(Received for publication, September 19, 1996)
From the Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, S-751 23 Uppsala, Sweden
Escherichia coli K4 bacteria
synthesize a capsule polysaccharide (GalNAc-GlcA(fructose))n
with the carbohydrate backbone identical to chondroitin. GlcA- and
GalNAc-transferase activities from the bacterial membrane were assayed
with acceptors derived from the capsule polysaccharide and radiolabeled
UDP-[14C]GlcA and UDP-[3H]GalNAc,
respectively. It was shown that defructosylated oligosaccharides (chondroitin) could serve as substrates for both the GlcA- and the
GalNAc-transferases. The radiolabeled products were completely degraded
with chondroitinase AC; the [14C]GlcA unit could be
removed by -D-glucuronidase, and the
[3H]GalNAc could be removed by
-N-acetylhexosaminidase. A fructosylated oligosaccharide
acceptor tested for GlcA-transferase activity was found to be inactive.
These results indicate that the chain elongation reaction of the K4
polysaccharide proceeds in the same way as the polymerization of the
chondroitin chain, by the addition of the monosaccharide units one by
one to the nonreducing end of the polymer. This makes the biosynthesis
of the K4 polysaccharide an interesting parallel system for studies of
chondroitin sulfate biosynthesis. In the biosynthesis of capsule
polysaccharides from E. coli, a similar mechanism has
earlier been demonstrated for polysialic acid (NeuNAc)n (Rohr,
T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342) and for the K5 polysaccharide (GlcA
1-4GlcNAc
1-4)n (Lidholt, K., Fjelstad, M., Jann, K., and Lindahl, U. (1994) Carbohydr. Res. 255, 87-101).
In contrast, chain elongation of hyaluronan
(GlcA
1-3GlcNAc
1-4)n is claimed to occur at the reducing
end (Prehm, P. (1983) Biochem. J. 211, 181-189).
The biosynthesis of heparin/heparan sulfate and chondroitin sulfate polysaccharides has been intensively studied. These glycosaminoglycan chains are synthesized as proteoglycans. The chains are attached to serine units in the core protein via a tetrasaccharide linkage, and the polysaccharides are formed by the addition of monosaccharide units, one by one, from the corresponding UDP sugars to the nonreducing end of the growing chain (1). The glycosyl transferases involved in the polymerization reaction of heparin/heparin sulfate, GlcNAc- and GlcA-transferase, have been studied in different tissues (2-5) and purified from bovine serum (6). One very interesting finding from these studies is that the two glycosyl transferase activities seem to be catalyzed by one single protein with two catalytic sites. The glycosyltransferases involved in chain elongation of chondroitin sulfate, GalNAc- and GlcA-transferase, have so far not been purified, although their activities have been studied (7-11).
Two bacterial strains, Escherichia coli K5 and E. coli K4, synthesize polysaccharide capsules with the same backbone
structures as heparin/heparan sulfate and chondroitin sulfate,
respectively (Fig. 1). The K5 bacteria produce a (GlcA-GlcNAc)n
polymer (12) and the K4 synthesizes a
(GalNAc-GlcA(Fru))n1 sequence (13)
having the same structure as chondroitin with the fructose units
removed (Fig. 1B). We have previously studied the biosynthesis of the K5 polysaccharide and found that chain elongation occurs in the same way as heparin/heparan sulfate
biosynthesis. We have also demonstrated that the bacterial enzymes have
the same substrate specificity regarding N-sulfate groups on
the oligosaccharide acceptor as the mammalian ones (14). Although the
K5 bacteria produce a nonsulfated polymer, the bacterial
GlcA-transferase, like its mammalian equivalent, preferred
oligosaccharides with an N-sulfate group on the penultimate
GlcNAc unit from the reducing end (14).
The structure of E. coli K4 capsule polysaccharide has been
characterized by Rodriguez et al. (13), but the biosynthesis of the polymer has not been investigated. There are so far no reports
of assays for the corresponding glycosyl transferases using endogenous
primers or exogenous oligosaccharide acceptors. The aim of this study
was to clarify whether polymerization of K4 polysaccharide occurred in
the same way as that of the chondroitin polysaccharide. With bacterial
K4 membranes as the enzyme source, we were able to assay the transfer
of both [14C]GlcA and [3H]GalNAc to
defructosylated K4 oligosaccharides (chondroitin oligosaccharides). The
radiolabeled oligosaccharide products were identified by enzyme digestions. Both [14C]GlcA- and
[3H]GalNAc-labeled products could be degraded by
chondroitinase AC, the [14C]GlcA could be removed by
-glucuronidase, and the [3H]GalNAc could be removed by
-N-acetylhexosaminidase. These results show that the
radiolabeled monosaccharide units are added to the nonreducing end of a
chondroitin oligosaccharide. On the other hand, when fructosylated K4
oligosaccharides (with a GalNAc unit at the nonreducing end) were
tested as acceptors for the GlcA-transferase, no transfer of
[14C]GlcA could be detected. Taken together, these
findings show that K4 polysaccharide polymerization is analogous to
that of chondroitin and that information about biosynthesis of
bacterial polysaccharide, both on protein and genetic levels, could be
useful for understanding of chain elongation reaction in mammalian
systems.
