Characterization of the substrate specificity of [alpha]1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides

Cheryl L.M. Stults4, Bruce A. Macher3, Ruhie Bhatti, Om P. Srivastava1 and Ole Hindsgaul2

Department of Chemistry and Biochemistry, San Francisco State University 1600 Holloway Avenue, San Francisco, CA 94132, USA, 1Alberta Research Council, PO Box 8330, Station F, Edmonton, Alberta, T6H 5X2, Canada and 2Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada

Received on September 24, 1998; revised on December 10, 1998; accepted on December 10, 1998

[alpha]1,3galactosyltransferase ([alpha]1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Gal[alpha]1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Gal[alpha]1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8 (Gal[beta]1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing [alpha]1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Gal[beta]1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 µM vs. Km = 190 µM for Gal[beta]1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-[beta]-d-galactopyranosyl-(1,4)-2-acetamindo-2-deoxy-[beta]-d-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of [alpha]1,3GalT. [alpha]1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 µM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that [alpha]1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for [alpha]1,3GalT activity.

Key words: [alpha]-galactosyltransferase/synthetic acceptors and inhibitors/amino acid modifiers

Introduction

[alpha]1,3-Galactosyltransferase ([alpha]1,3GalT) catalyzes the transfer of galactose (Gal) from UDP-Gal to glycoconjugate acceptors with a LacNAc (Gal[beta]1-4GlcNAc) nonreducing terminal disaccharide. The product of this reaction (i.e., Gal[alpha]1,3Gal[beta]1-4GlcNAc-R) is recognized by the naturally occurring human antibody, anti-Gal (Galili, 1989). The interaction of anti-Gal with Gal[alpha]1,3Gal[beta]1-4GlcNAc-R epitopes leads to acute rejection of tissue in xenotransplantations (Cooper et al., 1993; Galili, 1993). Thus, it has been demonstrated that nonprimate mammals and New World monkeys express glycoconjugates carrying Gal[alpha]1,3Gal[beta]1-4GlcNAc-R epitopes, whereas these epitopes are not found in human tissues (Galili et al., 1987). The basis for the evolutionarily restricted distribution of Gal[alpha]1,3Gal[beta]1-4GlcNAc-R epitopes correlates with the expression of [alpha]1,3GalT (Galili et al., 1988). Thus, [alpha]1,3GalT activity is not found in tissues from humans or Old World monkeys (Galili et al., 1988). The absence of [alpha]1,3GalT in these species is the result of mutations occurring in the gene(s) encoding the enzyme. Interestingly, these species do not express mRNA encoding the mutated form of [alpha]1,3GalT. Therefore, these sequences represent pseudogenes for [alpha]1,3GalT (Joziasse et al., 1989; Larsen et al., 1990).

Recently, we began an investigation of recombinant forms of this enzyme to obtain information on its structure, with the goal of identifying the catalytic domain and amino acids which are involved in substrate binding and catalysis. We have identified the catalytic domain of the enzyme from mouse and New World monkey; demonstrating that only 285 of a total of 376 amino acids are required for enzyme activity (Henion et al., 1994).

In the present study, substrate analogs were used to (1) probe the acceptor substrate binding site of [alpha]1,3GalT, (2) aid in the design of photoprobes to identify amino acids which participate in substrate binding, and (3) enzymatically produce compounds that can be used to analyze the binding specificity of anti-Gal and other Gal[alpha]1-3Gal binding proteins (Clark et al., 1986; Krivan et al., 1986). We have examined the ability of the enzyme to bind a series of acceptor substrate analogs (based on the minimum acceptor substrate, LacNAc). In cases in which the analogs were found to be acceptors, the products of the reactions were structurally characterized. A range of nucleotide sugars also have been evaluated as potential donor substrates. Finally, amino acid modifiers have been utilized to identify residues that are essential for enzyme activity.

Results

Characterization of enzyme activity

Preliminary experiments were done to determine the optimal reaction conditions for the recombinant [alpha]1,3GalT utilized in this study. In these experiments the following components were examined: divalent cation, buffer pH, detergent, UDP-Gal and acceptor concentration. There was an absolute requirement for a divalent cation. This was demonstrated by a lack of activity in the absence of Mn2+, or when EDTA was present in the reaction mixture. Addition of 40 mM Mg2+ ( 40%), Co2+ (15%), or (5%) Zn2+ resulted in a lower level of activity when compared to Mn2+. As shown in Figure 1, the pH optimum for the recombinant [alpha]1,3GalT was pH 6.0. Membrane bound forms of [alpha]1,3GalT have been shown to be activated by TX-100 (Elices et al., 1986). In contrast, the activity of the soluble recombinant [alpha]1,3GalT was not affected by TX-100 (0.8%). The kinetic parameters for UDP-Gal and two known acceptors, LacNAc-C8 and nLc4-C8, were determined. The Km values were 710 µM, 190 ± 10 µM and 835 ± 144 µM for UDP-Gal, LacNAc-C8, and nLc4-C8, respectively.


