The DXD motif is required for GM2 synthase activity but is not critical for nucleotide binding

Jianghong Li3, David M. Rancour4, Maria Laura Allende1,3, Christopher A. Worth5, Douglas S. Darling3, J. Bradley Gilbert3, Anant K. Menon4 and William W. Young Jr.2,3

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We tested the importance of the aspartate–any residue–aspartate (DXD) motif for the enzymatic activity and nucleotide binding capacity of the Golgi glycosyltransferase GM2 synthase. We prepared point mutations of the motif, which is found in the sequence 352-VLWVDDDFV, and analyzed cells that stably expressed the mutated proteins. Whereas the folding of the mutated proteins was not seriously disrupted as judged by assembly into homodimers, Golgi localization, and secretion of a soluble form of the enzyme, exchange of the highly conserved aspartic acid residues at position 356 or 358 with alanine or asparagine reduced enzyme activity to background levels. In contrast, the D356E and D357N mutations retained weak activity, while the activity of V352A and W354A mutants was 167% and 24% that of wild-type enzyme, respectively. Despite the major effect of the DXD motif on enzymatic activity, nucleotide binding was not altered in the triple mutant D356N/D357N/D358N as revealed by binding to UDP-beads and labeling with the photoaffinity reagent, P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP). In summary, rather than being critical for nucleotide binding, this motif may function during catalysis in GM2 synthase, as has been proposed elsewhere for the SpsA glycosyltransferase based on its crystal structure.

Key words: DXD motif/glycosyltransferase/GM2 synthase/ganglioside/Golgi


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ganglioside synthesis is regulated during differentiation, development, and malignant transformation (Van Echten and Sandhoff, 1993; Hakomori, 1996Go; Birklé et al., 1999Go) and occurs in the Golgi apparatus by the stepwise addition of monosaccharides to glycolipid acceptors by membrane-bound glycosyltransferases. The simple gangliosides GM3, GD3, and GT3 are the precursors of the a-, b-, and c-series of gangliosides, respectively, and are synthesized by the addition of one, two, or three molecules of sialic acid to lactosylceramide. Complex gangliosides are formed by the addition of GalNAc to simple gangliosides by the action of UDP-GalNAc:lactosylceramide/GM3/GD3 ß-1,4-N-acetylgalactosaminyltransferase (GM2 synthase) followed by the attachment of Gal and additional sialic acid residues (Van Echten and Sandhoff, 1993). Thus, GM2 synthase is a key enzyme in ganglioside biosynthesis, controlling the balance between the expression of simple and complex gangliosides (Van Echten-Deckert and Sandhoff, 1998Go). Genetic ablation of GM2 synthase in mice resulted in male sterility (Takamiya et al., 1998Go) as well as decreased myelination and axonal degeneration of the central and peripheral nervous system (Sheikh et al., 1999Go).

To date, little structural information is available concerning the catalytic site of GM2 synthase. Recently, however, an aspartate–any residue–aspartate (DXD) motif was found to be conserved in 13 families of glycosyltransferases, including GM2 synthase (Breton et al., 1998Go; Wiggins and Munro, 1998Go), and in several of those families, this motif was found to be critical for enzymatic activity (Busch et al., 1998Go; Wiggins and Munro, 1998Go; Keusch et al., 2000aGo,b; Moloney et al., 2000Go; Munro and Freeman, 2000Go). This motif is hhhhDxDxh, where h is a hydrophobic residue and x is any residue (Wiggins and Munro, 1998Go), and in human GM2 synthase the motif is found in the sequence 352-VLWVDDDFV (Nagata et al., 1992Go; Wiggins and Munro, 1998Go). 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., 1998Go). 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., 1998Go). In striking contrast, mutations of the DXD motif in the Fringe signaling molecule eliminated biological activity (Moloney et al., 2000Go; Munro and Freeman, 2000Go) but did not alter photolabeling by a UDP analog (Munro and Freeman, 2000Go). 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, 1999Go), 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, 2000Go) and dissimilar to those for the large clostridial cytotoxins (Busch et al., 1998Go).


