Co-purification of galactosyltransferases from chick-embryo liver

We inserted a full-length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS-1 cells to characterize the pCMGTl -directed enzyme. The galactosyltransferase activity toward asialo-agalacto-transferrin (AsAg-Tf) in the pCMGTl -transfected cells was approximately eightfold higher than that in mock-or non-transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGTl -transfected cells and mock-or non-transfected cells when asialo-ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg-Tf was released by digestion with streptococcal j-galactosidase, most of the linkage created by this enzyme was in the Galbl-4GlcNAc group.The acceptor specificity of the pCMGTl -directed enzyme was changed from N-acetylglucosamine to glucose by adding a-lactalbumin in the reaction mixture. a-Lactalbumin also partially inhibited the galactose transfer to AsAg-Tf. The kinetic study revealed that the apparent K, values of the pCMGT1-directed enzyme for N-acetylglucosamine, AsAg-Tf and UDP-Gal are 2 mM, 60 pM and 24 pM, respectively.These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N-acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a j1-4 linkage The carbohydrate moieties of glycoproteins, glycolipids and proteoglycans are synthesized through a series of sugar chain elongation reactions which are catalyzed by specific glycosyltransferases [l]. A fl1-4galactosyltransferase (UDPGal :N-acetylglucosamine /?1-4galactosyltransferase ; PI -4-GalT is a member of the glycosyltransferase family. This enzyme catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine (GlcNAc) with the specific intersugar linkage, Galpl-4GlcNAc. During the last two decades, a number of investigators have reported the purification and characterization of jl-4GalTs from various sources such as human and bovine milk [2, 31, swine [ll]. Among these /31-4GalTs, a milk b1-4GalT (lactose synthetase A protein) is capable of recognizing glucose as an acceptor in the presence of a-lactalbumin (lactose synthetase B protein) [12]. The fll-4GalT activities purified from other tissues toward N-acetylglucosamine were also reported to be inhibited by a-lactalbumin, and the acceptor specificities of these b1-4GalTs were analogous to thatCorrespondence to H. Narimatsu,

Section: Activation Q F U Fli-4gnlt H-v Mn2' Ionmentioning

confidence: 99%

Characterization of a murine β1‐4galactosyltransferase expressed in COS‐1 cells

Nakazawa

Furukawa

Kobata

et al. 1991

“…The activity was detected in many mammalian cells and should also be present in nonmammalian tissues, according to published structures [4]. The enzyme has been partially purified from chick liver [5], pig trachea [6] and rat liver [7].…”

mentioning

confidence: 99%

UDPgalactose:glycoprotein–N‐acetyl‐d‐galactosamine 3‐β‐d‐galactosyltransferase activity synthesizing O‐glycan core 1 is controlled by the amino acid sequence and glycosylation of glycopeptide substrates

Granovsky

Bielfeldt

Peters

et al. 1994

In order to investigate the role of the peptide moiety of glycoproteins in the control of 0-glycan biosynthesis, UDPga1actose:glycoprotein-N-acetyl-D-galactosamine 3-P-D-galactosyltransferase (core 1 P3-Gal-T) from rat liver was tested for its specificity towards GalNAc-containing glycopeptide substrates. Series of glycopeptides have been synthesized by solid-phase synthesis, protected with an acetyl group on the amino terminal and an amide group on the carboxy terminal, based on variations of the repeat sequences of human intestinal mucin. Most glycopeptides were excellent substrates for core 1 /33-Gal-T compared to benzyl a-D-galactosamine as indicated by their relatively high V,,,,IK,,,. The enzyme preferred threonine a-D-galactosamine Thr(Ga1NAc) to serine a-D-galactosamine. Pro on the carboxy-terminal side adjacent to Thr(Ga1NAc) was inhibitory. Negatively charged amino acids on either side showed a low K,; substrates with negatively charged amino acids on the amino-terminal side were highly efficient substrates, suggesting charge-charge interactions between enzyme and substrate. GalPl-3GalNAca residues adjacent to Thr(Ga1NAc) reduced the activity. Product analysis using glycopeptide substrates with three adjacent GalNAc residues showed incorporation of one, two and a small amount of three Gal residues per molecule with an uneven distribution of the potential di-galactosylated isomers. These studies indicate that, in addition to initial glycosylation, the second step in the glycosylation pathways of 0-glycans is also controlled by the structure and glycosylation of the peptide core of substrates.

“…1 a. The 1,4 activity is represented by closed circles; the 1,3 activity by open circles result from differences in the degrees ofsialylation. To test this possibility, both adult and embryonic galactosyltransferases from UDP-hexanohmine -Sepharose were treated with neuraminidase.…”

Section: Previous Work [ L ]mentioning

confidence: 99%

Embryonic and adult forms of two galactosyltransferases differ in their degrees of sialylation

Furukawa

Roth

1985

Embryonic and adult chicken liver galactosyltransferases behave differently on DEAE-Sepharose chromatography. After solubilization, two embryonic activities (one transfers galactose in a p 1 -4 linkage to asialo-agalactoal-acid glycoprotein and to N-acetylglucosamine; the other transfers galactose in a p 1 -3 linkage to asialo-ovine submaxillary mucin) elute after the bulk of the protein, and after free glucose. The same two enzymes in adults elute more rapidly, almost coincident with the bulk of the protein, and before free glucose. The difference in elution patterns occurs when the column buffer contains 0.1 M NaCI. Without salt, both embryonic and adult transferases bind to the column, but with 0.5 M NaCI, the embryonic and adult transferases elute identically, and with the bulk of the protein. After treatment with neuraminidase, the embryonic transferase activities elute significantly earlier on a DEAE-Sepharose column in the presence of 0.1 M NaCl. The embryonic forms migrate more rapidly than do the adult forms on cellulose acetate electrophoresis, but neuraminidase treatment renders both enzyme forms immobile in this system. Neuraminidase treatment also inhibits the binding of the embryonic transferases to a wheat-germ-agglutinin -Sepharose column. Kinetically, the embryonic and adult transferases are indistinguishable.Previous work [l] extensively purified an embryonic chick liver UDP-Gal : GlcNAc asialo-agalacto-ccl-acid glycoprotein galactosyltransferase (1,4-transferase) and a UDPGal : GalNAc asialo-ovine submaxillary mucin galactosyltransferase (1,3-transferase). Both enzymes behave identically on a variety of chromatographic and affinity columns, and show very similar molecular masses. Ultimately, however, they could be separated from one another by acceptor affinity chromatography. Within the limits of assay sensitivity, the acceptor specificities of the enzymes appear absolute.For comparisons, the same, two galactosyltransferase activities from adult chicken livers were subjected to the identical purification procedures. The adult enzymes were extracted from the microsomal pellets as were the embryonic enzymes. On DEAE-Sepharose chromatography, however, the adult enzymes eluted quite differently.The data presented suggest that the two embryonic galactosyltransferases are more negatively charged than are the corresponding enzymes from adult livers. Neuraminidase treatment affects the behavior of the embryonic enzymes when subsequently tested on cellulose acetate electrophoresis followed by visualization with activity stains, and WGASepharose affinity chromatography. Therefore, the charge differences may be due entirely to differences in sialic acid content of the embryonic and adult transferases. Kinetic