From the Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093-0687
Received for publication, January 30, 2001, and in revised form, March 7, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
While studying the cellular localization and
activity of enzymes involved in heparan sulfate biosynthesis, we
discovered that the published sequence for the glucuronic acid
C5-epimerase responsible for the interconversion of
D-glucuronic acid and L-iduronic acid residues encodes a truncated protein. Genome analysis and 5'-rapid amplification of cDNA ends was used to clone the full-length
cDNA from a mouse mastocytoma cell line. The extended cDNA
encodes for an additional 174 amino acids at the amino terminus
of the protein. The murine sequence is 95% identical to the human
epimerase identified from genomic sequences and fits with the general
size and structure of the gene from Drosophila melanogaster
and Caenorhabditis elegans. Full-length
epimerase is predicted to have a type II transmembrane topology with a
17-amino acid transmembrane domain and an 11-amino acid cytoplasmic
tail. An assay with increased sensitivity was devised that detects
enzyme activity in extracts prepared from cultured cells and in
recombinant proteins. Unlike other enzymes involved in
glycosaminoglycan biosynthesis, the addition of a c-myc
tag or green fluorescent protein to the highly conserved
COOH-terminal portion of the protein inhibits its activity. The
amino-terminally truncated epimerase does not localize to any cellular
compartment, whereas the full-length enzyme is in the Golgi, where
heparan sulfate synthesis is thought to occur.
Heparan sulfate proteoglycans are located on the cell surface and
in the extracellular matrix, where they play important roles in cell
adhesion, differentiation, and growth in vitro and in vivo (1-3). To a large extent, these biological activities depend on the heparan sulfate chains attached to the core protein. Heparan sulfate, a type of glycosaminoglycan, initially assembles by the copolymerization of N-acetyl-D-glucosamine
(GlcNAc) and D-glucuronic acid (GlcA). The backbone then
undergoes extensive modification initiated by the
N-deacetylation and N-sulfation of subsets of GlcNAc residues. Subsequently, D-GlcA residues adjacent to
the N-sulfated sugars are converted to
L-IdoUA1 by a
C5-epimerase and are sulfated at C-2 by a specific sulfotransferase. The glucosamine units also can be sulfated at C-6 and to a lesser extent at C-3. The blocklike arrangement of the modified residues confers specific binding properties to the chains for protein ligands,
which in turn facilitate various biological activities.
Many of the enzymes involved in heparan sulfate and heparin formation
seem to be members of multienzyme gene families. Two exceptions are the
C5-epimerase that interconverts D-GlcA and L-IdoUA and the 2-O-sulfotransferase that adds
sulfate to C-2 of IdoUA residues and to a lesser extent GlcA residues.
The C5-epimerase has been partially purified from mouse mastocytoma (4)
and purified to homogeneity from bovine liver (5). A bovine cDNA for the epimerase has been cloned as well (6). Kinetic studies have
clarified the substrate specificity of the epimerase, showing that the
enzyme will react with both D-GlcA (forward reaction) and
L-IdoUA (reverse reaction) when these residues are located toward the reducing side of N-sulfated glucosamine residues,
but it will not react with uronic acids that are O-sulfated
or that are adjacent to O-sulfated glucosamine residues (7,
8). This specificity is consistent with the overall order of
modification, suggesting that epimerization begins to occur after
GlcNAc N-deacetylation and N-sulfation but before
glucosamine residues undergo 6-O-sulfation and
3-O-sulfation (7, 9). The fact that the epimerase seems to
be represented only once in vertebrate and invertebrate genomes suggests that the extent of uronic acid epimerization depends on the
level of enzyme expression and production of the N-sulfated tracts.
In an attempt to study the cellular localization and potential
interaction of the epimerase with other enzymes in the pathway, we
discovered that the published bovine sequence encodes a truncated protein.2 This report
provides the full-length sequence from mouse and human, an improved set
of conditions for assaying the epimerase in cell extracts, and a
demonstration that the enzyme is localized to the Golgi in vertebrate cells.
Cell Culture--
Chinese hamster ovary cells (CHO-K1) were
obtained from the American Type Culture Collection (CCL-61, Manassa,
VA). MST cells were derived from the Furth murine mastocytoma (10). CHO
cells were grown in Ham's F-12 medium (Life Technologies, Inc.), and MST cells were grown in RPMI 1640 medium. Both media were supplemented with 10% (v/v) fetal bovine serum (Hyclone Laboratories), 100 µg/ml
streptomycin sulfate, and 100 units/ml penicillin G. The cells were
cultured at 37 °C under an atmosphere of 5% CO2 in air
at 100% relative humidity.