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Effect of alteration of the DXD motif on the life cycle of GM2 synthase
To determine the importance of residues in the DXD motif to the activity of GM2 synthase, we mutated not only the highly conserved aspartates at positions 356 and 358 (Wiggins and Munro, 1998Go) but also D357, V352, and W354 (Table I). Wild-type GM2 synthase forms a membrane-bound homodimer in the endoplasmic reticulum (ER) (Zhu et al., 1997Go) and then moves to the Golgi, where it is cleaved to release the stem and catalytic domains as a soluble fragment (Jaskiewicz et al., 1996aGo). If any of these mutations seriously disrupted the folding of GM2 synthase, the resulting aberrant proteins would be unlikely to follow the normal life cycle of the enzyme. Therefore, after transfecting CHO cells with each mutant construct, we first determined by immunofluorescence if each mutated protein reached the Golgi. For clarity we should note that in all of the results described below, cell clones were analyzed for the D356A, D358A, DDD/ADA, DDD/NNN, V352A, and W354A mutations, whereas uncloned cell mixtures were used for the D356E, D356N, D357N, and D358N mutations.


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Table I. Summary of wild-type and mutated GM2 synthase.
 
As we reported previously (Jaskiewicz et al., 1996bGo), wild-type GM2 synthase was located primarily in the Golgi, which consists of a cluster of punctate structures appearing in CHO cells as a small circle or half moon located over the nucleus (Figure 1A). In addition a small portion of wild-type enzyme was retained in the ER (Figure 1A) as indicated by staining of the nuclear membrane plus a lacy reticular network that extends throughout the cytoplasm (Pagano et al., 1981Go; Terasaki et al., 1984Go). A similar staining pattern was seen when D356 and D358 were mutated simultaneously to Ala (Figure 1B; referred to below as the DDD/ADA mutation) or individually to Ala (data not shown). Costaining with anti-mannosidase II (Figure1D), a well-characterized Golgi marker (Dunphy and Rothman, 1983Go), and anti-myc (Figure 1B) verified that the cluster of anti-myc-reactive punctate perinuclear dots stained in the cells expressing the DDD/ADA mutation was actually the Golgi. Mutation of D356 to Glu, the individual replacement of D356, D357, or D358 to Asn (data not shown) or the simultaneous mutation of all three Asp to Asn (DDD/NNN; Figure 1C) all resulted in strong Golgi staining, but in addition the ER staining intensity was greater than that for cells expressing wild-type enzyme (Figure 1A). Thus, folding sufficient to allow passage to the Golgi was accomplished for mutation of the conserved Asp to Ala, Glu, or Asn.



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Fig. 1. Localization of GM2 synthase mutants. CHO cells expressing wild-type or mutant GM2 synthase were fixed, permeabilized, and stained with mouse anti-myc 9E10 followed by FITC-conjugated anti-mouse Ig. For clone C2 cells expressing the doubly mutated DDD/ADA enzyme (panels B and D), cells were double-labeled with rabbit anti-mannosidase II and anti-myc followed by the corresponding FITC-conjugated goat anti-rabbit Ig and Texas red–conjugated goat anti-mouse Ig. The same field was viewed through the Texas red filter (panel B) and the fluorescein filter (panel D). Panels: A, wild-type clone C5; C, DDD/NNN triple mutant clone F7; E, V352A mutant clone C1; and F, W354A mutant clone D3. The arrowhead denotes the plasma membrane to indicate that the lacy reticular network characteristic of the ER extends up to the cell surface; arrows denote the nuclear membrane. The width of each panel represents 80 microns.

 
Finally, to determine if residues other than the aspartates in the motif were critical for activity, we also prepared V352A and W354A mutants. The V352A (Figure 1E) and W354A (Figure 1F) expressing cells exhibited strong staining of both the ER and the Golgi; in both of these cells the lacy reticular pattern characteristic of the ER was especially apparent, whereas Golgi staining was more difficult to detect due to the heavy staining of the nuclear membrane.

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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Fig. 2. Homodimerization of GM2 synthase/myc mutants and release of soluble forms. Panel A, cell extracts were Western blotted with anti-myc under nonreducing conditions. The ratios of GM2 synthase dimer to monomer were determined by quantitative densitometry and are shown in Table I. Panel B, conditioned media were Western blotted with anti-myc under nonreducing conditions.

