Inhibition of hyaluronan synthesis in
Streptococcus equi
FM100
by 4-methylumbelliferone
Ikuko Kakizaki
1
, Keiichi Takagaki
1
, Yasufumi Endo
1
, Daisuke Kudo
1
, Hitoshi Ikeya
2
, Teruzo Miyoshi
2
,
Bruce A. Baggenstoss
3
, Valarie L. Tlapak-Simmons
3
, Kshama Kumari
3
, Akio Nakane
4
, Paul H. Weigel
3
and Masahiko Endo
1
Departments of
1

of the membrane phospholipids from FM100 cells treated
with MU, changes were observed in the distribution of only
cardiolipin species but not of the other major phospholipid,
PtdGro. These results suggest that MU treatment may cause
a decrease in hyaluronan synthase activity by altering the
lipid environment of membranes, especially the distribution
of different cardiolipin species, surrounding hyaluronan
synthase.
Keywords: hyaluronan; synthesis; Streptococcus; 4-methyl-
umbelliferone; phospholipids.
Hyaluronan (HA) is a high molecular weight glycosamino-
glycan composed of repeating disaccharide units of
GlcNAc-b(1fi4)-GlcUA-b(1fi3) [1]. HA is one of the
major components of the extracellular matrix together with
proteoglycans and collagens, and is involved in many
biological processes, including tissue organization, wound
healing, tumor invasion and cancer metastasis, through its
interactions with other extracellular matrix components
[2,3].
It has long been suggested that HA may be implicated
in malignant transformation and tumor progression [4].
There are many reports that HA production is increased in
various tumor tissues including mesothelioma and Wilm’s
tumor. Recently, a direct correlation between HA and
tumorigenesis, and cancer metastasis was shown in studies
using genetic manipulations to create mutant cells that were
either overproducing HA or HA-deficient [5,6]. Overpro-
duction of HA is also observed in diseases associated with
inflammation and fibroses [3].
Many strains of group A and C Streptococci are able to

GlcNAc, N-acetylglucosamine; GlcUA, glucuronic acid; HA,
hyaluronan (hyaluronic acid); HABP, hyaluronan binding protein;
HAS (Has), hyaluronan synthase; MU, 4-methylumbelliferone;
spHAS, S. pyogenes HAS; seHAS, S. equisimilis HAS.
Note: A web site is available at http://www.med.hirosaki-u.ac.jp/
bioche1/test/Biochem-top/Biochem-top1.html
(Received 20 June 2002, revised 14 August 2002,
accepted 29 August 2002)
Eur. J. Biochem. 269, 5066–5075 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03217.x
cloning of the HAS genes, it has been possible to genetically
manipulate the production of HA, and consequently,
correlations between HA production and various biological
processes have now been brought to light. For example, the
effects of antisense inhibition of HA production on the
organization of the extracellular matrix in human articular
chondrocytes has been examined [15]. Studies using targeted
deletion of HAS genes have also been made to investigate
theroleofHAin vivo.ItwasreportedthatHas2
+
embryos,
which lack HA production by Has2, exhibit severe cardiac
and vascular abnormalities and die during fetal develop-
ment [16]. However the details about the multiple functions
of HA have not been fully established.
We found that HA synthesis in cultured human skin
fibroblasts was inhibited by 4-methylumbelliferone (MU,
7-hydroxy-4-methyl-2H-1-benzopyran-2-one) with no effect
on the synthesis of any other glycosaminoglycan and that an
HA-deficient extracellular matrix was formed [17,18]. Some
agents have been reported to inhibit HA synthesis, however,

