From the Department of Medical Biochemistry and Microbiology, the University of Uppsala, the Biomedical Center, Box 582, SE-751 23 Uppsala, Sweden and § BioTie Therapies Corp., Turku Technology Center, BioCity, FIN-20520 Turku, Finland
Received for publication, December 29, 2001, and in revised form, March 22, 2001
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ABSTRACT |
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The murine gene for the glucuronyl C5-epimerase
involved in heparan sulfate biosynthesis was cloned, using a previously
isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter,
Å., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997)
J. Biol. Chem. 272, 28158-28163) as probe. The
~11-kilobase pair mouse gene contains 3 exons from the first ATG to
stop codon and is localized to chromosome 9. Southern analysis of the
genomic DNA and chromosome mapping suggested the occurrence of a single
epimerase gene. Based on the genomic sequence, a mouse liver cDNA
was isolated that encodes a 618-amino acid residue protein, thus
extending by 174 N-terminal residues the sequence deduced from the
(incomplete) bovine cDNA. Comparison of murine, bovine, and human
epimerase cDNA structures indicated 96-99% identity at the amino
acid level. A cDNA identical to the mouse liver species was
demonstrated in mouse mast cells committed to heparin biosynthesis.
These findings suggest that the iduronic acid residues in heparin and
heparan sulfate, despite different structural contexts, are generated
by the same C5-epimerase enzyme. The catalytic activity of the
recombinant full-length mouse liver epimerase, expressed in insect
cells, was found to be >2 orders of magnitude higher than that of the
previously cloned, smaller bovine recombinant protein. The ~52-kDa,
similarly highly active, enzyme originally purified from bovine liver
(Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén,
L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem.
269, 26953-26958) was found to be associated with an ~22-kDa peptide
generated by a single proteolytic cleavage of the full-sized protein.
Heparin and heparan sulfate (HS)1 are both linear,
sulfated glycosaminoglycans based on a
common carbohydrate backbone of alternating D-glucosamine
(GlcN) and hexuronic acid (D-glucuronic (GlcUA) or
L-iduronic acid (IdoUA)) units. Both polymer types are
synthesized as proteoglycans. Heparin occurs in connective tissue-type
mast cells, whereas HS has a ubiquitous distribution and is produced by
almost all mammalian cells. The biosynthesis of heparin and HS follows
a common pathway that involves: (i) formation of a GlcUA-Gal-Gal-Xyl-Ser carbohydrate-protein linkage region; (ii) assembly of repeating -GlcUA-GlcNAc- disaccharide units; and (iii) modification of the [GlcUA-GlcNAc]n polymers (1, 2). The
latter process includes N-deacetylation and
N-sulfation of GlcNAc units, C5-epimerization of GlcUA to
IdoUA units, and O-sulfation at various sites of the sugar
residues. Heparin is highly modified, heavily N- and
O-sulfated and with more IdoUA than GlcUA units. Generally,
a predominant proportion of heparin chains consists of trisulfated
-IdoUA(2-OSO3)-GlcNSO3(6-OSO3)-
units. By comparison, HS has a more variable and heterogeneous
structure, with highly modified, less modified, and unmodified
sequences arranged in domain-type fashion (3, 4). The IdoUA units occur
in essentially two types of domains, composed of contiguous
N-sulfated, and of alternating N-sulfated and
N-acetylated disaccharide units, respectively (5). The
sequence variability of the chains is believed to reflect the
functional role of HS glycosaminoglycans (GAGs) in specific
interactions with different proteins (1-4, 6). IdoUA residues
generally appear to promote protein binding due to their conformational
flexibility and have been identified as invariable constituents of
protein-binding HS domains (4). The reaction catalyzed by the
C5-epimerase therefore is crucial for many biological functions of
heparin and HS.
The mechanisms in control of the structural diversity of heparin and,
in particular, of HS are only partly understood but clearly rely on the
substrate specificities of the enzymes involved (2, 4). The enzymes
required to synthesize a HS chain have all been cloned. Notably,
several of these proteins, including species committed to polymer
modification, occur in multiple forms that are encoded by different
genes. Some of these species have been shown to differ with regard to
kinetic properties and/or substrate specificity from the respective
homologous forms (7-10).
