RESUMO
Transgalactosylation of chitobiose and chitotriose employing beta-galactosidase from bovine testes yielded mixtures with beta1-3 linked galactose (type I) and beta1-4 linked galactose (type II) in a final ratio of 1:1 for the tri- and 1:1.4 for the tetrasaccharide. After 24 h incubations of the two purified oligosaccharide mixtures with large amounts (20-fold increase compared with standard conditions) of human alpha1,3/4-fucosyltransferase III (FucT III), the type I tri-/tetrasaccharides were completely converted to the Lewis(a) structure, whereas approximately 10% fucosylation of the type II isomers to the Lewis(x) oligosaccharides was observed in long-term incubations. Employing large amounts of human alpha1,3-fucosyltransferase VI (FucT VI), the type I trisaccharide substrate was exclusively fucosylated at the proximal O-4 substituted N-acetylglucosamine (GlcNAc) (20%) whereas almost all of the type II isomers was converted to the corresponding Lewis(x) product. 45% of the type I tetrasaccharide was fucosylated at the second GlcNAc solely by FucT VI. The type II isomer was almost completely alpha1-3 fucosylated to yield the Lewisx derivative with traces of a structure that contained an additional fucose at the reducing GlcNAc. The results obtained in the present study employing high amounts of enzyme confirmed our previous results that FucT III acts preponderantly as a beta1-4 fucosyltransferase onto GlcNAc in vitro. Human FucT VI attaches fucose exclusively in an alpha1-3 linkage to 4-substituted GlcNAc in vitro and does not modify any 3-substituted GlcNAc to yield Lewis(a) oligosaccharides. With 8-methoxycarbonyloctyl glycoside acceptors used under standard conditions, FucT III acts exclusively on the type I and FucT VI only on the type II derivative. With lacto-N-tetraose, lacto-N-fucopentraose I, or LS-tetrasaccharide as substrates, FucT III modified the 3-substituted GlcNAc and the reducing glucose; FucT VI recognized only lacto-N-neotetraose as a substrate.
Assuntos
Fucosiltransferases/metabolismo , Oligossacarídeos/biossíntese , Sequência de Carboidratos , Linhagem Celular , Dissacarídeos/metabolismo , Fucosiltransferases/genética , Galactose/metabolismo , Humanos , Antígenos do Grupo Sanguíneo de Lewis/biossíntese , Metilação , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transfecção , Trissacarídeos/metabolismoRESUMO
In this study beta1-3 linked analogues of the T-antigen determinant were synthesized in preparative scale by transgalactosylation using beta-galactosidase from bovine testes to give synthetic antigens. Acceptors with modifications of the sugar residue such as alpha-glycosylated spacers, as well as GlcNAc-alphaOR- and 2dGal-alphaOR-substrates opened further possibilities for galactosylation.
Assuntos
Antígenos Virais de Tumores/química , Dissacarídeos/síntese química , Galactosídeos/síntese química , Testículo/enzimologia , beta-Galactosidase/metabolismo , Animais , Bovinos , Epitopos/química , Glicosilação , Masculino , Especificidade por SubstratoRESUMO
The efficient chemoenzymatic synthesis of the Thomsen-Friedenreich antigen determinant is demonstrated under transglycosylation conditions employing beta-galactosidase from bovine testes.
Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Dissacarídeos/síntese química , Epitopos/química , Animais , Antígenos de Neoplasias , Bovinos , Dissacarídeos/química , Glicosilação , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Masculino , Estrutura Molecular , Testículo/enzimologia , beta-GalactosidaseRESUMO
Galactosyltransferase from bovine milk was found to be able to utilise UDP-Glc to transfer Glc onto GlcNAc and chitooligomers[-beta-GlcNAc-(1-->4)-]n, n = 2-4. beta-Glucosylated products were used in binding studies with NKR-P1A protein cloned from rat natural killer cells.
