Role of a conserved acidic cluster in bovine [beta]1,4 galactosyltransferase-1 probed by mutagenesis of a bacterially expressed recombinant enzyme

Yingnan Zhang, Vladimir A.Malinovskii, Tristan J.Fiedler and Keith Brew1

Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101, USA

Received on December 17, 1998; revised on February 8, 1999; accepted on February 8, 1999

The truncated catalytic domain of bovine [beta]1,4 galactosyltransferase-1 was expressed as inclusion bodies in E.coli and folded to generate 10-15 mg of active enzyme per liter of bacterial culture after extraction and purification under denaturing conditions. Mutations were introduced to investigate the roles of Trp312, Asp318, and Asp320, components of a highly conserved region of sequence in all known [beta]4GT-1 homologues that includes a cluster of acidic residues. Near and far UV CD spectra of the mutants indicate that the substitutions did not perturb the secondary and tertiary structure of [beta]4GT-1, and steady state kinetic studies indicate only minor effects on the response to an essential metal cofactor. However substitutions for the two aspartyl residues result in a reduction in catalytic efficiency of a magnitude that suggests they are important for catalysis. It seems possible that this anionic center may act in stabilizing a carbocation formed from the galactose component of the donor substrate in the transition state, reflecting a common reaction mechanism for [beta]-galactosyltransferase reactions.

Key words: [beta] 1,4 galactosyltransferase/expression in E.coli./mutagenesis/reaction mechanism

Introduction

Hetero-oligosaccharides play important roles in cellular recognition processes, transformation and biological structures (Varki, 1993). Although many of the glycosyltransferases that catalyze the synthesis of different linkages in glycoconjugates have been cloned and sequenced, there is limited information regarding their structures and mechanisms of action. UDP-galactose [beta]-N-acetylglucosaminide [beta]4-galactosyltransferase-1 ([beta]4GT-1; EC 2.4.1.38), a trans-Golgi membrane enzyme that catalyzes the attachment of [beta] galactose to the 4-position of [beta]-linked N-acetylglucosamine, is one of the most extensively characterized glycosyltransferases. Its specificity is not as strict as those of many other glycosyltransferases and it has consequently proved to be useful for the enzymatic synthesis of various natural and unnatural oligosaccharides (Palcic and Hindsgaul, 1991; Ichikawa et al., 1992; Öhrlein et al., 1992). In vivo, [beta]4GT-1 has a well-defined second function as the catalytic component of lactose synthase, the enzyme system that catalyzes and regulates the synthesis of lactose in the lactating mammary gland (Brew et al., 1968; Hill and Brew, 1975). The interaction of [beta]4GT-1 with the lactose synthase regulatory protein, [alpha]-lactalbumin (LA), results in a change in specificity for acceptor substrates so that glucose, a marginal acceptor substrate, is bound with an affinity that is increased by three orders of magnitude. The combination of the two proteins can consequently catalyze lactose synthesis efficiently. LA also promotes the binding of a range of other acceptors, including noncarbohydrate molecules to [beta]4GT-1, further enhancing its synthetic capabilities (Schanbacher et al., 1970; Yu et al., 1995). A fraction of [beta]4GT-1 is found on the surfaces of some cells which may act as an adhesion molecule by binding to oligosaccharides in the extracellular matrix and on the surfaces of other cells (Shur, 1991); in this context, roles have been attributed to [beta]4GT-1 in a wide range of cell-cell interactions (Shur, 1983; Bayna et al., 1988; Miller et al., 1992; Pratt et al., 1993).

The sequences of [beta]4GTs from different sources indicate, as for other glycosyltransferases (Appert et al., 1986; Narimatsu et al., 1986; Shaper et al., 1986, 1988), the presence of a short N-terminal cytosolic region, a helical hydrophobic transmembrane segment, a stem of about 60 residues and a C-terminal catalytic region (Paulson and Colley, 1989). Recently, five additional homologous [beta]4GTs have been identified in human ([beta]4GT -2 to -6) which differ in acceptor substrate specificity and sensitivity to [alpha]-lactalbumin (Almeida et al., 1997; Lo et al., 1998; Schwientek et al., 1998). Four [beta]4GT-1 homologues have been identified in invertebrates, a UDP-GlcNAc : [beta]-GlcNAc GlcNAc transferase from the pond snail (Bakker et al.,1994), a protein from C.elegans that has a similar domain structure to [beta]4GT-1, a second additional C.elegans clone that encodes a molecule that lacks the stem region and the N-terminal section of the catalytic domain; we have also recently identified a homologous EST from Drosophila (accession number AA142310 from the dbEST database). When all [beta]4GT homologues are compared, a few highly conserved regions of sequence are found that include a region corresponding to residues 305-324 of bovine [beta]4GT-1 [NGfpNnYwGWGgEDDdiynR] (Figure 1). This is located in a region of the catalytic domain that has been implicated in UDP-galactose binding by chemical (Yadav and Brew, 1990, 1991) and mutational studies (Aoki et al., 1990; Zu et al., 1995).


Figure 1. Alignment of the region conserved in (A) [beta]4GT (Shaper et al., 1997; Lo et al., 1998), (B) Lex1, (C) FNG, (D) BRN, and (E) [beta]1,3 galactosyltransferase subfamilies. Residues conserved within subfamilies are boldfaced. The conserved patches among all the sequences are shaded. Angled brackets indicate the omission of amino acids flanking the conserved sequences.