 
To confirm that dimerization and passage to the Golgi were in fact indications of proper folding, we performed subcellular fractionation on cells expressing a mutated protein with one of the lowest dimer to monomer ratios, the DDD/ADA double mutation (Figure 3). The ER fractions contained dimer at approximately 130,000, heavy bands at the position of monomer (ca. 65,000), and lower molecular weight bands indicative of proteolytic degradation. In contrast, the Golgi fractions contained only dimer, confirming that only properly folded and dimerized GM2 synthase was able to pass through the quality control system of the ER (Hammond and Helenius, 1995Go).



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Fig. 3. Golgi localization of the homodimer of the DDD/ADA double mutant of GM2 synthase/myc by subcellular fractionation. Cells were homogenized, and Golgi and ER membranes were separated on a discontinuous sucrose gradient. Aliquots of fractions 3–13 were analyzed by Western blotting with anti-myc under nonreducing conditions. The peak fractions for the Golgi marker enzyme galactosyl transferase were fractions 3 and 4 and for the ER marker enzyme {alpha}-glucosidase II fractions 9–11 (data not shown).

 
As a third measure of whether the mutants followed the normal life cycle of GM2 synthase, we determined if the mutated proteins were released from the cells as soluble forms following proteolytic cleavage (Figure 2B). Among the D to A mutation group, the D358A mutated enzyme was released, whereas the D356A and the doubly mutated DDD/ADA enzymes were not. This result, when combined with the fact that the ratio of dimer to monomer was higher for the D358A mutated enzyme than for D356A or DDD/ADA (Table I), indicated more normal folding of D358A than for D356A or DDD/ADA. The D356E, D356N, D357N, and D358N mutated enzymes were all released (Figure 2B), data that when combined with their high ratios of dimer to monomer (Table I) indicated that the overall folding of these mutated enzymes was similar to wild-type enzyme. Finally, the triple DDD/NNN mutation as well as W354A and V352A were released (Figure 2B) despite their relatively low ratios of dimer to monomer (Table I). These latter findings suggest that even though relatively small percentages of these mutated enzymes were able to dimerize, the portion that did reach the Golgi was susceptible to cleavage and release. Thus, the only mutated proteins that reached the Golgi but were not released were D356A and the doubly mutated DDD/ADA. In summary, based on the three parameters that we utilized to judge proper folding, the mutated enzymes that most closely resembled wild-type enzyme were the single N mutations and D356E. The D358A, DDD/NNN, V352A, and W354A mutations also dimerized, reached the Golgi, and were released but not as efficiently as the former group of mutations.

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., 1996bGo), 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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Fig. 4. Anti-GM2 flow cytometry. Intact cells were incubated with anti-GM2 antibody 10-11, followed by staining with FITC-conjugated goat anti-mouse Ig. Relative fluorescence is in arbitrary logarithmic units. Fluorescence profiles for the negative control antibody anti-LeY essentially overlapped the profile for anti-GM2 for CHO and all cells expressing mutated GM2 synthase except V352 and W354A. The samples shown on the bottom row were analyzed with a lower fluorescence gain setting to show the extremely bright staining of the W354A cells.

 


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Fig. 5. Cholera toxin flow cytometry. Intact cells were stained with FITC-cholera toxin B subunit. Relative flourescence is in arbitrary logarithmic units.

 
Rosales Fritz et al. (1997)Go found that CHO-K1 cells expressing cloned GM2 synthase converted a significant portion of GM2 into GM1 due to the action of an endogenous galactosyltransferase (GM1 synthase). Thus, it was possible that cells producing mutated GM2 synthase might produce small amounts of GM2 that would not accumulate but instead would be quantitatively converted to GM1. To test this point, we analyzed cells expressing the DXD mutant proteins by flow cytometry with fluoresceinated-cholera toxin B subunit to detect cell surface GM1. Clone C5 expressing wild-type GM2 synthase/myc displayed strong staining (Figure 5). None of the clones expressing the single or double D to A mutations displayed GM1 on the cell surface (Figure 5). Similarly, none of the cells expressing the single D to N mutations D356N and D358N or the triple mutation DDD/NNN were cholera toxin positive (Figure 5). Interestingly, however, at least 25% of the cell mixtures expressing the D357N mutation and the D356E mutation were strongly positive for cholera toxin B subunit (Figure 5). Because the percent of myc positive cells in these populations was approximately 20–30% (data not shown), these results indicated that nearly all cells expressing the mutated enzymes were able to produce a small amount of GM2, which was quantitatively converted to GM1.

