Glycobiology, Vol 8, 857-867, Copyright © 1998 by Society for Glycobiology
MB Sevigny, J Ye, S Kitazume-Kawaguchi and FA Troy 2nd
The alpha2,8-polysialyltransferases (polySTs) from embryonic chick brain
catalyze the alpha2,8-specific polysialylation of endogenous neural cell
adhesion molecules (N-CAMs). This posttranslation glycosylation decreases
N-CAM-dependent cell adhesion and migration. The enzymatic properties of
the membrane-bound form of the polyST activity was investigated in vitro.
Our results show that the polyST activity was developmentally expressed
with maximum specific activity appearing about 12 days after fertilization.
This time shortly precedes maximal expression of the cognate polysialylated
N-CAMs. Kinetic studies showed the KMand Vmaxfor CMP-Neu5Ac were 133 microM
and 0.13 microM/h, respectively, at pH 6.1, 33 degrees C. CMP-Neu5Gc was
not a donor substrate. PolyST activity was increased 5- to 6-fold in the
presence of 10 mM MnCl2,the preferred divalent cation, and 1 mM
dithiothreitol (DTT). Heparin (3 kDa) was a noncompetitive inhibitor of
polysialylation with a Kiof 9 microM. Based on the affinity of the enzyme
for heparin, the polyST activity was partially purified ( approximately
30-fold) by heparin-Sepharose affinity chromatography, after differential
solubilization with the zwitterionic detergent, CHAPS. DTT and chemical
modification studies using the thiol-directed alkylating reagents,
N-ethylmaleimide (NEM) and iodoacetamide (IAA), were used to show that at
least one cysteinyl residue in the polyST was of critical importance for
polysialylation, but of lesser importance for monosialylation, catalyzed by
the alpha2,3-, alpha2,6-, and alpha2,8-monosialyltransferases (monoSTs). A
sulfhydryl residue is implicated in chain initiation. Two important
structural differences between the mono- and polySTs were revealed by
sequence analyses. First, the polySTs contain heparin-like, positively
charged amino acid clusters upstream of both sialylmotif L and S. Second,
the polySTs contain a uniquely extended basic amino acid region (pI 11.
6-12.0) of 31 residues immediately upstream of sialylmotif S. This
extended, positively charged region may function in the processive
mechanism of polymerization by allowing nascent polySia chains to remain
bound to the polyST during the repetitive addition of each new Sia residue
to the nonreducing termini of the growing chain. The importance of these
studies is that they provide new information on the enzymatic basis of
polysialylation. They also reveal that sulfhydryl residues and extended
basic amino acid domains are two structural features unique to
polysialylation, in contrast to monosialylation. Both may be important
distinguishing features between the classes of distributive (monoSTs) and
processive polysialyltransferases, which have not been previously
described.
ORIGINAL ARTICLES
Developmental expression and characterization of the alpha2,8- polysialyltransferase activity in embryonic chick brain
Department of Biological Chemistry, University of California School of Medicine, Davis, CA 95616, USA.
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