Truncated forms of [beta]4GT-1 have been previously expressed in yeast (Krezdorn et al., 1993; Herrmann et al., 1995) and E.coli (Boeggeman et al., 1993; Nakazawa et al., 1993). With the yeast system, using fermentation technology, yields of secreted [beta]4GT-1 are about 700 mU/l (~0.3 mg). [beta]4GT-1 has also been expressed in E.coli as inclusion bodies, from which folded protein has been generated in yields of about 1 mg/ liter (Boeggeman et al., 1993); similar low yields have also been obtained using an E.coli secretion vector (Nakazawa et al., 1993). With these systems insufficient protein has been obtained for characterizing the effects of mutations on structure as well as activity. We describe here the expression of bovine [beta]4GT-1 using the pET15b vector, with which a truncated form of the catalytic domain is produced with a N-terminal 6-histidine tag, allowing the protein extracted from inclusion bodies with denaturants to be readily purified under denaturing conditions. Conditions for folding the protein in high yields have been devised, to provide up to 100 U/l of bacterial culture. After purification the recombinant enzyme has kinetic and regulatory properties as well as a CD spectrum that are closely similar to the wild-type enzyme. Mutagenesis studies with this system indicate that the conserved cluster of anionic residues is crucial for the catalysis and not for the binding of the metal cofactor or substrate; a possible role in transition state stabilization is discussed.

Results

Expression of [beta]4GT

[beta]4GT-1 was expressed as a His-Tag fusion protein using the pET15b vector to facilitate purification under denaturing conditions prior to folding. pET3a is an efficient vector for expressing recombinant truncated [beta]4GT-1 (designed as r[beta]4GT-1) as inclusion bodies from which the protein can be extracted with urea and other denaturants, but only low levels of folding (~2%) were achieved using a variety of procedures and large amounts of precipitate and low yields of active enzyme were obtained when folding was attempted by dialysis or dilution methods. This appears to result from the interaction of the highly cationic [beta]4GT-1 molecule (pI 9.6) and negatively charged nonprotein components, such as nucleic acids that are also present in inclusion bodies, since the truncated [beta]4GT-1 extracted from inclusion bodies with 8 M urea did not bind to cation exchange columns at pH 6 (Malinovskii and Brew, unpublished observations). However, protein extracted with 6 M guanidine HCl from inclusion bodies produced by expression from the pET15b vector was readily purified by Ni2+ chelate chromatography. Using the protein purified in this manner, 70-100 U of active enzyme were generated from the protein expressed in a liter of bacterial culture; this corresponds to 10-15 mg of folded r[beta]4GT-1 (Table II and Table III). The presence of a redox system is necessary for generating active enzyme, indicating the need for disulfide bond formation during folding.

Table I. Primers used for construction of pET15b_r[beta]4GT-1 and mutagenesis
Primer Sequence Orientation
XhoI-GT CCTTTGTATGTGCAATTCG Complementary
StuI-GT CAGTTAGACTATGGCATC Coding
D318N:D320N TGGGGAGGTGAAAACGATAACATTTATAACAGA Coding
D318N TGGGGAGGTGAAAACGATGACATTTATAACAGA Coding
D318E TGGGGAGGTGAAGAGGATGACATTTATAACAGA Coding
W312F TTTCCTAATAACTACTTCGGCTGGGGA Coding

Table II. Yields of r[beta]4GT-1 and its variants during purification
Protein Yield (mg/l of culture)
r[beta]4GT-1 10.0
D318N:D320N 5.3
D318N 6.1
D318E 7.0
W312F 11.0

Properties of recombinant [beta]4GT-1

The folded enzyme, isolated after chromatography in the absence of denaturant showed a single band on SDS-polyacrylamide gel electrophoresis with a mobility close to the 31 kDa marker, which is consistent with the predicted molecular weight of 33 kDa (Figure 2). The near and far UV CD spectra (Figure 3) are very similar to those previously reported for bovine [beta]4GT-1 (Geren et al., 1975). Analysis of the far UV CD spectrum to determine the secondary structure composition using the k2d neural network program (Andrade et al., 1993; Merelo et al., 1994) gave 29% [alpha]-helix and 21% [beta]-sheet, but the predicted spectrum had a poor fit with the experimentally determined spectrum. Although the far UV CD spectra of proteins are strongly influenced by contributions from secondary structure elements, the side chains of aromatic amino acids in fixed environments in the structure can also contribute to the spectrum in this region (Woody, 1995). The sequence of [beta]4GT-1 has a high content of aromatic amino acids, particularly of Tyr and Phe, which may account for the poor fit obtained for the secondary structure analysis.

Previous work has shown that [beta]4GT-1 binds two Mn2+ ions, with micromolar and millimolar affinities, respectively (Powell and Brew, 1976). The activity of the recombinant enzyme over a wide range of Mn2+ concentrations was found to fit best to an equation describing two-site binding in which saturation of the high affinity site produces activity V1 and at the second lower affinity site, a higher level of activity V2 (Table III).


Figure 2. SDS-PAGE analysis of wild type recombinant [beta]4GT-1 or mutants D318E, D318N, W312F, and D318N:D320N after folding; 1.5 µg/lane of sample in 20 mM Tris-HCl pH7.4 and 50% glycerol were loaded to the gel with sample loading buffer containing 0.36 M Tris-HCl, pH6.7/10% SDS/40% glycerol/50 mM DTT/0.005% bromophenol blue. Lanes 1-5 are wild type [beta]4GT-1, D318E, D318N, D318N:D320N, and W312F, respectively.


