Synthesis Of N-acetyllactosamine Research Articles

Use of transformed whole yeast cells expressing β-1,4-galactosyltransferase for the synthesis of N-acetyllactosamine

We report the use of transformed whole yeast cells for the synthesis of N-acetyllactosamine (LacNAc), which is the core-disaccharide of the anti-inflammatory agent Sialyl-Lewis X . Transformed whole yeast cells, which carry plasmids for the heterologous expression of a soluble human b-1,4-galactosyltransferase (GalT), proved to be a suitable catalyst for the synthesis of LacNAc by employing multi-enzyme-systems. This convenient use of the recombinant enzyme, taken together with the high availability of the transformed yeast through fermentation technology, will provide sufficient amounts of glycosyltransferase for chemoenzymatic synthesis of oligosaccharides.

Effects of Zn(II) on galactosyltransferase activity.

The enzyme beta-4-galactosyltransferase (GT) catalyzes the transfer of a galactosyl group from UDP-galactose to N-acetylglucosamine (GlcNAc) on glycoproteins. In the presence of alpha-lactalbumin (alpha-LA), galactosyltransferase catalyzes the transfer of galactose to glucose to yield lactose. It is known that, in the absence of alpha-lactalbumin, Zn(II) competes with Mn(II) for the same binding site(s) in galactosyltransferase, resulting in an increase in the apparent Michaelis constant, Km(app), for Mn(II)-activation of N-acetyllactosamine synthesis. In the presence of alpha-lactalbumin (i.e., lactose synthase), the Mn(II)-activation is biphasic and the initial phase is inhibited by increasing concentrations of Zn(II). The Zn(II) inhibition of lactose synthase plateaus at [Zn(II)]:[alpha-lactalbumin] approximately 1:1, while for N-acetyllactosamine synthesis there is no plateau at all. The results suggest that Zn(II) binding to alpha-lactalbumin effects lactose synthase. Kinetically, Zn(II) induces a decrease in both the Km(app) and Vm for Mn(II), which results in an apparent increase, followed by a decrease, in lactose synthase activity at Mn(II) concentrations below saturation of the first [Mn(II)] binding site. Increasing Zn(II) also decreases Km(app) and Vm for both glucose and UDP-galactose in the lactose synthase reaction with either both Ca(II)- or apo-alpha-lactalbumin, further suggesting novel interactions between Zn(II)-alpha-lactalbumin and the lactose synthase complex, presumably mediated via a Zn(II)-induced conformational change upon binding to alpha-lactalbumin. On the other hand, in N-acetyllactosamine synthesis, Zn(II) only slightly effects Km(app) for N-acetylglucosamine and has essentially no effect on Km(app) or Vm for UDP-galactose.

Regeneration of sugar nucleotide for enzymic oligosaccharide synthesis: use of Gal-1-phosphate uridyltransferase in the regeneration of UDP-galactose, UDP-2-deoxygalactose, and UDP-galactosamine

A new multiple enzymatic system containing galactose-1-phosphate uridyltransferase suitable for the regeneration of UDP-Gal, UDP-2-deoxy-Gal, and UDP-galactosamine for the synthesis of N-acetyllactosamine and analogs has been developed

Biochemical and Immunologic Characterization of Galactosyltransferase Purified From the Ascites of Ovarian Cancer Patients<xref ref-type="fn" rid="FN2">2</xref>

Galactosyltransferase appears to be a promising marker for ovarian carcinoma. For an understanding of its role in this cancer, the enzyme was purified from the ascites of ovarian cancer patients, and its biochemical and immunologic properties were studied. For adequate recovery and stability, Triton X-100 (0.01%) was necessary in all the buffers used for the purification of this enzyme. Immunoglobulins were not detectable in this preparation, which showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis under nondenaturing conditions resolved the enzyme into two to three active components. An antiserum in rabbits, however, produced a single precipitin line suggesting a single determinant. By chromatography in concanavalin A-Sepharose 4B, the enzyme can be resolved into two components (F-1 and F-2). Purified galactosyltransferase and components F-1 and F-2 all catalyzed the transfer of galactose from UDP-galactose to alkali-stable beta-N-glycosidic acceptors, as well as to alkali-labile beta-O-glycosidic mucin-type acceptors. In addition, they catalyzed the N-acetyllactosamine synthetase reaction and, in the presence of alpha-lactalbumin, the lactose synthetase reaction. Galactosyltransferase and components F-1 and F-2 differed in their sensitivity to alpha-lactalbumin-induced inhibition of N-acetyllactosamine synthesis. Galactosyltransferase in the malignant ascites exists as different isoforms, which do not differ significantly in major biochemical and immunologic properties.