Figure 1. Effect of pH on the activity of [alpha]1,3GalT. The pH values (range 4-7.2) are those measured in the final reaction mixture (4 ng/µl enzyme).

Table I. Kinetic parameters for Type 2 acceptors
Substitution Km (µM) Vmax/Vmax LacNAc-C8
LacNAc 192 ± 10 1.00
2-Azido 152 ± 36 1.13
2-Propionamido 230 ± 41 1.07
N-Succinimido 235 ± 29 1.03
N-Acrylamido 311 ± 23 1.10
3-Deoxy 404 ± 167 1.18
2-Hydroxy 650 ± 113 0.60
2-Formamido 1620 ± 220 1.46

Use of modified disaccharides to produce a range of Gal[alpha]1,3Gal trisaccharides

A range of disaccharides based on a type 2 core were tested as potential acceptor substrates for [alpha]1,3GalT. These included compounds with modifications at the C2 and C3 positions of the GlcNAc residue and the C6 position of the Gal residue (see Figure 2 for structures). In addition to LacNAc-C8, eight other type 2 disaccharides were found to serve as acceptors for the enzyme (Table I). However, the enzyme's relative affinity (reflected in the Km) for different compounds varied more than 20-fold (<200 to >1600 µM). For example, substitution of an azido group at the C2 position of Glc in the type 2 core produced an acceptor substrate with a Km of 150 µM, whereas substitution of a formamido group in the same position produced an acceptor with a Km more than 10 times larger (1620 µM). Despite this large variation in Km, these acceptors could be quantitatively converted to product as demonstrated in Figure 3.


Figure 2. Structures of compounds tested as acceptor substrates or inhibitors of [alpha]1,3GalT. (A) LacNAc-C8 disaccharide derivatives; (B) DTFP-LacNAc-C8.


Figure 3. TLC plate of products synthesized from type 2 compounds found to be acceptors for [alpha]1,3GalT. Products formed by [alpha]1,3GalT utilizing the following LacNAc-C8 disaccharide acceptor substrates: lane 1, LacNAc-C8; lane 2, N-acrylamido derivative; lane 3, N-succinimido derivative; lane 4, 3-deoxy derivative; lane 5, 2-azido derivative; lane 6, 2-propionamido derivative, lane S, LacNAc-C8 disaccharide starting material. The starting substrates ran just below the solvent front (i.e., 2-azido derivative), or between the solvent front and the position of LacNAc-C8 disaccharide.

NMR analyses demonstrated that each product contained the expected [alpha]-linked Gal residue (d, J. 3.7-4.0, [delta] 5.141-5.146) in addition to the two [beta]-linkages of the starting disaccharide (Table II). Additional resonances, to support the presence of the various substitutions (e.g., 2-propionamido) of the Gal or GlcNAc residues, were present in the NMR spectrum for each product. MALDI-TOF was used to further characterize some of the products. The expected molecular ions plus Na+ were obtained for the products formed from; LacNAc (m/z 738), 2-propionamido (m/z 752), N-acrylamido (m/z 750), and 3-deoxy (m/z 722).

Table II. 3GalT reactions utilizing modified Type 2 acceptors
LacNAc N-Acrylamido 3-deoxy N-Succinimido 2-Azido 2-Propionamido
  6.289
(dd, J. = 17.0, 1.4 Hz, vinyl)
       
  6.224
(dd, J. = 10.0, 17.0 Hz, vinyl)
       
  5.180
(dd, J. = 10.0, 1.4 Hz, vinyl)
       
5.146 5.146 5.141 5.145 5.144 5.144
(d, J. = 3.8 Hz, H-1[prime][prime]) (d, J. = 4.0 Hz, H-1[prime][prime]) (d, J. = 4.0 Hz, H-1[prime][prime]) (d, J. = 3.7 Hz, H-1[prime][prime]) (d, J. = 4.0 Hz, H-1[prime][prime]) (d, J. = 4.0 Hz, H-1)
4.544 4.581 4.524 4.545 4.576 4.576
(d, J. = 7.9 Hz, H-1[prime]) (d, J. = 8.2 Hz, H-1[prime]) (d, J. = 8.1 Hz, H-1[prime]) (d, J. = 7.9 Hz, H-1[prime]) (d, J. = 8.4 Hz, H-1[prime]) (d, J. = 8.4 Hz, H-1[prime])
4.526 4.554 4.490 4.540 4.527 4.527
(second order, J = 7.9 Hz, H-1) (d, J. = 7.9 Hz, H-1) (d, J. = 8.4 Hz, H-1) (second order, m, H-1) (d, J. = 7.7 Hz, H-1) (d, J. = 7.7 Hz, H-1)
    2.500
(ddd, J. = 12.6, 4.6, 4.6 Hz, H-3eq)
2.5-2.7
(m, C(O) CH2 CH2 C(O))
3.321
(dd, J. = 7.7, 10 Hz, H-2)
 
