CMP substitutions preferentially inhibit polysialic acid synthesis
Glycobiology vol. 18 no. 2 pp. 187–194, 2008
doi:10.1093/glycob/cwm132
Advance Access publication on December 12, 2007
CMP substitutions preferentially inhibit polysialic acid synthesis
Tatsuo Miyazaki2,3,4 , Kiyohiko Angata3,4 , Peter H.
Seeberger4 , Ole Hindsgaul4 , and Minoru Fukuda1,4
4 Tumor Microenvironment Program, Glycobiology Unit, Cancer Research
Center, Burnham Institute for Medical Research, La Jolla, CA 92037, USA
Received on August 15, 2007; revised on November 9, 2007; accepted on
December 3, 2007
It is widely reported that derivatives of sugar moieties
can be used to metabolically label cell surface carbohydrates or inhibit a particular glycosylation. However, few
studies address the effect of substitution of the cytidylmonophosphate (CMP) portion on sialyltransferase activities. Here we first synthesized 2 -O-methyl CMP and 5methyl CMP and then asked if these CMP derivatives
are recognized by α2,3-sialyltransferases (ST3Gal-III and
ST3Gal-IV), α2,6-sialyltransferase (ST6Gal-I), and α2,8sialyltransferase (ST8Sia-II, ST8Sia-III, and ST8Sia-IV).
We found that ST3Gal-III and ST3Gal-IV but not ST6Gal-I
was inhibited by 2 -O-methyl CMP as potently as by CMP,
while ST3Gal-III, ST3Gal-IV, and ST6Gal-I were moderately inhibited by 5-methyl CMP. Previously, it was reported
that polysialyltransferase ST8Sia-II but not ST8Sia-IV was
inhibited by CMP N-butylneuraminic acid. We found that
ST8Sia-IV as well as ST8Sia-II and ST8Sia-III are inhibited
by 2 -O-methyl CMP as robustly as by CMP and moderately
by 5-methyl CMP. Moreover, the addition of CMP, 2 -Omethyl CMP, and 5-methyl CMP to the culture medium resulted in the decrease of polysialic acid expression on the cell
surface and NCAM of Chinese hamster ovary cells. These
results suggest that 2 -O-methyl CMP and 5-methyl CMP
can be used to preferentially inhibit sialyltransferases, in
particular, polysialyltransferases in vitro and in vivo. Such
inhibition may be useful to determine the function of a carbohydrate synthesized by a specific sialyltransferase such as
polysialyltransferase.
Keywords: α2,8-sialyltransferase/2 -O-methyl CMP/5-methyl
CMP/polysialic acid
Introduction
Glycosylation requires several enzyme-catalyzed steps to
synthesize an activated form of a monosaccharide. For sialic
acid synthesis, N-acetylglucosamine is first converted to
N-acetylmannosamine by N-acetylglucosamine 2-epimerase.
1 To whom correspondence should be addressed: Tel: +858-646-3144;
Fax: +858-646-3193; e-mail:
2 Current address: Department of Applied Life Sciences, Niigata University of
Pharmacy and Applied Life Sciences, Niigata, 956-8603, Japan.
3 These authors contributed equally to this work.
N-Acetylmannosamine is then phosphorylated to Nacetylmannosamine 6-phosphate by N-acetylmannosaminekinase. The resulting N-acetylmannosamine 6-phosphate is
conjugated to phosphoenolpyruvate by N-acetylneuraminic
9-phosphate synthase, through aldol condensation, forming
N-acetylneuraminic 9-phosphate. The product in turn yields
N-acetylneuraminic acid (NeuAc) by N-acetylneuraminic acid
9-phosphatase. N-Acetylneuraminic acid is then activated by
conjugation to CTP by N-acetylneuraminyl cytidyltransferase,
forming cytidyl N-acetylneuraminic acid 5 -phosphate, cytidylmonophosphate (CMP)-NeuAc. These reactions occur in the
nucleus. In animal and plant cells, the transfer of sialic acid to
acceptor carbohydrates takes place in the Golgi apparatus; thus,
CMP-NeuAc needs to be transported to the lumen of the Golgi
by the CMP-NeuAc transporter. Once there, CMP-NeuAc is
transferred to acceptor glycans by various sialyltransferases
(Schauer 1982; Varki 1992). Mutation in various enzymes in
this multistep process results in genetic conditions known as
congenital disorders of glycosylation (CDGs) (Freeze 2006).
Indeed, in one CDG, the CMP-NeuAc transporter is mutated,
leading to thrombocytopenia and neutropenia (Willig et al.
2001; Martinez-Duncker et al. 2005).
Polysialic acid is a homopolymer of α2,8-linked sialic acid
attached to the neural cell adhesion molecule (NCAM) and
is highly expressed in embryonic brain (Angata and Fukuda
2005). Recently, polysialic acid-deficient mutant mice were generated by inactivating two polysialyltransferase genes (ST8SiaII and ST8Sia-IV). Polysialic acid-deficient mice die as neonates
(Weinhold et al. 2005) and exhibit impaired migration of neural
cells, resulting in apoptosis of those cells (Angata et al. 2007).
In certain tumors such as neuroblastoma and glioma, polysialic
acid is ectopically expressed even in the adult and is thought to
play a role in tumor invasion (Scheidegger et al. 1994; Suzuki
et al. 2005). Inhibiting polysialic acid synthesis in those tumors
could thus have an antitumorigenic effect. Indeed Bertozzi’s
group showed that feeding cells N-butylmannosamine can inhibit polysialyltransferase activity (Mahal et al. 2001). In their
study, it was critical that all of the enzymes required for CMPNeuAc synthesis noted above tolerate the increase in size of
the N-acyl group from an acetyl (O=C–CH3 ) to a butyl (O=C–
CH2 –CH2 –CH3 ) group. By contrast, polysialyltransferases, in
particular ST8Sia-II (STX), utilize CMP-N-butylmannosamine
much less efficiently than CMP-N-acetylneuraminic acid or
CMP-N-propanyl neuraminic acid. This inhibition leads to a decreased synthesis of polysialic acid by ST8Sia-II (Mahal et al.
2001). These observations suggest that glycosylation efficiency
may decrease substantially when the structure of a precursor
carbohydrate residue is modified.
Monosaccharide derivatives have also been used to tag glycans. A well-characterized example is the use of N-azido-acetyl
mannosamine containing O=C–CH2 –N3 instead of O=C–CH3
(Prescher et al. 2004). The azido group is intact during metabolic
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T Miyazaki et al.
Fig. 1. The structure of CMP, 2 -O-methyl CMP, and 5-methyl CMP. Cytidine
5 -monophosphate (CMP), 2 -O-methyl cytidine 5 -monophosphate
(2 -O-methyl CMP), and 5-methyl cytidine 5 -monophosphate (5-methyl
CMP) are shown.
conversion and the sialic acid-containing azido group can be
expressed on the cell surface. Cell surface glycoproteins expressing sialic acid can be tagged using the Staudinger ligation
of FLAG peptide to the azido group, which can be detected
by fluorescent anti-FLAG peptide antibody. In this approach,
it is essential that each metabolic step required to form sialylated glycoproteins, including sialylation by sialyltransferases,
tolerate the N-azido acetyl group (Prescher et al. 2004).
Monosaccharides with modified groups have been used in
other studies (Rabuka et al. 2006; Sawa et al. 2006), but only
a few address how modification of the nucleotide phosphate
portion influences enzymatic activity. Since the nucleotide portion is far from the activated monosaccharide, which is attached
to the nucleotide through a phosphate (...truncated)