Membranes from E. coli wild-type
05:K4:H4 (13) were obtained as described (15). In short, bacteria were
grown to late logarithmic phase and centrifuged (10,000 × g, 10 min, 4 °C). The sediment was suspended in 50 mM Hepes, pH 7.2, 30 mM magnesium acetate, and
1.5 mM dithioerythritol and centrifuged (10,000 × g, 10 min, 4 °C). The sediment was resuspended in the
same buffer, and the bacteria were disrupted by three passages of the
suspension through a French pressure cell. The homogenate was
centrifuged for 10 min at 10,000 × g to remove large
bacterial fragments and then for 60 min at 100,000 × g
at 4 °C. The membrane pellet was resuspended in the same buffer.
UDP-[14C]GlcA was prepared enzymatically from
D-[14C]glucose (uniformly labeled, 321 mCi/mmol; The Radiochemical Center) as described (16), and
UDP-[1-3H]GalNAc (6.3 Ci/mmol) was obtained from New
England Nuclear. UDP-GlcA, UDP-GalNAc, and bovine liver
-D-glucuronidase (type B-10) were from
Sigma, and chondroitinase AC I (Flavobacterium heparinum) and
-N-acetylhexosaminidase (Jack Bean) were
from Seikagaku Corp., Tokyo, Japan.
Oligosaccharides of the type
(GlcA-GalNAc)n-GlcA-2,5-anhydro-D-talose
(n 8) with alternating sugar residues as exist in
chondroitin were derived from the E. coli K4 polysaccharide (13) (provided by Italfarmaco S.p.A., Milan, Italy). The K4 polysaccharide was first hydrolyzed by acid treatment at pH 1.5 at
80 °C for 30 min to remove the fructose unit from the chondroitin backbone. For the cleavage of the polysaccharide and the separation of
the oligosaccharide substrates, a similar method was used as for the K5
polysaccharide (5). In short, partial N-deacetylation (hydrazinolysis) of the K4 polysaccharide followed by deaminative cleavage at the resulting N-unsubstituted galactosamine
units by treatment with nitrous acid at pH 3.9, gave a mixture of even numbered oligosaccharides. These were separated by gel chromatography on Sephadex G-50, and fractions larger than octasaccharides were recovered and desalted. These oligosaccharides have a GlcA unit at the
nonreducing end and can serve as substrates for the GalNAc-transferase. Substrates for the GlcA-transferase were created by
-glucuronidase digestion of the oligosaccharides. An alternative method used to make
substrates for the GlcA-transferase was also applied: K4 polysaccharide
(10 mg) with fructose units still retained in the polymer were digested
with chondroitinase AC I, 0.75 units in Tris-HAc buffer, pH 7.3, in a
final volume of 2.5 ml for 20 h. The polysaccharide chains were
cleaved at positions lacking fructose units, and oligosaccharides with
an unsaturated uronic acid at the nonreducing end,
-D-gluco-4-enepyranosyluronic
acid-GalNAc-(GlcA(Fru)-GalNAc)n (n
0)
were recovered. Unsaturated uronic acid was removed by treatment with
10 mM mercuric acetate for 30 min. These odd numbered oligosaccharides, still containing the fructose
GalNAc(GlcA(Fru)-GalNAc)n, were desalted on a PD-10
column (Pharmacia Biotech Inc., Uppsala, Sweden) and tested as
substrates for the GlcA-transferase. As a control, the Fru units were
then removed by acid hydrolysis at pH 1.5 at 80 °C for 30 min. The
defructosylated oligosaccharides GalNAc-(GlcA-GalNAc)n were
then desalted on a PD 10 column and used as substrates for the
GlcA-transferase.