The IdoUA units of heparin and HS chains occur in widely different
structural contexts, which range from the minimally sulfated -GlcNSO3-IdoUA-GlcNAc- sequence found in HS molecules to
the extensively sulfated
-GlcNSO3(6-OSO3)-IdoUA(2-OSO3)-GlcNSO3(6-OSO3)-
structure typical of heparin (4). These findings raise the question as to whether the C5-epimerase, similar to other enzymes in the same biosynthetic process, also occurs in genetically distinct forms. More
specifically, is the same epimerase committed to the formation of
heparin, in the mast cell, and to HS, in other cells? Following the
isolation of the GlcUA C5-epimerase from bovine liver, the corresponding cDNA was cloned from bovine lung, and a recombinant protein with significant catalytic activity was expressed in insect cells (11). In the present study we have addressed the question of
genetic polymorphism by cloning the murine C5-epimerase gene. Only one
form of the gene was found. Moreover, we have cloned the C5-epimerase
cDNA from mouse liver that generates HS, and from mouse mastocytoma
cells that produce heparin. The results strongly suggest that the same
enzyme protein is implicated in both biosynthetic processes. Finally,
analysis of the epimerase gene structure indicated that the bovine
cDNA previously cloned (11) was incomplete, as ~28% of the
5'-terminal coding region was missing. This conclusion was verified by
cloning and expression of the full-length protein, with the catalytic
activity much higher than that of the previously expressed truncated form.
Isolation and DNA Sequence Analysis of Mouse Genomic
Clones--
A mouse genomic library (Lambda FX-II from Stratagene) was
screened with a 1407-bp DNA probe from the bovine epimerase coding sequence, labeled with [32P]dCTP (PerkinElmer Life
Sciences). Approximately 2 × 106 phages were plated
at a density of 250,000 plaques per 20 × 20-cm plate, and
duplicate nylon filters were prepared from each plate. Hybridization
was performed at 60 °C in 5× Denhardt's hybridization solution,
containing 100 µg of salmon sperm DNA/ml. The final washes were in
0.1× SSC (1× SSC is 150 mM NaCl, 15 mM sodium
citrate, pH 7.0) containing 0.1% SDS. Plaques that produced positive
signals on both replicas were selected for second and third round
screening. Five positive clones were found. Two of the clones
were digested, clone 5a with EcoRI and clone 64 with
SacI, and the resultant fragments were cloned into pUC 119 and BlueScript vectors, respectively. The insert-containing plasmids
were purified using the Qiagen plasmid kit and subsequently sequenced.
Nucleotide sequencing was performed using the BigDye terminator method
and an ABI Prism 310 Genetic Analyzer according to the instructions of
the manufacturer (PerkinElmer Life Sciences). Exon/intron boundaries
were determined by primer walking sequencing on both strands of the
subclones. The size of introns was estimated by agarose gel
electrophoresis. The exons were sequenced from both strands.
Southern Blot Analysis--
Southern blot analysis was performed
according to Sambrook et al. (12). Mouse genomic DNA (20 µg), prepared from liver using an Easy Prep kit (Amersham Pharmacia
Biotech), was digested with restriction enzymes, and the products were
separated by electrophoresis on an 0.8% agarose gel. After separation,
the gel was treated with 0.5 M NaOH for 30 min and
neutralized in 0.5 M Tris-HCl buffer, pH 7.4, containing
1.5 M NaCl. The DNA fragments were transferred onto a
Hybond-N+ nylon transfer membrane (Amersham Pharmacia
Biotech). A fragment of an ~800-bp intron DNA from genomic clone 5a
was labeled with [32P]dCTP using Klenow enzyme from Roche
Molecular Biochemicals and applied as probe. Hybridization was
performed at 65 °C in ExpressHyb hybridization solution
(CLONTECH) for 1 h followed by washing with
0.5× SSC containing 0.5% SDS at room temperature twice for 20 min.
The membrane was exposed to a x-ray film for 3 days.