Figure 3. Far (A) and near (B) UV circular dichroism spectra of r[beta]4GT-1. These were determined at a protein concentration of 0.23mg/ml at 25°C. [solid line] Wild type; [-·-·-·-] W312F; [··-··-··] D318E; [········] D318N:D320N; [· · · · ·] D318N. Differences between the spectra can be accounted by the effects of the substitution itself on the CD spectrum, as opposed to structural perturbations. Near UV CD spectra have multiple peaks and troughs resulting from the fixed asymmetric environments of aromatic side chains and disulfide bonds while far UV CD spectra are strongly influenced by secondary structure (Woody, 1995). Differences between the single site mutants are small and appear to be effects of the substitutions on the environments of aromatic residues or, in the case of the Trp312 to Phe mutant, the direct contribution of the side chain of this residue to the spectrum. This mutant shows the largest change in the far UV region that is likely to reflect the contribution of Trp312 to the spectrum in this region. The near UV CD spectrum of the double mutant is weaker but similar in shape to those of the other proteins and is minor compared to changes observed in structurally perturbed mutants of other proteins (e.g., see Huang et al, 1997).

Analysis of steady state kinetic data at a fixed concentration of Mn2+ in which the concentrations of both UDP-galactose and GlcNAc substrates are varied or the concentrations of LA and glucose are varied at a fixed concentration of UDP-galactose gives values for kinetic parameters (Table III) that are similar to those previously reported for bovine [beta]4GT-1 (Powell and Brew, 1974) except the specific activity (the kcat) for r[beta]4GT-1 is 3- to 4-fold lower than that of the enzyme purified from milk.

Mutants of r[beta]4GT-1

As discussed previously, mutations were introduced into r[beta]4GT-1 to investigate the possible role of a conserved region of sequence W312GWGGEDDD320, which represents a plausible location for a binding site for the catalytically essential cation. Because of the highly conserved nature of this region, substitutions were designed to be structurally conservative and to introduce changes in size or charge; the goal was to perturb but not eliminate activity so that the roles of residues can be examined quantitatively through their effects on kinetic parameters. Phenylalanine was substituted Tryptophan 312 since this substitution is present in the homologous UDP-GlcNAc : [beta]-GlcNAc GlcNAc transferase from the pond snail (Bakker et al.,1994). Aspartates 318 and 320 were selected for mutagenesis since they form a DXD motif that is found in many glycosyltransferases and hydrolases (Breton et al., 1998). Substitutions were made of asparagine and glutamate for aspartate 318, and asparagine for both aspartates 318 and 320 using the mutagenic primers listed in Table I.

The entire sequences of the mutants were checked by DNA sequencing, confirming the presence of the desired mutation in each case and that no unwanted mutations were introduced by the PCR mutagenesis procedure. All proteins expressed at similar levels as inclusion bodies and behaved similarly during the folding process; folded forms of the proteins were isolated in final yields of 6-11 mg/l of culture after the chromatography and ammonium sulfate precipitation steps (Table II) and were homogeneous on SDS-gel electrophoresis (Figure 2).

Figure 3 shows the near and far UV CD spectra of the mutant proteins in comparison with wild type r[beta]4GT-1. Analysis of the far UV CD spectra indicate that the substitutions have little effect on the calculated secondary structure content; the near UV spectra are similar in detail, although the spectrum of the double mutant is less strong. Overall, the CD spectra indicate that the mutations do not result in gross changes in structure; however, their effects on local structure cannot be determined.

Table III. Kinetic parameters determined for r[beta]4GT-1 and variants, compared with those previously reported for bovine [beta]4GT-1 (Powell and Brew, 1974, 1976)
Ligand Parameter Bovine [beta]4GT-1 r[beta]4GT-1 D318N D318E D318N: D320N W312F
UDP-galactose Kia 0 0 0.12 ± 0.02 1.7 ± 0.5 NAa 0.15 ± 0.04
  Ka 0.28 mM 0.13 ± 0.02 mM 0.10 ± 0.03 mM 0.7 ± 0.2 mM   0. 9 ± 0.2 mM
GlcNAc Kb 25 mM 14 ± 2 mM 21 ± 3 mM 18 ± 5 mM NAa 64 ± 9 mM
Bovine LA Kc 0 0 NAa NAa NAa 0
  Kic 25.5 µM 27 ± 4 µM       36 ± 8 µM
Mn2+ b K1 20 µM 37 ± 8 µM 17 ± 1 µM 31 ± 21 µM NAa 17 ± 6 µM
  K2 1 mM 0.92 ± 0.06 mM 0.55 ± 0.09 mM 1.9 ± 0.7 mM   2.0 ±0.1 mM
glucose Kd 1.7 mM 2.7 ± 0.5 mM NAa NAa NAa 0.43 ± 0.09 mM
  Spec. activity 26 (µmol/min/mg) 7.7 ± 0.6 (µmol/min/mg) 0.049 ± 0.005 (µmol/min/mg) 0.051 ± 0.005 (µmol/min/mg) 0.003 (µmol/min/mg) 8.1 ± 0.7 (µmol/min/mg)
These values were determined at 37°C in the presence of 10 mM MnCl2 as described in Materials and methods.
aThe activity is too low to measure the relevant parameter.
b[beta]4GT-1 has two Mn2+ binding sites.