Cloning the Murine C5-Epimerase--
A murine epimerase cDNA
fragment corresponding to the published bovine sequence
(GenBankTM accession number AF003927) was cloned from an
MST cDNA library using PCR, the forward primer
5'-ATGTCCTTTGAAGGCTACAATGTGG-3', and the reverse primer
5'-CTAGTTGTGCTTTGCCCGGCTGCC-3', which anneal with the first and last 24 bases of the partial bovine sequence (6). The PCR product was blunt-end
cloned into pGEM (Promega) for sequencing. Subsequently, the primers
XhoEpi5' (5'-CCCCGGCTCGAGGCCGCCATGTCCTTTGAAGCCTACAATG-3') and BamEpi3'
(5'-CTGGATCCTAGTTGTGCTTTGCCCGG-3') were used to amplify the cDNA
from the pGEM epimerase clone for transfer into pCDNA3.1 (Invitrogen) using the XhoI and BamHI sites. To
generate the YFP-tagged truncated epimerase, the primers XhoEpi5' and
3'Epi-GFPBam (5'-CTGGATCCCCGTTGTGCTTTGCCCGG-3') were used to amplify
the epimerase cDNA, which was cloned into the XhoI and
BamHI sites of pEYFP-N1.
The cDNA containing the full-length epimerase was cloned using a
primer designed to the proposed 5' end deduced from the human genomic
DNA sequence (GenBankTM accession number AC026992). This
primer, 5EpiXhoI (5'-CTCGAGCCATGCGTTGCTTGGCAGCTCGG-3'), was used
with an internal reverse primer, 3EpiBam
(5'-GGATCCGAGATTCCATGCCGCGCTCGTACAAG-3'), to amplify the 5' 900 base pairs of the cDNA from the murine MST cDNA library. The
cDNA encoding the full-length epimerase was then constructed by
digesting the truncated epimerase in pCDNA3.1 with XhoI
and HindIII and then by inserting the extended 5' end amplified by PCR. The GFP-tagged full-length epimerase was generated by
amplifying the full coding region from pCDNA3.1 with the primers 5EpiXhoI and 3'Epi-GFPBam and by cloning into the XhoI and
BamHI sites of pEGFP-N1. The coding sequence was verified by
directly sequencing PCR products from three independent amplifications from the MST cDNA library. All PCR amplifications were done using Vent DNA polymerase (New England Biolabs), and clones were sequenced on
an ABI 373 DNA sequencer using dye terminator cycle sequencing.
Generation of Full-length Murine Epimerase--
5'-Rapid
amplification of cDNA ends was performed according to the
manufacturer's instructions (CLONTECH) with
mRNA isolated from MST cells. Two gene-specific primers were used
based on the murine sequence for the epimerase described above,
3EpiBam (5'-GGATCCGAGATTCCATGCCGCGCTCGTACAAG-3') and BC15
(5'-ACATTGGTGGATCTAGACTT-3'). Analysis of five independent clones, each with the same 5' end, yielded a consensus sequence.
The sequence of the 3' end of the murine coding sequence was determined
by amplifying the 3' end from an MST cDNA library using BC11
(5'-GGAGACCACAGAAAAGAATC-3') and BC42 (5'-GGAGACCACAGAAAAGAATC-3'). BC11 was designed to anneal between nucleotides 1164 and 1183 of the
mouse epimerase cDNA, whereas BC42 was designed to anneal to the
3'-untranslated region (UTR) and was designed based on nucleotides
1832-1855 of the partial human sequence (GenBankTM
accession number AB020643). The PCR product was cloned, and three
independent isolates were sequenced. All three clones contained two
silent changes from the published human sequence. The
GenBankTM accession numbers for the murine cDNA and
encoded protein are AF325532 and AAG42004, respectively.