Chromosome Slide Preparation--
Mouse lymphocytes
were isolated from normal spleen and cultured at 37 °C in RPMI 1640 medium supplemented with 15% fetal calf serum (FCS), 3 µg/ml
concanavalin A, 10 µg/ml lipopolysaccharide, and 5 × 10 Probe Labeling and in Situ Hybridization--
The
full-length mouse epimerase cDNA was biotinylated with dATP using
Life Technologies, Inc., BioNick labeling kit (15 °C, 1 h)
(13). The procedure for fluorescence in situ hybridization (FISH) detection was as described (13, 14). Briefly, chromosome slides
were baked at 55 °C for 1 h and were then treated with RNase A,
denatured in 70% formamide in 2× SSC for 2 min at 70 °C, and
finally dehydrated with ethanol. The probe was denatured at 75 °C
for 5 min in a hybridization mixture consisting of 50% formamide and
10% dextran sulfate. The slides were pre-hybridized for 15 min at
37 °C, and the probe was loaded on the denatured slides. After
overnight hybridization, slides were washed, and signals were detected
as well as amplified using the published method (13). FISH signals and
the 4'-6-diamidino-2-phenylindole (DAPI) banding patterns were recorded
by separate photographs, and the assignment of the FISH mapping data
with chromosomal bands was achieved by superimposing FISH signals with
DAPI-banded chromosomes (14).
Northern Blot Analysis--
Mouse multiple tissue
Northern blot was purchased from CLONTECH. A DNA
probe of 837 bp from the C-terminal end region of bovine cDNA clone
was labeled with [32P]dCTP by use of Klenow enzyme. The
hybridization was carried out in ExpressHyb hybridization solution at
60 °C for 1 h, and the blot was then washed with 0.1× SSC
containing 0.5% SDS at 60 °C. The membrane was exposed to a Kodak
x-ray film for 2 days.
Cloning of C5-epimerase cDNA--
Primers for
cloning of mouse cDNA were designed based on the nucleotide
sequence obtained by sequencing the exons of the genomic clones. The
sense primer corresponds to bp 1-26 of the open reading frame
(5'-ATGCGTTGTTTGGCAGCTCGGGTCAA). The antisense primer corresponds to
the 3'-end of the coding sequence (bp 1829-1854), excluding the stop
codon (5'-GTTGTGCTTTGCCCTACTGCCTTTAA). PCR was performed using a
mouse liver QUICK-CloneTM cDNA
(CLONTECH) as template under the following
conditions: 1 cycle of 94 °C for 1 min, 30 cycles each of 94 °C
for 30 s, 60 °C for 45 s, and 72 °C for 1 min, and a
final extension at 72 °C for 10 min.
Mouse mastocytoma mast cells (denoted "mast cells" in this paper),
established in culture after passage through an ascites stage (15),
were grown in Dulbecco's modified Eagle's medium containing 50 µg/ml penicillin, 50 µg/ml streptomycin, and 10% FCS until
confluent. Total RNA was extracted from one flask (75 ml) of cultured
mast cells according to the LiCl/urea/SDS procedure of Sambrook
et al. (12). About 1 µg of total RNA was used as template
for RT-PCR. The single strand cDNA was prepared in a volume of 20 µl with 1st Strand cDNA Synthesis Kit for RT-PCR (Roche Molecular
Biochemicals). A portion (5 µl) of the RT-PCR reaction mixture was
used for amplification of C5-epimerase cDNA using the primer pair
described above under the same conditions.
For cloning of the 5'-terminal portion of bovine C5-epimerase cDNA,
PCR was performed using a bovine lung gt10 cDNA library (CLONTECH) as template under the conditions
described above. The sense primer is 5'-ATGCGTTGTTTGGCAGCTCGGGTCAA,
corresponding to bp 1-26 of the open reading frame of mouse cDNA.
The antisense primer is 5'-GCAGCCCTTGGGCACAGTCCAGTCATTGGGCTTGC
corresponding to bp 287-321 of the bovine cDNA reported
earlier (11).
All PCR products were directly cloned into a TOPOTM-TA
Cloning vector (Invitrogen) according to the protocol provided by the manufacturer and were subsequently sequenced. The sequencing was carried out as described for the genomic DNA.