2.384 2.378 2.385 2.388 2.382 2.383
(t, J. = 7.2Hz, CH2COO) (t, J. = 7.5 Hz, CH2COO) (t, J. = 7.3 Hz, CH2COO) (t, J. = 7.4 Hz, CH2COO) (t, J. = 7.5Hz, CH2COO) (t, J. = 7.5Hz, CH2COO)
2.03
(s, 3H, NAc)
  1.984
(s, 3H, NAc)
    2.295
(t, J. = 7.8 Hz, CH2 CH3)
    1.664
(ddd, J. = 12.6, 12.0, 12.0 Hz, H-3a)
    1.128
(t, J. = 7.8 Hz, CH2CH3)

The NMR spectra of both 2-N-succinimido-LacNAc-C8 and its 3[prime]-[alpha]-galactosylated trisaccharide product were initially confusing in that small additional high-field peaks were present and these peaks increased in intensity when the samples were recovered from the NMR tubes and relyophilized before re-recording the spectra. In the case of the trisaccharide, the new peaks could clearly be assigned to the product where the N-succinate had dehydratively cyclized to the N-succinimide derivative based on the very high field signal for H-1 of the [beta] GlcN-succinimide residue ([delta] 5.226, J. 8.5 Hz), whereas the [alpha]-Gal H-1 signal ([delta] 4.543, J. 7.9 Hz) and the new [alpha]-Gal H-1 signal ([delta] 5.141, J. 3.7 Hz) retained the expected normal chemical shifts. Lemieux et al. (Lemieux et al., 1976) originally reported such unusual high-field signals for H-1 and H-3 of the related [beta]-GlcN-phthalimdo derivatives. Indeed, inspection of the NMR spectrum of the N-succinimido trisaccharide revealed the presence of the expected upfield shifted H-3 of the GlcN residue at [delta] 4.401 (J 10.8 Hz).


Figure 4. Summary of the functional substitutions of LacNAc that are and are not tolerated by [alpha]1,3GalT.

Additional disaccharides were tested as acceptor substrates for [alpha]1,3GalT including one with an -OSO3 substituted at C6 of the Gal residue, and others with an -OSO3 substituent at C3, or an NH2 at C2 or C3, or a formamido at C3 of the GlcNAc residue (see Figure 2). In addition to these type 2 disaccharides, the core type 1 (Gal[beta]1,3GlcNAc-C8) disaccharide was evaluated as an acceptor. None of these compounds served as acceptors. Figure 4 summarizes the types of substitutions that are and are not tolerated by [alpha]1,3GalT.

Evaluation of nonacceptor substrates as inhibitors

Although several of the tested compounds did not serve as acceptor substrates, they were potential inhibitors and could have value in designing photoprobes for the acceptor binding site of [alpha]1,3GalT. Thus, each of the nonacceptors were evaluated as inhibitors of [alpha]1,3GalT. Only the type 1 disaccharide functioned as an inhibitor of [alpha]1,3GalT. Figure 5 demonstrates that the type 1 disaccharide acts as a competitive inhibitor of [alpha]1,3GalT when LacNAc-C8 is used as the acceptor substrate for the enzyme. The Ki obtained was only slightly higher (270 vs. 190 µM) than the Km for LacNAc-C8, indicating that the enzyme has a similar affinity for the type 1 and type 2 disaccharides.


Figure 5. Inhibition of [alpha]1,3GalT by Gal[beta]1,3GlcNAc-C8. [alpha]1,3GalT (43 ng/µl enzyme) activity was measured with different concentrations of LacNAc-C8 in the presence of various concentrations (indicated in figure) of Gal[beta]1,3GlcNAc-C8. (A) Initial velocity vs. substrate concentration. (B) Lineweaver-Burk plot.

Developing photoprobes for [alpha]1,3GalT

In a previous study we (Helland et al., 1995) demonstrated that a derivative (substituted with an NH2 group at C3 of Gal and designated C3-NH2-LacNAc-C8) of LacNAc-C8 is an effective inhibitor of [alpha]1,3GalT. The availability of an amino function in the compound provided an opportunity to incorporate a photoaffinity label (2-diazo-3, 3, 3-trifluoropropionyl) at this position of the inhibitor, producing DTFP-LacNAc-C8. To determine if the DTFP substitution interferes with binding to [alpha]1,3GalT, a kinetic analysis was performed using LacNAc-C8 as the acceptor substrate. As demonstrated in Figure 6, DTFP-LacNAc-C8 was an inhibitor of [alpha]1,3GalT with a Ki of 300 µM. This Ki value is approximately three times greater than the Ki for C3-NH2-LacNAc-C8. As we (Helland et al., 1995) have previously reported for the C3-NH2-LacNAc-C8, the kinetics observed with DTFP-LacNAc-C8 do not appear to be competitive with respect to LacNAc. This has also been observed with other enzyme systems in which the binding site of an inhibitor displaying similar kinetics has been shown by crystallographic analysis is the substrate binding site (see references in Helland et al., 1995).


