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.

Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase).

1. Manganese(II) buffers were set up with inorganic triphosphate, trimethylenediaminetetraacetate and tetramethylenediaminetetraacetate to study the Mn dependence of beta 1,4-galactosyltransferase (lactose synthetase) in preparations of rat mammary gland. 2. In intact particulate preparations, treated with the calcium ionophore A23187, lactose synthesis was abolished by chelators and restored by bivalent transition metal ions in a manner characteristic of activation site I of the pure enzyme. Ni(II) also activated, as did Mg at high concentration. 3. Only Mn(II) could restore endogenous rates, giving an apparent Km of 0.1-0.2 microM, and eliciting about 70% full activity without addition of a site II activator. 4. In purified Golgi membrane vesicles, Mn gave an apparent Km of 0.4 microM. This increased sharply to about 10 microM on permeabilization with filipin, lysis with detergents, solubilization with Triton X-100, or in the pure enzyme. Preparations of chemically undamaged Golgi vesicles, known to include a proportion of the enzyme on exposed membranes, exhibited both low-Km and high-Km components. 5. The response of particulate galactosyltransferase to apparently physiological concentrations of free Mn(II) ion is interpreted as either due to a sensitizing factor within the Golgi lumen, or to the accumulation of Mn at elevated concentrations. Alternatively, the high Km of the soluble enzyme may reflect proteolytic damage.

Plasma membrane association, purification, and partial characterization of mouse sperm beta 1,4-galactosyltransferase.

Gamete recognition in the mouse is mediated, in part, by the binding of sperm surface galactosyltransferase (GalTase) to appropriate substrates in the egg zona pellucida. In this paper, sperm GalTase is shown to be an externally oriented, integral plasma membrane component. GalTase is not peripherally adsorbed to the cell surface, nor is it bound to cell surface glycoside substrates. GalTase can be released from the surface of intact sperm by either mild proteolysis or by detergent under conditions in which the sperm membranes remain intact as judged by double-label indirect immunofluorescence. Detergent-solubilized sperm GalTase has been purified to apparent homogeneity by affinity chromatography and characterized as a beta 1,4-GlcNAc:GalTase by substrate and kinetic analyses. Purified and membrane-bound GalTase both show an unusual thermal inactivation above 39-40 degrees C, whereas other sperm enzyme activities as well as GalTase activity from other cell types are temperature-dependent. Purified sperm GalTase inhibits sperm binding to the egg zona pellucida, consistent with its proposed role during gamete recognition.

Ehrlich ascites tumor cell UDP-Gal:N-acetyl-D-glucosamine beta(1,4)-galactosyltransferase. Purification, characterization, and topography of the acceptor-binding site.

A UDP-Gal:N-acetylglucosamine beta(1,4)-galactosyltransferase which catalyzes the synthesis of beta-D-Gal(1,4)-D-GlcNAc units has been purified 17,560-fold from Ehrlich tumor cells to apparent electrophoretic homogeneity. The enzyme appears to be a monomeric protein with Mr = 56,000-58,000. Enzymatic activity requires the presence of MnCl2, is stimulated by detergent, and exhibits a pH optimum at 6.9. The Km values for GlcNAc and UDP-Gal are 1.89 and 0.046 mM, respectively. The Ehrlich cell beta-galactosyltransferase acts efficiently on glycoproteins and glycolipids terminating in GlcNAc, but is inactive toward glycoconjugates possessing terminal GalNAc units. The oligosaccharides beta-D-GlcNAc(1,3)-D-Gal and beta-D-GlcNAc(1,3)[beta-D-GlcNAc(1,6)]-D-Gal are good acceptors for the beta-galactosyltransferase from Ehrlich cells, suggesting that the enzyme may participate in the biosynthesis of i/I structures. In addition, other linear and branched sugars presenting GlcNAc residues at their nonreducing termini also act as acceptors for the enzyme. The activity of Ehrlich cell beta-galactosyltransferase both in the presence and absence of alpha-lactalbumin has been studied using a series of derivatives of Glc and GlcNAc which were substituted at various positions of the pyranose ring. This study has provided a map of the molecular contacts necessary for enzymatic activity in the presence and in the absence of alpha-lactalbumin.

Purification and properties of N-acetylglucosaminide beta 1----4 galactosyltransferase from embryonal carcinoma cells.