Enzymatic transfer of GalNAc and GlcA units were studied by incubating acceptor oligosaccharides (0.5 mM corresponding to 90 µg of GlcA assay, as determined by the carbazole reaction), 2 µCi of UDP-[3H]GalNAc (0.5 mM UDP-GalNAc) or 1.35 µCi of UDP-[14C]GlcA (0.5 mM UDP-GlcA), and 400 µg of bacterial membrane protein in a total volume of 100 µl of 10 mM MnCl2, 10 mM MgCl2, 5 mM CaCl2, 50 mM Hepes, pH 7.2, and 1% Triton X-100. After incubation at 37 °C for 30 min, the reactions were stopped by the addition of 100 µl of 10% trichloroacetic acid, and the mixtures were centrifuged. The supernatants were neutralized with 50 µl of 1 M NaOH, mixed with 0.5 mg of carrier chondroitin sulfate, recentrifuged, and finally applied to gel filtration on a high pressure liquid chromatography (1.6 × 60 cm) Superdex 30 column, (Pharmacia) and eluted with 1 M NaCl, 0.1% Triton X-100, 0.05 M Tris-HCl, pH 8.0, at a rate of 60 ml/h, and fractions of 1 ml were collected. For quantification of the radioactive product, the samples were applied to gel filtration on Sephadex G-25 (1 × 100 cm) (Pharmacia) and eluted with 1 M NaCl, 0.1% Triton X-100, 0.05 M Tris-HCl, pH 8.0, at a rate of 20 ml/h, and fractions of ~1 ml were collected. Appropriate controls without added oligosaccharide acceptors were done in parallel for all incubations.
General MethodsHexuronic acid was determined by the carbazole method (17) using D-glucuronolactone as a standard. The concentrations of the defructosylated K4-derived oligosaccharides were estimated, assuming that GlcA accounted for half of the weight. Protein quantifications were made by BCA reagent (Pierce). Radioactivity was determined by liquid scintillation spectrometry using a Beckman Model LS 6000IC apparatus.
Preparation of Oligosaccharide Acceptors
Substrates for the GalNAc-transferaseThe K4 polysaccharide
was subjected to acidic hydrolysis, which removed the fructose units
from the polymer. These polysaccharide products were then partially
N-deacetylated with hydrazine and cleaved at the resulting
free amino groups with nitrous acid, pH 3.9 (18). The resulting
oligosaccharides, which have a GlcA unit at the nonreducing end, were
separated by gel filtration. Oligosaccharides with 8 monosaccharide
units were collected and used as acceptor oligosaccharides for the
GalNAc-transferase.
Oligosaccharide
substrates for the GlcA-transferase were created in two different ways.
1) The oligosaccharides used for the assay of the GalNAc-transferase
were treated with -D-glucuronidase, which removes the
GlcA unit at the nonreducing end of the oligosaccharide, converting the
oligosaccharides to a GlcA acceptor substrate. 2) In an alternative
approach, where the purpose was to create a substrate in which fructose
units were retained, the fructosylated K4 polysaccharide was digested
with chondroitinase AC. The polysaccharide was partially degraded (Fig.
2), probably due to random loss of fructose units.
Chondroitinase AC degrades chondroitin, but it does not degrade the
fructosylated K4 polysaccharide. The unsaturated hexuronic acid that
appears at the nonreducing end of the oligosaccharides after the
digestion was removed by mercuric acetate, and the remaining fructosylated oligosaccharides of GalNAc(GlcA(Fru)-GalNAc)n type could be tested as acceptors for the GlcA-transferase. These oligosaccharides were then defructosylated by acidic hydrolysis, and
substrates with the structure GalNAc-(GlcA-GalNAc)n were
recovered (defructosylation of the polymer has been investigated in an
earlier paper (13). After digestion with chondroitinase AC, these
defructosylated oligosaccharides appeared as disaccharides on the gel
filtration Superdex 30 column (Fig. 2). On this occasion the enzyme
completely degraded the oligosaccharides, showing that the fructose
units were removed from the chains.
Experimental Conditions
The experimental conditions were chosen to optimize the incorporation of radioactive sugar to oligosaccharide substrates in order to get a sufficient amount of radiolabeled product for further analysis and were based on earlier experiences of chain elongation reactions in E. coli K5 (14) membranes and microsomal membranes from mouse mastocytomas (5).
Because the incorporation of both [3H]GalNAc and [3H]GlcA to their specific substrates when incubated with E. coli K4 membranes solubilized in 1% Triton X-100 increased with incubation times of up to 30 min, this time was selected for the experiments described below.
The effect of enzyme concentration was tested. It was found that in 15-min incubations transfer of both [3H]GalNAc and [3H]GlcA was linear with protein concentrations up to 4 mg/ml (data not shown). The membrane concentration was thus set to 4 mg of membrane protein/ml of incubation volume.