Figure 6. Inhibition of [alpha]1,3GalT by DTFP-LacNAc-C8. [alpha]1,3GalT (8 ng/µl enzyme) activity was measured with different concentrations of LacNAc-C8 in the presence of various concentrations (indicated in figure) of DTFP-LacNAc-C8. Initial velocity vs. substrate concentration and Lineweaver-Burk plot (inset).

Nucleotide sugar donor specificity of [alpha]1,3GalT

Previous analyses of glycosyltransferases have demonstrated that some have a strict specificity for the nucleotide sugar donor substrate, whereas others tolerate structural variants of the natural substrate. Those with flexibility can be used as versatile synthetic tools. Therefore, it was of interest to evaluate the nucleotide sugar donor specificity of [alpha]1,3GalT. Several UDP-sugars (UDP-Glc, -GlcNAc, -GlcA, -GalNAc) were evaluated as substrates. None were found to substitute for UDP-Gal when examined in either large scale synthesis reactions, or in the case of UDP-GlcNAc, in the radioactive-SepPak assay.

Inhibition of [alpha]1,3GalT by amino acid modifiers

We and others have previously demonstrated the value of probing glycosyltransferases with amino acid modifying reagents as an approach to identify amino acid residues near the substrate binding site (Holmes et al., 1995, and citations therein). As a first step to investigate [alpha]1,3GalT in a similar manner, we have analyzed a series of amino acid modifiers (Table III) for their effect on enzyme activity. Among eight compounds tested, only one (DEPC) caused a reduction (50%) in enzyme activity. Inhibition may result from the modification of an amino acid residue in or near the substrate binding, as previously demonstrated for [alpha]1,3 fucosyltransferases (Holmes et al., 1995), or for other reasons. To test the former possibility, protection studies were done with UDP-Gal and LacNAc-C8. Preincubation of [alpha]1,3GalT with either substrate did not significantly reduce the level of inhibition by DEPC.

Table III. Amino acid modifying reagents used with [alpha]1,3GalT
Reagent Amino acid modified Final concentration (mM)
N-Acetylimidizole Tyrosine 5
1,2-Cyclohexanedione Arginine 5
Diacetyl Arginine 20
Diethylpyrocarbonate Histidine, tyrosine 5
N-Ethylmaleimide -SH 5
Iodoacetamide -SH 5
Pyridoxal phosphate Lysine 2.5
Succinic anhydride Lysine 5

Discussion

We have presented a range of results related to the substrate specificity of [alpha]1,3GalT, and additional information on the effects of amino acid modifiers on the activity of this enzyme. In a previous study, we described the first synthetic inhibitor of this enzyme and have demonstrated the feasibility of extending this work to the synthesis of an inhibitor that incorporates a photolabel substituent. Our present results are discussed in the context of previous analyses of other galactosyltransferases which have been analyzed similarly.

A substantial database of information is available on the acceptor substrate specificity of [beta]1,4 galactosyltransferase ([beta]1,4GT; see a recent review by Palcic and Hindsgaul, 1996) and the blood group B [alpha]1,3 galactosyltransferase (B-transferase) (Lowary and Hindsgaul, 1993, 1994). [beta]1,4GT, an enzyme that transfers galactose from UDP-Gal to the 4-OH of an acceptor with inversion of configuration, has been extensively studied in terms of its ability to utilize modified forms of its substrate. This enzyme utilizes a wide range of GlcNAc and Glc analogs as acceptors, and can tolerate substitutions at all positions except the 4-OH. This includes substituents at C2 such as azido and alkylamido, C3 deoxy and O-methyl, and C6 substituents such as O-methyl, fucose. [beta]1,4GT also utilizes a range of nucleotide sugar donors including UDP-GalNAc, UDP-GlcNH2 and a range of deoxy analogs of UDP-Gal. However, it does not utilize UDP-GlcNAc or UDP-Man.

B-Transferase, an enzyme that transfers galactose from UDP-Gal to the 3-OH of an acceptor with retention of configuration, has been evaluated with deoxy, deoxyfluro, O-methyl, epimeric, and amino analogs of its disaccharide acceptor, Fuc[alpha]1,2Gal-R. Disaccharides with substitutions at C6 of Gal are substrates, those with substitutions at C3 are inhibitors, and those with substitutions at C4 do not bind to the enzyme. Thus, the OH at C4 appears to be essential for binding and is therefore a key polar group, whereas other substitutions are tolerated. The nucleotide sugar donor specificity of the B-transferase has been investigated, and it is clear that this enzyme can bind both UDP-Gal and UDP-GalNAc. However, there is a large difference in the enzyme's affinity for the these two nucleotide donors (15). Yamamoto and Hakomori (Yamamoto and Hakomori, 1990) have presented evidence that alterations of one or more amino acids that differ between the A- and B-transferases leads to an alteration in the resulting protein's ability to utilize UDP-Gal vs. UDP-GalNAc. A more recent kinetic analysis of A-, B-transferases, and recombinant variants which contain different combinations of the four amino acids that distinguish the parent enzymes has shown that the amino acid differences not only alter nucleotide sugar donor specificity, but also acceptor substrate binding (Seto et al. 1997).

