From the Department of Biochemistry, Kobe
Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan and the
§ Departments of Pediatrics and Cell Biology, Harvard
Medical School, Children's Hospital, Boston, Massachusetts 02115
Received for publication, September 12, 2002, and in revised form, November 15, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Drosophila melanogaster genome
contains three putative glucuronyltransferases homologous to human
GlcAT-I and GlcAT-P. These enzymes are predicted to be
Proteoglycans (PGs)1
play an essential role in a variety of biological processes such as
cell-cell adhesion, cell proliferation, and tissue morphogenesis (1,
2). PGs consist of a core protein and sulfated glycosaminoglycan (GAG)
side chains. PGs can be classified into three groups based on the
nature of their GAGs: heparan sulfate (HS)-type PGs, chondroitin
sulfate (CS)-type PGs, and keratan sulfate PGs. There is increasing
evidence that uniquely sulfated domain structures of GAG side chains
are critically involved in various functions of these PGs (3-5), and
defective synthesis of GAGs causes aberrant morphology and even
embryonic lethality during development (6).
In biosynthesis, HS or CS linear chains are differentially assembled on
the common linkage region tetrasaccharide
GlcUA The synthesis of the linkage region is initiated by the addition of a
Xyl residue by xylosyltransferase (19) from UDP-Xyl to specific serine
residues, followed by sequential additions of two Gal residues from
UDP-Gal by galactosyltransferases I (20, 21) and II (22), respectively.
The reaction is completed by a GlcUA transfer from UDP-GlcUA catalyzed
by glucuronyltransferase I (GlcAT-I) (reviewed in Ref. 4). GlcAT-I was
the first member cloned by degenerate PCR using primers derived from
the conserved domain sequences of glucuronyltransferase P
(GlcAT-P) (23) required for the addition of GlcUA to the terminal
Gal The presence of GAGs, including HS, CS, and nonsulfated chondroitin,
have been demonstrated in the invertebrate organisms Drosophila
melanogaster and Caenorhabditis elegans (29, 30). Furthermore, use of a conventional linkage region tetrasaccharide sequence was recently established for these invertebrate GAG chains (31), suggesting that their fundamental structures and biosynthetic mechanisms are similar to the mammalian GAG chains. A single syndecan and two glypicans, all of which are HSPGs whose mammalian homologs function as a receptor or co-receptor for extracellular effector molecules, have been identified in Drosophila (32, 33).
Accumulating evidence suggests that these GAGs play critical roles in
development through the mediation of signaling processes of various
morphogens such as decapentaplegic, Hedgehog,
fibroblast growth factor, and Wingless (6). Also in C. elegans, defects in galactosyltransferase I (21, 22) and GlcAT-I
(34), which are involved in the synthesis of the linkage region
tetrasaccharide, cause morphological abnormality such as squashed
vulva. Drosophila mutants defective in the synthesis of
GAGs, especially HS chains (35-38) or a core protein of glypican HS-PG
(33), also show severe morphological abnormality. The HNK-1
glycoepitope has been demonstrated in Drosophila (40), and its critical roles in the fasciculation and pathfinding in the
developing nerve were recently reported for embryonic zebrafish (39). Although targeted deletion of the GlcAT-P gene in mice was
recently reported to cause deficiency of the HNK-1 antigen and
attenuation of the brain function (41), no relevant
Drosophila mutant has been reported despite a large panel of
existing mutants (40). Here, we report our identification of three
GlcAT-I homologs in the D. melanogaster genome and our
biochemical characterization of the expressed enzymes. All three
proteins appear to be involved in the synthesis of the GAG-protein
linkage region. However, two enzymes have broader specificity toward
the linkage region, the HNK-1 carbohydrate epitope and glycosphingolipids.
Materials--
UDP-[U-14C]GlcUA (285.2 mCi/mmol) was purchased from PerkinElmer Life Sciences. Unlabeled
UDP-GlcUA, Gal Data Base Search for GlcAT-I Homologs--
The amino acid
sequence of human GlcAT-I was used for BLASTP search of
GenBankTM and the homepage of the Berkeley
Drosophila Genome Project (www.fruitfly. org), which
identified three putative glucuronyltransferases. Their
GenBankTM accession numbers are CAA21824, AAF50082 (the
CG6207 gene product), and AAF52795 (the CG3881 gene product) in the
order of higher homology to human GlcAT-I, which were tentatively named
CAA21824, CG6207, and CG3881. The corresponding Drosophila EST clones were purchased from Research Genetics, Inc. (Huntsville, AL).
Construction of Soluble Forms of the Three Putative
Glucuronyltransferases--
The mammalian expression vector pEF-BOS/IP
was generated as described previously (10). The truncated form of
CAA21824, lacking the amino-terminal 27 amino acids, was amplified by
PCR with the cDNA from the Drosophila EST clone GH05057
using a 5'-primer (5'-CGGGATCCAACGGGAAGCGCACATGCC-3') containing an in-frame BamHI site and a 3'-primer
(5'-CGGGATCCTTAGACCTCCATGCCGCC-3') containing a
BamHI site just after the stop codon. The soluble form of
CG3881, lacking the first 61 amino acids, was amplified with the
obtained cDNA from the GH22332 clone as a template using a
5'-primer (5'-ATGGATCCGTTCACATATGCAGCGAGAGTT-3') containing an in-frame BamHI site and a 3'-primer
(5'-ATGGATCCGTGGATTCGAGATTTGTTTTAG-3') containing a
BamHI site located 59 base pairs downstream of the stop
codon. The soluble form of CG6207, lacking the first 59 amino acids at
the amino-terminal end, was amplified with the plasmids from RE26967
using a 5'-primer (5'-CGGGATCCTTCGCCGCCAGCGAGGTTGT-3') containing an in-frame BamHI site and a 3'-primer
(5'-CGGGATCCACGGCGAGCTCCTTGCTAAT-3') containing a
BamHI site at 28 base pairs downstream of the stop codon.