)1
)
was purchased from American Radiolabeled Chemicals
(St. Louis, MO, USA). UDP-GlcNAc, ATP, dithiothreitol,
bovine testicular hyaluronidase and bovine heart CL were
purchased from Sigma (St. Louis, MO, USA). HA from
human umbilical cords and Streptomyces hyaluronidase
were obtained from Seikagaku Corporation (Tokyo,
Japan). Actinase E was from Kaken Pharmaceutical
(Tokyo, Japan). Hyaluronic Acid ÔChugaiÕ quantitative test
kit for the sandwich binding protein assay was purchased
from Chugai Pharmaceutical (Tokyo, Japan) [26]. The
random-primed DNA labeling kit was from Amersham
Pharmacia Biotech (Tokyo, Japan) and [a-
32
P] dCTP was
from NEN Life Science Products (Boston, MA, USA).
Antiserum raised against the whole HAS from Streptococcus
pyogenes (spHAS) was described previously [10,27]. Anti-
rabbit Ig conjugated to horseradish peroxidase was from
Dako Japan (Kyoto, Japan) and n-dodecyl-b-
D
-maltoside
(DDM) was from Nakarai Tesque (Kyoto, Japan).
Culture of Streptococci and treatment with MU
Encapsulated group C S. equi FM100 was derived from
S. equi (ATCC9527) and maintained at 33 °C in a synthetic
solid medium (pH 8), which we have modified from the
medium reported by van de Rijn and Kessler [28]. Cells were
precultured in liquid medium (1.5% polypeptone-S/0.5%

were 1.0, 3.0, 4.1, 8.0, 12 and 19 · 10
5
[29].
Preparation of a membrane-rich fraction
and solubilization
The membrane-rich fraction was prepared by following the
method of Sugahara et al. [30]. Briefly, exponential phase
cell cultures were harvested by centrifugation at 18 000 g for
30 min. Then, cells were suspended in 0.05
M
sodium and
potassium phosphate buffer, pH 7.4, containing 5 m
M
dithiothreitol. The cell suspension was sonicated on ice
using a Branson Sonifer (model 250) for 1 min. The
disrupted cells were centrifuged at 10 000 g for 10 min
and the supernatant fluid was withdrawn. Following
centrifugation of the 10 000 g supernatant fluid at
105 000 g for 60 min, the resultant pellet was washed with
fresh buffer by centrifugation at 229 000 g for 45 min. The
pellet was suspended in 0.033
M
sodium and potassium
phosphate buffer, pH 7.4 containing 5 m
M
dithiothreitol,
and used as an enzyme source for the cell-free HA synthesis
assay. Solubilization of membranes using DDM was
performed as previously reported by Tlapak-Simmons et al.
[14]. Each cell-free assay was standardized for the amount of

GlcUA (1.8 lCi), 5 m
M
ATP, and 12.5–250 lgofmem-
brane-rich fractions or DDM extracts as the enzyme source.
MU was added as indicated to the assay mixture or the
culture medium. For certain experiments, the enzyme was
preincubated with 2 m
M
bovine heart CL. The assay
mixture was incubated at 37 °Cfor1.5h,andthereaction
was terminated by boiling for 2 min. Because the reaction
was linear up to 1.5 h of incubation time, this time point
wasused.AfteractinaseEdigestion(2.5mgÆmL
)1
,16h
at 45 °C), 1/6 volume of 50% trichloroacetic acid was
added with mixing, the reaction mixture was cooled on
ice, and the supernatant was withdrawn after centrifugation.
In the presence of 50 lg of carrier HA, ethanol precipitation
was performed five times to remove the unincoporated,
free radioisotopes. The final precipitate was dissolved in
water, digested with Streptomyces hyaluronidase and then
precipitated with ethanol. The radioactivity remaining in
the supernatant following ethanol precipitation, which
represents the digestion products specifically derived from
HA, was then determined using a liquid scintillation
counter.
Isolation of
S. equi
FM100