The reactions above were carried out in a buffer containing 5 mM Ca2+, 10 mM Mn2+,
and 10 mM Mg 2+, as used for studies of chain
elongation of the E. coli K5 polysaccharide. The requirement
for metal ions in the E. coli K4 system was tested in both
glycosyltransferase reactions (Fig. 3), and a mixture of
all three metal ions showed the highest activity. These conditions were
thus maintained in the following experiments.
Transfer of GalNAc
Oligosaccharides with 8 monosaccharide units with a GlcA at the
nonreducing end were tested as substrates for the bacterial GalNAc-transferase. Incubations with UDP-[3H]GalNAc,
oligosaccharides, and bacterial K4 membranes solubilized in 1% Triton
X-100 resulted in a radiolabeled product that was not found in control
incubations without the addition of oligosaccharides (Fig.
4A). Furthermore, the labeled product was
completely degraded by chondroitinase AC, and after digestion, the
label eluted as a monosaccharide on the gel filtration chromatogram
(Fig. 4B). The released monosaccharide confirms that a
single monosaccharide unit has been added to the nonreducing end of the
chondroitin oligosaccharide (Fig. 1C). The main part of the
incorporated [3H]GalNAc could also be removed by
treatment with
-N-acetylhexosaminidase (Fig.
4B), an exo enzyme that removes
-linked GalNAc from the nonreducing end (Fig. 1C).
The addition of UDP-GlcA, the other nucleotide sugar, to these assays had no influence on the amount of incorporated radioactivity, and we could only detect the transfer of one GalNAc unit to the exogenous oligosaccharide acceptors (data not shown).
The incubations were also carried out with intact bacterial membranes, without the addition of Triton X-100. No polymerization was detected in any of these incubations in terms of higher incorporation of radioactivity or changed profiles in the gel chromatograms, but transfer of one GalNAc unit could be assayed in the same way as for the solubilized system (data not shown). No differences could be seen between solubilized and intact membranes regarding GalNAc transfer.
Transfer of GlcA
GlcA-transferase activity was tested essentially as described for
GalNAc-transferase, by the incubation of bacterial membrane with
oligosaccharide acceptors with a terminal GalNAc (nonreducing end) and
UDP-[14C]GlcA in 1% Triton X-100. Two different
oligosaccharide substrates were tested (preparation method 2), one
defructosylated chondroitin acceptor and one acceptor containing
fructose units in the chain. A striking difference in the
GlcA-transferase activity was noticed between the two different
oligosaccharide substrates (Fig. 5A). The
GlcA was readily transferred to the defructosylated acceptor, whereas
the fructosylated acceptor did not incorporate GlcA at all (Fig. 5).
From the elution profile of the product derived from the
defructosylated substrate, it can also be shown that a heptasaccharide
is large enough to serve as a substrate for the bacterial enzymes. The
differences in the size of the substrate seen in Fig. 2 and in the
product in Fig. 5 are mainly due to the detection method used. The gel
filtration pattern of the substrate (Fig. 2) illustrates the content of
hexuronic acid, whereas only the radiolabeled nonreducing ends are
detected in the product. However, one cannot exclude the possibility
that the difference might partly be due to GlcA-transferase having a
preference for shorter oligosaccharide substrates. Digestion of the
radiolabeled product by -glucuronidase yielded labeled
monosaccharides (Fig. 5B), demonstrating that the GlcA
unit is added with a
-linkage to the nonreducing end of the
oligosaccharide (Fig. 1D). Furthermore, when the
oligosaccharide products were treated with chondroitinase AC, only
labeled disaccharides could be detected (Fig. 5B), showing that the cleavage site of the enzyme is a disaccharide unit from the
nonreducing end (Fig. 1D).
The GlcA-transferase activity was also tested in incubations with the addition of UDP-GalNAc. No change in incorporated radioactivity could be seen due to this addition, and no polymerization was detected in the gel chromatograms (data not shown). Intact membrane fractions, assayed in the absence of Triton X-100, showed the same GlcA-transferase activity as did solubilized systems (data not shown).
Degradation of 3H-Labeled Product
In order to test endogenous activity of polysaccharide degrading
enzymes, oligosaccharides enzymatically labeled with
[3H]GalNAc were recovered and incubated with fresh
membrane enzymes. The radiolabeled product was recovered after
separation from the UDP-[3H]GalNAc on a G-25 Sephadex gel
filtration column (Fig. 6A). Four fractions,
as indicated in the chromatogram, were pooled, concentrated, desalted
on PD-10, evaporated, and added back to a membrane incubation with the
same buffer used in the previous incubations. After 30 min at 37 °C,
the sample was applied to the same gel filtration column (Fig.