All the polymerase chain reactions were carried out using Pfu polymerase (Promega). The amplified fragments were
digested with BamHI and cloned into the BamHI
site of pEF-BOS/IP, resulting in fusion proteins with a cleavable
insulin signal sequence for secretion and protein A for purification of
the expressed fusion proteins.
Expression of the Soluble Forms of Three Glucuronyltransferases
and Enzyme Assays--
The respective expression vector (6.7 µg) was
introduced into COS-1 cells using FuGENETM 6 (Roche
Molecular Biochemicals) according to the provided instructions. The
transfected cells were incubated at 30 °C for 48 h, and a 1-ml
aliquot of the culture medium was used for incubation with 10 µl of
an IgG-Sepharose suspension for 2 h at 4 °C. The enzyme-bound beads were recovered by centrifugation, washed with and suspended in
the assay buffer for enzyme assay. A glucuronyltransferase assay
mixture contained 10 µl of the resuspended IgG-Sepharose beads, 50 mM MES-NaOH buffer, pH 6.5, 171 µM ATP, 10 mM MnCl2, 14.3 µM
UDP-[14C]GlcUA (1.1 × 105 dpm), and 1 nmol of each acceptor in a total volume of 30 µl. The tested
acceptors were Gal Characterization of the Glucuronyl Linkages Formed by
Glucuronyltransferases--
The reaction products obtained with two
different acceptors, Gal Determination of Expression Levels of Three
Glucuronyltransferases by Semiquantitative RT-PCR--
A
Drosophila Rapid-ScanTM gene expression panel
purchased from OriGene Technologies, Inc. (Rockville, MD) provides a
semiquantitative method for determining the Drosophila gene
expression level. The panel contained first strand cDNAs from
different Drosophila tissues and developmental stages, which
have been normalized against the RP49 transcript, the mRNA encoding
a constitutively expressed ribosomal protein. The following primer
pairs for each gene were used to amplify DNA fragments of 639, 497, and
715 bp, respectively: a 5'-primer (5'-CGGTTTTTACCAACTGCC-3') and a
3'-primer (5'-GAATGCCTCCTACCGTGAT-3') for CAA21824, a 5'-primer
(5'-GTTGTACGATTCCTGGACTC-3') and a 3'-primer (5'-AGCGAGCAGTGGATTCGAGA-3') for CG3881, and a 5'-primer
(5'-ATGAAGGGCGGCAACTACAC-3') and a 3'-primer
(5'-AGAACTTGACGCTCACTGC-3') for CG6207. PCR was carried
out, with Taq polymerase (Promega), under the conditions described in the provided protocol, pre-denaturing at 94 °C for 3 min, and 35 cycles of 95 °C for 30 s, 50 °C for 30 s,
and 72 °C for 60 s.
Identification of Drosophila Homologs of Human GlcAT-I--
BLASTP
analysis of the D. melanogaster genome, using the amino acid
sequence of human GlcAT-I, identified three putative
glucuronyltransferases (CAA21824, CG6207, and CG3881). These three
predicted proteins consisted of 313, 479, and 443 amino acid residues,
respectively. To determine whether they are expressed in
Drosophila, RT-PCR was carried out using
Drosophila embryonic mRNA (BD Biosciences) as a
template, and the individual amplified fragments of putative full-lengths were cloned into a pGEM®-T Easy vector
(Promega) for sequence analysis. The sequencing results suggested that
all three glucuronyltransferase-like proteins are expressed in
Drosophila; however, the GenBankTM sequences
include an intronic sequence. To obtain more detailed sequence
information for the mature mRNAs, in particular for 5'- and
3'-untranslated regions, a panel of corresponding EST clones were
purchased and their sequences were determined. Using the combined
sequence information, mRNA sequences without intron sequences were
generated, resulting in proteins with slightly reduced sizes. A
Kyte-Doolittle hydropathy analysis suggested that all of these proteins
have type II transmembrane topology typical of endoplasmic reticulum-Golgi resident glycosyltransferases. The three
putative glucuronyltransferases of the corrected sequences derived from CAA21824, CG3881, and CG6207 were designated DmGlcAT-I, DmGlcAT-BSI, and DmGlcAT-BSII, respectively, based on their homology to mammalian GlcAT-I and the broad specificity for the
acceptor recognition of the others (see below).
The cDNA of DmGlcAT-I, derived from EST clone GH05057, consisted of
an open reading frame encoding 306 amino acid residues with an
NH2-terminal cytoplasmic tail of 9 residues, a putative transmembrane domain of 17 residues, and one potential
N-glycosylation site (Fig. 1).
The cDNA of DmGlcAT-BSI derived from EST clone GH22332 was
predicted to encode 366 residues with an NH2-terminal cytoplasmic tail of 9 amino acid residues and a putative transmembrane domain of 22 amino acid residues and three potential
N-glycosylation sites (Fig.
2). The DmGlcAT-BSII cDNA was
obtained from EST clone RE26967 and encoded 316 amino acid residues
with a relatively long NH2-terminal cytoplasmic tail of 35 residues, a putative transmembrane domain of 15 residues, and two
potential N-glycosylation sites, although another potential
initiation codon predicted a 9-residue cytoplasmic tail (Fig.
3).
Using the ClustalW alignment method from the DNA Data Bank of Japan
(DDBJ) homepage, multiple sequence alignment for the reported glucuronyltransferases was performed. Fig.
4A shows the aligned sequences
of the three Drosophila proteins and the
glucuronyltransferases from human, rat, and C. elegans.