The hasA DNA probe was labeled by the random
priming method [34]. Hybridization was performed with
a
32
P-labeled probe at 42 °C for 18 h in 50% formamide,
3· NaCl/Cit, 0.05
M
Tris/HCl (pH 7.5), 1 m
M
EDTA,
0.02% BSA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone,
20 lgÆmL
)1
tRNA, 20 lgÆmL
)1
herring sperm DNA. Then
the filters were washed twice with 3· NaCl/Cit, 0.1% SDS
at 37 °C for 30 min, and twice with 0.1· NaCl/Cit, 0.1%
SDS at 50–65 °C for 30 min. Autoradiography was carried
out by exposure to X-ray film (Kodak X-Omat AR) at
)80 °C using an intensifying screen. Results of autoradio-
graphy were quantified using
NIH IMAGE
(version 1.62)
software.
SDS/PAGE and immunoblotting
SDS/PAGE was performed in 10% acrylamide gels by the
method of Laemmli [32]. Protein was stained with the
Coomassie brilliant blue R-250. For immunoblotting,
proteins were transferred to a polyvinylidene fluoride

delayed extraction of 200 ns, a grid voltage of 79%, and
were subjected to a 20-kV accelerating voltage. An external
calibration was obtained using bovine heart CL, which has
a mass of 1448.97. The matrices used were 6-aza-2-
thiothymine or 2,4,6-trihydroxyacetophenone at 5 mgÆmL
)1
in chloroform/methanol (2 : 1, v/v) containing 10 m
M
dibasic ammonium citrate. Samples were diluted with two
volumes of chloroform/methanol (2 : 1, v/v) and then mixed
1 : 1 with the matrix solution prior to spotting on a sample
plate and air drying. Spectra are an average of 80–100 scans.
In some cases the identity of specific m/z species was
confirmed by post source decay analysis in both the positive
and negative ion modes. Total amounts of CL or PtdGro
recovered from FM100 cells were assessed based on their
5068 I. Kakizaki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
signal intensities relative to appropriate phospholipids that
were used as standards.
RESULTS
Inhibition of HA production of FM100 cells
To examine the effect of MU on the growth of FM100 cells,
the cells were cultured in liquid medium with or without MU
for various periods, and cell numbers were estimated by
measuring absorbance at 660 nm at each time point. No
significant effect on the growth was observed in the range of
0–2.0 m
M
MU (data not shown). Absorbance at 660 nm was
measured in all subsequent experiments, however, inhibition

peaks disappeared after digestion with the very specific
Fig. 1. Effect of MU treatment on the HA coat formation of S. equi FM100 cells. Microscopic photograph of HA coats (arrowheads) on cell surfaces
of FM100. A–C, untreated cultures; D–F, treated with 0.5 m
M
MU; G–I, with 1.0 m
M
MU. A, D and G, cultured for 3 h; B, E and H, cultured for
5.5 h; C, F and I, cultured for 8 h. Original magnification, · 1000. Magnification bar represents 10 lm.
Fig. 2. Analysis of HA released into the culture medium. FM100 cells
were cultured with various concentrations of MU (0–2.0 m
M
)for22 h.
(A) Micrograph of the HA coats on cell surfaces cultured for 8 h (a)
andfor22h(b)withoutMU.(B)HPLCanalysisofHAreleasedinto
the culture medium. A Shodex OHpak KB-805 column (8 · 300 mm)
was used and eluted with 0.2
M
NaCl at a flow rate of 0.5 mLÆmin
)1
.
Eluted fractions were monitored at a wavelength of 215 nm. Arrow-
heads indicate the peak of HA.
Ó FEBS 2002 Inhibition of HA synthesis in Streptococcus by MU (Eur. J. Biochem. 269) 5069
Streptomyces hyaluronidase (data not shown). However,
when the secretion of HA into the culture medium by
FM100 cells was quantified by measuring the HA peak
areas, it was found that HA production was clearly
decreased by MU. To verify this effect and study it further,
FM100 cells were cultured in the presence of MU for
various periods (0, 3, 5.5, 8 and 22 h), and the HA