Enzyme Localization--
Epimerase constructs were generated
with COOH-terminal c-myc and GFP tags by subcloning into
pCDNA3.1 containing myc/His6 and
pEGFP (CLONTECH), respectively. An
amino-terminal c-myc-tagged clone was generated by annealing
the oligonucleotides BC46
(5'-ATGTCTAGAGAACAAAAACTCATCTCAGAAGAGGATCTGTCTAGAGCA-3') and
BC47 (5'-TGCTCTAGACAGATCCTCTTCTGAGATGAGTTTTTGTTCTCTAGACAT-3'), which codes for a myc tag. The oligonucleotides were
boiled for 1 min, cooled on ice, phosphorylated with polynucleotide
kinase (New England Biolabs), and cloned in-frame into the
EcoRV site in the polylinker region of pcDNA3.1
containing the full-length epimerase. The clone used in these
experiments actually contained two Myc tags in a tandem repeat at the
amino terminus.
CHO cells were transiently transfected with 2 µg of plasmid DNA using
LipofectAMINE according to the manufacturer's directions (Life
Technologies, Inc.). Cells were grown on 24-well glass microscope slides and were processed for enzyme localization studies 24-36 h
after transfection. After the cells were fixed for 1 h with 2%
paraformaldehyde in 75 mM phosphate buffer, pH 7.5, they
were rinsed several times with phosphate-buffered saline (PBS) (11). Cells were then permeabilized with 1% Triton X-100 (v/v) and 0.1% bovine serum albumin (BSA) (w/v) in PBS. The primary antibody, mouse
anti-Myc (Invitrogen) monoclonal antibody, and rabbit polyclonal anti- Epimerase Assay--
Normal and transfected cells were washed
twice with cold PBS and once with cold 0.25 M sucrose in 20 mM Tris, pH 7.4, and were then scraped with a rubber
policeman into 100 µl of cold buffer containing 0.25 M
sucrose, 20 mM Tris-HCl, pH 7.4, 20 µM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml pepstatin. Cells were lysed by sonication with a microtip sonicator, and the protein concentration was quantitated with the Bradford assay
(Bio-Rad) using BSA as the standard. The extracts were stable when
stored at
The epimerase substrate consisted of modified
N-acetylheparosan and was prepared as described (12).
Briefly, Escherichia coli K5 capsular polysaccharide was
labeled in vivo with
D-[5-3H]glucose (PerkinElmer Life Sciences)
and purified from the growth medium. The GlcNAc residues in the labeled
polysaccharide were N-deacetylated to near completion with
anhydrous hydrazine and hydrazine sulfate (Sigma) and were
N-sulfated with trimethylamine sulfur trioxide complex
(Sigma). The concentration of N-acetylheparosan was
determined by a carbazol assay for uronic acids (13), which yielded a
radiospecific activity of 76 cpm/pmol GlcA (43 Ci/mol).
Detection of epimerase activity was based on the release of
3H from [5-3H]GlcA units in the
polysaccharide and recovery as 3H2O (12).
Initial assays were set up according to the published reaction
conditions ("original"), which contained 50 mM HEPES, 15 mM EDTA, 100 mM KCl, and 0.015% Triton
X-100, pH 7.4 (12). Protein, substrate, and various ancillary factors
were adjusted to maximize the activity detected in normal and
transfected cells. The "revised" assay consisted of 25 mM HEPES, pH 7.0, 0.1% Triton X-100, 300 pmol of
3H-sulfated heparosan substrate, and 2 µg of cell protein
in a total volume of 20 µl. Some assays contained 40 mM
CaCl2, but divalent cations were later found not to be
required. The reactions were incubated for 2 h at 37 °C and
halted by the addition of 50 µl of cold 50 mM sodium
acetate buffer, pH 4.0, containing 50 mM LiCl. The sample
and a 100-µl rinse of the tube with buffer (25 mM HEPES,
pH 7.0, and 0.1% Triton X-100) were transferred to a 0.4-ml column of
DEAE-Sephacel (Amersham Pharmacia Biotech) that was equilibrated with
the same buffer. The column was washed with 0.9 ml of assay buffer, and
the 3H2O recovered in the flow-through
fractions was counted by liquid scintillation spectrometry using Ultima
Gold (Packard Instrument Co.). A reagent blank containing everything
except a source of enzyme was included as a control. This yielded
values of ~200 cpm, which were subtracted from the experimental
values that ranged from 300 to 3000 counts. All assays were done in
duplicate with comparable results from three or more independent experiments.