N-Acetylglucosaminide beta 1----4 galactosyltransferase was chromatographically purified about 1,700-fold from F9 embryonal carcinoma cells after solubilization with Triton X-100, using N-acetylglucosamine as the acceptor. As the last step of the purification, affinity chromatography was performed either on N-acetylglucosamine-Sepharose or on alpha-lactalbumin-Sepharose: in both cases, two protein bands with molecular weights of around 68,000 and 59,000 were detected by SDS-polyacrylamide gel electrophoresis of the purified preparations. The enzymological properties including behavior toward alpha-lactalbumin were very similar to those of the enzyme from other sources. The specificity of the enzyme was confirmed by determining the structure of the product; it was mostly Gal beta 1----4GlcNAc. beta-Galactosidase-treated embryoglycan (poly-N-acetyllactosamine) and asialo-agalactofetuin could serve as acceptors with the purified enzyme. Thus, the embryonic enzyme, apparently involved in the synthesis of poly-N-acetyllactosamines, has properties similar in several respects to those of the beta-galactosyltransferases so far studied.

Structure identification of the complex-type, asparagine-linked sugar chains of beta-D-galactosyl-transferase purified from human milk.

The asparagine-linked sugar chains of human milk galactosyltransferase were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted into radioactive oligosaccharides by sodium borotritiate reduction after N-acetylation, and fractionated by paper electrophoresis and by Bio-Gel P-4 column chromatography after sialidase treatment. Structural studies of each oligosaccharides by sequential exoglycosidase digestion and methylation analysis indicated that the galactosyltransferase contains bi, tri-, and probably tetra-antennary, complex-type oligosaccharides having alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D- GlcpNac-(1----4)-alpha-L-[Fucp-(1----6)]-D-GlcNAc as their common core. Variation is produced by the different locations and numbers of the five different outer chains: beta-D-Galp-(1----4)-D-GlcNAc, alpha-L-Fucp-(1----3)-[beta-D-Galp-(1----4)]-D-GlcNAc, alpha-NeuAc-(2----6)-beta-D-Galp-(1----4)-D-GlcNAc, alpha-L-Fucp-(1----3)-[beta-D-Galp-(1----4)]-beta-D-GlcpNAc-(1---- 3)-beta- D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-GlcNAc, and alpha-NeuAc-(2----6)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D - Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcNAc.

Galactosyltransferase purified from rat milk is distinct from the human and bovine enzyme.

N-Acetylglucosaminide beta 1, 4-galactosyltransferase (EC 2.4.1.22) was purified from rat milk by affinity chromatography on N-acetylglucosamine-Sepharose and alpha-lactalbumin-Sepharose columns. The purified enzyme migrated as three polypeptides of relative molecular weight 59,000, 54,000, and 27,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antiserum raised against the 54K rat protein immunoprecipitated all three polypeptides, suggesting that they share common antigenic sites. The human milk galactosyltransferase, purified under similar conditions, was electrophoretically homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with relative molecular weight 54K, and was not immunoprecipitated by the antiserum to the 54K rat milk protein. In addition, Michaelis constants for the enzyme from rat and human milk differed. The apparent Michaelis constant for N-acetylglucosamine and uridine 5'-diphosphate-galactose were 4.8 and .3 mM, respectively, for the rat enzyme, and 1.8 and .028 mM, respectively, for the human enzyme.

Two distinct UDP-galactose: 2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferases in porcine trachea.

In previous studies on glycosyltransferase activities in porcine trachea, we demonstrated the presence of two galactosyltransferases which transfer galactose from UDP-galactose to N-acetylglucosamine (Sheares, B.T. and Carlson, D.M. (1983) J. Biol. Chem. 258, 9893-9898). One enzyme, UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase, synthesized galactosyl-beta 1,3-N-acetylglucosamine while the other, UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase, synthesized galactosyl-beta 1,4-N-acetylglucosamine. A third galactosyltransferase has now been demonstrated utilizing a solubilized membrane preparation from pig trachea, which also synthesizes galactosyl-beta 1,4-N-acetylglucosamine as determined by gas-liquid chromatography and Diplococcus pneumoniae beta-galactosidase treatment. This new UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase is distinct from the lactose synthetase A protein in that it does not bind to alpha-lactalbumin-agarose or to N-acetylglucosamine-agarose. The enzyme is separable from the UDP-galactose:N-acetylgalactosaminyl-mucin 3 beta-galactosyltransferase by affinity chromatography on asialo ovine submaxillary mucin adsorbed to DEAE-Sephacel. This newly discovered 4 beta-galactosyltransferase binds to UDP-hexanolamine-Sepharose and is partially separated from UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase by Sephacryl S-200 gel filtration chromatography. Neither high concentrations of N-acetylglucosamine (200 mM) nor alpha-lactalbumin inhibits the incorporation of galactose into galactosyl-beta 1,4-N-acetylglucosamine by this enzyme.

