackiewicz, E., Darling, D.S., Macher, B.A., and Young, W.W., Jr. (2000) Disulfide bonds of GM2 synthase homodimers: antiparallel orientation of the catalytic domains. J. Biol. Chem., 275, 4147641486.
Moloney, D.J., Panin, V.M., Johnston, S.H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K.D., Haltiwanger, R.S., and others (2000) Fringe is a glycosyltransferase that modifies Notch. Nature, 406, 369375.[ISI][Medline]
Munro,S. and Freeman,M. (2000) The Notch signalling regulator Fringe acts in the Golgi apparatus and requires the glycosyltransferase signature motif DxD. Curr. Biol., 10, 813820.[ISI][Medline]
Nagata, Y., Yamashiro, S., Yodoi, J., Lloyd, K.O., and Furukawa, K. (1992) Expression cloning of beta 1,4 N-acetylgalactosaminyltransferase cDNAs that determine the expression of GM2 and GD2 gangliosides. J. Biol. Chem., 267, 1208212089.
Pagano, R.E., Longmuir, K.J., Martin, O.C., and Struck, D.K. (1981) Metabolism and intracellular localization of a fluorescently labelled intermediate in lipid biosynthesis within cultured fibroblasts. J. Cell. Biol., 91, 872877.
Rancour, D.M. and Menon, A.K. (1998) Identification of endoplasmic reticulum proteins involved in glycan assembly: synthesis and characterization of P3-(4-azidoanilido)uridine 5'-triphosphate, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins. Biochem. J., 333, 661669.[ISI][Medline]
Rosales Fritz, V.M., Daniotti, J.L., and Maccioni, H.J.F. (1997) Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase. Biochim. Biophys. Acta Gene Struct. Exp., 1354, 153158.[ISI][Medline]
Ruan, S. and Lloyd, K.O. (1992) Glycosylation pathways in the biosynthesis of gangliosides in melanoma and neuroblastoma cells: Relative glycosyltransferase levels determine ganglioside patterns. Cancer Res., 52, 57255731.[Abstract]
Sheikh, K.A., Sun, J., Liu, Y.J., Kawai, H., Crawford, T.O., Proia, R.L., Griffin, J.W., and Schnaar, R.L. (1999) Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc. Natl Acad. Sci. USA, 96, 75327537.
Smith, P.L. and Lowe, J.B. (1994) Molecular cloning of a murine N-acetylgalactosamine transferase cDNA that determines expression of the T lymphocyte-specific CT oligosaccharide differentiation antigen. J. Biol. Chem., 269, 1516215171.
Takamiya, K., Yamamoto, A., Furukawa, K., Zhao, J.M., Fukumoto, S., Yamashiro, S., Okada, M., Haraguchi, M., Shin, M., Kishikawa, M., and others (1998) Complex gangliosides are essential in spermatogenesis of mice: possible roles in the transport of testosterone. Proc. Natl Acad. Sci. USA, 95, 1214712152.
Tatewaki, K., Yamaki, T., Maeda, Y., Tobioka, H., Piao, H.Z., Yu, H.W., Ibayashi, Y., Sawada, N., and Hashi, K. (1997) Cell density regulates crypticity of GM3 ganglioside on human glioma cells. Exp. Cell Res., 233, 145154.[ISI][Medline]
Terasaki, M., Song, J., Wong, J.R., Weiss, M.J., and Chen, L.B. (1984) Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes. Cell, 38, 101108.[ISI][Medline]
Tong, P.Y., Gregory, W., and Kornfeld, S. (1989) Ligand interactions of the cation-independent mannose 6-phosphate receptor. The stoichiometry of mannose 6-phosphate binding. J. Biol. Chem., 264, 79627969.
Urdal, D. and Hakomori, S. (1983) Characterization of tumor-associated ganglio-N-triaosylceramide in mouse lymphoma and the dependency of its exposure and antigenicity on the sialosyl residues of a second glycoconjugate. J. Biol. Chem., 258, 68696874.
Van Echten, G. and Sandhoff, K. (1993) Ganglioside metabolism. Enzymology, topology, and regulation. J. Biol. Chem., 268, 53415344.
Van Echten-Deckert, G. and Sandhoff, K. (1998) in Pinto, B.M. (ed) Comprehensive natural products chemistry, Pergamon, Elsevier Science, New York, Chapter 5,.
Wessel, D. and Flugge, U.I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem., 138, 141143.[ISI][Medline]
Wiggins, C.A.R. and Munro, S. (1998) Activity of the yeast MNN1 -1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc. Natl Acad. Sci. USA, 95, 79457950.
Yanagisawa, K., Taniguchi, N., and Makita, A. (1987) Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver. Biochim. Biophys. Acta, 919, 213220.[ISI][Medline]
Zhu, G., Jaskiewicz, E., Bassi, R., Darling, D.S., and Young, W.W. Jr. (1997) Beta1,4 N-acetylgalactosaminyltransferase (GM2/GD2/GA2 synthase) forms homodimers in the endoplasmic reticulum: a strategy to test for dimerization of Golgi membrane proteins. Glycobiology, 7, 987996.[Abstract]
Zhu, G., Allende, M.L., Jaskiewicz, E., Qian, R., Darling, D.S., Worth, C.A., Colley, K.J., and Young,W .W. Jr. (1998) Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts. Glycobiology, 8, 831840.