Expression of the Murine C5-epimerase Recombinant
Protein--
Based on the cDNA sequence of the mouse C5-epimerase,
new primers were designed and used for generation of a fragment without transmembrane domain for recombinant expression. PCR was performed by
using the full-length mouse C5-epimerase cDNA as a template under
the conditions described above. The resulting fragment was inserted
into a modified (by introducing EGT signal peptide, FLAG epitope,
enterokinase cleavage site, and His tag at the 5'-end) pIZ/V5
expression vector (Invitrogen) and subsequently sequenced. The
expression construct was introduced into Sf9 insect cells, and
the cells were cultured according to the manufacturer's instructions. The medium was collected and analyzed for epimerase activity.
Enzyme Purification and Characterization--
Medium containing
recombinant epimerase (100 ml) was applied to a 20-ml column of
O-desulfated heparin immobilized on Sepharose (16),
equilibrated in 50 mM HEPES buffer, pH 7.4, 100 mM KCl, 15 mM EDTA. After extensive washing
with the same buffer, the bound material was eluted with the same HEPES
buffer containing 300 mM KCl, 15 mM EDTA. The
eluted material was concentrated by centrifugation in an Ultrafree-MC
10,000 filter unit (Millipore).
Bovine liver C5-epimerase was extracted and purified to homogeneity as
described (16). Peptide sequences were determined as described (11).
Purified enzyme preparations were analyzed by SDS-PAGE (17). After
electrophoresis, gels were stained with silver and documented.
Determination of Epimerase Activity--
Epimerase assay was
based on the release of 3H (recovered as
3H2O) from a C5-3H-labeled
polysaccharide substrate, as described (11). For determination of
enzyme activity, tissues were freshly dissected from a (A/Sn × Leaden) F1 mouse that had been inoculated previously with
Furth mastocytoma cells in a hind leg (18). The tissues were
immediately homogenized in 10 volumes of 50 mM HEPES, pH
7.4, containing 100 mM KCl, 15 mM EDTA, 1%
Triton X-100, and protease inhibitors (16). Lysates were shaken at
4 °C for 30 min and centrifuged. The supernatants were collected and
assayed for enzyme activity (11) and for total protein (19). Controls
were performed to ascertain that all determinations of epimerase
activity in tissue extracts fell within the linear range of the assay.
Enzymatic conversion of GlcUA to IdoUA units was directly demonstrated
using purified recombinant mouse C5-epimerase (~50 ng of protein in
100 µl of incubation mixture) and a metabolically [1-3H]glucose-labeled
[4GlcUA Labeling and Preparation of Glycosaminoglycans from Mast
Cells--
Mastocytoma cells established from the Furth tumor (15)
were cultured in RPMI 1640 medium (Life Technologies, Inc.) containing 50 µg/ml penicillin, 50 µg/ml streptomycin, and 10% FCS. To label the cells, 50 µCi of Na35SO4 (Amersham
Pharmacia Biotech) was added per ml of culture medium. After incubation
for 24 h, the medium was collected, and the cells were lysed in 50 mM HEPES, pH 7.4, containing 0.1 M KCl and 1% Triton X-100. The cell lysate was kept on ice for 30 min and
centrifuged. The supernatant and medium were pooled and treated with
0.5 M NaOH at 4 °C overnight and subsequently
neutralized. The sample, containing released GAG chains, was diluted
and applied to a DEAE-Sephacel column (4 ml) equilibrated in 0.05 M NaAc, pH 4.0, containing 0.05 M NaCl. Labeled
GAGs were eluted with a gradient of 0.05-1.5 M NaCl in the
same buffer. The fractions containing labeled GAGs were pooled and
concentrated. Analytical chromatography on DEAE-Sephacel was performed
using the same buffer and salt gradient but in a high pressure liquid
chromatography system. Treatments of GAGs with chondroitinase ABC (20)
or with nitrous acid at pH 1.5 (21) were carried out as described.
Unlabeled heparin and chondroitin sulfate standards were detected by
the carbazole reaction for hexuronic acid (22).
Organization of the Mouse Epimerase Gene--
Screening a genomic
library from mouse liver with the previously described
32P-labeled bovine epimerase cDNA (11) as a probe
yielded five positive clones that were purified and further
characterized. The two largest clones, 5a and 64, were 16-18 kb in
size, whereas the remaining three ranged 8-12 kb. All clones showed
sequence overlap with at least three of the other clones (Fig.