Isolation and characterization of UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase activity induced in rat parotid glands treated with isoproterenol.

Chronic injection of the beta-adrenergic receptor agonist isoproterenol causes a 6- to 10-fold increase in the specific activity of the Golgi membrane marker enzyme UDP-galactose:D-glucose 4-beta-D-galactosyltransferase (EC 2.4.1.22) in rat parotid glands. Using a combination of size exclusion chromatography and affinity chromatography on alpha-lactalbumin-agarose, the Triton X-100-solubilized enzyme was purified to homogeneity from treated and control animals. The enzyme purified from both sources had a single Mr = 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme isolated from control and isoproterenol-treated animals had identical amino acid compositions and isoelectric points (pI = 5.9). Neutral sugar content as determined by the phenol-sulfuric acid assay was 10%. UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase activity purified by alpha-lactalbumin column chromatography was inhibited by the presence of 200 mM N-acetylglucosamine and by the inclusion of alpha-lactalbumin in assays using 10 mM N-acetylglucosamine as acceptor. Increased enzyme activity was immune precipitated from Triton X-100-solubilized membrane prepared from isoproterenol-treated animals versus control animals. This was also true of protein synthesized by in vitro translation of poly(A+) RNA prepared from parotid glands, indicating an increase in mRNA levels for the enzyme. The molecular mass of the immune-precipitated, in vitro translation product was determined to be 45,000 Da.

Use of concanavalin A as a topographical probe for protein-protein interaction. Application to lactose synthase.

Galactosyltransferase (EC 2.4.1.38) has been shown to bind to Con A-Sepharose. Concentrations of methyl-alpha-mannoside greater than 0.7 M were required to release the enzyme from the immobilized lectin. Molecular weight determination by gel filtration revealed that galactosyltransferase formed a 1:1 complex with concanavalin A. Preincubation of the enzyme with excess concanavalin A did not affect its catalytic activity either in the presence or absence of alpha-lactalbumin. The galactosyltransferase-concanavalin A complex was retained on an alpha-lactalbumin-Sepharose column in the presence of N-acetylglucosamine and manganese chloride and was eluted from the column in their absence. Galactosyltransferase immobilized onto a Con A-Sepharose was still active either in the presence or absence of alpha-lactalbumin. Lactose synthase activity was also observed when the galactosyltransferase-concanavalin A complex was assayed with alpha-lactalbumin immobilized on Sepharose. These data indicate that the carbohydrate moiety of galactosyltransferase is involved in neither the catalytic process nor the binding of alpha-lactalbumin and must be linked to the enzyme at a location where it does not present any steric hindrance on the binding of concanavalin A, either free or immobilized on Sepharose.

Purification and characterization of human serum galactosyltransferase (lactose synthetase A protein).

A galactosyltransferase, which transfers galactose from UDP-galactose to N-acetylglucosamine, was purified 286,000-fold to homogeneity with 40% yield from human plasma by repeated affinity chromatography on alpha-lactalbumin-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with molecular weight of 49,000. The enzyme is a glycoprotein with 11% by weight carbohydrate, which seems to have only asparagine-N-acetylglucosamine linkage-type carbohydrate chains. The enzyme showed characteristic changes in activity at different alpha-lactalbumin concentrations, indicating that the enzyme is the A protein of lactose synthetase. Km values for the substrates were found to be 0.056 mM for UDP-galactose, 3.2 mM for GlcNAc, and 0.44 mM for Mn2+, and in the presence of alpha-lactalbumin, 3.4 mM for Glc, and 0.20 mM for Mn2+. The activity of the enzyme was neutralized by anti-enzyme antibody, but the antibody did not neutralize the bovine milk galactosyltransferase (A protein) activity.

Purification by affinity chromatography and some properties of microsomal galactosyltransferase from pig thyroid.