Western Blotting--
Cells were harvested, and 25 µg of
protein for each sample was analyzed by SDS-polyacrylamide gel
electrophoresis on a 10% gel. The samples were transferred to a
nitrocellulose membrane using the Bio-Rad Mini Protean II system. The
membrane was blocked at 4 °C overnight with 4% BSA in Tris-buffered
saline (10 mM Tris, pH 8.0, and 150 mM NaCl)
with 0.1% Tween 20 (TBST). Mouse anti-GFP (CLONTECH) and mouse anti-Myc (Invitrogen) were
diluted 1:1000 and 1:5000 in TBST, respectively, and incubated for
1 h with the membrane at room temperature with shaking. The
membrane was washed three times with TBST before the application of the
secondary antibody, goat anti-mouse horseradish peroxidase (Bio-Rad)
diluted 1:3000 in TBST. The membrane was washed six times with TBST and developed with SuperSignal West Pico chemiluminescent substrate (Pierce).
The GlcA C5-epimerase was previously purified from murine
mastocytoma and bovine liver (4, 5). Sequencing the purified protein
yielded proline as the amino-terminal amino acid, suggesting that the
protein had been proteolytically cleaved during the purification process (6). Several internal peptides also were generated by
controlled proteolysis, and the peptide sequences were used to design
oligonucleotides for screening a bovine lung cDNA library. A
cDNA sequence was obtained that was 3085 base pairs and contained an open reading frame corresponding to a 444-amino acid protein from
the first in-frame ATG codon. The deduced amino acid sequence predicted
a 49,905-Da protein with a potential transmembrane domain located near
the amino terminus. Expression of this clone in the baculovirus system
yielded the expected activity (6).
Further analysis of the epimerase sequence suggested that it was
incomplete. First, computer-aided analysis using PSORT did not indicate
the predicted transmembrane domain and in fact suggested that the
protein was most likely soluble (14). As described below, this was
confirmed in localization studies with GFP-tagged constructs. Second,
alignment of the published bovine epimerase sequence with other
orthologs in the GenBankTM data base suggested that the
bovine sequence was potentially missing a large domain from the amino
terminus (Fig. 1). A BLAST search
revealed a human genomic DNA clone (GenBankTM accession
number AC026992) containing an extended open reading frame that more
closely matched the size of the epimerase orthologs found in
Caenorhabditis elegans and Drosophila
melanogaster. In addition, a partial human cDNA also was
identified in the sequence data bases (GenBankTM accession
number AB020643).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-mannosidase II antiserum (a gift from Marilyn G. Farquhar, University of California, San Diego) were diluted 1:400 in PBS with 1%
BSA and incubated for 1 h with the fixed cells. To remove the
unbound primary antibody, the cells were washed several times for 30 min with PBS containing 0.1% BSA. The samples were then incubated for
1 h with the secondary antibody, anti-rabbit Cy5 (Qccuvate
Chemicals, NY) or anti-mouse-TRITC (Sigma), diluted 1:200 in PBS
containing 1% BSA. After several washes, the cells were mounted with
Vectashield containing 4',6-diamidino-2-phenylindole for nuclear
staining (Vector Laboratories). The photomicrographs shown in Fig. 5,
A-D, were captured with a Photometrics
charge-coupled device mounted on a Nikon microscope adapted to a
DeltaVision (Applied Precision, Inc.) deconvolution imaging system. The
data sets were deconvolved and analyzed using SoftWorx software
(Applied Precision, Inc.) on a Silicon Graphics Octane work station.
The photomicrograph shown in Fig. 5E was captured with a
Hamamatsu C5810 three-color chilled charge-coupled device camera
mounted on a Zeiss Axiophot (×100 lens) microscope.
20 °C.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (64K):
[in a new window]
Fig. 1.
Amino acid alignment of epimerase
orthologs. Alignment of GlcA C5-epimerase orthologs (bovine,
AAB72083; human, BAA74859; murine, AAG42004; D. melanogaster, AAF57373; and C. elegans, P46555)
suggested that the 5' end of the bovine epimerase sequence was missing.
The triangle indicates the putative initiating Met residue
in the original bovine sequence (6). The asterisks indicate
conserved lysine residues in the COOH terminus that may be involved in
catalysis (17).
