Use of site-directed mutagenesis to identify the galactosyltransferase binding sites for UDP-galactose.

Site-directed mutagenesis was utilized to identify binding sites for UDP-galactose in galactosyltransferase (EC 2.4.1.22). Mutant cDNAs were generated by a procedure based on PCR, and the mutated enzymes were expressed in E.coli cells. The mutant enzymes were purified by Ni-NTA Sephadex, and the degree of purification was judged by SDS-PAGE. Purified mutant GTs, F305L, P306V, N307S, N308S, showed dramatic decreases in activities in comparison with the activity of the wild-type GT. Enzyme kinetic analysis revealed that the Km values of F305L, P306V, N307S and N308S for UDP-galactose were, respectively, 9-, 11-, 50- and 20-fold higher than the Km of wild-type GT, but the Km values for manganese were not significantly different from that of the wild-type GT. The quartet mutant F305L/P306V/N307S/N308S showed no activity. From the results of this study it is concluded that amino acids, Phe-305, Pro-306, Asn-307 and Asn-308, in GT are most probably involved in GT catalysis or are located close to the UDP-galactose binding region but are not involved in the binding of manganese.

Identification of a region of UDP-galactose:N-acetylglucosamine beta 4-galactosyltransferase involved in UDP-galactose binding by differential labeling.

The location of regions in the primary structure of UDP-galactose:N-acetylglucosamine beta 4-galactosyl-transferase (GT) that are involved in binding UDP-galactose has been investigated by differential chemical modification with two different reagents in the presence and absence of UDP-galactose. Treatment with periodate-cleaved UDP and NaCNBH3 resulted in a loss of 80% of GT activity, which was largely prevented by UDP-galactose. Stoichiometry of labeling and peptide maps of the modified enzyme samples indicated partial labeling at many sites. A major site of reaction in the absence of UDP-galactose that was essentially unmodified in its presence was found to correspond to Lys341 in the cDNA sequence of GT. As a second approach, the reactivities of the amino groups of GT were compared in the presence and absence of saturating levels of UDP-galactose by trace acetylation with [3H]acetic anhydride. UDP-galactose binding was found to perturb the reactivities of a number of lysines in the C-terminal region of GT, the most pronounced effect being a reduction in the reactivity of Lys351. The two procedures thus identified a region between residues 341 and 351 as being associated with UDP-galactose binding. This region overlaps a small section in the sequence of GT that was previously noted to be similar to part of bovine alpha-1,3-galactosyltransferase (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Sequence comparisons indicate that extended regions at the C terminus of each enzyme encompassing this area may represent homologous UDP-galactose-binding domains.

Identification of a soluble UDP-Gal: Gal (beta 1-4)Glc (or GlcNAc) (alpha 1-3) galactosyltransferase of bovine colostrum.

A soluble UDP-Gal: Gal (alpha 1-3) galactosyltransferase was first detected in bovine colostrum and this enzyme activity was simply assayed by using rho-nitrophenyl-beta-lactoside (Gal(beta 1-4)Glc-C6H5NO2, rho NP-lactoside) as an acceptor. Treating the radioactive product with alpha- or beta-galactosidase, the radioactivity (greater than 95%) was released by only alpha-galactosidase and was identified as [3H]galactose. This shows that galactosyl residue was alpha-linked to rho-nitrophenyl-beta-lactoside. Methylation, hydrolysis, thin layer chromatography and fluorography of the reaction product (Gal(alpha 1-)-[3H]Gal(beta 1-4)Glc-rho NP) yielded 2,4,6-tri-O-methyl[3H]galactose, indicating that galactosyl residue had been transferred to the carbon-3 position of the terminal nonreducing beta-galactosyl residue in rho-nitrophenyl-beta-lactoside. These results confirmed that the structure of the reaction product was Gal(alpha 1-3)Gal(beta 1-4)Glc-rho NP. The enzyme requires Mn2+ for its activity, and shows pH optimum from 6.5 to 7.5. rho-Nitrophenyl-beta-lactoside and asialo alpha 1-acid glycoprotein were more effective as an acceptor than N-acetyllactosamine. The bovine colostrum (alpha 1-3) galactosyltransferase could not convert human O red cells into B active cells, indicating that this enzyme preparation did not contain the activity to synthesize human blood group B erythrocytes.

Stimulation of bovine milk galactosyltransferase activity by bovine colostrum N-acetylglucosaminyltransferase I.