Membrane-bound 4-beta-galactosyltransferase (lactose synthase; UDP galactose: D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) was purified 1500-fold to near homogeneity from pig thyroid microsomes with about 30% yield. The purified enzyme behaved as a lipophilic protein, rapidly losing activity and aggregating if not supplemented with either Triton X-100 or serum albumin (both of these were equally effective for long-term stabilization). The enzyme preparation showed an absolute requirement for Mn2+, which could not be replaced by other cations. Catalytic properties were very similar to those reported for soluble forms of the enzyme in biological fluids. The purified galactosyltransferase showed a major protein band of approx. 74,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity was eluted at approx. 70,000 daltons. It is concluded that the membrane-bound thyroid galactosyltransferase is a monomeric protein significantly larger than the soluble forms of this enzyme described earlier; but it resembles recently reported galactosyltransferases from sheep mammary Golgi membranes and liver microsomes.

Lactose synthetase. The isolation and characterisation of the protein-protein complex.

Galactosyl transferase and alpha-lactalbumin were concurrently isolated from bovine milk. The galactosyl transferase had an S20,w of 3.0 S and D20,w of 60 mum2/s. It exists as a monomer of 46000 molecular weight, as determined by sedimentation equilibrium and dodecylsulphate-polyacrylamide electrophoresis. Aggregation of the enzyme was promoted by N-acetylglucosamine. Sedimentation velocity experiments show an association between galactosyl transferase and alpha-lactalbumin in the presence of N-acetylglucosamine. In contrast, UDP-galactose, UDP or lactose do not promote protein-protein association. A complex between galactosyl transferase and alpha-lactalbumin was isolated by gel filtration in the presence of excess alpha-lactalbumin and either N-acetylglucosamine or glucose. The complex was stable over a range of concentrations of these components. The complex is a discrete homogeneous entity with a molecular weight of 60000, corresponding to one molecule of galactosyl transferase and one molecule of alpha-lactalbumin. The estimated association constants for the ternary complexes of the two proteins and either of the sugars, suggest that alpha-lactalbumin enhances equally the binding of N-acetylglucosamine or glucose to the galactosyl transferase.

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.

Cross-linking of the components of lactose synthetase with dimethylpimelimidate.

The cross-linking of the two components of lactose synthetase, alpha-lactalbumin and a galactosyltransferase, with dimethylpimelimidate was examined. The extent of the cross-linking at pH 8.1 was found to be dependent upon the presence of substrates or inhibitors for the galactosyltransferase. N-acetylglucosamine and mixtures of either N-acetylglucosamine, Mn-2+ and UDP, or UDP-galactose and Mn-2+ promoted the formation of cross-linked species. Glucose or a mixture of UDP and Mn-2+ were much less effective in promoting cross-linking. Two types of intermolecularly cross-linked species of alpha-lactalbumin and the galactosyltransferase were obtained. Each was a 1:1 cross-linked complex of alpha-lactalbumin and either of the two forms of the transferase with molecular weights of about 42,000 and 48,000, respectively. Cross-linked complexes were not observed with more than 1 molecule each of alpha-lactalbumin and the transferase. The cross-linked complexes were obtained in homogeneous form by gel filtration on Sephadex and absorption of uncross-linked enzyme by affinity chromatography on alpha-lactalbumin-Sepharose in the presence of N-acetylglucosamine. They migrated on gel electrophoresis in sodium dodecyl sulfate with mobilities in accord with their predicted molecular weights as 1:1 complexes of alpha-lactalbumin and the transferase. The amino acid composition of the cross-linked complex was in reasonable agreement with the expected composition of a 1:1 mixture of alpha-lactalbumin and galactosyltransferase. The enzymic properties of the cross-linked and uncross-linked enzymes were compared. The cross-linked complex had a much higher intrinsic lactose synthetase activity than did uncross-linked enzyme although only about 1% of the potential activity of uncross-linked enzyme in the presence of optimal concentrations of alpha-lactalbumin. The lactose synthetase activity of the cross-linked complex, however, was unaffected by exogenous alpha-lactalbumin. In addition, the complex readily catalyzed the transfer of galactose from UDP-galactose to xylose in the absence of exogenous alpha-lactalbumin. The N-acetyllactosamine synthetase activity of the complex was low compared to its activity with other monosaccharides. Ovalbumin, which is a good acceptor for the uncross-linked transferase, was not an acceptor for the cross-linked complex. Kinetic studies of the complex suggest that its modified catalytic activity is not the result of the modification by dimethylpimelimidate but reflects the expected effects of is provided, and that