Functional properties of mutants of r[beta]4GT-1

[beta]4GT-1 mutants with substitutions for Asp318 and 320 were much less active than the wild-type protein and were assayed at higher protein concentrations. Mutant enzymes were analyzed with respect to Mn2+-activation in comparison with the wild-type protein. Data fitted to an equation describing two metal-binding sites (Powell and Brew, 1976) indicate insignificant differences between the mutants and wild-type protein (Table III). Other kinetic analyses were conducted at an essentially saturating concentration of metal ion (10 mM). Initial velocity data obtained with varying concentrations of UDP-galactose at a series of fixed concentrations of GlcNAc give essentially parallel lines in double reciprocal plots for the wild-type protein and fit best to a rate equation that lacks a Kia term (Equation 2). The parameters for these mutants are summarized in Table III. Compared to wild type r[beta]4GT-1, the two mutants with substitutions for Asp318, asparagine, and glutamic acid have only 0.1% of the activity of the wild-type protein, and the double mutant, D318N:D320N had ~0.01% activity of wild type r[beta]4GT-1. In contrast, the Trp312 to Phe mutant has a similar activity to the wild type protein. Steady state kinetic parameters determined by a detailed analysis of the single-site mutants are summarized in Table III; however, the extremely low level of activity of the double mutant precluded this type of analysis. The results indicate that the similar kinetic parameters associated with Mn2+, GlcNAc and UDP-Gal, are similar to those of the wild-type protein except in the case of D318E, Kia, the equilibrium dissociation constant for UDP-Gal binding from the enzyme·Mn2+ complex was much greater. This change is illustrated in Figure 4. The principle effect of the substitutions for D318 and D320 are therefore a major decrease in kcat.


Figure 4. Comparison of steady state kinetic properties between mutants (A) D318N and (B) D318E. The double reciprocal plots show variation of N-acetyl-lactosamine synthase activity with the concentrations of UDP-galactose and GlcNAc at 10 mM Mn2+. UDP-galactose is plotted as the variable substrate at the following fixed concentrations of GlcNAc. (A) solid circles, 6 mM; open circles, 7.5 mM; inverted solid triangles, 10 mM; inverted open triangles, 15 mM; solid squares, 30 mM. (B) solid circles, 6 mM; open circles, 10 mM; inverted solid triangles, 15 mM; inverted open triangles, 30 mM; solid squares, 50 mM.

Discussion

Previous mutational studies of soluble truncated forms of [beta]4GT-1 have utilized low yield bacterial secretion expression systems (Aoki et al., 1990; Zu et al., 1995). Substitutions of Gly for Tyr286, Tyr311, or Trp312 (sequence numbering is given in Figure 1), near the acidic cluster, were found to perturb the binding of a disaccharide acceptor substrate, N, N[prime] diacetylchitobiose, but replacement of Tyr311 by Gly perturbs kinetic parameters for UDP-galactose and chitobiose (Aoki et al., 1990). Substitutions of Gly for Tyr289 or Trp312 generated enzyme variants with insufficient activity for characterization, but a Phe for Tyr289 mutation had insignificant effects on kinetic parameters for the donor and acceptor substrates. Unfortunately, neither specific activities nor kcat values were given for these variants and the Km values for acceptor substrate reported for two mutants exceed the highest concentration used in the assays. Consequently, the roles of the residues in [beta]4GT-1 action require further investigation and the kinetic parameters are of questionable validity. Mutations were also introduced for Phe307, Pro308, Asn309, and Asn310 that were reported to affect catalytic activity (5- to 10-fold) and the Km for UDP-galactose. However, these parameters were apparent values, determined at a fixed concentration of acceptor substrate and do not necessarily report changes in the true kinetic parameter (Zu et al., 1995). The structures of these mutants of [beta]4GT-1 were also not characterized in these studies. Thus, although mutations in the region between residues 286 and 312 in [beta]4GT-1 perturb activity, the exact basis of this has not been established.

Our results show that conservative substitutions for Asp318 and 320 do not alter the ability of r[beta]4GT-1 to fold to a correct native structure and produce relatively small changes in the binding of the Mn2+ cofactor and acceptor and donor substrates. However, there is a large reduction in kcat with a magnitude comparable to that obtained when residues of the catalytic triad are mutated in serine proteases (Fersht and Sperling, 1973). kcat/(Kia*Kb), a parameter for bisubstrate reaction that is related to transition state stabilization and is a measure of catalytic efficiency, for D318N and D318E is decreased by 3 and 5 orders, respectively, compared to that of human milk [beta]4GT-1 (Khatra et al., 1974). The substitution of Phe for W312, six amino acids away from the highly conserved DDD (318-320) patch, did not perturb the enzyme properties. These results suggest that these highly conserved aspartates in [beta]4GT-1 have a critical role in catalysis.

The kinetic mechanism of [beta]4GT-1 is sequential, in keeping with a displacement mechanism through which the 4-OH of the acceptor displaces the phosphoryl-galactose bond in the donor substrate, resulting in the inversion of configuration at the galactose C1. Secondary deuterium isotope effects observed with [1-2H]-UDP-galactose (Kim et al., 1988) and competitive inhibition by UDP-(2-deoxy-2-fluoro)-galactose (Hayashi et al., 1997) indicate that the galactose transfer reaction mechanism involves an intermediate in which the anomeric C of galactose has sp2 character and the UDP to galactose bond is substantially cleaved in the transition state. The stabilization of a cationic galactose moiety by this anionic region of [beta]4GT-1 in the transition state provides a plausible, but not unique, explanation of the results reported here. Aspartates 318 and 320 do not directly affect substrate and metal ion binding, but do affect the interaction between the enzyme and the transition state in the catalytic mechanism.