Synthesis of N-acetyllactosamine containing a d-[6- 3H]galactopyranosyl group

A simple and convenient method for the introduction of radiolabel onto C-6′ of N-acetyllactosamine is described. 1- N-Benzyl-3- O-β- d-galactopyranosyl- d-arabinosylamine ( 1) was synthesized from 3- O-β- d-galactopyranosyl- d-arabinose as described by Lee and Lee. Compound 1 was oxidized with d-galactose oxidase, and the product reduced with KB 3H 4 to introduce the label at C-6′. After dilution with unlabeled material, the N-benzyl-3- O-β- d-[6- 3H]galactopyranosyl- d-arabinosylamine was converted into 2-(benzylamino)-2-deoxy-4- O- d-[6- 3H]galactopyranosyl- d-glucononitrile, which was subjected to simultaneous hydrogenolysis of the benzylamino and nitrile groups. N-Acetylation of the amino group as described by Alais and Veyrières afforded the crystalline title compound in 63% yield.

The sulfhydryl group microenvironment of lactose synthase from bovine milk.

Galactosyltransferase from bovine milk was inactivated by a series of sulfhydryl group specific reagents of different structures and sizes. The inactivation rate constants suggest that the thiol is located in a nonpolar microenvironment. The ESR spectrum of a spin labeled galactosyltransferase showed that the sulfhydryl group is in a region of non-restricted rotation, consistent with its broad reactivity towards various thiol reagents. Galactosyltransferase immobilized onto agarose through its sulfhydryl group retained its ability to catalyze the synthesis of N-acetyllactosamine and lactose. Thus the residual activity of the sulfhydryl group modified enzyme is not due to an isozyme lacking such a group. In addition, the active thiol can not be located at the active site nor the protein-protein interaction site between galactosyltransferase and alpha-lactalbumin.

Enzyme-catalyzed synthesis of N-acetyllactosamine with in situ regeneration of uridine 5'-diphosphate glucose and uridine 5'-diphosphate galactose

Enzyme-catalyzed synthesis of N-acetyllactosamine with in situ regeneration of uridine 5'-diphosphate glucose and uridine 5'-diphosphate galactose

Galactosyltransferase from buffalo milk: Further characterization

Buffalo milk galactosyltransferase is a single poly-peptide of molecular weight 55,000 to 56,000. The enzyme is specific for glucose as an acceptor substrate in the presence of 8-lactalbumin, L-Arabinose. L-xylose, D-ribose and D-fructose did not serve as acceptor substrates even at concentration as high as 0.13 M, while N-acetylglucosamine and ovalbumin served as good acceptors of galactosyl moiety in the absence of ∞ -lactalbumin. UDP-galacturonic acid did not serve as a donor substrate; on the contrary, it inhibited the reaction. Lactose synthetase reaction was inhibited by D-ribose, L-arabinose and L-xylose, whereas D-fructose did not show any inhibition. Buffalo milk ∞ -lactalbumin enhanced the synthesis of lactose but inhibited the synthesis of N-acetyllactosamine. Cations like Ca2+, Mg2+, Cu2+, Ba2+ and Co2+ could not replace Mn2+ in the N-acetyllactosamine synthetase reaction. Except Co2+, these cations had no effect on this reaction. Co2+ was found to be a competitive inhibitor of Mn2+. The observed inhibition of the reaction by-EDTA also confirmed the absolute requirement of Mn2+ for the reaction. Lactose synthetase reaction had an optimum pH of 8.5, whereas N-acetyllactosamine synthetase reaction was maximal at pH 8.0.

The kinetic mechansim of bovine milk galactosyltransferase. The role of alpha-lactalbumin.

