Identification and characterization of a novel UDP-GalNAc:GlcAbeta-R alpha1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line.

Abstract

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.

• Authors: H Kitagawa, Y Kano, H Shimakawa, F Goto, T Ogawa, H Okabe, K Sugahara
• Journal: Glycobiology (Glycobiology) Vol. 9 Issue 7 Pg. 697-703 (Jul 1999) ISSN: 0959-6658 [Print] England
• PMID: 10362839 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Oligosaccharides
• N-Acetylgalactosaminyltransferases
• UDP-GalNAc - GlcA beta-R alpha1,4-N-acetylgalactosaminyltransferase

Topics

• Animals
• Carbohydrate Sequence
• Cattle
• Histiocytoma, Benign Fibrous (enzymology)
• Humans
• In Vitro Techniques
• Kinetics
• Magnetic Resonance Spectroscopy
• Mice
• Molecular Sequence Data
• N-Acetylgalactosaminyltransferases (chemistry, isolation & purification, metabolism)
• Oligosaccharides (chemistry, metabolism)
• Substrate Specificity
• Tumor Cells, Cultured