Based on this information, we cloned an extended cDNA from mouse
mastocytoma mRNA using primers based on the human sequence. This
fragment was further extended using 5'-rapid amplification of cDNA
ends. The additional sequence added 753 nucleotides including 231 nucleotides in a 5'-UTR and 522 coding nucleotides. The 5'-UTR contains
an in-frame termination codon (TGA) 21 base pairs upstream of the new
initiation codon, suggesting that the cDNA encodes the full-length
epimerase (Fig. 2). The context of the
new start codon conforms to an "adequate" Kozak sequence (AATatgC,
consensus RNNatgY, where R = A or G and Y = T or C) (15),
whereas the previously suggested start codon lacks A or G at 3
(tttatgt). Previous studies reported a 3'-UTR sequence of ~1.6
kilobases (6). Because the mRNA was found to be ~5 kilobases, an
additional untranslated sequence of ~1.4 kilobases apparently exists,
but its location is unknown.
|
The revised sequence adds 174 amino acids to the amino terminus of the previous sequence of the bovine epimerase for a total of 618 amino acids. The full-length epimerase predicts a protein of 70,099 Da with a relatively basic isoelectric point (pI = 8.95). The protein contains a stretch of 17 hydrophobic residues located 11 amino acids from the amino terminus. As expected, PSORT predicts this as a transmembrane domain, and therefore the protein would most likely have a type II transmembrane topology like other enzymes involved in polysaccharide biosynthesis. The full-length clone contains three potential N-linked glycosylation sites at residues 225, 304, and 394, consistent with previous studies suggesting that the protein contained one or more Asn-linked chains. Using the extended sequence to perform a BLAST search of the GenBankTM data bases did not reveal additional homologs, suggesting that there may be only one heparin/heparan sulfate C5-epimerase (16).
A comparison of C. elegans, D. melanogaster, mouse, bovine, and human epimerase sequences indicated weak homology in the amino-terminal domain (residues 1-171) followed by a region of high identity (62%, residues 172-223). However, neither of these regions seems critical for catalytic activity because the purified protein from bovine liver was truncated at residue 248 (6). The COOH-terminal domain was also highly conserved across phylogeny (60% identity, residues 497-618), suggesting that this may represent an important functional part of the protein.
Expression studies of the truncated and full-length epimerase
were undertaken to determine whether the additional 174 amino acids had
any effect on catalysis. Initial attempts to assay the basal enzyme
activity in cell extracts prepared from CHO and MST cells met with
limited success. Expression of the truncated or full-length enzyme gave
variable results, suggesting that the assay originally described for
the bovine enzyme might not be optimal for cultured cells or
recombinant enzymes expressed in cultured cells (4, 5). Variation of
each component improved the activity additively. Monovalent salts were
inhibitory in contrast to previous findings (Fig.
3A) (5, 7). Divalent cations, such as Ca2+ or Mg2+, and EDTA (up to 40 mM) had no effect (4, 7). The activity was highly dependent
on detergent, even in sonicated extracts, with maximal effects obtained
with 0.1% Triton X-100 (Fig. 3B). However, other detergents
inhibited the reaction, suggesting that the effect was not merely
because of solubilization of the protein from membranes. The pH optimum
was ~7.0, which is in general agreement with previous findings (Fig.
3C) (4, 5, 7), but the activity showed marked sensitivity to
the type of buffer (Fig. 3D). HEPES was found to be optimal.
Under the revised conditions, the reaction was proportional with time
for over 2 h and with protein concentration in the range of 1-30
µg. The Km of the enzyme for the N-deacetylated/N-sulfated heparosan was estimated
to be 25 µM GlcA equivalents (~500 pmol of GlcA/assay).
With 300 pmol of GlcA/assay, a 4.5-fold increase in epimerase activity
in MST cell extracts and a 6.5-fold enhancement in CHO cell extracts
were observed compared with the original conditions (Fig.
3E). At lower concentrations of substrate, the difference
was even more dramatic (data not shown).
|
Transient transfection of CHO cells revealed 4-fold greater activity
associated with the full-length protein compared with the truncated
enzyme in the revised assay (Fig. 4).
Increasing the substrate 10-fold did not enhance the rate of reaction
for either recombinant enzyme (data not shown). These findings
indicated that the natural amino terminus was not a prerequisite to
detect activity, which is consistent with previous findings showing
that the truncated protein purified from liver and mastocytoma had substantial activity (4, 5, 12, 17). Extracts prepared from cells
transfected with epimerase containing a COOH-terminal GFP or YFP tag
were analyzed by Western blotting with an anti-GFP monoclonal antibody.