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., 1994Go; Tatewaki et al., 1997Go). 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, 1998Go). Although Mn2+ has been commonly used for GM2 synthase activity assays (Ruan and Lloyd, 1992Go; Jaskiewicz et al., 1996bGo), 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+ {approx} Co2+, Mg2+, and Ca2+ (Yanagisawa et al., 1987Go), the order for the enzyme purified from mouse liver was Mn2+ > Co2+ > Ca2+ >= Mg2+ >= Fe2+ > Ni2+ (Hashimoto et al., 1993Go). To clarify this discrepancy, we determined the activity of the soluble form of wild-type GM2 synthase from clone GTm1 (Zhu et al., 1998Go) 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., 1998Go). However, mutation of the aspartates of the DXD motif in Fringe had minimal effect on labeling with a UDP analog (Munro and Freeman, 2000Go). 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., 1989Go).



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Fig. 6. Binding of soluble forms of GM2 synthase to UDP-beads. UDP-beads were incubated with sample, centrifuged, boiled in sample buffer, and electrophoresed as described in Materials and methods. Lanes 1–4, wild-type GM2 synthase; lanes 5–8, the DDD/NNN mutant. Lanes 1, 2, 5, and 6 were incubated in the presence of Mn2+; lanes 3, 4, 7, and 8 in the presence of EDTA. Lanes 2, 4, 6, and 8 were incubated in the presence of 22 mM UDP (final), which represented a 1000-fold excess over the content of UDP bound to the beads. A, Anti-myc Western blot. B, Coomassie stain. The arrow indicates the location of the GM2 synthase dimer. C, quantitation of the Coomassie-stained GM2 synthase dimer band shown in panel B. For both the wild-type and the mutant enzyme, binding is plotted as the percent of maximum binding for that protein which was the result of incubation in the presence of Mn2+ (set as 100% in the bar graph). The averaged results of two separate experiments are shown. The error bars represent the range of results. Maximum binding for wild-type enzyme was 60% and 26% of the input enzyme in the two separate experiments and for the mutant enzyme 40% and 14%.

 
UDP inhibited wild-type enzyme binding in the presence of EDTA as well (Figure 6, lane 4) although the extent of inhibition was not as great as in the presence of Mn2+. To demonstrate the specificity of this inhibition, we compared inhibition by UDP with that by dADP. Whereas dADP inhibited GM2 synthase activity approximately fivefold poorer than UDP (data not shown), dADP inhibited binding of wild-type GM2 synthase to UDP-beads approximately twofold less than UDP (data not shown). Thus, at least a portion of the binding of GM2 synthase to UDP-beads was the result of specific recognition of the uridine moiety.

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, 2000Go).

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, 1998Go). 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, 1992Go)]. 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[{alpha}-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[{alpha}-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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Fig. 7. Photoaffinity labeling of wild-type GM2 synthase with AAUTP[{alpha}-32P]. Wild-type, soluble GM2 synthase was photolabeled with AAUTP[{alpha}-32P] as described in Materials and methods. The top of the figure shows the autoradiogram and corresponding anti-myc immunoblot for samples labeled in the absence or presence of UV irradiation and in the absence or presence of UDP and GDP. Quantitation of the results was achieved by normalizing the autoradiogram signal to the immunoblot signal for each reaction. The averaged results of two separate experiments (including the experiment shown in the autoradiogram and immunoblot) are shown in the histogram in the lower part of the figure. The error bars represent the range of results.

 
We next determined the efficiency with which AAUTP labeled GM2 synthase. This was done by measuring labeling intensity as a function of increasing AAUTP[{alpha}-32P] concentration. A direct comparison using both autoradiography and liquid scintillation counting was made between GM2 synthase samples that were incubated with AAUTP[{alpha}-32P] in the presence or absence of UV irradiation. The difference between the two treatments was taken as the specific labeling. The data are shown in Figure 8A. A single-phase exponential fit of the data points resulted in a curve with a goodness of fit (R2) value of 0.99. The calculated half-maximal concentration was 36 µM and saturation at 58% of the targets labeled assuming (1) one binding site per monomer component and (2) a molecular weight per monomer of 64 kDa. Thus, labeling of GM2 synthase by AAUTP is saturable, indicating that a finite number of binding sites is available for labeling. Because binding saturates at close to 50%, it would appear that only a single subunit of the GM2 synthase dimer contains a functional nucleotide sugar binding site.