In order to examine whether the addition of MU to a
membrane-rich fraction could inhibit HAS activity, a
cell-free HA synthesis experiment was performed. A mem-
brane-rich fraction was prepared from cultured FM100 cells
by sonication and ultracentrifugation, and used as an
enzyme source. UDP-[U-
14
C] GlcUA and UDP-GlcNAc
were used as donors, and the transfer of UDP-[U-
14
C]
GlcUA to newly synthesized HA was analyzed. The activity
in the membrane-rich fraction was hardly inhibited by MU
up to 1.0 m
M
(data not shown). This result suggest that the
inhibition of HA production was not caused by direct
inhibition of HAS activity.
Effect of MU on HAS activity in FM100 cells treated
with MU
The activity of the HAS in FM100 cells cultured with
various concentrations of MU for 12 h was also measured.
Membrane-rich fractions were prepared, and their ability to
support cell-free HA synthesis was determined. HA pro-
duction by these isolated membranes was decreased by MU
treatment of the live cells in a dose-dependent manner (data
not shown). At 2.0 m
M
, HA production was decreased to
about 10% of control value.

staining pattern is shown at the bottom.
Fig. 3. Effect of MU treatment of S. equi FM100 cells on HA accu-
mulation in culture medium. FM100 cells were cultured with or without
MU for various periods (0, 3, 5.5, 8 and 22 h). HA production and
release into culture medium of the FM100 cells was quantitated by the
sandwich binding protein assay. Time represents hours after addition
of a 1/100 volume of inoculum into fresh medium. Symbols are as
follows; d,0;h,0.2m
M
; j,0.5m
M
; n,1.0m
M
; m,2.0m
M
MU.
5070 I. Kakizaki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The anti-spHAS antibody used here is also strongly
cross-reactive with the seHAS, the HAS from group C
S. equisimilis [10]. In this experiment the antibody recog-
nized a protein of M
r
48 000, the size expected for the
calculated relative molecular mass (M
r
47 778) of seHAS
[10] (Fig. 5A). The HAS protein level, however, did not
change by treatment of cells with up to 1.0 m
M
MU. At

ity in whole membranes. The enhancement of HAS activity
when membranes are solubilized by DDM has already been
reported [14]. Further stimulation of HAS activity by
addition of exogenous CL to the DDM extract resulted in
recovery of HAS activity to about the level in samples not
treated with MU (Fig. 6, solid bars). Even when cells were
treatedwith2.0m
M
MU, subsequent stimulation of HAS
by CL was observed in DDM extracts, although the
stimulated activity did not reach that of MU-untreated
samples. This deficiency of reconstituting HA synthesis
activity at 2 m
M
MU can be explained by the decreased level
of HAS protein noted above. CL addition did not cause a
significant increase in HAS activity in the extracts from
untreated cells.
A key question in the experiment of Fig. 6 is whether
there might be a differential solubilization of some CL
species by the detergent DDM, so that the membrane
composition of CL is not reflected in the DDM extract. To
address this issue, Folch extractions were performed on
untreated membrane preparations and on the DDM
Fig. 6. Effects of detergent solubilization and addition of cardiolipin on
HAS activities in membranes from MU-treated cells. FM100 cells were
cultured with or without various concentrations of MU (0–2.0 m
M
)for
12 h. Then, the effect of solubilization with DDM and addition of

relative peak heights in each Folch extract typically varied
from 0.2 to 0.7 and their standard errors were ±5–15% of
these values. There were no statistically significant differ-
ences between the two Folch extracts for any of the CL
species detected (not shown). The pattern and relative
intensities of CL species was the same in membranes or in
the DDM extract derived from those membranes.
MALDI-TOF mass spectrometric analysis was then
performed to determine whether the phospholipid profile
of cell membranes was altered by MU treatment. Phos-
pholipids were extracted from the membranes of FM100
cells cultured with 0–2.0 m
M
MU for various times, and the
lipids present in the extracts were then analyzed. No change
in the total amount of CL was observed after MU treatment
(data not shown). Only two major classes of phospholipids
were detected by MALDI-TOF MS analysis of Folch
extracts, CL and PtdGro. Other phospholipids including
PtdEtn, PtdCho, PtdSer and PtdIns were not detected. As
reported by others, Gram-positive bacteria such as S. equi
typically have essentially only these two phospholipids [39].
Although only two phospholipids were present, their
diversity was striking, as at least 60 variants of CL and
PtdGro could be identified. The pattern and relative
abundance of the minor and major PtdGro species was
not altered by treatment with MU (data not shown).
Unexpectedly, there were no obvious or reproducible
changes in the composition of the multiple CL species, when
cells were treated with MU. One of these experiments is