As shown in the inset of Fig. 4, the tagged full-length
protein was present at higher levels than the truncated enzyme. Both
forms were engineered into a near perfect Kozak sequence in the
expression vector, suggesting that their expression was similar. Thus,
we believe that the lower amount of the truncated enzyme was caused by
decreased stability. As shown below, the truncated enzyme was also
mislocalized, which may add to its instability. Thus, the
amino-terminal domain does not seem to enhance the intrinsic activity
of the enzyme.
|
Fusing c-myc or GFP to the COOH terminus resulted in a dramatic reduction of enzyme activity (<1 pmol/min/mg), but when a c-myc tag was placed at the amino terminus, enzyme activity was normal (23 pmol/min/mg versus 26 pmol/min/mg, respectively). These findings suggested that the highly conserved COOH terminus plays an important role in binding, conformation, or catalysis. Recent investigations into the catalytic mechanism of the C5-epimerase implicated two polyprotic bases in the proton exchanges at C-5 (17) that are possibly mediated by two lysine residues. Interestingly, two lysine residues (amino acids 547 and 616) in the COOH-terminal domain of the epimerase are highly conserved across phylogeny (Fig. 1, asterisks). Adding GFP to the COOH terminus of other enzymes involved in heparin/heparan sulfate biosynthesis does not result in loss of activity (18).2
The Full-length Epimerase Localizes to the Golgi--
To study the
intracellular localization of epimerase, cDNAs encoding the
truncated and full-length enzymes were fused to GFP or c-myc
and expressed in CHO cells. Full-length epimerase was located in a
juxtanuclear position, co-localizing with the Golgi marker,
-mannosidase II (Fig. 5,
A-C). This localization was observed with tags
on either the C or amino terminus, indicating that the location of the
tag did not interfere with subcellular localization signals in the
protein (Fig. 5E). When the truncated epimerase was
expressed, it behaved as a soluble protein exhibiting diffuse
cytoplasmic staining (Fig. 5D). This is not an unexpected result given that the protein lacks a signal peptide. The
mislocalization of the truncated enzyme may act to destabilize its
structure and activity (Fig. 4, inset).
|
Future studies of the epimerase will be greatly expedited by having the
full-length sequence. Interestingly, very little information is
available about the function of IdoUA in the biological activity of
heparin and heparan sulfate. In general, it is assumed that the greater
conformational flexibility of IdoUA will enhance the binding
opportunities for heparin and heparan sulfate (19). The best studied
example is the interaction of antithrombin with a heparin
pentasaccharide, in which a critical IdoUA residue located to the
reducing side of a central 3-O-sulfated glucosomine
unit confers high affinity binding to antithrombin (20). Fibroblast growth factor-2 also apparently requires at least one IdoUA unit for
binding and activation (21, 22). In the former case, the addition of
the 2-O-sulfate group to the IdoUA residue seems to be
dispensable (20, 23), whereas in the latter it is essential for binding
(24-26). These findings suggest that in some cases the IdoUA may play
a direct role in binding to the ligand, whereas in others it may simply
serve as a scaffold for placement of a critical sulfate residue. In
both cases, the epimerase plays an essential role in creating the
preferred binding site for the ligand. With full-length recombinant
enzyme now available, it should be possible to engineer binding sites
in isolated oligosaccharides and to explore the function of epimerase
in vivo by creating mutants in cells and model organisms.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank James Feramisco and Brian Smith from the Digital Imaging Shared Resource at the University of California, San Diego Cancer Center for their help in the deconvolution microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R37GM33063 (to J. D. E.), a fellowship from the PEW Latin American Fellows Program in the Biomedical Sciences (to M. A. S. P.), and National Institutes of Health Training Grants CA67754 (to B. E. C.) and GM08666 (to S. K. O.).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.
To whom correspondence should be addressed: Universidade Federal
De Sao Paulo, Vila Clementino, CEP 04044020, Sao Paulo, Brazil. E-mail:
maspinhal.bioq@epm.br.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M100880200
2 M. A. S. Pinhal, B. Smith, J. Aikawa, K. Kimata, and J. D. Esko, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IdoUA, L-iduronic acid; CHO, Chinese hamster ovary; GFP, green fluorescent protein; PCR, polymerase chain reaction; YFP, yellow fluorescent protein; UTR, untranslated region; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Lindahl, U.,
Kusche-Gullberg, M.,
and Kjellén, L.