1A). Digestion of clone 5a
with EcoRI released seven fragments that were subsequently cloned into pUC119. Clone 64 was cleaved with SacI, and the
resultant 3 major fragments were cloned into BlueScript. Analysis of
exon-intron organization revealed that the C5-epimerase is encoded by
only 3 exons, of which the largest one (exon 3) encodes more than 50% of the protein (Fig. 1B). The genomic sequences defining the
exon/intron boundaries (splice sites) follow the gt/ag consensus rule
(Table I). The precise match between the
open reading frame (ORF) sequence in the exons and the cDNA (data
not shown) suggests that the identified genomic clone represents the
functional gene for the C5-epimerase.
Southern Analysis and Chromosome Localization--
In order to
address whether the C5-epimerase is encoded by a single gene in the
mouse genome, we performed Southern blot hybridization of genomic DNA
that had been digested with restriction enzymes as indicated in Fig.
2. The probe used was an intron fragment (indicated by the asterisk in Fig. 1A) generated
from clone 5a by digestion with EcoRI. This ~800-bp probe
detected a single band of the same size in the sample cleaved with
EcoRI and single bands of the expected sizes (estimated
based on restriction enzyme mapping) upon cleavage with
HindIII (~3.5 kb), NcoI (~5 kb),
SacI (~6.5 kb), and PstI (~8 kb).
By using the full-length mouse epimerase cDNA as probe, a single
chromosomal locus was detected by FISH mapping in both mouse and human
chromosomes. The FISH detection efficiency with this probe was 85% on
mouse chromosomes and 63% on human chromosomes. The mouse epimerase is
localized to chromosome number 9 (Fig. 3A) and the human epimerase to
chromosome number 15 (Fig. 3B), in accordance with the
homology relationship between human and mouse genomes. The loci were
further defined based on the combined information from 10 photos each.
The epimerase probe thus was mapped to mouse chromosome 9, region C and
D (Fig. 3C), and to human chromosome 15, region q23-q24
(Fig. 3D). These findings along with the Southern analysis
indicate that the C5-epimerase is encoded by a single gene.
Expression of the C5-epimerase Transcript and Enzyme Activity in
Mouse Tissues--
Northern analysis of a mouse multiple tissue
Northern mRNA blot by hybridization with a 32P-labeled
cDNA probe corresponding to the C-terminal region of bovine
C5-epimerase revealed a single transcript of ~5 kb in all tissues
examined. The highest expression level was seen in liver, whereas small
amounts of transcript were seen in spleen (Fig. 4A). Yet epimerase activity in
spleen was similar to that in kidney or lung (Fig. 4B).
Although we have no explanation to this discrepancy, we note that two
other enzymes involved in the biosynthesis of HS,
N-deacetylase/N-sulfotransferase 1 and
2-O-sulfotransferase, showed similar low levels of mRNA
expression in spleen (23, 24). Mouse mastocytoma showed high epimerase
activity (Fig. 4B) and contained the same ~5-kb epimerase
transcript (11) as the other murine tissues.
Assessment of Epimerase Structure and Activity--
Previous
studies in our laboratory led to the isolation of a highly active
~52-kDa epimerase from bovine liver (16) and to the subsequent
cloning of a bovine lung cDNA (11). This cDNA encoded a protein
consisting of 444 amino acid residues. N-terminal analysis of the
purified liver enzyme showed that it lacked 73 amino acid residues
predicted from the cDNA and thus represented a truncated form. Yet
the recombinant protein, expressed in insect cells, had a catalytic
activity much lower than that of the purified liver enzyme. Although
this discrepancy could have several reasons, we noted that also the
cloned protein could be incomplete, since the 5'-end of the cDNA
open reading frame was not defined with certainty. With the gene
structure for the epimerase at hand it became possible to reassess this
question, by renewed cDNA isolation based on the genomic sequence
information. Appropriate oligonucleotide primers were therefore
designed based on the genomic sequences, as described under
"Experimental Procedures", and used for PCR amplification using
mouse liver cDNA as template. Agarose gel electrophoresis of the
PCR product revealed one strong band of ~2 kb in size, and this
product was directly inserted into a TOPTM-TA cloning
vector. Double-strand sequencing of six selected clones revealed an
epimerase cDNA encompassing the entire ORF in exon sequences of the
corresponding genomic DNA. The ORF of the 1854-bp sequence encodes a
protein of 618 amino acid residues, with a strongly hydrophobic domain
close to the N terminus of the deduced polypeptide (indicated in Fig.