SQV-8 is orthologous to C. elegans GlcAT-I and possesses a
functional enzyme activity (34, 45). DmGlcAT-I, DmGlcAT-BSI, and
DmGlcAT-BSII showed 36-40, 27-30, and 28-30% identities,
respectively, to mammalian glucuronyltransferases and 24-35% amino
acid identity to one another. Interestingly, DmGlcAT-I showed much
higher homology (39%) to SQV-8 (C. elegans GlcAT-I) than
the other two homologs: CG6207 (27%) and CG3881 (25%), suggesting
that DmGlcAT-I might be a GlcAT-I ortholog in Drosophila. This idea was also supported by the phylogenetic
tree generated using the whole amino acid sequences of these proteins, in which DmGlcAT-I was grouped together with human GlcAT-I, whereas DmGlcAT-BSI and DmGlcAT-BSII were separately categorized (Fig. 4B).
All Three Proteins Possess Glucuronyltransferase
Activities--
To determine whether the three predicted proteins are
functional glucuronyltransferases, their putative catalytic domains were expressed as chimeric proteins fused to an IgG-binding domain of
bacterial protein A. The secreted proteins were purified with IgG-Sepharose beads to eliminate endogenous glycosyltransferases, and
the enzyme-bound beads were subjected to glucuronyltransferase assays.
When the linkage trisaccharide Gal
DmGlcAT-BSI and DmGlcAT-BSII transferred GlcUA from UDP-GlcUA not only
to Gal Characterization of the Anomeric Configuration of the Glucuronyl
Linkages Produced by Drosophila Glucuronyltransferase
Reactions--
The anomeric configuration of the GlcUA residues in the
transferase reaction products was characterized by Expression Profile of Three Drosophila
Glucuronyltransferases--
To investigate spatiotemporal expression
of DmGlcAT-I, DmGlcAT-BSI, and DmGlcAT-BSII, their expression
profile was determined by RT-PCR using a commercial cDNA panel, in
which first strand cDNAs from different tissues and developmental
stages of Drosophila were serially diluted over a 4-log
range with the highest concentration (×1000) corresponding to 1 ng of
cDNA in a PCR-ready tube. Although this method is semiquantitative,
it was possible to grossly compare expression patterns of three
Drosophila genes. The results obtained from the highest
cDNA concentration only are displayed in Fig. 7 because the amplified bands observed
below this concentration were faint for all three genes. The
transcripts of all three glucuronyltransferases were ubiquitously
expressed; a measurable amount of expression was detected even in the
earliest embryonic stage (0-4-h embryo) for all enzymes. In
particular, DmGlcAT-BSI and DmGlcAT-BSII transcripts were expressed
more strongly as compared with that of DmGlcAT-I, which may suggest
their relative importance during embryonic development of
Drosophila.
In this study, we have identified three putative
glucuronyltransferases in the D. melanogaster genome and
demonstrated that they are catalytically active enzymes likely involved
in the biosynthesis of various glycoconjugates. DmGlcAT-I showed strict
acceptor substrate specificity toward the trisaccharide
Gal In contrast to DmGlcAT-I, DmGlcAT-BSI and DmGlcAT-BSII showed broad
acceptor specificity, utilizing all tested acceptor substrates (Table
I). Interestingly, both enzymes were potently active toward the linkage
region trisaccharide Gal DmGlcAT-BSI and DmGlcAT-BSII transferred GlcUA to Gal The recently solved crystal structure of human GlcAT-I revealed the
essential amino acid residues in the active site (46, 47), which are
well conserved in Drosophila glucuronyltransferases. The
five amino acid residues (marked by closed circles)
corresponding to Asp194, Arg156,
Arg161, Asp256, and His308 in human
GlcAT-I, which interact with the donor GlcUA, are completely conserved
in all aligned sequences (Fig. 4A). Also important are the
four residues corresponding to Glu227, Arg247,
Asp252, and Glu281 of human GlcAT-I, which
interact with the acceptor sugar (marked by arrows) (46).
Among these, completely conserved are the Glu at the corresponding
position of Glu281 of human GlcAT-I (marked by closed
square), which acts as a catalytic base (46), as well as two other
residues corresponding to Arg247 and Asp252,
which are involved in recognition of the non-reducing terminal Gal of
Gal All three Drosophila glucuronyltransferases exhibited
evident transferase activity toward the linkage region trisaccharide, indicating that GAG biosynthesis in Drosophila appear to be
regulated at the level of the linkage region differently from those of
mammalian systems. However, it remains to be investigated how these
three enzymes play their individual roles in terms of the synthesis of
the GAG-protein linkage region of different types of GAG chains or
different types of PGs in various tissues and during development. Comprehensive and thorough analysis of glycoconjugates of mutants deficient in each one of these enzymes or double mutants would also be
required for better understanding of functional roles of these enzymes
in GAG biosynthesis.
1,3-glucuronyltransferases involved in the synthesis of the
glycosaminoglycan (GAG)-protein linkage region of proteoglycans and the
HNK-1 carbohydrate epitope of glycoproteins, respectively. The genes
encode active enzymes, which we have designated DmGlcAT-I, DmGlcAT-BSI,
and DmGlcAT-BSII (where BS stands for "broad
specificity"). Protein A-tagged truncated soluble forms
of all three enzymes efficiently transfer GlcUA from UDP-GlcUA to the
linkage region trisaccharide Gal
1-3Gal
1-4Xyl. Strikingly,
DmGlcAT-I has specificity for Gal
1-3Gal
1-4Xyl, whereas DmGlcAT-BSI and DmGlcAT-BSII act on a wide array of substrates with
non-reducing terminal
1,3- and
1,4-linked Gal residues. Their
highest activities are obtained with asialoorosomucoid with a
terminal Gal
1-4GlcNAc sequence, indicating their possible
involvement in the synthesis of the HNK-1 epitope in addition to the
GAG-protein linkage region. Gal
1-3GlcNAc and Gal
1-3GalNAc,
disaccharide structures widely found in N- and
O-glycans of glycoproteins and glycolipids, also serve as
acceptors for DmGlcAT-BSI and -BSII. Transcripts of all three enzymes
are ubiquitously expressed throughout the developmental stages and in
adult tissues of Drosophila. Thus, all three
glucuronyltransferases are likely involved in the synthesis of the
GAG-protein linkage region in Drosophila, and DmGlcAT-BSI and -BSII appear to be involved in various GlcUA transfer reactions for
the synthesis of proteoglycans, glycoproteins, and glycolipids. This
activity distinguishes these glucuronyltransferases from their
mammalian homologs GlcAT-P and GlcAT-D (or -S). Sequence alignment of
the Drosophila glucuronyltransferases with homologs in
human, rat, and Caenorhabditis elegans demonstrates the
conservation of a majority of the critical amino acid residues in the
active sites of the three Drosophila enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3Gal
1-3Gal
1-4Xyl
1-O-, which takes
place on a specific serine residue in a given core protein (7).