The present results demonstrate that [alpha]1,3GalT can utilize a range of type 2 disaccharide substrates and can bind an additional compound (i.e., Gal[alpha]1,3GlcNAc-R; see Figure 4). The results obtained are similar to those reported for [beta]1,4GT. Thus, both enzymes can tolerate substitutions at C2 of the GlcNAc residue of the LacNAc disaccharide, including those that incorporate functional groups (i.e., azido, hydroxyl, formamido, propionamido, succinimido, and N-acrylamido) of various sizes and hydrophobicity. Interestingly, both enzymes have a high affinity for a disaccharide with a C2 azido group on Glc. It is also important to note that a 2-amino substitution at this position was not tolerated by the [alpha]1,3GalT. Thus, groups larger or smaller than the naturally occurring N-acetyl do not eliminate binding to the enzyme, but the presence of a positively charged group at this position does. Both enzymes utilize the 3-deoxy-GlcNAc-containing disaccharide as a substrate, indicating that the OH at this site is not required for enzyme recognition by either enzyme.

Both [alpha]1,3-galactosyltransferases ([alpha]1,3GalT and B-transferase) bind disaccharides with modifications at C3 of the Gal residue of their respective acceptors. In the present study, we expand on our observation (Helland et al., 1995) that, like the B-transferase, [alpha]1,3GalT can bind a disaccharide substrate in which the 3-OH of Gal has been replaced by an amino group. Both enzymes are inhibited by these compounds and bind them with relatively high affinity compared to their natural acceptors (i.e., LacNAc and Fuc[alpha]1,2Gal). In the present study, we have shown that a relatively large (DTFP) functional group is tolerated by [alpha]1,3GalT. The observation that [alpha]1,3GalT has a relatively high affinity for DTFP-LacNAc-C8 provides an opportunity for studies that could ultimately lead to the identification of amino acids that function in binding of the acceptor substrate by [alpha]1,3GalT.

Previous studies by Blanken et al. (Blanken and Van den Eijnden, 1985) and Elices et al. (Elices et al., 1986) showed that naturally occurring forms of [alpha]1,3GalT can utilize a type 1 disaccharide as an acceptor substrate, but the rate of catalysis was very low (<2%) compared to that obtained with LacNAc. We did not detect activity with a modified form of the type 1 disaccharide containing an aliphatic aglycon moiety (i.e., Gal[alpha]1-3GlcNAc-C8) with our recombinant enzyme. However, [alpha]1,3GalT could bind the type 1 core disaccharide and produced a competitive inhibition profile in the presence of LacNAc-C8. The enzyme bound the type 1 compound with a relatively high affinity; the Ki obtained was only slightly higher (270 vs. 190 µM) than the Km for LacNAc-C8.

[alpha]1,3GalT appears to have a rather narrowly restricted specificity for nucleotide sugar donors; however, further analyses are required for full characterization. Substitutions of the sugar residue at either C2 (NAc for OH or C2 epimerization), or at C2 and C6 (UDP-GlcA) are not tolerated. Thus, [alpha]1,3GalT has a more limited nucleotide sugar specificity than [beta]1,4GT or B transferase. Further studies are required to determine if deoxygenated forms of UDP-Gal can act as substrates, as with [beta]1,4GT.

To further characterize [alpha]1,3GalT, compounds known to modify amino acids were tested as inhibitors. A broad range of amino acid modifiers were tested, but only DEPC caused a reduction of [alpha]1,3GalT activity. The observed inhibition by DEPC, but not N-acetylimidizole, suggests that a His may be important for catalytic activity. Thus, the functional groups associated with Tyr, Arg, Cys, and Lys are either not important for substrate binding and/or catalytic activity, or are not accessible to modification by compounds used in this study.

Materials and methods

Reagents

Nucleotide sugar donors were obtained from Sigma (St. Louis, MO). UDP-[3H]Gal (15 mCi/mmol) was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Hydrofluor liquid scintillation cocktail was obtained from National Diagnostics (Manville, NJ). C8-disaccharide analogs were synthesized by standard procedures similar to those described previously (Field et al. 1995; Helland et al. 1995), details will be reported elsewhere. DTFP-LacNAc-C8 is a modification of methyl 3-amino-3-deoxy-[beta]-d-galactosylpyranosyl-(1-4)-2-acetoamido-2-deoxy-[beta]-d-glucopyranoside which was synthesized by acylation using p-nitrophenyl [alpha]-diazo-3, 3, 3,-trifluoropropionate from Pierce Chemical. All other reagents were of the highest grade available.