5).
Mouse mast cell C5-epimerase cDNA was derived through the same
procedure as was used to clone the liver cDNA. The two sequences are identical (Fig. 6). Low stringency
PCR (annealing at lower temperatures with degenerate primers) using the
mast cell cDNA as template failed to reveal any additional, related
cDNA (data not shown). BLAST search yielded a cDNA sequence
from human brain (AB020643 in GenBankTM) with 93% identity
to the mouse C5-epimerase at the nucleotide level and 96% identity at
the amino acid level. The previously described incomplete bovine lung
epimerase cDNA structure was extended by cloning of the
missing 5'-terminal portion, revealing a total sequence highly similar
to those of the murine and human structures (90% identity to the mouse
sequence at the nucleotide level and 96% identity at the amino acid
level). The deduced amino acid sequences of epimerase from mouse liver,
mouse mast cells, bovine lung, and human brain are aligned in Fig.
6.
Following expression in insect cells, purification of the recombinant
mouse protein on a column of immobilized O-desulfated heparin yielded a product that migrated as a single ~70-kDa protein on SDS-PAGE (Fig. 7), thus in agreement
with the molecular mass calculated from the amino acid sequence (70,096 Da). By contrast, the purified bovine liver epimerase gave two bands
under the same reducing conditions, corresponding to ~52- and
~22-kDa polypeptides (Fig. 7). Tryptic digestion of the latter
component yielded a peptide (not shown) with an N-terminal sequence
identical to residues 119-131, common to all cloned epimerase species
(Fig. 6). We conclude that the ~22-kDa polypeptide represents a
cleavage product of the original enzyme that remained associated with
the ~52-kDa polypeptide throughout the purification
procedure.2
The recombinant full-length mouse epimerase, expressed in insect
cells, showed a catalytic activity of ~1 × 109 cpm
3H/h/mg protein, thus almost as high as that of the
purified bovine liver enzyme (9.6 × 109 cpm
3H/h/mg protein) (16), and much higher than that of the
recombinant (truncated) bovine lung enzyme (~1 × 106
3H cpm H/h/mg protein) previously reported (11).
The ability of the recombinant mouse epimerase to actually convert
GlcUA to IdoUA units was ascertained by incubation with a chemically
N-sulfated, metabolically radiolabeled K5 polysaccharide substrate. The product was depolymerized by treatment with
HNO2, and the resultant disaccharides were reduced,
purified, and analyzed by paper chromatography. About one-third of the
GlcUA residues had been converted to IdoUA (Fig.
8B), indicating equilibrium of
C5-epimerization (25), whereas GlcUA only was found in control incubations (Fig. 8A).
Heparin Production in Murine Mast Cells--
The murine mast cells
shown to express the epimerase gene were analyzed with regard to GAG
biosynthesis. Following metabolic labeling with
[35S]sulfate, polysaccharides were isolated and subjected
to anion-exchange chromatography (Fig.
9A). Part of the highly
retarded, labeled material was eliminated by digestion with
chondroitinase ABC (Fig. 9B), in accord with previous
findings of "oversulfated" chondroitin sulfate in similar cells
(26). The remaining, major polysaccharide component emerged at the same
elution position as commercial heparin and was susceptible to
degradation by nitrous acid (Fig. 9C), thus demonstrating
that the mast cells had indeed synthesized heparin. No significant
amounts of nitrous acid-sensitive material appeared at the elution
position expected for HS. These findings indicate that the GlcUA
C5-epimerase committed to heparin biosynthesis is the same as that
involved in the formation of HS in other cells.
The murine GlcUA C5-epimerase gene consists of 3 exons and spans
11 kb from the first ATG to the stop codon. The five genomic clones
obtained all relate to the same gene, as indicated by restriction enzyme mapping (not shown in detail; results outlined in Fig. 1).