MU for 12 h. The phospholipid profile in the CL
mass region was then analyzed by MALDI-TOF MS. The matrix used was 6-aza-2-thiothymine. Multiple CL peaks, which are labeled A through I,
were detected. The distribution of these multiple CL species (as a percent of the total CL) is summarized in Table 1. Minor peaks that differ from a
more major peak by 1 m/z unit represent mass variants due to natural isotope abundance (e.g. one more
13
C present in the molecule instead of
12
C
as in a neighboring peak).
5072 I. Kakizaki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
added in the cell-free HA synthesis experiment (Fig. 6) was
also analyzed, and was compared with that of the CL in the
FM100 cells. The bovine heart CL has the same compo-
sition as that reported in a previous paper (14). It contained
one major species with m/z-value of 1448.97. This bovine
CL species is relatively large and, although it may not be
present in FM100 cells, is intermediate in mass between two
of the major CL species (i.e. peaks E and F in Fig. 7).
DISCUSSION
As shown previously in fibroblasts, MU also did not affect
the molecular size distribution of HA produced by FM100
cells. This suggests that MU acts to inhibit the HA synthesis
pathway but not to stimulate the HA degradation pathway.
In the present study, we showed that MU did not directly
inhibit HAS activity even at a relatively high concentration,
and this result agrees with that obtained using cultured
human skin fibroblasts. It seems likely therefore that the
mechanisms of inhibition of HA synthesis by MU are very
similar if not identical between eukaryote and prokaryote
cells.

However, we did not detect any metabolites of MU either in
the culture supernatant or in the cell extract from FM100
cells cultured with MU (data not shown). We believe
therefore there is no or little involvement of MU-meta-
bolites in the inhibition of HA synthesis by MU in FM100
cells. These above results indicate that something required
for a fully functional HA synthesis system is down-regulated
or inhibited in intact cells exposed to MU. The presence
of post-translational modifications in the native enzyme
in Streptococci has not been addressed. Thus, it is likely
that MU may alter either a required event needed to
generate active HAS enzyme or the availability of a required
activator such as CL or some other event needed to generate
active enzyme.
It has been suggested by other investigators that HA
synthesis in mammalian cells and Streptococcal cells is
strictly controlled by complicated mechanisms, including
phosphoryl modification of HAS [21,41–43]. It should also
be noted that multiple consensus sequences for phosphory-
lation by some kinases are found in HASs [44,45]. Based on
protein motif analysis using the
PROSITE
database (release
16.22), multiple potential phosphorylation sites could also
be found in S. equi FM100 HAS. However, no change in
the phosphorylation-level of HAS protein by MU-treat-
ment was observed when it was examined by Western
analysis using anti-phosphoamino acid antibodies (data not
shown). Furthermore, previous MALDI-TOF analysis of
purified recombinant Streptococcal HAS demonstrated that

G 9.56 0.86 12.44 0.18 14.39 0.14
H 7.91 0.28 7.64 0.50 8.06 0.88
I 7.92 0.97 6.33 0.41 6.65 1.00
Ó FEBS 2002 Inhibition of HA synthesis in Streptococcus by MU (Eur. J. Biochem. 269) 5073
stabilization of the enzymatic activity was again suggested
[46]. If this pore model is correct, then another possibility for
how MU inhibits HA synthesis is that this HA translocation
process may be blocked by MU through subtle changes in
the steric conformation of the HAS protein or the mem-
brane bilayer. Because MU is very lipid soluble the
membrane-bound HAS or the organization of lipids in the
membrane itself may be very sensitive to this compound.
Alternatively, the glycosyltransferase activities of HAS or its
HA translocation activity, all of which are very dependent
on the proper conformation of this membrane protein, may
be adversely affected by MU in an indirect way.
Our results suggest that a possible mechanism of
inhibition is that MU alters the phospholipid distribution
of the cell membrane, which could then destabilize the HAS
activity. MALDI-TOF mass spectrometric analysis indi-
cates that FM100 cells contain predominantly only PtdGro
and CL as their major phopholipids. In fact, it may not be a
coincidence that CL is a major membrane lipid, as it is
required by HAS. Natural selection of cells able to
synthesize large HA coats may have resulted in a compo-
sition of membrane phopholipids compatible with high
HAS activity. A key finding in the present study is that HAS
inhibition, in membranes isolated from MU-treated cells, is
rescued by solubilizing the enzyme in DDM and then
providing endogenous CL. Even without MU treatment,