Enzyme preparation

A preparation of the murine enzyme used in these studies was obtained as follows (Henion et al., 1994). A cDNA PCR product corresponding to amino acids 62-376 was inserted in to the pPROTA expression vector (deVries et al., 1995). This construct was transfected into COS-7 cells from which the supernatant was collected. The secreted [alpha]1,3GalT-protein A chimera was recovered from the media by collection on IgG-agarose beads. The beads were rinsed and stored in PBS at 4°C. The chimeric protein was subsequently eluted from the beads to obtain the enzyme solution used in the experiments. This was done by rinsing an aliquot of beads (15 µl) with bis-TRIS propane buffer (1 ml; 1 mM, pH 8.0) followed by addition of citrate buffer (50 µl; 0.1 M, pH 4.4). The supernatant, which contained the enzyme, was then combined with bis-TRIS propane buffer (20 µl; 1 M, pH 8.2, 0.15 M NaCl, 1 mg/ml BSA). Protein concentration was determined using a Western blot method as described previously (Holmes et al., 1995).

C18 Sep-Pak assay

A Sep-Pak assay was developed to measure the product from the [alpha]1,3GalT reaction. The following components were added to a microtiter plate in a final volume of 50 µl: 20-40 mM MnCl2, 1 mM UDP-Gal, 0.2 µCi UDP-[3H]Gal, 100 mM cacodylate, pH 6.0, 0.4% TX-100, 400 µM LacNAc-C8. A reaction mixture without acceptor was used as a control. After incubation at 37°C for 3 h the reaction was terminated by removing the enzyme reaction mixture from each well and rinsing the microtiter wells with distilled, deionized water (DDW; 100 µl, three times). The sample mixture and well rinses were passed through a C18 Sep-Pak. The product and any unreacted acceptor were eluted with methanol (6 ml) after rinsing the column with DDW (20 ml). The methanol, containing the radiolabeled product, was dried under a stream of nitrogen in a scintillation vial. Hydrofluor (5 ml plus 0.5 ml DDW) was added to the dry vial, which was counted in a liquid scintillation counter (Beckman LS 3801). The enzyme reaction was linear up to 4 h under these conditions.

For the enzyme characterization experiments the reaction components were varied over the specified ranges: Mn2+, 0-120 mM; UDP-Gal, 0-8 mM; TX-100, 0-0.8%; cacodylate, pH 5-9; LacNAc-C8, 0-500 µM. For the inhibitor/acceptor experiments, the concentration of the C8-disaccharide derivatives were varied from 0 to 640 µM.

Amino acid modifying reagents

Further characterization of [alpha]1,3GalT was done with amino acid modifying reagents. An enzyme mixture was made: 3 µl enzyme, 2 µmol cacodylate (pH 6.0), and either 50 nmol UDP-Gal plus 0.2 µCi UDP-[3H]Gal, or LacNAc-C8, in a total volume of 19 µl; a second mixture identical to this was made except that water was substituted for UDP-Gal or LacNAc-C8. (In some assays with UDP-Gal 1 µmol MnCl2 was included in the preincubation rather than adding it to the final reaction mixture. The results were the same in either case.) Each mixture was incubated for 0.5 h on ice; subsequently, 1 µl of each reagent was added to an aliquot of each solution and incubated on ice for 0.5 h. Each reagent solution was made fresh and added to the enzyme mixture. After the incubations on ice, the following components were added: 1 µmol MnCl2 (if not added in the preincubation mixture), 3 µmol cacodylate (pH 6.0), 2 µl TX-100 (10%, w/v), and 20 nmol LacNAc-C8 (if not added in the preincubation mixture). The complete mixture (total volume 50 µl) was incubated at 37°C for 3 h in the dark and the radioactive product isolated as described for the C18 Sep-Pak assay.

Kinetic analysis

The kinetic constants given here were determined with the computer program KinetAssyst. The data were fit to the Michaelis-Menten equation by an iterative fitting routine.

Scale-up of the [alpha]1,3GalT reaction

To facilitate product characterization, the reaction was scaled to produce 1 mg product. A 1 ml reaction mixture (containing 0.5 mg acceptor, 4 µmol UDP-Gal, 40 µl TX-100 (10%), 50 µmol MnCl2, 5 µmol cacodylate (pH 6.0)) was added to 38 µl of the IgG-agarose beads with bound [alpha]1,3GalT. The reaction tubes were incubated at 37°C for 24 h and then stored at 4°C until the products were recovered by elution from a C18 Sep-Pak with 10 ml MeOH after rinsing with 40 ml DDW. To determine the completeness of the reaction, aliquots representing 3 nmol of each nonreacted acceptor and eluted reaction product were applied on separate HPTLC plates (Silica Gel 60, EM Separations). Chromatographic separation was done with CHCl3/MeOH/DDW (60:35:6), the acceptors and products were visualized with orcinol reagent.