Initial rate parameters obtained with bovine galactosyltransferase at saturating Mn2+ concentrations, and a variety of acceptors including N-acetylglucosamine, glucose, ovalbumin, and di-N-acetylglucosamine are inconsistent with an ordered addition of UDP-galactose and acceptor substrates to the enzyme-Mn2+ complex. Inhibition patterns with N-acetylglucosamine or UDP-glucose as inhibitors of the galactosylation of ovalbumin indicated that either UDP-galactose or N-acetylglucosamine can bind to an enzyme-Mn2+ complex by a random equilibrium mechanism. Initial rate studies also indicate that alpha-lactalbumin may bind to either an enzyme-Mn2+-acceptor complex or an enzyme-Mn2+-UDP-galactose complex, suggesting that lactose synthesis also proceeds by a random equilibrium addition of substrates and alpha-lactalbumin. From the initial rate data assuming the random equilibrium mechanism, the dissociation constants for UDP-galactose, acceptor substrates, and alpha-lactalbumin from the appropriate complexes have been calculated. These values are in good agreement with those obtained independently by nonkinetic methods, providing additional support for the proposed random equilibrium mechanism. From similar studies with a cross-linked complex of alpha-lactalbumin and transferase, dissociation constants for UDP-galactose and acceptor substrates from the enzyme-Mn2+-alpha-lactalbumin complex were calculated. Comparison of each of the dissociation constants in the substrate addition phase shows that the binding of acceptor substrates and alpha-lactalbumin to enzyme-Mn2+ complexes is highly synergistic; the affinity of alpha-lactalbumin for the enzyme-Mn2+ acceptor complex is about 2 orders of magnitude greater than for the enzyme-Mn2+ complex. Similarly, the affinity of the acceptor for the enzyme-Mn2+-alpha-lactalbumin complex is about 2 orders of magnitude greater than the enzyme-Mn2+ complex. Synergism is also observed between alpha-lactalbumin and UDP-galactose binding but the synergism is much less than that observed with acceptor substrates and alpha-lactalbumin. Thus, the large decrease in the Michaelis constant for glucose in the presence of alpha-lactalbumin, which is observed for lactose synthesis by the galactosyltranferase, is primarily the result of the high degree of synergism in the binding of alpha-lactalbumin and glucose to enzyme-Mn2+ complexes. This synergism also accounts for the activation of N-acetyllactosamine synthesis by alpha-lactalbumin at low concentrations (less than 2 mM) of N-acetylglucosamine. An abortive enzyme-Mn2+-UDP-acceptor complex in the product release phase of the reaction appears to account for the inhibition of either lactose, or N-acetyllactosamine synthesis at a high concentration of either N-acetylglucosamine or glucose. This abortive complex is further stabilized by alpha-lactalbumin, thus the resulting substrate inhibition is observed at much lower acceptor concentrations in the presence of alpha-lactalbumin.

Puromycin does not inactivate the galactosyltransferase of golgi membranes

The effects of puromycin on galactosyltransferase from four sources, rat liver and lactating sheep mammary Golgi membranes, bovine colostrum and human serum have been investigated. We do not find that the synthesis of N-acetyllactosamine is much inhibited, even in the presence of 3mM puromycin. This is in contrast to previously reported results for rat liver Golgi membranes. We interpret the low level of inhibition observed in terms of pH effects.

Some kinetic properties of human-milk galactosyl transferase.

Reactions catalyzed by human milk N-acetyl lactosamine synthetase in the presence and absence of ä-lactalbumins have been investigated by steady-state kinetics. N-Acetyl lactosamine synthesis and lactose synthesis in the absence of α-lactalbumin appear to proceed by an ordered sequential reaction, with substrates attaching in the order: Mn2+, UDP-galactose and monosaccharide. Under the conditions used (pH 7.4, 37 °C) the attachment of Mn2+ is not at thermo-dynamic equilibrium and it appears that the enzyme can accept either free UDP-galactose or its Mn2+ complex as substrate. Evidence is presented which suggests that the Mn2+ complex of UDP may be the final product released from the enzyme. Reactions in the presence of α-lactalbumin proceed by a similar ordered mechanism. Kinetic effects observed in the presence of human α-lactalbumin with three different monosaccharide acceptors, and in the presence of bovine α-lactalbumin with glucose, can be reasonably explained only by assuming that α-lactalbumin attaches to the enzyme immediately before monosaccharides, contrary to suggestions by other workers. It is proposed that α-lactalbumin attaches to an enzyme Mn2+ UDP-galactose complex at thermodynamic equilibrium, producing a new enzyme form with increased affinity for monosaccharides. The inhibitory effects of ä-lactalbumin on N-acetyl lactosamine synthesis are attributed to inhibition resulting from attachment of the protein to a central complex in an alternative pathway in the reaction scheme. The kinetic effects of four α-lactalbumins with human galactosyl transferase are characterized and intrinsic differences shown to be independent of the source of galactosyl transferase. The function deduced for α-lactalbumin in the lactose synthetase system is discussed in relation to its structure and a procedure is indicated for quantitatively measuring activity differences in α-lactalbumins.