6B). No degradation could be detected.
The mechanisms regarding polysaccharide biosynthesis has earlier been investigated for mammalian heparin/heparan sulfate, chondroitin sulfate, and hyaluronan synthesis. The hyaluronan polysaccharide, which does not form proteoglycans and is synthesized at the cell membrane (19), is claimed to elongate at the reducing end (20). For the hyaluronan producing bacterium Streptococcus pyogenenes, the mechanism for chain elongation has so far not been studied. The biosynthesis of polysialic acid from capsule-producing E. coli bacteria has been intensively investigated, and Rohr and Troy (21) showed that the polymer, although its polymerization only involves one sugar unit, is elongated at the nonreducing end.
The biosynthesis of the K4 polymer has not previously been studied, and it is not clear what primes the polysaccharide or how it is anchored to the membrane, and it is not obvious that it proceeds in the same way as chondroitin biosynthesis. In the biosynthesis of mammalian chondroitin, the polysaccharide is attached to a core protein, and chain elongation of the polysaccharides occurs in the Golgi vesicles. In E. coli bacteria, polymerization of the K5 and K4 polysaccharides occurs at the cytoplasmic membrane, and the polysaccharides are then translocated through the bacterial membranes and transported out of the organism (22). The attachment site of the reducing end of the bacterial polysaccharide is not known. K5 polysaccharides isolated from the bacterial capsule have been shown to contain 2-keto-3-deoxyoctonate and phosphatidic acid at the reducing end (15), but polysaccharides isolated from mutated K5 bacteria deficient in the transportation/translation mechanisms with the polysaccharide appearing inside the cell lack phosphatidyl-2-keto-3-deoxyoctonate (23). It is thus likely that the reducing 2-keto-3-deoxyoctonate is added to the polymer after synthesis during transportation and translocation of the polysaccharide out of the cell.
We have previously studied the glycosyltransferase activities in the bacterial membranes from K5 bacteria and have found that chain elongation of the K5 polysaccharide behaves in much the same way as the mammalian enzymes involved in heparin biosynthesis (14). The sugar units are added one by one from the corresponding UDP sugars to the nonreducing end of the oligosaccharide, and like the mammalian enzyme, the GlcA-transferase also has preferences for specific N-sulfated oligosaccharides (5, 14). E. coli K5 bacteria are thus a good parallel system for studies of the polymerization reaction in heparin/heparan sulfate biosynthesis.
The study of polymerization of the K4 polysaccharide is rather
ambiguous, because the role of the fructose units in the polymer is
obscure. The sugar donor for the fructose units is still unknown, and
it is not clear how fructoses are added to the polymer during biosynthesis. The results of this study show that both the GlcA and the
GalNAc units can be added to exogenous chondroitin oligosaccharides that lack fructose (Figs. 4 and 5). We have also used bacterial-derived chondroitin oligosaccharides in assays with mammalian enzymes, and we
have found that these oligosaccharides work as well as chondroitin
oligosaccharides derived from other sources (data not shown). Similar
oligosaccharide substrates have previously been used in studies of
chondroitin polymerization using enzymes from chick embryo chondrocytes
(7) where these oligosaccharides were shown to be acceptors for the
transfer GalNAc and GlcA units. Bacterial glycosyltransferases seem to
have similar requirements for the acceptor substrates as for
chondroitin sulfate-forming enzymes. The fact that fructosylated
oligosaccharides were not acceptors for the GlcA-transferase (Fig. 5)
strongly indicates that fructose units are added to the polymer either
after the formation of the chondroitin backbone or as a second step
after the addition of GlcA and of the GalNAc units further downstream in the polymer. Digestion of the radiolabeled products with
-D-glucuronidase and
-N-acetylhexosaminidase proves that the GlcA and the
GalNAc monosaccharides were added to the nonreducing end of the
oligosaccharides, because radiolabeled monosaccharides were released
from the enzymatically labeled product (Figs. 4B and
5B). The complete degradation of the
[14C]GlcA- and [3H]GalNAc-labeled products
with chondroitinase AC shows that the products are chondroitin (Figs.
4B and 5B).
These results suggest that bacterial biosynthesis of K4 polysaccharide proceeds in the same way as chondroitin biosynthesis and that the addition of fructose units is not critical for chain elongation. This makes the E. coli K4 bacterium a suitable parallel system for studies of chondroitin biosynthesis in the same way as E. coli K5 polysaccharide biosynthesis is a parallel system for studies of heparin biosynthesis.
We are grateful to Dr. Klaus Jann for support by providing us with E. coli K4 bacteria.