Transfer of either
1,4-GlcNAc or
1,4-GalNAc to the
tetrasaccharide linkage region terminus mediated by the GAG-specific
hexosaminyltransferases determines and initiates HS (8-10) or CS
assembly, respectively (Refs. 11-13; reviewed in Ref. 14). HS and CS
chains are polymerized as relatively simple structures composed of
repeating disaccharide units, -4GlcNAc
1-4GlcUA
1- and
-3GalNAc
1-4GlcUA
1-, by HS polymerases (15, 16) or chondroitin synthase (17), respectively. These structures are further modified by
the cooperative action of specific epimerases and sulfotransferases to
produce mature, biologically active chains (reviewed in Ref. 18).
1-4GlcNAc- sequence of glycoprotein oligosaccharides, producing
the HNK-1 carbohydrate epitope precursor sequence
GlcUA
1-3Gal
1-4GlcNAc-. The trisaccharide sequence then serves
as the acceptor site for the 3-O-sulfotransferase (24, 25)
that subsequently generates the HNK-1 epitope. Both GlcAT-I and GlcAT-P
are
1,3-glucuronyltransferases and share a high degree of homology.
They form a unique gene family that apparently plays several critical
roles during development. GlcAT-I is constitutively expressed in all
tissues, being consistent with the wide distribution and a huge array
of biological functions of GAGs (3). In contrast, GlcAT-P is
specifically expressed in neural cells (26), possibly reflecting a
specific role in neural development. Although it seems that in mammals,
GlcAT-I and GlcAT-P play specific roles in the synthesis of PGs and
glycoproteins, respectively, overexpression of GlcAT-I can produce the
HNK-1 epitope in mammalian cells (27, 28). Therefore, it remains obscure whether there is some overlap between their specificities.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3GlcNAc, and Gal
1-3GalNAc were obtained from
Sigma. N-Acetyllactosamine and lactose were purchased from
Seikagaku Corp. (Tokyo, Japan). Gal
1-3Gal
1-4Xyl was a gift from
Dr. Nancy B. Schwartz (University of Chicago, Chicago, IL).
Asialoorosomucoid was prepared as described previously (42).
-Glucuronidase (EC 3.2.1.31), homogeneously purified from
Ampullaria (freshwater apple shell) hepatopancreas, was
obtained from Tokyo Zouki Chemical Co. (Tokyo, Japan). A
SuperdexTM Peptide HR10/30 column and IgG-Sepharose were
obtained from Amersham Biosciences.
1-3Gal
1-4Xyl, N-acetyllactosamine, lactose, Gal
1-3GlcNAc, Gal
1-3GalNAc, and asialoorosomucoid
(Gal
1-4GlcNAc-R, where R represents the remainder of the
N-linked oligosaccharide chain). All the reaction mixtures
were incubated at 25 or 37 °C. The radiolabeled product obtained
with asialoorosomucoid was separated from the donor
UDP-[14C]GlcUA by gel filtration on a
SuperdexTM Peptide column. The isolation of the
14C-labeled products obtained with the other acceptor
substrates was carried out by applying the reaction mixtures on to
Pasteur pipette columns packed with Dowex 1-X8 (a
PO
1-3Gal
1-4Xyl and asialoorosomucoid,
were characterized by
-glucuronidase digestion. Isolation of the
products from the reaction mixtures was carried out as described above.
The individual isolated products were digested with 20 milliunits of
Ampullaria
-glucuronidase in a total volume of 20 µl of
50 mM sodium citrate buffer, pH 4.5, at 37 °C overnight
and then analyzed by gel-filtration chromatography on a
SuperdexTM Peptide column.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (51K):
[in a new window]
Fig. 1.
Structure of DmGlcAT-I
cDNA. The complete nucleotide sequence of
DmGlcAT-I cDNA and the predicted amino acid sequence of
DmGlcAT-I are shown. The putative transmembrane domain and
polyadenylation signal are underlined. A potential
N-glycosylation site is marked by asterisk.
View larger version (61K):
[in a new window]
Fig. 2.
Structure of DmGlcAT-BSI
cDNA. The complete nucleotide sequence of
DmGlcAT-BSI cDNA and the predicted amino acid sequence
of DmGlcAT-BSI are shown. The putative transmembrane domain and
polyadenylation signal are underlined. Three potential
N-glycosylation sites are marked by
asterisks.
View larger version (76K):
[in a new window]
Fig. 3.
Structure of DmGlcAT-BSII
cDNA. The complete nucleotide sequence of
DmGlcAT-BSII cDNA and the predicted amino acid sequence
of DmGlcAT-BSII are shown. The presumptive two start codons, putative
transmembrane domain, and polyadenylation signal are
underlined. Two potential N-glycosylation sites
are marked by asterisks.
View larger version (58K):
[in a new window]
Fig. 4.