Previously, a conserved DXD motif has been noted in many prokaryotic and eukaryotic galactosyltransferases (Breton et al., 1998) and other glycosyltransferases (Wiggins and Munro, 1998). It has been speculated that these residues may constitute a binding site for the divalent metal ion that is required for catalysis by many of these enzymes (Wiggins and Munro, 1998). [beta]4GT-1 and its homologues also have a conserved DVD motif that is located about 60 residues N-terminal to the acidic cluster investigated here. Our results show that the EDDD sequence of [beta]4GT-1 is not the binding site for the essential metal cofactor and suggest that it is possible that not all of the conserved acidic clusters in the various glycosyltransferases are functionally and structurally equivalent. The region investigated here appears more similar to one that is highly conserved among the FNG, BRN, Lex1 families (Yuan et al., 1997) and [beta]1,3 galactosyltransferase subfamilies (Amado et al., 1998; Kolbinger et al., 1998) (Figure 1). Two proteins of the Lex1 family have been characterized as galactosyltransferases that add galactose to glucose or GlcNAc residues of the lipooligosaccharides (LOS) (Gotschlich, 1994; Jennings et al., 1995), and the Fringe and Brainiac gene families may also encode glycosyltransferases (Yuan et al., 1997). Further mutational and structural studies are needed to determine if these similarities are a coincidence, reflect the presence of a common domain with [beta]4GT-1, or evolutionary convergence associated with a similarity in catalytic mechanism.

Materials and methods

Materials

pET3a and pET15b expression vectors and T7 promoter and T7 terminator primers were obtained from Novagen, Madison, WI. Restriction enzymes and DNA ligase were supplied by New England BioLabs, Beverly, MA. UDP-[3H]-galactose was from NEN Products, Boston, MA. Guanidine hydrochloride (Ultrapure) was purchased from J.T. Baker, Inc., Jackson, Tenn. His-Bind resin was from Novagen.

Expression

The region of the cDNA for bovine [beta]4GT-1 encoding residues 129-402 of the protein sequence was amplified by PCR using the synthetic primers:

1. (N-terminal; coding)

Ndel

5[prime] GTG CCC TCC ACC CAT ATG CGC TCG CTG ACC GCA3[prime]
                             Met Arg Ser Leu Thr Ala

2. (C-terminal; complementary)

BamHI

5° GAT CAG TGC ACC GGA TCC CTA GCT GCT CGG CGT CCC3[prime]
                          (stop)

PCR reactions were performed using a Perkin Elmer/Cetus thermocycler for 25 cycles. The product was gel purified, digested with NdeI and BamHI and cloned into preparations of the pET15b vector that had been previously treated with the same enzymes. The product was transformed into E.coli.DH5[alpha] competent cells. The selected clones were grown and used for plasmid minipreps. The purified plasmid construct, designed as pET15b-r[beta]4GT-1, was used for DNA sequencing to confirm that no mutations were introduced by PCR. The pET15b-r[beta]4GT-1 was used to transform BL21(DE3) cells and the recombinant [beta]4GT-1 was expressed using previously described methods (Grobler et al., 1994). Cells were harvested and lysed using lysozyme and deoxycholate. Inclusion bodies were collected by centrifugation and washed.

Purification and Folding of r[beta]4GT-1

Inclusion bodies were dissolved in 6 M guanidine hydrochloride containing 20 mM Tris-HCl pH 7.9, 0.5 M NaCl, and 5 mM imidazole. The extract is applied to a column containing 10 ml Ni2+-charged His-Bind resin. After washing, with 20 mM imidazole, protein is eluted with 200 mM imidazole. All buffers contain 6 M guanidine hydrochloride, 0.5 M NaCl, and 20 mM Tris-HCl pH 7.9.

To determine the protein concentration, the absorbance of enzyme solutions were measured at 280 nm and the concentration was determined using a value for E280nm0.1% of 1.23 calculated from the amino acid composition (Mach et al., 1992).

For folding, the solution containing purified r[beta]4GT-1 is diluted to a concentration of 0.1mg/ml with the same buffer. Dialysis is carried out at 4°C in a Macro DiaCell system (InstruMed Inc., Union Bridge, MD) fitted with 6-8000 molecular weight cutoff membranes against 5 volumes of Tris-HCl buffer pH 7.5 containing 20 mM imidazole, 10% glycerol, 10 mM [beta]-mercaptoethanol, and 1 mM 2-hydroxyethyl disulfide. The dialysis solution is changed four times at 18-24 h intervals. Up to 400 ml of r[beta]4GT-1 solution can be accommodated in one apparatus producing, reproducibly about 50 units. Folded r[beta]4GT-1 is precipitated between 10 and 80% ammonium sulfate and stored at -20° in 50% glycerol. Further purification can be carried out by affinity chromatography with LA-Sepharose in the presence of N-acetyl-glucosamine (Trayer and Hill, 1971).

Mutagenesis of [beta]4GT-1

Mutations were introduced by PCR using the "megaprimer" method (Sarker and Sommer, 1990) with pET15b-r[beta]4GT-1 construct as the template. The amplification to generate the megaprimer was performed with the synthetic StuI primer or XhoI primer together with an appropriate mutagenic primer. Table I lists the StuI and XhoI primers together with the mutagenic primers that were used for the different substitutions. The megaprimer was purified by agarose gel electrophoresis and used in a second amplification with the same template and the cognate StuI or XhoI primer. After purification by agarose gel electrophoresis and a Promega PCR purification kit, the final amplified product was digested with StuI and XhoI and cloned into the pET15b-r[beta]4GT-1 vector which was previously made by cleaving pET15b-r[beta]4GT-1 construct with the same enzymes. The product was transformed into E.coli DH5[alpha] competent cells, and selected clones were grown and used for minipreps to provide plasmid for characterization by restriction mapping and DNA sequencing.

Enzyme assays

Galactosyltransferase assays with GlcNAc and glucose (in the presence of LA) were performed by a radiochemical method as described previously (Brew et al., 1968). r[beta]4GT-1 and the Trp312Phe mutant were assayed at a final concentration of 4.6 µg/ml and other mutants at concentrations of 92 µg/ml. For characterization of metal-dependence, Mn2+ was added in a concentration range of 2 µM to 20 mM in the presence of 0.3 mM UDP-galactose and 10 mM GlcNAc.