result indicated that with increasing time of MU treatment
there was a decrease in the proportion of smaller CL species
and an increase in the larger species. Although we do not
know exactly what this observation means for the activity of
HAS in membranes of MU-treated live cells, the results
provide a possible explanation for the inhibition of HAS by
MU and the rescue of solubilized inhibited HAS by CL,
because the enzyme is lipid-dependent and relatively
CL-specific for its activity. Our interpretation at this point
is that in order to be optimally active, the HAS may require
or prefer to interact with CL species containing fatty acids
with a particular chain length and unsaturation pattern, and
that the MU treatment of cells decreases the availability of
these favorable CL species. Additionally, the interaction of
HAS with CL species that have quite different fatty acid
components (e.g. larger or with a different number and
location of double bonds) may actually inhibit the enzyme,
so that in live cells the HAS activity could be decreased by
MU treatment as the cellular distribution of CL species
changed. The isolated membranes from the cells treated
with MU show a similar inhibition of HAS activity because
the enzyme is still associated with these ÔbadÕ CL species.
However, when these membranes are solubilized and
exogenous CL is added, the enzyme can then interact with
the CL species it prefers and become reactivated. Further
study will be required to confirm this interpretation and to
understand fully the mechanisms for inhibition of HA
synthesis by MU. This information may be useful in the
treatment of diseases involving excess production of HA.
ACKNOWLEDGMENTS

(1991) Hyaluronic acid capsule is a virulence factor for mucoid
group A Streptococci. Proc. Natl Acad. Sci. USA 88, 8317–8321.
9. Weigel, P.H., Hascall, V.C. & Tammi, M. (1997) Hyaluronan
synthases. J. Biol. Chem. 272, 13997–14000.
10. Kumari, K. & Weigel, P.H. (1997) Molecular cloning, expression,
and characterization of the authentic hyaluronan synthase from
group C Streptococcus equisimilis. J. Biol. Chem. 272, 32539–
32546.
5074 I. Kakizaki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
11. Ward, P.N., Field, T.R., Ditcham, W.G., Maguin, E. & Leigh,
J.A. (2001) Identification and disruption of two discrete
loci encoding hyaluronic acid capsule biosynthesis genes
hasA, hasB,andhasC. Streptococcus uberis. Infect. Immun. 69,
392–399.
12. Itano, N., Sawai, T., Yoshida, M., Lenas, P., Yamada, Y.,
Imagawa, M., Shinomura, T., Hamaguchi, M., Yoshida, Y.,
Ohnuki, Y., Miyauchi, S., Spicer, A.P., McDonald, J.A. &
Kimata, K. (1999) Three isoforms of mammalian hyaluronan
synthases have distinct enzymatic properties. J. Biol. Chem. 274,
25085–25092.
13. Tlapak-Simmons, V.L., Kempner, E.S., Baggenstoss, B.A. &
Weigel, P.H. (1998) The active Streptococcal hyaluronan syn-
thases (HASs) contain a single HAS monomer and multiple car-
diolipin molecules. J. Biol. Chem. 273, 26100–26109.
14. Tlapak-Simmons, V.L., Baggenstoss, B.A., Clyne, T. & Weigel,
P.H. (1999) Purification and lipid dependence of the recombinant
hyaluronan synthases from Streptococcus pyogenes and Strepto-
coccus equisimilis. J. Biol. Chem. 274, 4239–4245.
15. Nishida, Y., Knudson, C.B., Nietfeld, J.J., Margulis, A. &
Knudson, W. (1999) Antisense inhibition of hyaluronan synthase-