Southern analysis of mouse genomic DNA by hybridization with a genomic
DNA probe detected a single band for the products of each restriction
enzyme applied (Fig. 2). Further evidence for a single gene encoding
the epimerase was obtained by FISH mapping of murine and human
chromosomes (Fig. 3). The human gene, located at chromosome 15, also
contains 3 exons (AC026992 in GenBankTM), which are similar
in size to those of the murine gene. Alignment of polypeptide sequences
deduced from the epimerase cDNAs isolated from bovine and murine
tissues and from the reported human brain cDNA (AB020643 in
GenBankTM) points to highly conserved structures (Fig. 6),
with more than 90% similarity between any two different species at the
nucleotide level and 96-99% similarity at the amino acid level.
Heparin is synthesized exclusively by mast cells, whereas HS is
produced by a large variety of cells. Whereas the two biosynthetic pathways involve basically similar reactions, the heparin chain is more
extensively modified. Recent findings indicate that several of the
polymer-modifying enzymes occur in various genetically distinct forms.
The N-deacetylase/N-sulfotransferase (NDST) that converts N-acetylated to N-sulfated glucosamine
units thus has been found in four variant forms, encoded by different
genes (7, 10, 27-29). Similarly, there are at least five distinct
glucosamine 3-O-sulfotransferases (8) and three
6-O-sulfotransferases (9). By contrast, only one hexuronyl
2-O-sulfotransferase has been reported so far, and this
enzyme has been demonstrated to catalyze the 2-O-sulfation
of both GlcUA and IdoUA residues (30, 31) (notably, an analogous
bifunctional 2-O-sulfotransferase has been implicated in
dermatan sulfate biosynthesis (32)). Results obtained after targeted
gene disruption of NDST-1 and NDST-2 suggest that these isoforms may be
preferentially committed to the biosynthesis of HS and heparin,
respectively. An NDST-2 knockout mouse thus was unable to synthesize
heparin and showed abnormal mast cells but seemed otherwise healthy and
produced apparently normal HS (33, 34), whereas elimination of NDST-1
resulted in a severely compromised HS biosynthesis and a lethal
phenotype (35, 36). The IdoUA units generated in the C5-epimerase
reaction occur in different structural contexts in heparin and HS. In
heparin the vast majority of such units are located between two
adjacent N-sulfated GlcN residues, whereas in HS about half
of the IdoUA units occur in domains composed of alternating
N-sulfated and N-acetylated GlcN residues (5). We
therefore anticipated also that the epimerase might present in
different forms, committed to the formation of IdoUA units in different
types of sequences. On the contrary, the results obtained in the
present study point to the involvement of the same epimerase enzyme in
heparin and HS biosynthesis. A single gene was implicated, as discussed
above. A single ~5-kb transcript was detected in all mouse tissues
analyzed, including mastocytoma (11). Moreover, identical cDNA
structures were expressed in a murine mast cell, found to produce
heparin but no significant amounts of HS, and in liver, known to
synthesize HS (37). In accord with these findings, epimerase
preparations derived from mouse mastocytoma and mouse liver showed
highly similar kinetic properties (38).
Cloning and expression of mouse liver cDNA, based on the epimerase
gene structure, yielded a protein composed of 618 amino acid residues,
thus considerably larger than either the recombinant bovine lung enzyme
(444 residues (11)) or the original preparation purified from bovine
liver (371 residues (16)). Given the structure of the full-length
protein reported here, these discrepant results can all be explained in
terms of the same epimerase gene. As shown in Fig.
10, the bovine lung cDNA thus was
incomplete, lacking a 5'-terminal sequence corresponding to 173 amino
acid residues, whereas the purified bovine liver enzyme was truncated
due to proteolytic cleavage. Interestingly, both the full-length
recombinant (Fig. 10C) and the truncated liver (Fig.
10A) enzymes showed essentially similar, high catalytic
activities, more than 2 orders of magnitude higher than that of the
recombinant, intermediate-sized, bovine lung enzyme (Fig.