The data were fitted to a single substrate Michaelis-Menten equation and also to an equation describing metal binding to a high affinity site (K1) to generate an enzyme form with a low kcat (V1) and to a second lower affinity site (K2) to generate a form with a higher kcat (V2):

Kinetic parameters for UDP-galactose and GlcNAc were determined using concentrations varying from 0.1-2.0 mM and 6-50 mM, respectively, at 10 mM MnCl2. Kinetic parameters associated with the action of LA using LA in a concentration range of 0-40 µM as an inhibitor of galactose transfer to N, N[prime]-diacetylchitobiose to determine the dissociation constant (Ki) of LA from the Enzyme·Mn2+·UDP-galactose·LA complex (Grobler et al., 1994; Malinovskii et al., 1996). The promotion of lactose synthase at a fixed concentration of glucose (10 mM) by 0.5-3.6 µM LA and the data were deconvoluted as described previously (Grobler et al., 1994; Malinovskii et al., 1996) to determine the Km for glucose at a saturating concentration of LA.

Kinetic data were fitted to appropriate rate equations using the Curvefitter algorithm of SigmaPlot (Jandel Corp.). The following rate equations were used for different sets of data.

1. General equation for sequential symmetrical initial velocity pattern (ordered or random equilibrium sequential mechanism):

2. Equation for an asymmetric initial velocity pattern associated with a ping pong mechanism or sequential mechanism in which substrate A does not dissociate well from the E·S complex:

CD spectroscopy

Near and far UV CD spectra of recombinant proteins were determined with a JASCO J-710/720 spectropolarimeter. Twenty spectra were scanned for each sample at a speed of 100 nm/min which were subsequently averaged and smoothed. Near UV CD spectra (250-320 nm) were determined using a cell with a path length of 1 cm, and far UV spectra (200-250 nm) using a cell with a path length of 0.1 cm. Proteins were dissolved in 20 mM Tris-HCl, pH 7.4, containing 50% glycerol at concentration between 0.15 and 0.5 mg/ml.

Other methods

Oligonucleotide synthesis and DNA sequencing was carried out by Dr. Rudolf Werner, Department of Biochemistry and Molecular Biology, University of Miami.

Acknowledgments

We thank Dr. Joel H.Shaper, Department of Oncology, John Hopkins University, School of Medicine for providing the cDNA for bovine [beta]1,4 galactosyltransferase-1. This work was partially supported by grant GM21363 from NIH. This work was supported in part by Research Grant GM21363 from the National Institutes of Health. The costs of publication were defrayed in part by the payment of page charges. This article must thereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Abbreviations

[beta]4GT-1, UDP-galactose [beta]-N-acetylglucosaminide [beta] 1,4 galactosyltransferase; LA, [alpha]-lactalbumin; CD, circular dichroism.

References

Almeida ,R., Amado,M., David,L., Levery,S.B., Holmes,E.H., Merkx,G., Kessek,A.G.V., Rygaard,E., Hassan,H., Bennett,E. and Clausen,H. (1997) A family of human [beta]4-galactosyltransferases: cloning and expression of two novel UDP-galactose:[beta]-N-acetylglucosamine [beta]1,4-galactosyltransferases, [beta]4Gal-T2 and [beta]4Gal-T3. J. Biol. Chem., 272, 31979-31991. MEDLINE Abstract

Amado ,M., Almeida,R., Carneiro,F., Levery,S.B., Holmes,E.H., Nomoto,M., Hollingsworth,M.A., Hassan,H., Schwientek,T., Nielsen,P.A., Bennett,E.B. and Clausen,H. (1998) A family of human [beta]3-galactosyltransferases. Characterization of four members of a UDP-galactose: [beta] -n-acetyl-glucosamine/[beta] -nacetyl-galactosamine [beta]-1,3 galactosyltransferase family. J. Biol. Chem., 273, 12770-12778 MEDLINE Abstract

Andrade ,M.A., Chacon,P., Merelo,J.J. and Moran,F. (1993) Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng., 6, 383-390. MEDLINE Abstract

Aoki ,D., Appert,H.E., Johnson,D., Wong,S.S. and Fukuda,M.N. (1990) Analysis of the substrate binding sites of human galactosyltransferase by protein engineering. EMBO J., 9, 3171-3178. MEDLINE Abstract

Appert ,H.E., Rutherford,T.J., Tarr,G.E., Wiest,J.S., Thomford,N.R. and McCorquodale,D.J. (1986) Isolation of a cDNA coding for human galactosyltransferase. Biochem. Biophys. Res. Commun., 139, 163-168. MEDLINE Abstract

Bakker ,H., Agterberg,M., Van Tetering,A., Koeleman,C.A.M., Van den Eijnden,D.H. and Van Die,I. (1994) A Lymnaea stagnalis gene, with sequence similarity to that of mammalian [beta]1->4-galactosyltransferases, encodes a novel UDP-GlcNAc:GlcNAc[beta]-R [beta]1->4-N-acetylglucosaminyltransferase. J. Biol. Chem., 269, 30326-30333. MEDLINE Abstract

Bayna ,E.M., Shaper,J.H. and Shur,B.D. (1988) Temporally specific involvement of cell surface [beta]-1,4 galactosyltransferase during mouse embro morula compaction. Cell, 53, 145-157. MEDLINE Abstract

Boeggeman ,E.E., Balaji,P.V., Sethi,N., Masibay,A.S. and Qasba,P.K. (1993) Expression of deletion constructs of bovine [beta]-1,4-galactosyltransferase in Escherichia coli: importance of Cys134 for its activity. Protein Eng., 6, 779-785. MEDLINE Abstract

