(Received for publication, May 23, 1995; and in revised form, July 13, 1995)
From the
During the course of a study to elucidate the role of
modification of the common polysaccharide-protein linkage structure,
GlcA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser,
in biosynthetic sorting mechanisms of the different sulfated
glycosaminoglycan chains, a novel N-acetylgalactosamine
(GalNAc) transferase was discovered in fetal bovine serum. The enzyme
catalyzed the transfer of [
H]GalNAc from
UDP-[
H]GalNAc to linkage tetrasaccharide and
hexasaccharide serines synthesized chemically and to various regular
oligosaccharides containing terminal D-glucuronic acid (GlcA),
which were prepared from chondroitin and chondroitin sulfate using
testicular hyaluronidase digestion. The labeled products obtained with
the linkage tetra- and hexasaccharide serines and with the
tetrasaccharide (GlcA
1-3GalNAc)
were resistant
to digestion with chondroitinase AC-II and
-N-acetylhexosaminidase but sensitive to
-N-acetylgalactosaminidase digestion, indicating that the
enzyme is an
-N-acetylgalactosaminyltransferase. This
finding is in contrast to that of Rohrmann et al. (Rohrmann,
K., Niemann, R., and Buddecke, E.(1985) Eur. J. Biochem., 148,
463-469), who reported that a corresponding product was
susceptible to digestion with
-N-acetylhexosaminidase.
The presence of a sulfate group at C4 of the penultimate GalNAc or Gal
units markedly inhibited the transfer of GalNAc to the terminal GlcA,
while a sulfate group at C6 of the GalNAc had little effect on the
transfer. Moreover, a slight but significant transfer of
GalNAc was observed to an oligosaccharide
containing terminal 2-O-sulfated GlcA as acceptor, whereas no
incorporation was detected into oligosaccharides containing terminal
unsaturated or 3-O-sulfated GlcA units. These results suggest
that this novel serum enzyme is a UDP-GalNAc:chondro-oligosaccharide
1-3- or
1-4-N-acetylgalactosaminyltransferase. The possibility
of involvement of this enzyme in glycosaminoglycan biosynthesis is
discussed.
Sulfated glycosaminoglycans including heparin/heparan sulfate,
chondroitin sulfate and dermatan sulfate are covalently bound to Ser
residues in the core proteins through the common carbohydrate-protein
linkage structure,
GlcA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
(for reviews, see (1) and (2) ). Heparin/heparan
sulfate is synthesized once GlcNAc is transferred to the common linkage
region, while chondroitin sulfate is formed if GalNAc is first added.
The two distinct transferases, which catalyze the transfer of GlcNAc or
GalNAc, (
)respectively, to the common linkage region are the
key enzymes that determine the type of glycosaminoglycans to be
synthesized. The transferases for the first GlcNAc and GalNAc residues
are thought to be different from the glycosyltransferases catalyzing
the elongation steps, which transfer GlcNAc or GalNAc to the
corresponding repeating disaccharide region. Molecular mechanisms are
unknown, however, for the synthesis of different glycosaminoglycans on
the common linkage region.
We have been investigating the structure
of the linkage region of various glycosaminoglycans to search for
possible structural differences that may determine the characteristics
of the glycosaminoglycan species to be synthesized. These structural
studies revealed that sulfation of C6 on both Gal residues and C4 of
Gal adjacent to GlcA was characteristic of chondroitin
sulfate(3, 4, 5, 6, 7) .
Sulfation of C4 of the Gal residue was also demonstrated in the linkage
region of bovine aorta dermatan sulfate(8) . Sulfated Gal
residues have not been found to date in the linkage region of heparin
or heparan sulfate(9, 10) . In view of these
structural variations in the linkage region, we investigated the
effects of sulfation in the linkage region on the specificity of GalNAc
transferase, which is involved in the biosynthesis of chondroitin
sulfate using chemically synthesized linkage tetrasaccharide serines,
GlcA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
and GlcA
1-3Gal(4-sulfate)
1-3Gal
1-4Xyl
1-O-Ser, and hexasaccharide serines,
GlcA
1-3GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser,
GlcA
1-3GalNAc(4-sulfate)
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser,
and
GlcA
1-3GalNAc(4-sulfate)
1-4GlcA
1-3Gal(4-sulfate)
1-3Gal
1-4Xyl
1-O-Ser,
as acceptor substrates(11, 12, 13) . During
studies to characterize the enzyme products, we unexpectedly found that
the products contained the transferred [
H]GalNAc
-linked to the nonreducing terminal GlcA of the acceptor
substrates employed. Since the occurrence of such an enzyme has not
been previously documented, we investigated its substrate specificity
further using a series of oligosaccharides derived from various
chondroitin sulfate isomers.
