Glycobiology, Vol 9, 697-703, Copyright © 1999 by Oxford University Press
H Kitagawa, Y Kano, H Shimakawa, F Goto, T Ogawa, H Okabe and K Sugahara
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.
ORIGINAL ARTICLES
Identification and characterization of a novel UDP-GalNAc:GlcAbeta-R alpha1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada- ku, Kobe 658-8558, Japan.
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