Comparison of three putative
Drosophila glucuronyltransferases with the reported
glucuronyltransferases involved in synthesis of either the GAG-protein
linkage region or the HNK-1 carbohydrate epitope. A,
multiple sequence alignment of DmGlcAT-I, DmGlcAT-BSI and DmGlcAT-BSII
with the glucuronyltransferases reported to date among human (23, 26),
rat (43, 44), and C. elegans (34, 45). Introduced gaps are
shown by hyphens. Closed boxes
indicate identical amino acids in all proteins. Amino acids identical
in more than four proteins are shaded. The conserved
DXD motifs are underlined. The amino acid
residues that interact with uracil and GlcUA, are indicated by
asterisks and closed circles, respectively (46,
47). Arrows indicate the amino acids interacting with
non-reducing terminal Gal of the acceptor trisaccharide
Gal 1-3Gal
1-4Xyl (46). Glu marked with closed square
is a catalytic base (46). Arg marked by open circle
interacts with a
-phosphate of UDP (47). Arg marked by
triangle interacts with 6-hydroxyl group of the terminal Gal
of the acceptor (46). B, the phylogenetic tree based on the
above alignment. The multiple sequence alignment and the phylogenetic
tree were produced using ClustalW. hGlcAT-I, human GlcAT-I;
hGlcAT-P, human GlcAT-P; rGlcAT-D, rat
GlcAT-D.
1-3Gal
1-4Xyl, an authentic
substrate for GlcAT-I, was used as an acceptor and UDP-GlcUA as a donor
substrate, significant GlcUA transferase activities were detected for
all three proteins (Table I). Although
DmGlcAT-I showed a relatively low efficiency compared with DmGlcAT-BSI
and DmGlcAT-BSII, it did not utilize any other acceptor substrates, showing strict acceptor specificity toward Gal
1-3Gal
1-4Xyl. In
this regard, DmGlcAT-I resembled human GlcAT-I. Interestingly, unlike
the other two enzymes, DmGlcAT-I showed no detectable GlcAT-I activity
at 37 °C. Accordingly, although DmGlcAT-BSI and DmGlcAT-BSII showed
their highest activities at 37 °C, all the enzyme activities were
measured at 25 °C for comparison, which would be closer to the body
temperature of Drosophila.
Comparison of the acceptor substrate specificity of truncated forms of
the three Drosophila glucuronyltransferases
1-3Gal
1-4Xyl but also to N-acetyllactosamine (Gal
1-4GlcNAc) and asioloorosomucoid (Gal
1-4GlcNAc-R) (Table I). These reactions are considered to represent the
glucuronyltransferase reaction involved in the synthesis of the
nonsulfated precursor for the 3-O-sulfated GlcUA-containing
HNK-1 carbohydrate epitope on glycoproteins (26). In addition,
DmGlcAT-BSI and DmGlcAT-BSII could utilize Gal
1-3GalNAc,
Gal
1-3GlcNAc, and lactose (Gal
1-4Glc), with the highest
activity toward Gal
1-3GalNAc. The trisaccharide sequence
GlcUA
1-3Gal
1-3GalNAc of one of these products has been reported
for glycosphingolipids in three dipterans including D. melanogaster (48-50), indicating that the detected
glucuronyltransferase activity is involved in the synthesis of acidic
glycosphingolipids as well. Trisaccharide sequences of the reaction
products, GlcUA
1-3Gal
1-3GlcNAc and GlcUA
1-3Gal
1-4Glc,
have not been reported so far. These findings indicate that
DmGlcAT-BSI and DmGlcAT-BSII have a broad spectrum in their
substrate specificity, which clearly distinguishes these enzymes from
DmGlcAT-I. It should be noted that none of the three
glucuronyltransferases transferred GlcUA from UDP-GlcUA to either
N-acetylheparosan oligosaccharides with terminal GlcNAc (16)
or chondroitin (data not shown), excluding the possibility that they
might be involved in the synthesis of the repeating disaccharide
regions of HS or CS chains. The medium recovered from the COS-1 cells
transfected with the empty vector showed no glucuronyltransferase
activity, confirming that all the observed activities are attributable
to the expressed recombinant enzymes.
-glucuronidase digestion. The GlcAT-I reaction products obtained using
Gal
1-3Gal
1-4Xyl as an acceptor and each of the three
glucuronyltransferases and the GlcAT-P reaction products obtained using
asialoorosomucoid as an acceptor and each of the two
glucuronyltransferases (DmGlcAT-BSI and DmGlcAT-BSII) were separated
from the unutilized UDP-[14C]GlcUA as described under
"Experimental Procedures." Ten pmol each of the reaction product
was digested with Ampullaria
-glucuronidase and analyzed
by gel filtration chromatography on a SuperdexTM Peptide
column. The radiolabels of both GlcAT-I and GlcAT-P reaction products
were completely released by
-glucuronidase digestion, resulting in
the shift of the radioactive peak to the free GlcUA position (Figs.
5 and 6),
suggesting that the GlcUA residues had been transferred to the
non-reducing termini of the respective acceptor substrates by all three
enzymes through
-configuration.
View larger version (22K):
[in a new window]
Fig. 5.
Characterization of GlcAT-I reaction products
obtained with
Gal 1-3Gal
1-4Xyl as
acceptor and DmGlcAT-I, DmGlcAT-BSI, and DmGlcAT-BSII as enzyme
proteins. Each GlcAT-I reaction product obtained with
Gal
1-3Gal
1-4Xyl by DmGlcAT-I (A), DmGlcAT-BSI
(B), and DmGlcAT-BSII (C) was digested by
-glucuronidase (closed square), and each digest or an
undigested sample (open circle) was gel-filtrated on a
SuperdexTM Peptide HR 10/30 column as described under
"Experimental Procedures." The separated fractions (0.4 ml each)
were measured for radioactivity. An arrow indicates the
elution position of free GlcUA, and a closed
arrowhead and an open arrowhead show
the void volume and the total volume of the column, respectively.
View larger version (21K):
[in a new window]
Fig. 6.