Purified bovine milk galactosyltransferase was stimulated by purified bovine colostrum N-acetylglucosaminyltransferase I by more than 10-fold. Only slight stimulation of the N-acetylglucosaminyltransferase I by galactosyltransferase was observed. Heat inactivation destroyed the ability of the N-acetylglucosaminyltransferase I to stimulate the galactosyltransferase. The stimulation of galactosyltransferase was accompanied by a decrease in Km of this enzyme from 9.7 to 3.3. mM and an increase in Vmax from 1.87 to 3.71 nmol galactose transferred/min per mg galactosyltransferase when GlcNAc was the substrate. When the Km for UDPgalactose was determined, it increased from 0.19 to 0.42 mM in the presence of N-acetylglucosaminyltransferase I and the Vmax increased from 0.66 to 2.76 nmol galactose transferred/min per mg galactosyltransferase. In phosphatidylcholine vesicles, no effect on Km values with GlcNAc as substrate was noted, while an increase in the Km of UDPgalactose was observed. The Vmax values were generally higher in the lipid vesicles. Complex formation between galactosyltransferase and N-acetylglucosaminyltransferase I was demonstrated both by glycerol density gradient centrifugation and Bio-Gel P-100 column chromatography. An approximate molecular weight for the complex was obtained on a calibrated Sephadex G-200 column and found to be about 75 000, consistent with a 1:1 complex. The stimulation of galactosyltransferase involved the N-acetyllactosamine synthetase activity of this enzyme and not the lactose synthetase activity, since the latter activity was only slightly affected. Since N-acetylglucosaminyltransferase I is not involved in the lactose synthetase reaction, the stimulation is consistent with the known biosynthetic role of N-acetylglucosaminyltransferase I in the biosynthesis of asparagine-linked oligosaccharides.

Branch specificity of bovine colostrum and calf thymus UDP-Gal: N-acetylglucosaminide beta-1,4-galactosyltransferase.

The branch specificity of bovine colostrum and calf thymus UDP-Gal:N-acetylglucosaminide beta-1----4-galactosyltransferase toward several branched oligosaccharides, which form part of the complex-type N-glycans of glycoproteins, was investigated. A novel method was used based on acetolysis of the bi[14C,3H] galactosylated oligosaccharide products formed by the enzymes in vitro and analysis of the acetolysis fragments by high-pressure liquid chromatography. It could be established that the galactosylation of different oligosaccharide branches occurred in a preferred order. No difference in branch specificity was observed between the soluble bovine colostrum galactosyltransferase and the enzyme that had been solubilized from calf thymus membranes. A preferential pathway for the biosynthesis of bisialylated biantennary glycans is proposed.

The lactose synthase acceptor site: a structural map derived from acceptor studies.

A pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either alpha or beta-, but alpha- is somewhat preferred. In the absence of alpha-lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH2) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of alpha-lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (beta-configuration). However, upon alpha-lactalbumin binding the aglycone specificity allows for axial (alpha-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that alpha-lactalbumin binds to a hydrophobic site some distance from the C-2 group.

Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk.

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4 degrees C and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined. Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose N-acetylglucosamine beta-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes. In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions, When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes. Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.

Purification and properties of galactosyltransferase from human colostrum.

The galactosyltransferase (Uridine diphosphate-D-galactose: D-glucose 1-galactosyltransferase EC 2.4.1.22) was purified from human colostrum by chromatography on DEAE-cellulose, cellulose phosphate, sephadex G-100, and hydroxylapatite after removal of caseins by centrifugation. The final preparation showed two forms of protein on polyacrylamide disc gel electrophoresis, and both of them exhibited galactosyltransferase activity. The molecular weights of the two forms of the protein were estimated as 44,000 to 45,000 and 55,000 to 57,000 by polyacrylamide disc gel electrophoresis containing sodium dodecyl sulfate. General properties of galactosyltransferase were investigated.

On the interaction of alpha-lactalbumin and galactosyltransferase during lactose synthesis.

The regulatory effect of alpha-lactalbumin in the lactose synthase system has been ascribed to its reversible association with a complex of galactosyltransferase with Mn2+ and UDP-galactose, prior to the binding of monosaccharides; the resulting complex has a higher affinity for various monosaccharides. Two steps in the postulated catalytic cycle have been investigated; UDP-galactose binding to enzyme-Mn2+ by equilibrium dialysis and alpha-lactalbumin binding to enzyme-Mn2+-UDP-galactose by sedimentation velocity and kinetics. There is a single binding site for UDP-galactose on the enzyme-Mn2+ complex, and the dissociation constant for UDP-galactose from enzyme-Mn2+-UDP-galactose was found to be 72 muM at 37 degrees. The formation of a complex between galactosyltransferase and alpha-lactalbumin in the presence of Mn2+ and UDP-galactose was observed as an increase in sedimentation coefficient of enzyme activity So20,w from 3.25 +/- 0.03 in the absence of alpha-lactalbumin to 4.22 +/- 0.03 at saturating concentrations of alpha-lactalbumin, a value closely similar to that of a cross-linked 1:1 complex of the proteins under the same conditions (4.35 +/- 0.03). No interaction was observed in the absence of substrates or with UDP-galactose and EDTA. From the ultracentrifuge data and steady state kinetics, dissociation constants for alpha-lactalbumin from the enzyme-Mn2+-UDP-galactose-alpha-lactalbumin complex were determined at several temperatures and salt concentrations. These showed good internal agreement. The free energy change delta G degrees for the association of the two proteins is calculated, and the results are discussed in relation to the nature of the interaction.