Other nucleotide sugar donors

To evaluate the possibility that [alpha]1,3GalT could utilize other nucleotide sugar donors the following scaled-up reaction mixtures were prepared. A 100 µl reaction mixture (containing 80 nmol LacNAc-C8, 1.6 µmol UDP-sugar, 3.2 µl TX100 (10%), 4 µmol MnCl2, 8 µmol cacodylate (pH 6.0)) was added to 30 µl of the IgG-agarose beads with bound [alpha]1,3GalT. (In the case of UDP-GlcNAc a separate reaction was done as described under C18 Sep-Pak assay above, utilizing UDP-3H-GlcNAc.) Each reaction mixture was incubated in a microfuge tube at 37°C for 3 days and then stored at 4°C. The products were recovered by elution from a C18 Sep-Pak with 6 ml MeOH after rinsing with 20 ml DDW. The nonradioactive product was dissolved in 100 µl of chloroform/methanol (1:1), and 10 µl was spotted on a glass HPTLC plate (Silica Gel 60, EM Separations). Chromatographic separation was done with C/M/W 60:35:8 (v/v/v).

NNR and MALDI mass spectrometry

1H-NMR spectra were recorded in D2O on Varian spectrometers operating at either 500 or 600 MHz at ambient at 30°C. MALDI mass spectra were recorded on a Kratos Linear I instrument using gentisic acid (2,5-dihydroxybenzoic acid) as the matrix.

Acknowledgments

We thank Dr. Albin Otter and Ms. Shumei Wang for assistance with the NMR and MALDI analyses. This work was supported by grants to B.A.M. from the National Institutes of General Medicine (GM40205), and National Center for Research Resources (Research Infrastructure in Minority Institutions (P20 RR11805) with funding from the Office of Research on Minority Health), National Institutes of Health, and to O.H. from the Natural Sciences and Engineering Research Council of Canada.

Abbreviations

DEPC, diethylpyrocarbonate; DDW, distilled, deionized water; EDTA, ethylene diamine tetraacetic acid; HPTLC, high performance thin layer chromatography; LacNAc-C8, N-acetyllactosamine-O-(CH2)8COOCH3; MeOH, methanol; nLc4-C8, Gal[beta]1,4GlcNAc[beta]1,3Gal[beta]1,4Glc-O-(CH2)8COOCH3; PBS, phosphate-buffered saline; DTFP-LacNAc-C8, N-2-diazo-3, 3, 3-trifluoropropionyl-3-amino-3-deoxy-[beta]-d-galacto-pyranoside-(1,4)-2-acetoamido-2-deoxy-[beta]-d-glucopyranoside; TLC, thin layer chromatography; TX-100, Triton X-100.

References

Blanken ,W.M. and Van den Eijnden,D.H. (1985) Biosynthesis of terminal Gal[alpha]->3Gal[beta]1-4GlcNAc-R oligosaccharide sequence on glycoconjugates: purification and acceptor specificity of a UDP-Gal: N-acetyllactosaminide [alpha]1-3 galactosyltransferase. J. Biol. Chem., 260, 12927-12934. MEDLINE Abstract

Clark ,G.F., Krivan,H.C., Wilkins,T.D. and Smith,D.F. (1986) Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal[alpha]1-3Gal[beta]1-4GlcNAc sequence. Arch. Biochem. Biophys., 257, 217-229.

Cooper ,D.K.C., Good,A.H., Koren,E., Oriol,R., Malcolm,A.J., Ippolito,R.M., Neethling,F.A., Ye,Y., Romano,E. and Zuhdi,N. (1993) Identification of [alpha]-galactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies: relevance to discordant xenografting in man. Transplant Immunol., 1, 198-205.

deVries ,T., Srnka,C.A., Palcic,M.M., Swiedler,S.J., van den Eijnden,D.H. and Macher,B.A. (1995) Acceptor specificity of different length constructs of human recombinant [alpha]1,3/4-fucosyltransferases. Replacement of the stem region and the transmembrane domain of fucosyltransferase V by protein A results in an enzyme with GDP-fucose hydrolyzing activity J. Biol. Chem., 270, 8712-8722. MEDLINE Abstract

Elices ,M.J., Blake,D.D. and Goldstein,I.J. (1986) Purification and characterization of a UDP-Gal:[beta]-d-Gal (1,4)-d-GlcNAc [alpha] (1-3) galactosyltransferase from Ehrlich ascites tumor cells. J. Biol. Chem., 261, 6064-6072. MEDLINE Abstract

Field ,R.A., Otter,A., Fu,W. and Hindsgaul,O. (1995) Synthesis and 1H-NMR characterization of the six isomeric non-sulfates of 8-methoxycarbonyloct-1-yl O-[beta]-galactopyranosyl- (1,4)-2-acetamindo-2-deoxy-[beta]-glucopyranoside. Carb. Res., 276, 347-363.