suramin. Oncol. Res. 5, 415–422.
23. Ueki, N., Taguchi, T., Takahashi, M., Adachi, M., Ohkawa, T.,
Amuro, Y., Hada, T. & Higashino, K. (2000) Inhibition of hya-
luronan synthesis by vesnarinone in cultured human myofibro-
blasts. Biochim. Biophys. Acta 1495, 160–167.
24. Endo, Y., Takagaki, K., Takahashi, G., Kakizaki, I., Funahashi,
M., Yokoyama, M. & Endo, M. (2000) Formation of hyaluronic
acid-knock-down extracellular matrix using 4-methylumbellifer-
one. In Progress in Transplantation (Munakata, A., ed.), pp. 1–7.
Elsevier Science B.V., Amsterdam, the Netherlands.
25. Sohara, Y., Ishiguro, N., Machida, K., Kurata, H., Thant, A.A.,
Senga, T., Matsuda, S., Kimata, K., Iwata, H. & Hamaguchi, M.
(2001) Hyaluronan activates cell motility of v-Src-transformed
cells via Ras-mitogen-activated protein kinase and phosphoinosi-
tide 3-kinase-Akt in a tumor-specific manner. Mol. Biol. Cell 12,
1859–1868.
26. Chichibu, K., Matsuura, T., Shichijo, S. & Yokoyama, M.M.
(1989) Assay of serum hyaluronic acid in clinical application. Clin.
Chim. Acta 181, 317–323.
27. DeAngelis, P.L. & Weigel, P.H. (1994) Immunochemical con-
firmation of the primary structure of Streptococcal hyaluronan
synthase and synthesis of high molecular weight product by the
recombinant enzyme. Biochemistry 33, 9033–9039.
28. van de Rijn, I. & Kessler, R.E. (1980) Growth characteristics of
group A Streptococci in a new chemically defined medium. Infect.
Immun. 27, 444–448.
29. Tanaka, K., Nakamura, T., Ikeya, H., Higuchi, T., Tanaka, A.,
Morikawa,A.,Saito,Y.,Takagaki,K.&Endo,M.(1994)Hya-
luronate depolymerization activity induced by progesterone in
cultured fibroblasts derived from human uterine cervix. FEBS

40. Tlapak-Simmons, V.L., Baggenstoss, B.A., Kumari, K.,
Heldermon, C. & Weigel, P.H. (1999) Kinetic characterization of
the recombinant hyaluronan synthases from Streptococcus pyo-
genes and Streptococcus equisimilis. J. Biol. Chem. 274, 4246–4253.
41. Klewes, L. & Prehm, P. (1994) Intracellular signal transduction for
serum activation of the hyaluronan synthase in eukaryotic cell
lines. J. Cell. Physiol. 160, 539–544.
42. Nickel, V., Prehm, S., Lansing, M., Mausolf, A., Podbielski, A.,
Deutscher, J. & Prehm, P. (1998) An ectoprotein kinase of group
C Streptococci binds hyaluronan and regulates capsule formation.
J. Biol. Chem. 273, 23668–23673.
43. Jacobson, A., Brinck, J., Briskin, M.J., Spicer, A.P. & Heldin, P.
(2000) Expression of human hyaluronan synthases in response to
external stimuli. Biochem. J. 348, 29–35.
44. Spicer, A.P., Augustine, M.L. & McDonald, J.A. (1996) Mole-
cular cloning and characterization of a putative mouse hyaluronan
synthase. J. Biol. Chem. 271, 23400–23406.
45.Itano,N.&Kimata,K.(1996)Molecularcloningofhuman
hyaluronan synthase. Biochem. Biophys. Res. Commun. 222,
816–820.
46. Heldermon, C., DeAngelis, P.L. & Weigel, P.H. (2001) Topo-
logical organization of the hyaluronan synthase from Strepto-
coccus pyogenes. J. Biol. Chem. 276, 2037–2046.
Ó FEBS 2002 Inhibition of HA synthesis in Streptococcus by MU (Eur. J. Biochem. 269) 5075