Characterization of GlcAT-P reaction products
obtained with asioloorosomucoid as acceptor and DmGlcAT-BSI and
DmGlcAT-BSII as enzyme proteins. GlcAT-P reaction product produced
by either DmGlcAT-BSI (A) or DmGlcAT-BSII (B)
using asioloorosomucoid as an acceptor was digested with
-glucuronidase (closed square), and each digest or an
undigested sample (open circle) was analyzed by gel
filtration chromatography using a SuperdexTM Peptide HR
10/30 column as described under "Experimental Procedures." The
separated fractions (0.4 ml each) were quantified for radioactivity in
a liquid scintillation counter. An arrow indicates the
elution position of free GlcUA, and a closed
arrowhead and an open arrowhead show
the void volume and the total volume of the column, respectively.
View larger version (23K):
[in a new window]
Fig. 7.
Expression profiles of three
Drosophila glucuronyltransferases. RT-PCR
analysis was performed using first cDNAs prepared from different
developmental stages and different tissues of Drosophila
under the conditions described under "Experimental Procedures."
Lane 1, 0-4-h embryos; lane 2, 4-8-h embryos;
lane 3, 8-12-h embryos; lane 4, 12-24-h
embryos; lane 5, first instar; lane 6, second
instar; lane 7, third instar; lane 8, pupae;
lane 9, male head; lane 10, female head;
lane 11, male body; lane 12, female body. The
results shown were obtained with a 1000-fold concentration (~1
ng).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3Gal
1-4Xyl derived from the protein linkage region of
GAGs, as in the case of human GlcAT-I (27). It is noteworthy that
SQV-8, GlcAT-I ortholog in C. elegans, shows significant
activities toward several artificial substrates representing sugar
sequences found in glycoproteins (34). These different substrate
specificities of the two GlcAT-I orthologs suggest that DmGlcAT-I may
have diverged earlier than SQV-8 in the evolutionary tree, which can
also be expected from the phylogenetic tree shown in Fig.
4B.
1-3Gal
1-4Xyl, showing ~2-fold greater
activity than DmGlcAT-I. However, their highest activity was
obtained with asialoorosomucoid (Table I), an acceptor substrate for
GlcAT-P involved in the synthesis of the HNK-1 carbohydrate epitope
precursor oligosaccharide on glycoproteins. The HNK-1 epitope is
exclusively found on membrane-bound cell recognition molecules in
nervous tissues of vertebrates and is involved in cell-cell and
cell-matrix adhesion. Recent studies showed that the HNK-1 carbohydrate
antigen is also present in insects including D. melanogaster
(40, 49-51). As in the case of Drosophila, two kinds of
HNK-1 epitope-synthesizing glucuronyltransferases have been described
for mammals: GlcAT-P and GlcAT-D (or GlcAT-S), both of which were
initially thought to be specific for glycoprotein oligosaccharides (26,
43), but were subsequently shown to act on glycolipids such as
paragloboside as well (41, 44). However, if compared with DmGlcAT-BSI
and DmGlcAT-BSII, these mammalian enzymes with dual acceptor
specificity still have the rigid substrate specificity in that neither
utilizes lactose (27), and GlcAT-P does not act on the linkage region
trisaccharide Gal
1-3Gal
1-4Xyl (27). In contrast, DmGlcAT-BSI
and DmGlcAT-BSII exhibited broader acceptor specificities than these
mammalian enzymes as discussed below, utilizing all tested acceptor
oligosaccharide substrates containing terminal
1,3- and
1,4-linked Gal residues, which are found in glycoproteins,
glycolipids, or PGs (Table I).
1-3GalNAc
(Table I), producing the terminal sequence GlcUA
1-3Gal
1-3GalNAc of the acidic glycosphingolipids reported for dipterans including Drosophila (48-50). Interestingly, such glycosphingolipids
were shown in larvae and in the head of adult females of
Drosophila, where the carbohydrate epitope was detected in
non-neural as well as neural tissues (40). The same trisaccharide
sequence has been reported for mucin-type O-glycans in
C. elegans as well (51), although no such glycosphingolipids
or mucin-type oligosaccharides that contain GlcUA have been reported
for vertebrates. The trisaccharide sequences,
GlcUA
1-3Gal
1-3GlcNAc and GlcUA
1-3Gal
1-4Glc, of the
glucuronyltransfer reaction products observed in this study (Table I),
have not previously been reported in other organisms. However, a
glucuronyltransfer reaction to lactose was previously reported for
cell-free extracts from embryonic chick cartilage (52), which may
indicate the existence of such glycoconjugates in vertebrates. Although
GlcUA
1-3Gal
1-3GlcNAc structure has not been demonstrated for
any organisms, the revealed unequivocal enzyme activity toward
Gal
1-3GlcNAc, which is a part of widespread type 1 glycan chains,
may suggest the existence of its glucuronylated products at least in
Drosophila. Considering the catalytic activities toward
various types of acceptor oligosaccharides and ubiquitous expression of
DmGlcAT-BSI and DmGlcAT-BSII throughout the developmental stages and in
adult tissues, it is speculated that their catalytic activities have
pivotal roles in forming diverse GlcUA-containing glycoconjugates
including GAGs, N- and O-linked glycoproteins, and glycolipids in Drosophila, and are involved in neuronal
events. Comprehensive and vigorous analysis of chemical structure
of various types of glycoconjugates in Drosophila
is required to understand in depth biological functions of
these glycosyltransferases.