Breton ,C., Bettler,E., Joziasse,D.H., Geremia,R.A. and Imberty,A. (1998) Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases. J. Biochem., 123, 1000-1009. MEDLINE Abstract

Brew ,K., Vanaman,T.C. and Hill,R.L. (1968) The role of [alpha]-lactalbumin and the A protein in lactose synthesis: a unique mechanism for the control of a biological reaction. Proc. Natl. Acad. Sci., USA, 59, 491-497. MEDLINE Abstract

Fersht ,A.R. and Sperling,J. (1973) The charge relay system in chymotrypsin and chymotrypsinogen. J. Mol. Biol., 74, 137-149 MEDLINE Abstract

Geren ,C.R., Magee,S.C. and Ebner,K.E. (1975) Circular diochroism changes in galactosyltransferase upon substrate binding. Biochemistry, 14, 1461-1463. MEDLINE Abstract

Gotschlich ,E.C. (1994) Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide. J. Exp. Med., 180, 2181-2190. MEDLINE Abstract

Grobler ,J.A., Wang,M., Pike,A.C.W. and Brew,K. (1994) Study by mutagenesis of the role of two aromatic clusters of [alpha]-lactalbumin in aspects of its action in the lactose synthase system. J. Biol. Chem., 269, 5106-5114. MEDLINE Abstract

Hayashi ,T., Murray,B.W., Wang,R. and Wong,C.H. (1997) A chemoenzymatic synthesis of UDP- (2-deoxy-2-fluoro)-galactose and evaluation of its interaction with galactosyltransferase. Bioorg. Med. Chem., 5, 497-500. MEDLINE Abstract

Herrmann ,G.F., Krezdorn,C., Malissard,M., Kleene,R., Paschold,H., Weuster-Botz,D., Kragl,U., Berger,E.G. and Wandrey,C. (1995) Large-scale production of a soluble human [beta]1,4-galactosyltransferase using a Saccharomyces cerevisiae expression system. Protein Expr. Purif., 6, 72-78. MEDLINE Abstract

Hill ,R.L. and Brew,K. (1975) Lactose synthetase. Adv. Enzymol. Relat. Areas Mol. Biol., 43, 411-490. MEDLINE Abstract

Huang ,W., Meng,Q., Suzuki,K., Nagase,H. and Brew,K. (1997) Mutational study of the amino-terminal domain of human tissue inhibitor of metalloproteinases 1 (TIMP-1) locates an inhibitory region for matrix metalloproteinases. J. Biol. Chem., 272, 22086-22091. MEDLINE Abstract

Ichikawa ,Y., Look,G.C. and Wong,C.-H. (1992) Enzyme-catalyzed oligosaccharide synthesis. (Review). Anal. Biochem., 202, 215-239. MEDLINE Abstract

Jennings ,M.P., Hood,D.M., Peak,I.R.A., Virji,M. and Moxon,E.R. (1995) Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. Mol. Microbiol., 18, 729-740. MEDLINE Abstract

Khatra ,B.S., Herries,D.G. and Brew,K. (1974) Some kinetic properties of human milk galactosyltransferase. Eur. J. Biochem., 44, 537-560. MEDLINE Abstract

Kim ,S.C., Singh,A.N. and Raushel,F.M. (1988) An analysis of the galactosyltransferase reaction mechanism by positional isotope exchange and secondary deuterium effects. Arch. Biochem. Biophys., 267, 54-59. MEDLINE Abstract

Kolbinger ,F., Streiff,M.B. and Katopodis,A.G. (1998) Cloning of a human UDP-galactose:2-acetamido-2-deoxy-d-glucose 3 [beta]-galactosyltransferase catalyzing the formation of type 1 chains. J. Biol. Chem., 273, 433-440. MEDLINE Abstract

Krezdorn ,C.H., Watzele,G., Kleene,R.B., Ivanov,S.X. and Berger,E.G. (1993) Purification and characterization of recombinant human [beta]-1,4-galactosyltransferase expressed in Saccharomyces cerevisiae. Eur. J. Biochem., 212, 113-120. MEDLINE Abstract

Lo ,N.W., Shaper,J.H., Pevsner,J. and Shaper,N.L. (1998) The expanding [beta]4-galactosyltransferase gene family: message from the databanks. Glycobiology, 8, 517-526. MEDLINE Abstract

Mach ,H., Middaugh,C.R. and Lewis,R.V. (1992) Statistical determination of the average values of the extinction coefficients of tryptonphan and tyrosine in native proteins. Anal. Biochem., 200, 74-80. MEDLINE Abstract

Malinovskii ,V.A., Tian,J., Grobler,J.A. and Brew,K. (1996) Functional site in alpha-lactalbumin encompasses a region corresponding to a subsite in lysozyme and parts of two adjacent flexible substructures. Biochemistry, 35, 9710-9715. MEDLINE Abstract

Merelo ,J.J., Andrade,M.A., Prieto,A. and Moran,F. (1994) Proteinotopic feature maps. Neurocomputing, 6, 1-12.

Miller ,D.J., Macek,M.B. and Shur,B.D. (1992) Complementarity between sperm surface [beta]-1,4 galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding. Nature, 357, 589-593. MEDLINE Abstract

Nakazawa ,K., Furukawa,K., Narimatsu,H. and Kobata,A. (1993) Kinetic study of human beta-1,4-galactosyltransferase expressed in E.coli. J. Biochem. (Tokyo), 113, 747-753. MEDLINE Abstract

Narimatsu ,H., Sinha,S., Brew,K., Okayama,H. and Qasba,P.K. (1986) Cloning and sequencing of cDNA of bovine N-acetylglucosamine [beta]-1,4 galactosyltransferase. Proc. Natl. Acad. Sci. USA, 83, 4720-4724. MEDLINE Abstract

Öhrlein ,R., Ernst,B. and Berger,E.G. (1992) Galactosylation of non-natural glycosides with human [beta]-d-galactosyltransferase on a preparative scale. Carbohydr. Res., 26, 335-338.