Figure 1:
Gel filtration analysis of GalNAc
transferase reaction products obtained with the tetrasaccharide serines
as acceptors. The GalNAc transferase reaction was carried out in the
presence (solid line) or absence (broken lines) of
the linkage tetrasaccharide serine
GlcA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser as an
acceptor as described under ``Experimental Procedures.'' The
sulfated tetrasaccharide serine
GlcA
1-3Gal(4-sulfate)
1-3Gal
1-4Xyl
1-O-Ser
was also tested as an acceptor, the pattern of which was
indistinguishable from that of the control (broken lines).
Effluent fractions were analyzed for
radioactivity.
To assess the
effects of C4 sulfation of the Gal and/or the first GalNAc residue on
the transferase reaction of the second GalNAc residue, a nonsulfated, a
monosulfated, and a disulfated hexasaccharide serine were used as
acceptor substrates for the serum enzyme. As shown in Fig. 2,
the nonsulfated compound
GlcA1-3GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser
was the best substrate (A) followed by the disulfated compound
GlcA
1-3GalNAc(4-sulfate)
1-4GlcA
1-3Gal(4-sulfate)
1-
3Gal
1-4Xyl
1-O-Ser (C), while little
incorporation into the monosulfated compound
GlcA
1-3GalNAc(4-sulfate)
1-
4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (B) was observed. These results indicate that C4 sulfation of
the first GalNAc residue is inhibitory to the transfer of the second
GalNAc residue and that C4 sulfation of the Gal residue also has some
regulatory effects on the reaction. All the above results seemed to
suggest that fetal bovine serum contains GalNAc transferase(s) involved
in the chain initiation and elongation of chondroitin.
Figure 2:
Gel filtration analysis of GalNAc
transferase reaction products obtained with the hexasaccharide serines
as acceptors. The linkage hexasaccharide serines
GlcA1-3GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (A),
GlcA
1-3GalNAc(4-sulfate)
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (B), or GlcA
1-
3GalNAc(4-sulfate)
1-4GlcA
1-3Gal(4-sulfate)
1-3Gal
1-4Xyl
1O-Ser (C) were tested as acceptors for GalNAc transferase in the
serum as described under ``Experimental Procedures.''
Effluent fractions were analyzed for
radioactivity.
Figure 3:
Enzymatic characterization of the anomeric
configuration of the transferred GalNAc using chondroitinase AC-II,
-N-acetylhexosaminidase, or
-N-acetylgalactosaminidase. The GalNAc transferase
reaction was conducted using non-heat-treated serum as an enzyme
source, and the linkage tetrasaccharide serine
GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (A), the linkage hexasaccharide serine
GlcA
1-3GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-O-Ser (B), or polymeric chondroitin (C) was used as an
acceptor. Each product was then isolated by gel filtration and
subjected to treatment with chondroitinase AC-II (a),
-N-acetylhexosaminidase (b), or
-N-acetylgalactosaminidase (c) as described
under ``Experimental
Procedures.''
Figure 4:
Analysis of the anomeric configuration of
the GalNAc transferred onto the regular chondro-oligosaccharides.
Chondro-oligosaccharide (GlcA1-3GalNAc)
(A), (GlcA
1-3GalNAc)
(B), or (GlcA
1-3GalNAc)
(C) was used as an acceptor substrate for the GalNAc
transferase reaction with the non-heat-treated serum. Each product was
then purified and characterized in terms of the anomeric configuration
of the transferred [
H]GalNAc using either
-N-acetylgalactosaminidase or
-N-acetylhexosaminidase as described under
``Experimental Procedures.''
As mentioned above, GalNAc incorporation into acceptor
substrates through an - or a
-linkage could readily be
distinguished by the differential thermostability of these two enzymes.
Accordingly, the products obtained by incubation of the heat-treated
serum with (GlcA
1-3GalNAc)
,
(GlcA
1-3GalNAc)
and
(GlcA
1-3GalNAc)
were tested in terms of their
sensitivity to digestion with
-N-acetylgalactosaminidase.
The [
H]GalNAc-labeled products obtained with all
three acceptors were completely digested with
-N-acetylgalactosaminidase (data not shown). Thus, the
heat treatment was useful to preclude the effect of the
-N-acetylgalactosaminyltransferase activity in bovine
serum.
In this paper, we describe for the first time a unique
-N-acetylgalactosaminyltransferase (
-GalNAc T),
which catalyzes the transfer of an
-GalNAc to the linkage
tetrasaccharide and hexasaccharide serines derived from chondroitin
sulfate proteoglycans and to the even-numbered regular
condro-oligosaccharides. Since no
-GalNAc-terminated structure has
been so far reported in naturally occurring glycosaminoglycan chains, a
question may arise whether the
-GalNAc T in the serum is a minor
curiosity. Based upon the specific activity of the nucleotide sugar
substrate, it is calculated that about 4 pmol of the
-GalNAc T
reaction product was obtained when 1 nmol of the linkage
tetrasaccharide was used as an acceptor substrate, which is comparable
with about 6 pmol of the GalNAc transferase II product obtained with
300 µg (>10 nmol) of chondroitin as an acceptor substrate under
the same incubation conditions described under ``Experimental
Procedures.'' Thus, the
-GalNAc T in the serum has a
comparable activity with that of the GalNAc transferase II and may play
a role in galactosaminoglycan biosynthesis.