(1998)
J. Biol. Chem.
273,
24979-24982 |
2. | Selleck, S. B. (2000) Trends Genet. 16, 206-212[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Park, P. W.,
Reizes, O.,
and Bernfield, M.
(2000)
J. Biol. Chem.
275,
29923-29926 |
4. |
Malmström, A.,
Rodén, L.,
Feingold, D. S.,
Jacobsson, I.,
Bäckström, G.,
and Lindahl, U.
(1980)
J. Biol. Chem.
255,
3878-3883 |
5. |
Campbell, P.,
Hannesson, H. H.,
Sandbäck, D.,
Rodén, L.,
Lindahl, U.,
and Li, J.
(1994)
J. Biol. Chem.
269,
26953-26958 |
6. |
Li, J. P.,
Hagner-McWhirter, Å.,
Kjellén, L.,
Palgi, J.,
Jalkanen, M.,
and Lindahl, U.
(1997)
J. Biol. Chem.
272,
28158-28163 |
7. | Jacobsson, I., Bäckström, G., Höök, M., Lindahl, U., Feingold, D. S., Malmström, A., and Rodén, L. (1979) J. Biol. Chem. 254, 2975-2982[Medline] [Order article via Infotrieve] |
8. |
Jacobsson, I.,
Lindahl, U.,
Jensen, J. W.,
Rodén, L.,
Prihar, H.,
and Feingold, D. S.
(1984)
J. Biol. Chem.
259,
1056-1063 |
9. | Lindahl, U., Jacobsson, I., Höök, M., Bäckström, G., and Feingold, D. S. (1976) Biochem. Biophys. Res. Commun. 70, 492-499[Medline] [Order article via Infotrieve] |
10. | Montgomery, R. I., Lidholt, K., Flay, N. W., Liang, J., Vertel, B., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11327-11331[Abstract] |
11. | Dulbecco, R., and Vogt, M. (1954) J. Exp. Med. 99, 167-182[Medline] [Order article via Infotrieve] |
12. |
Hagner-McWhirter, Å.,
Hannesson, H. H.,
Campbell, P.,
Westley, J.,
Rodén, L.,
Lindahl, U.,
and Li, J. P.
(2000)
Glycobiology
10,
159-171 |
13. | Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4, 330-334 |
14. | Nakai, K., and Kanehisa, M. (1992) Genomics 14, 897-911[Medline] [Order article via Infotrieve] |
15. | Kozak, M. (1996) Mamm. Genome 7, 563-574[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
17. | Hagner-McWhirter, Å., Lindahl, U., and Li, J. P. (2000) Biochem. J. 347, 69-75[CrossRef][Medline] [Order article via Infotrieve] |
18. |
McCormick, C.,
Duncan, G.,
Goutsos, K. T.,
and Tufaro, F.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
668-673 |
19. |
Mulloy, B.,
and Forster, M. J.
(2000)
Glycobiology
10,
1147-1156 |
20. | Atha, D. H., Lormeau, J. C., Petitou, M., Rosenberg, R. D., and Choay, J. (1985) Biochemistry 24, 6723-6729[Medline] [Order article via Infotrieve] |
21. |
Maccarana, M.,
Casu, B.,
and Lindahl, U.
(1993)
J. Biol. Chem.
268,
23898-23905 |
22. |
Guimond, S.,
Maccarana, M.,
Olwin, B. B.,
Lindahl, U.,
and Rapraeger, A. C.
(1993)
J. Biol. Chem.
268,
23906-23914 |
23. | Bjork, I., and Lindahl, U. (1982) Mol. Cell. Biochem. 48, 161-182[Medline] [Order article via Infotrieve] |
24. |
Bai, X. M.,
and Esko, J. D.
(1996)
J. Biol. Chem.
271,
17711-17717 |
25. | Pellegrini, L., Burke, D. F., Von Delft, F., Mulloy, B., and Blundell, T. L. (2000) Nature 407, 1029-1034[CrossRef][Medline] [Order article via Infotrieve] |
26. | Schlessinger, J., Plotnikov, A. N., Ibrahimi, O. A., Eliseenkova, A. V., Yeh, B. K., Yayon, A., Linhardt, R. J., and Mohammadi, M. (2000) Mol. Cell 6, 743-750[Medline] [Order article via Infotrieve] |