Identification and characterization of a novel UDP-GalNAc:GlcAbeta-R alpha1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line.
We recently discovered a novel alpha-N-acetylgalactosaminyltransferase in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995) and also in mouse mast cytoma cells (Lidholt et al., Glycoconjugate J., 14, 737-742, 1997), which catalyzed the transfer of an alpha-GalNAc residue to the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, derived from proteoglycans. In this study, we characterized this enzyme using a preparation obtained from the serum-free culture medium of a human sarcoma (malignant fibrous histiocytoma) cell line by phenyl-Sepharose chromatography. Structural characterization by1H NMR spectroscopy of the reaction product using the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, as a substrate demonstrated that the enzyme was a UDP-GalNAc:GlcAbeta1-R alpha1,4-N -acetylgalactosaminyltransferase. This is the first identification of an alpha1,4-N-acetylgalactosaminyltransferase. Using N -acetylchondrosine GlcAbeta1-3GalNAc as an alternative substrate, the enzyme required divalent cations for the transferase reaction, with maximal activity at 20 mM Mn2+and exhibited a dual optimum at pH 6.5 and pH 7.4 depending upon the buffers used, with the highest activity in a 50 mM 2-( N -morpholino)ethanesulfonic acid buffer at pH 6.5. The apparent Km values obtained for N -acetylchondrosine, the linkage tetrasaccharide-serine, and UDP-GalNAc were 1060 microM, 188 microM, and 27 microM, respectively. This suggested that the linkage tetrasaccharide-serine was a good acceptor substrate for the enzyme. In addition, the enzyme utilized glucuronylneolactotetraosylceramide GlcAbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcbeta1-1Cer but not sulfoglucuronylneolactotetraosylceramide GlcA(3-O -sulfate)beta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cbeta1-1Cer as acceptor substrates. The possibility of involvement of this enzyme in the biosynthesis of glycosaminoglycan as well as other GlcA-containing glycoconjugates is discussed.
Kitagawa
H
H
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
Kano
Y
Y
Shimakawa
H
H
Goto
F
F
Ogawa
T
T
Okabe
H
H
Sugahara
K
K
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D018219:Histiocytoma, Benign Fibrous / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D009844:Oligosaccharides; D017350:N-Acetylgalactosaminyltransferases; C120485:UDP-GalNAc - GlcA beta-R alpha1,4-N-acetylgalactosaminyltransferase