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Fig. 8. Efficiency and site of AAUTP[{alpha}-32P] labeling. Panel A, incorporation of AAUTP into wild-type GM2 synthase as a function of AAUTP concentration. The line through the points represents a single-phase exponential fit of the data indicating a plateau at ~58% pmol AAUTP/pmol GM2 synthase. Panel B, identification of AAUTP-modified peptide region: CNBr fragmentation of photoaffinity-labeled GM2 synthase. Silver-stained gel (left panel) and autoradiogram (right panel) of photoaffinity-labeled GM2 synthase that was either incubated with cyanogen bromide (CNBr+) or mock treated (CNBr–). The star highlights the peptide fragment modified by AAUTP[{alpha}-32P].

 
A final assessment of the specificity of AAUTP labeling was to elucidate the site of covalent incorporation of AAUTP into GM2 synthase. The soluble form of wild-type GM2 synthase was photoaffinity labeled with AAUTP[{alpha}-32P] and fragmented by cyanogen bromide treatment (Figure 8B). Soluble GM2 synthase contains only three methionine residues at positions 330, 515, and 530 (amino acid numbers for the intact enzyme); hence, CNBr cleavage is expected to produce fragments of GM2 synthase of ca. 40 kDa (amino acids 21–330, with the precise molecular weight depending on the actual Asn-linked oligosaccharide structures attached at residues 79, 179, and 274), 20.5 kDa (amino acids 331–515), 1.9 kDa (amino acids 516–530), and 1.6 kDa (amino acids 531–544, which includes the myc epitope). GM2 synthase was indeed fragmented by CNBr, albeit inefficiently, resulting in one fragment of ~40 kDa and a set of at least three fragments near 20 kDa (Figure 8B, left). The low efficiency of cleavage may be due to the unorthodox conditions used for fragmentation, which were chosen to ensure retention of the radioactive label in the target peptide. The set of bands near 20 kDa reflects sluggish cleavage of the C-terminal peptides. In separate experiments we have found that the largest of these fragments is myc positive by Western blotting, indicating some fragments in which only cleavage at position 330 occurred (Li et al., 2000Go). The MetThr site at position 530 previously was found to be cleaved by CNBr with a low yield (Leal et al., 1999Go). Autoradiography of the peptide fragments indicated that AAUTP was incorporated preferentially into the set of peptides near 20 kDa (Figure 8B, right). A lower level of labeling was apparent in the 40-kDa peptide as well. Although it is entirely possible for a region of the 40-kDa fragment to be located near the catalytic site in the native enzyme and, therefore, to be specifically labeled by AAUTP[{alpha}-32P], at least a portion of this labeling was the result of nonspecific labeling for the following reasons. In control experiments when labeling was performed in the presence of UDP, only label incorporation into either the intact protein or the 20-kDa fragment was significantly altered (data not shown). Therefore, it was concluded that specific labeling of GM2 synthase by AAUTP occurs in the C-terminal third of the protein, a region containing the DXD motif (amino acids 352–360) (Figure 8B).

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 80–90% 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[{alpha}-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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Fig. 9. Photoaffinity labeling with AAUTP[{alpha}-32P]. Soluble forms of wild-type GM2 synthase and the DDD/NNN mutant were photoaffinity labeled with 1 µM AAUTP[{alpha}-32P]. The data are averaged from two separate experiments with the wild-type value set to 100% and with the error bar showing the range of results. The histogram represents normalized data as obtained in Figure 7.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Effect of DXD mutations on GM2 synthase activity
The effects on enzyme activity of mutations of the DXD motif of GM2 synthase (Table I; Figures 4 and 5) were similar to those of the yeast MNN1 mannosyltransferase (Wiggins and Munro, 1998Go) and the large clostridial cytotoxins (Busch et al., 1998Go). In all three cases mutation of the two highly conserved Asp residues had the greatest effect on activity, with single mutations of these residues to Ala, Asn, or Glu in MNN1 and GM2 synthase causing the loss of detectable activity. In the case of the cytotoxins, exchange of either Asp to Ala or Asn decreased glucosyltransferase activity by about 5000-fold and completely eliminated glucohydrolase activity, whereas simultaneous conversion of both Asp to Ala destroyed all detectable activity. The aromatic residue two positions on the amino terminal side of the first invariant Asp of the motif (Phe in MNN1, Tyr in the cytotoxins, and Trp in GM2 synthase) appears to be of similar importance to enzyme activity in all three enzymes because in each case mutation of this residue to Ala reduced activity approximately four- to fivefold. Another similarity was that mutation to Ala of the valine (Val427) three positions on the amino terminal side of the first invariant Asp in MNN1 and the valine (Val352) four positions from the first invariant Asp in GM2 synthase resulted in enzyme activity greater than wild-type enzyme.