1-3Gal
1-4Xyl (46, 47). The catalytic Glu residue activates
the C3 hydroxyl group of the acceptor sugar (Gal) for nucleophilic
attack at C1 by deprotonation. Thus, the major amino acids in the
active site have been maintained irrespective of species. Taken
together, the findings in the present study strongly suggest that the
GlcUA transferase reaction of the three Drosophila glucuronyltransferases proceeds in the same inversion mechanism (47)
among all the enzymes. Three amino acid residues in the conserved
domains have been placed by other residues in DmGlcAT-BSI and
DmGlcAT-BSII. The Arg310 of human GlcAT-I (marked by
triangle), which interacts with a
-phosphate of UDP (47),
has been replaced by Gln in DmGlcAT-BSI and DmGlcAT-BSII. However, the
other residues interacting directly (Asp252) or indirectly
(Asp194, Asp196, and Asn197)
through manganese ion and a water molecule with the
-phosphate are
well conserved. In the acceptor-interacting regions of DmGlcAT-BSI and
DmGlcAT-BSII, both enzymes had Ser in place of conserved Glu (Glu227 in human GlcAT-I). Because this Glu (marked by
arrow and open circle) appears to interact with
6-hydroxyl of the terminal Gal (46), it is speculated that this change
from Glu to Ser might be responsible for their unique substrate
tolerance. In COOH-terminal region, one Cys residue is conserved in all
enzymes (Fig. 4A, double-underlined), and
replacement of this Cys with Ala totally inactivates human GlcAT-I;
therefore, this amino acid residue has been suggested to exist in the
catalytic site (53). Because, however, crystallographic data indicate
that this Cys residue does not exist in the vicinity of the catalytic
sites (46, 47), the role of this Cys remains controversial.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from Human Frontier Science Program, the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and by Grant-in-aid for Scientific Research C 14572086 (to H. K.) and Grant-in-aid for Scientific Research on Priority Areas 14082207 (to K. S.) from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB080695 (DmGlcAT-I), AB080696 (DmGlcAT-BSI), and AB080697 (DmGlcAT-BSII).
To whom correspondence should be addressed: Dept. of
Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. Tel.:
81-78-441-7570; Fax: 81-78-441-7569; E-mail:
k-sugar@kobepharma-u.ac.jp.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M209344200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PG, proteoglycan; CS, chondroitin sulfate; DmGlcAT-I, D. melanogaster glucuronyltransferase I; DmGlcAT-BSI, D. melanogaster glucuronyltransferase with broad specificity I; DmGlcAT-BSII, D. melanogaster glucuronyltransferase with broad specificity II; GAG, glycosaminoglycan; GlcAT-I, glucuronyltransferase I; GlcAT-P, glucuronyltransferase P; GlcUA, D-glucuronic acid; HS, heparan sulfate; RT, reverse transcriptase; MES, 2-(N-morpholino)ethanesulfonic acid; EST, expressed sequence tag.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bernfield, M., Götte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729-777[CrossRef][Medline] [Order article via Infotrieve] |
2. | Hartmann, U., and Maurer, P. (2001) Matrix Biol. 20, 23-35[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Salmivirta, M.,
Lidholt, K.,
and Lindahl, U.
(1996)
FASEB J.
10,
1270-1279 |
4. | Sugahara, K., and Kitagawa, H. (2000) Curr. Opin. Struct. Biol. 10, 518-527[CrossRef][Medline] [Order article via Infotrieve] |
5. | Sugahara, K., and Yamada, S. (2000) Trends Glycosci. Glycotech. 12, 321-349 |
6. | Perrimon, N., and Bernfield, M. (2000) Nature 404, 725-728[CrossRef][Medline] [Order article via Infotrieve] |
7. | Lindahl, U., and Rodén, L. (1972) in Glycoproteins (Gottschalk, A., ed) , pp. 491-517, Elsevier Science Publishing Co., Inc., New York |
8. |
Fritz, T. A.,
Gabb, M. M.,
Wei, G.,
and Esko, J. D.
(1994)
J. Biol. Chem.
269,
28809-28814 |
9. |
Kitagawa, H.,
Shimakawa, H.,
and Sugahara, K.
(1999)
J. Biol. Chem.
274,
13933-13937 |
10. |
Kim, B.-T.,
Kitagawa, H.,
Tamura, J.,
Saito, T.,
Kusche-Gullberg, M.,
Lindahl, U.,
and Sugahara, K.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
7176-7181 |
11. | Rohrmann, K., Niemann, R., and Buddecke, E. (1985) Eur. J. Biochem. 148, 463-469[Abstract] |
12. | Nadanaka, S., Kitagawa, H., Goto, F., Tamura, J., Neumann, K. W., Ogawa, T., and Sugahara, K. (1999) Biochem. J. 340, 353-357[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Uyama, T.,
Kitagawa, H.,
Tamura, J.,
and Sugahara, K.
(2002)
J. Biol. Chem.
277,
8841-8846 |
14. | Sugahara, K., and Kitagawa, H. (2002) IUMBM Life 54, 163-175 |
15. |
Lind, T.,
Tufaro, F.,
McCormick, C.,
Lindahl, U.,
and Lidholt, K.
(1998)
J. Biol. Chem.
273,
26265-26268 |
16. |
Senay, C.,
Lind, T.,
Muguruma, K.,
Tone, Y.,
Kitagawa, H.,
Sugahara, K.,
Lidholt, K.,
Lindahl, U.,
and Kusche-Gullberg, M.
(2000)
EMBO Rep.
1,
282-286 |
17. |
Kitagawa, H.,
Uyama, T.,
and Sugahara, K.
(2001)
J. Biol. Chem.
276,
38721-38726 |
18. | Habuchi, O. (2000) Biochim. Biophys. Acta 1474, 115-127[Medline] [Order article via Infotrieve] |
19. | Gotting, C., Kuhn, J., Zahn, R., Brinkmann, T., and Kleesiek, K. (2001) J. Mol. Biol. 304, 517-528[CrossRef] |
20. |
Okajima, T.,
Yoshida, K.,
Kondo, T.,
and Furukawa, K.
(1999)
J. Biol. Chem.
274,
22915-22918 |
21. |
Almeida, R.,
Levery, S. B.,
Mandel, U.,
Kresse, H.,
Schwientek, T.,
Bennett, E. P.,
and Clausen, H.