Palcic ,M.M. and Hindsgaul,O. (1991) Flexibility in the donor substrate specificity of [beta], 1,4-galactosyltransferase: application in the synthesis of complex carbohydrates. Glycobiology, 1, 205-209. MEDLINE Abstract

Paulson ,J.C. and Colley,K.J. (1989) Glycosyltransferases. Structure, localization and control of cell type-specific glycosylation. J. Biol. Chem., 264, 17615-17618. MEDLINE Abstract

Powell ,J.T. and Brew,K. (1974) Isolation and characterization of two forms of bovine galactosyltransferase. Eur. J. Biochem., 48, 217-228. MEDLINE Abstract

Powell ,J.T. and Brew,K. (1976) Metal ion activation of galactosyltrasferase. J. Biol. Chem., 251, 3645-3652. MEDLINE Abstract

Pratt ,S.A., Scully,N.F. and Shur,B.D. (1993) Cell surface [beta]1,4 galactosyltransferase on primary spermatocytes facilitates their initial adhesion to Sertoli cells in vitro. Biol. Reprod., 49, 470-482. MEDLINE Abstract

Sarker ,G. and Sommer,S.S. (1990) The "megaprimer" method of site-directed mutagenesis. BioTechniques, 8, 404-407. MEDLINE Abstract

Schanbacher ,F.L. and Ebner,K.E. (1970) Galactosyltransferase acceptor specificity of lactose synthetase A protein. J. Biol. Chem., 245, 5057-5061. MEDLINE Abstract

Schwientek ,T., Almeida,R., Levery,S.B., Holmes,E.H., Bennett,E. and Clausen,H. (1998) Cloning of a novel member of the UDP-galactose:beta-N-acetylglucosamine beta1,4-galactosyltransferase family, beta4Gal-T4, involved in glycosphingolipid biosynthesis. J. Biol. Chem., 273, 29331-29340. MEDLINE Abstract

Shaper ,N.L., Shaper,J.H., Meuth,J.L., Fox,J.L., Chang,H., Kirsch,I.R. and Hollis,G.F. (1986) Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library. Proc. Natl. Acad. Sci. USA, 83, 1537-1577.

Shaper ,N.L., Hollis,G.G., Douglas,J.G., Kirsch,I.R. and Shaper,J.H. (1988) Characterization of the full length cDNA for murine [beta]-1,4 galactosyltransferase. Novel features at the 5[prime] end predict two translational start sites at two in-frame AUGs. J. Biol. Chem., 263, 10420-10428. MEDLINE Abstract

Shaper ,N.L., Meurer,J.A., Joziasse,J.H., Chou,T.-D.D., Smith,E.J., Schnaar,R.L. and Shaper,J.H. (1997) the chicken genome contains two functional nonallelic [beta]-1,4-galactosyltransferase genes: chromosomal assignment to syntenic regions tracks fate of the two gene lineages in the human genome. J. Biol. Chem., 272, 31389-31399. MEDLINE Abstract

Shur ,B.D. (1983) Embryonal carcinoma cell adhesion: the role of surface galactosyltransferase and its 90K lactosaminoglycan substrate. Dev. Biol., 99, 360-372. MEDLINE Abstract

Shur ,B.D. (1991) Cell surface [beta]1,4 galactosyltransferases: twenty years later. Glycobiology, 1, 563-575. MEDLINE Abstract

Trayer ,I.P. and Hill,R.L. (1971) The purification and properties of the A protein of lactose synthetase. J. Biol. Chem., 246, 6666-6675. MEDLINE Abstract

Varki ,A. (1993) Biological roles of oligosaccharides: all of the theries are correct. Glycobiology, 3, 97-130. MEDLINE Abstract

Wiggins ,C.A.R. and Munro,S. (1998) Activity of the yeast MNN1 [alpha]-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc. Natl. Acad. Sci. USA, 95, 7945-7950. MEDLINE Abstract

Woody ,R.W. (1995) Circular dichroism. Methods Enzymol., 246, 34-71. MEDLINE Abstract

Yadav ,S.P. and Brew,K. (1990) Identification of a region of UDP-galactose: N-acetylglucosamine [beta]-4 galactosyltransferase involved in UDP-galactose binding by differential labeling. J. Biol. Chem., 265, 14163-14169. MEDLINE Abstract

Yadav ,S.P. and Brew,K. (1991) Structure and function in galactosyltransferase; sequence locations of [alpha]-lactalbumin binding site, thiol groups and disulfide bond. J. Biol. Chem., 266, 698-703. MEDLINE Abstract

Yu ,L., Cabrera,R., Ramirez,J., Wang,P., Malinovskii,V.A. and Brew,K. (1995) Chemical and enzymatic synthesis of glycoconjugates. 1. Enzymatic galactosylation of conduritol B. (1995) Tetrahedron Lett., 36, 2897-2900.

Yuan ,Y.P., Schultz,J., Moldzik,M. and Bork,P. (1997) Secreted Fringe-like signaling molecules may be glycosyltransferases. Cell, 88, 9-11. MEDLINE Abstract

Zu ,H., Fukuda,M.N., Wong,S.S., Wang,Y., Liu,Z., Tang,Q. and Appert,H.E. (1995) Use of site-directed mutagenesis to identify the galactosyltransferase binding sites for UDP-galactose. Biochem. Biophys. Res. Commun., 206, 362-369. MEDLINE Abstract


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