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., 2000Go; Munro and Freeman, 2000Go). 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., 1998Go). 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., 2000Go). 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., 2000Go; Munro and Freeman, 2000Go) but had no effect on labeling by a UDP analog (Munro and Freeman, 2000Go). 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., 1997Go). GM2 synthase was placed in family 12, which consists only of GM2 synthase and the closely related GALGT2 enzyme (Smith and Lowe, 1994Go). Therefore, family 12 may have a novel fold. The recently available crystal structures of family 2 member SpsA (Charnock and Davies, 1999Go) and family 7 member galactosyltransferase (ß4GalT-1; Gastinel et al., 1999Go) 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, 1999Go). 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., 1999Go) and SpsA (Charnock and Davies, 1999Go) 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, 1999Go).

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., 1998Go). 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., 1996aGo; Zhu et al., 1997Go). 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., 2000Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Chimeric constructs
CHO cell clone C5 expressing wild-type, full-length GM2 synthase/myc and clone GTm1 expressing a soluble form of GM2 synthase/myc were described previously (Jaskiewicz et al., 1996bGo; Zhu et al., 1998Go). Site-directed mutagenesis of GM2 synthase was performed on a pcDNA3 plasmid containing full-length GM2 synthase/myc cDNA using the TransformerTM Site-Directed Mutagenesis Kit (Clontech), according to the manufacturer’s instructions. The sequences of mutated constructs at the mutation sites were confirmed by DNA sequencing (EPSCoR Sequencing Center, University of Louisville and Biomolecular Research Facility, University of Virginia). Prior to cell transfection, each construct was tested for production of full-length GM2 synthase in vitro using the TnT-coupled transcription/translation system (Promega, Madison, WI). Wild-type CHO cells were transfected, and clones stably expressing mutated GM2 synthase/myc were selected by limiting dilution and anti-myc immunofluorescence screening as previously described (Jaskiewicz et al., 1996bGo).

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., 1998Go). 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., 1996bGo) 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., 1996aGo). The in vitro assay for GM2 synthase activity was performed as described elsewhere (Jaskiewicz et al., 1996bGo) 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)Go 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 0–0.25 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 SDS–polyacrylamide gels.

Photoaffinity labeling of GM2 synthase
[{alpha}-32P]-Labeled P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP) was prepared as described previously (Rancour and Menon, 1998Go). 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 solvent–precipitated as described previously (Wessel and Flugge, 1984Go). Dried protein pellets were dissolved in 25 µl 2x SDS–PAGE sample buffer by heating at 100°C for 5 min. The entire sample was resolved by SDS–PAGE 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[{alpha}-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, 1984Go). 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 SDS–PAGE sample buffer by heating at 100°C for 5 min and resolved by SDS–PAGE (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).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. K. Furukawa for providing wild-type GM2 synthase plasmid and K. O. Lloyd for anti-GM2 10-11. This work was supported by National Institutes of Health Grants GM42698 (to W.W.Y.) and GM55427 (to A.K.M.), NSF grant EPS-9874764 (to W.W.Y.), and a grant from the Mizutani Foundation for Glycoscience (to W.W.Y.).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
AAUTP, P3-(4-azidoanilido)uridine 5'-triphosphate; DXD, aspartate–any residue–aspartate; ER, endoplasmic reticulum.


    Footnotes
 
1 Current address: NIH, Building 10 Room 9-D16, 9000 Rockville Pike, Bethesda, MD 20892, USA Back

2 To whom correspondence should be addressed Back


    References
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 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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