(1999)
J. Biol. Chem.
274,
26165-26171 |
22. |
Bai, X.,
Zhou, D.,
Brown, J. R.,
Crawford, B. E.,
Hennet, T.,
and Esko, J. D.
(2001)
J. Biol. Chem.
276,
48189-48195 |
23. |
Kitagawa, H.,
Tone, Y.,
Tamura, J.,
Neumann, K. W.,
Ogawa, T.,
Oka, S.,
Kawasaki, T.,
and Sugahara, K.
(1998)
J. Biol. Chem.
273,
6615-6618 |
24. |
Ong, E.,
Yeh, J.,
Ding, Y,
Hindsgaul, O.,
and Fukuda, M.
(1998)
J. Biol. Chem.
273,
5190-5195 |
25. |
Bakker, H.,
Friedmann, I.,
Oka, S.,
Kawasaki, T.,
Nifant'ev, N.,
Schachner, M.,
and Mantei, N.
(1997)
J. Biol. Chem.
272,
29942-29946 |
26. |
Terayama, K.,
Oka, S.,
Seiki, T.,
Miki, Y.,
Nakamura, A.,
Kozutsumi, Y.,
Takio, K.,
and Kawasaki, T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6093-6098 |
27. | Tone, Y., Kitagawa, H., Imiya, K., Oka, S., Kawasaki, T., and Sugahara, K. (1999) FEBS Lett. 459, 415-420[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Wei, G.,
Bai, X.,
Sarkar, A. K.,
and Esko, J. D.
(1999)
J. Biol. Chem.
274,
7857-7864 |
29. | Yamada, S., Van Die, I., Van den Eijnden, D. H., Yokota, A., Kitagawa, H., and Sugahara, K. (1999) FEBS Lett 459, 327-331[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Toyoda, H.,
Kinoshita-Toyoda, A.,
and Selleck, S. B.
(2000)
J. Biol. Chem.
275,
2269-2275 |
31. |
Yamada, S.,
Okada, Y.,
Ueno, M.,
Iwata, S.,
Deepa, S. S.,
Nishimura, S.,
Fujita, M.,
Van Die, I.,
Hirabayashi, Y.,
and Sugahara, K.
(2002)
J. Biol. Chem.
277,
31877-31886 |
32. | Spring, J., Paine-Saunders, S. E., Hynes, R. O., and Bernfield, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3334-3338[Abstract] |
33. |
Filmus, J.,
and Selleck, S. B.
(2001)
J. Clin. Invest.
108,
497-501 |
34. | Bulik, D. A., Wei, G., Toyoda, H., Kinoshita-Toyoda, A., Waldrip, W. R., Esko, J. D., Robbins, P. W., and Selleck, S. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 98, 7176-7181 |
35. |
Binari, R. C.,
Staveley, B. E.,
Johnson, W. A.,
Godavarti, R.,
Sasisekharan, R.,
and Manoukian, A. S.
(1997)
Development
124,
2623-2632 |
36. |
Haecker, U.,
Lin, X.,
and Perrimon, N.
(1997)
Development
124,
3565-3573 |
37. | Lin, X., and Perrimon, N. (1997) Nature 400, 281-284 |
38. | Bellaiche, Y., The, I., and Perrimon, N. (1998) Nature 394, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
39. | Becker, T., Becker, C. G., Schachner, M., and Bernhardt, R. R. (2001) Mech. Dev. 109, 37-49[CrossRef][Medline] [Order article via Infotrieve] |
40. | Dennis, R. D., Martini, R., and Schachner, M. (1991) Cell. Tissue Res. 265, 589-600[Medline] [Order article via Infotrieve] |
41. |
Miyamoto, M.,
Asano, M.,
Sakagami, J.,
Sudo, K.,
Iwakura, Y.,
and Kawasaki, T.
(2002)
J. Biol. Chem.
277,
27227-27231 |
42. | Kawasaki, T., and Ashwell, G. (1977) J. Biol. Chem. 252, 6536-6543[Abstract] |
43. | Seiki, T., Oka, S., Terayama, K., Imiya, K., and Kawasaki, T. (1999) Biochem. Biophys. Res. Commun. 255, 182-187[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Shimoda, Y.,
Tajima, Y.,
Nagase, T.,
Harii, K.,
Osumi, N.,
and Sanai, Y.
(1999)
J. Biol. Chem.
274,
17115-17122 |
45. |
Herman, T.,
and Horvitz, H. R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
974-979 |
46. |
Pedersen, L. C.,
Tsuchida, K.,
Kitagawa, H.,
Sugahara, K.,
Darden, T. A.,
and Negishi, M.
(2000)
J. Biol. Chem.
275,
34580-34585 |
47. |
Pedersen, L. C.,
Darden, T. A.,
and Negishi, M.
(2002)
J. Biol. Chem.
277,
21869-21873 |
48. |
Sugita, M.,
Itonori, S.,
Inagaki, F.,
and Hori, T.
(1989)
J. Biol. Chem.
264,
15028-15033 |
49. | Weske, B., Dennis, R. D., Helling, F., Keller, M., Nores, G. A., Peter-Katalinic, J., Egge, H., Dabrowski, U., and Wiegandt, H. (1990) Eur. J. Biochem. 191, 379-388[Abstract] |
50. |
Seppo, A.,
Moreland, M.,
Schweingruber, H.,
and Tiemeyer, M.
(2000)
Eur. J. Biochem.
267,
3549-3558 |
51. | Guérardel, Y., Balanzino, L., Maes, E., Leroy, Y., Coddeville, B., Oriol, R., and Strecker, G. (2001) Biochem. J. 357, 167-182[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Helting, T.,
and Rodén, L.
(1969)
J. Biol. Chem.
244,
2799-2805 |
53. |
Quzzine, M.,
Gulberti, S.,
Netter, P.,
Magdalou, J.,
and Fournel-Gigleux, S.
(2000)
J. Biol. Chem.
275,
28254-28260 |