Galili ,U. (1993) Interaction of the natural anti-Gal antibody with [alpha]-galactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol. Today, 14, 480-482. MEDLINE Abstract

Galili ,U. (1989) Abnormal expression of [alpha]-galactosyl epitopes in man: a trigger for autoimmune processes? Lancet, II, 358-361.

Galili ,U., Clark,M.R., Shohet,S.B., Buehler,J. and Macher,B.A. (1987) Evolutionary relationship between the anti-Gal antibody and the Gal[alpha]1->3Gal epitope in primates. Proc. Natl Acad. Sci. USA, 84, 1369-1373. MEDLINE Abstract

Galili ,U., Kobrin,E., Shohet,S.B., Stults,C.L.M. and Macher,B.A. (1988) Man, apes and Old World monkeys differ from other mammals in the expression of [alpha]-galactosyl epitopes on nucleated cells. J. Biol. Chem., 263, 17755-17762. MEDLINE Abstract

Helland ,A.-C., Hindsgaul,O., Palcic,M.M., Stults,C.L.M. and Macher,B.A. (1995) Methyl 3-amino-3-deoxy-[beta]-d-galactopyranosyl- (1->4)-2-acetamido-2-deoxy-[beta]-d-glucopyranoside: an inhibitor of UDP-d-galactose: [beta]-d-galactopyranosyl- (1->4)-2-acetamido-2-deoxyl-d-glucose (1->3)-[alpha]-d-galactopyranosyltransferase. Carbohydr. Res., 276, 91-98. MEDLINE Abstract

Henion ,T.R., Macher,B.A., Anaraki,F. and Galili,U. (1994) Defining the minimal size of catalytically active primate [alpha]1,3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme. Glycobiology, 4, 193-201. MEDLINE Abstract

Holmes ,E.H., Xu,Z., Sherwood,A.L. and Macher,B.A. (1995) Structure-function analysis of human [alpha]1->3fucosyltransferase. J. Biol. Chem., 270, 8145-8151. MEDLINE Abstract

Joziasse ,D.H., Shaper,J.H., Van den Eijnden,D.H., Van Tunen,A.H. and Shaper,N.L. (1989) Bovine [alpha]1-3 galactosyltransferase: isolation and characterization of cDNA clone. Identification of homologous sequences in human genomic DNA. J. Biol. Chem., 264, 14290-14297. MEDLINE Abstract

Krivan ,H.C., Clark,G.F., Smith,D.F. and Wilkins,D.T. (1986) Cell surface binding site for Colstridium difficile enterotoxin: evidence of a glycoconjugate containing the sequence Gal[alpha]->3Gal[beta]1->4GlcNAc. Infect. Immun., 53, 573-581. MEDLINE Abstract

Larsen ,R.D., Rivera-Marrero,C.A., Ernst,L.K., Cummings,R.D. and Lowe,J.D. (1990) Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal[beta]-d-Gal (1,4)-d-GlcNAc[alpha] (1,3) galactosyltransferase cDNA. J. Biol. Chem., 265, 7055-7061. MEDLINE Abstract

Lemieux ,R.U., Takeda,T. and Chung,B.Y. (1976) Synthesis of 2-amino-2-deoxy-[beta]-d-glucopyranosides. Properties and use of 2-deoxy-2-phthalimidoglycosyl halides. American Chemical Society Symposium Series No., 39, Synthetic Methods for Carbohydrates, pp. 90-115

Lowary ,T.L. and Hindsgaul,O. (1993) Recognition of synthetic deoxy and deoxyfluoro analogs of the acceptor [alpha]-l-Fucp-(1->2)-[beta]-d-Galp-OR by the blood-group A and B gene-specified glycosyltransferases. Carbohydr. Res., 249, 163-195. MEDLINE Abstract

Lowary ,T.L. and Hindsgaul,O. (1994) Recognition of synthetic O-methyl, epimeric and amino analogues of the acceptor [alpha]-l-Fucp- (1->2)-[beta]-d-Galp-OR by the blood-group A and B gene-specified glycosyltransferases. Carbohydr. Res., 251, 33-67. MEDLINE Abstract

Palcic ,M.M. and Hindsgaul,O. (1996) Glycosyltransferases in the synthesis of oligosaccharide analogs. Trends Glycosci. Glycotechnol., 8, 37-49.

Seto ,N.O.L., Palcic,M.M., Compston,C.A., Li,H., Bundle,D.R. and Narang,S.A. (1997) Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferases boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes. J. Biol. Chem., in press.

Yamamoto ,F. and Hakomori,S. (1990) Sugar-nucleotide donor specificity of histo-blood group A and B transferases is based on amino acid substitutions. J. Biol. Chem., 265, 19257-19262. MEDLINE Abstract


3To whom correspondence should be addressed at: Department of Chemistry/Biochemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132
4Present address: Inhale Therapeutic Systems, 150 Industrial Road, San Carlos, CA 94407


This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification: 10 Jun 1999
Copyright©Oxford University Press, 1999.