Despite the synthesis of
sulfated glycosaminoglycans on the common carbohydrate-protein linkage
region
GlcA1-3Gal
1-3Gal
1-4Xyl
1-O-Ser,
the biosynthetic sorting mechanisms that determine whether
galactosaminoglycans (chondroitin/dermatan sulfate) or
glucosaminoglycans (heparin/heparan sulfate) assemble on the linkage
region remain to be elucidated. We have been working on the hypothesis
that there may be some structural differences in the common linkage
regions among the different glycosaminoglycans. Structural studies of
the linkage oligosaccharides isolated from various glycosaminoglycans
revealed that the 4-sulfated and the 6-sulfated galactose residues
could only be detected in the linkage region of chondroitin and/or
dermatan
sulfate(3, 4, 5, 6, 7, 8) .
Therefore, one can suspect that the sulfate groups inhibit
heparin/heparan sulfate biosynthesis and/or promote the
chondroitin/dermatan sulfate pathway. In this regard, it is interesting
that glycoserines and regular oligosaccharides with 4-sulfate but not
6-sulfate group(s) on the penultimate Gal or GalNAc residue showed
markedly lower acceptor activity for the present novel
-GalNAc T
(see Table 2). Although the relationship between the novel
-GalNAc T and chondroitin and/or dermatan sulfate biosynthesis is
unclear, the observed modulation of the enzyme activity by the sulfated
substrates implies that the enzyme might play an important role in the
regulation of glycosaminoglycan biosynthesis.
Rohrmann et al.(21) reported that the -GalNAc transferase (GalNAc
T-I), which catalyzes the addition of a GalNAc to the linkage
tetrasaccharide, is different from that (GalNAc T-II) which catalyzes
the polymerization of a chondroitin sulfate chain. This conclusion was
mainly based on the differential thermostability of the two activities, i.e. GalNAc T-I was quite thermostable while GalNAc T-II was
not. Interestingly, the present novel
-GalNAc T, which catalyzed
the addition of an
-GalNAc to the linkage tetrasaccharide and
hexasaccharide serines, was also resistant to thermodenaturation (50
°C for 60 min) as described under ``Results,'' while the
serum
-GalNAc transferase(20) , which utilized polymeric
chondroitin as an acceptor substrate, underwent rapid
thermodenaturation. Thus, the thermostability and substrate
specificities of these two enzymes, GalNAc T-I and
-GalNAc T, are
very similar regardless of their allegedly different linkage
specificities. The discrepancy between the two enzymes regarding
anomeric linkage specificity remains to be demonstrated.
Although
the positional assignment of GalNAc attachment to the terminal GlcA was
not accomplished in this study owing to the limited availability of
authentic acceptor substrates, it seems likely that this novel enzyme
may be an 1-4-N-acetylgalactosaminyltransferase.
Very recently, Etchison et al.(24) reported that when
several different cell lines were labeled with
[
H]galactose in the presence of 4-methyl
umbelliferyl
-D-xyloside (Xyl
4MU), a small portion
of the labeled products contained the carbohydrate-protein linkage
region of chondroitin sulfate terminating with an
-GalNAc residue
instead of a typical
-GalNAc residue. Moreover, structural
analysis of the labeled product by
H NMR spectroscopy
revealed that the GalNAc was linked to the non-reducing terminal GlcA
residue in an
1-4 linkage(25) . Presumably, the
-GalNAc T discovered in the present study catalyzes the formation
of a product similar to that terminating with an
1-4-linked
GalNAc unit in cultured cells. Indeed, the nonsulfated linkage
tetrasaccharide serine served as an acceptor only for the
-GalNAc
T, despite the presence of a chondro-oligosaccharide
-N-acetylgalactosaminyltransferase activity in fetal
bovine serum. In addition, it should be noted that when the
tetrasaccharide serine was tested as an acceptor using the solubilized
microsomal fraction from mouse mastocytoma, it served as an acceptor
only for GalNAc transferase but not for GlcNAc transferase, accepting a
GalNAc residue exclusively through an
-linkage.
The
role, if any, of the -GalNAc T in relation to glycosaminoglycan
biosynthesis is unclear. The addition of an
-GalNAc unit to the
tetrasaccharide core of the linkage region of proteoglycans may serve
as a stop signal that precludes further chain elongation. On the other
hand, it may be noted that 4-epimerization of the transferred
-GalNAc to an
-GlcNAc unit may provide a primer for
heparin/heparan sulfate biosynthesis ((24) ; however, see the
GlcNAc transfer to
GlcA
1-3Gal
1-O-naphthalenemethanol described by
Fritz et al.(26) ). Purification, characterization,
and molecular cloning of this transferase should reveal whether it
participates in the formation of glycosaminoglycan chains.