10B). This intriguing observation relates to the fact that
the bovine liver enzyme apparently remained associated with the
truncated N-terminal ~22-kDa peptide throughout the isolation procedure. The purified bovine liver epimerase and the recombinant mouse liver enzyme thus are similar, except for a proteolytic cleavage
in the polypeptide, between residues Ser-246 and Lys-247 of the bovine
protein (Figs. 6 and 10). Since both of these forms are highly active
compared with the shorter, recombinant bovine lung epimerase, the
N-terminal domain of the native enzyme molecule would seem to be
essential for the generation of catalytic activity. This domain
contains the transmembrane region and, presumably, a lumenal stem
portion but may conceivably contribute directly to the catalytic
mechanism, irrespective of whether the 618-residue polypeptide is
intact or cleaved. Alternatively, the N-terminal portion may be
essential for proper folding of the protein but not for the catalytic
activity per se. So far we cannot discriminate between these
possibilities, since we have been unable to separate the ~52- and
~22-kDa components of the extracted bovine liver enzyme, except by
SDS-PAGE. The enzyme activity was not affected by inclusion of reducing
agents such as dithiothreitol in the assay medium. Furthermore, a
non-reduced sample of purified bovine epimerase showed the same two
~52- and ~22-kDa components on SDS-PAGE as were seen with reduced
samples (data not shown). These findings suggest that the two fragments
of the protein are tightly associated by non-covalent linkage. Attempts
to renature epimerase activity from single or combined fractions
following SDS-PAGE have been unsuccessful. The cleaved ~52-kDa
component was consistently seen in several different enzyme
preparations. Moreover, it was noted that >90% of the epimerase
activity solubilized in buffer containing 1% Triton X-100 (and
protease inhibitors) could be released from freshly homogenized bovine
liver in the absence of any added
detergent.3 These
observations raise the possibility that the proteolytic cleavage had
occurred in vivo and not during purification of the epimerase. Similar degradation was not observed in the insect cells
used to express the recombinant mouse liver enzyme (Fig. 7).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 M mercaptoethanol for 44 h. Human lymphocytes were isolated from blood and cultured in
-minimal essential medium supplemented with 10% FCS and
phytohemagglutinin at 37 °C for 68-72 h. Both lymphocyte cultures
were synchronized by treatment with bromodeoxyuridine (0.18 mg/ml). The
synchronized cells were washed three times with serum-free medium and
recultured at 37 °C for 4-6 h in
-minimal essential medium with
thymidine (2.5 µg/ml). The cells were harvested, and chromosome
slides were made by hypotonic treatment, fixation, and air drying.
1-4GlcNSO3
1-]n polysaccharide substrate, as described (11). Briefly, the partially epimerized polysaccharide was subjected to deaminative cleavage with nitrous acid
(pH 1.5 reaction) followed by reduction, and the resultant labeled
GlcUA-aManR (glucuronyl-2,5-anhydromannitol) and
IdoUA-aManR (iduronyl-2,5-anhydromannitol) disaccharides
were separated by paper chromatography and quantified by scintillation counting.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Diagrammatic representation of the mouse
C5-epimerase gene. A, alignment of the five genomic
clones studied. The restriction sites in clones 5a (EcoRI)
and 64 (SacI) are indicated by vertical lines.
B, organization of the gene. The coding sequences of the
exons are shown as black boxes, and the non-coding sequences
as shadowed boxes. Introns are represented by
horizontal lines. The intron fragment marked by *,
generated by digestion of clone 5a with EcoRI, was used as a
probe in Southern analysis (Fig. 2).
Intron-exon boundary sequences of the mouse C5-epimerase gene
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Fig. 2.
Autoradiogram of Southern analysis.
Mouse genomic DNA was digested with the restriction enzymes indicated,
and the products were separated by agarose gel electrophoresis and
blotted onto a Hybond N+ nylon transfer membrane. The
membrane was hybridized with a probe containing intron sequence (see
Fig. 1), under the conditions described under "Experimental
Procedures."
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Fig. 3.
Localization of the C5-epimerase gene in
murine and human chromosomes. The FISH experiment was performed as
described under "Experimental Procedures," using the full-length
mouse epimerase cDNA as probe. The epimerase gene is located at
murine chromosome 9 (A) and human chromosome 15 (B) identified by DAPI staining. The diagrams of FISH
mapping results show that the epimerase is localized to region
C and D in murine chromosome 9 (C) and
region q23-q24 in human chromosome 15 (D).
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Fig. 4.
Distribution of C5-epimerase mRNA and
enzyme activity in adult mouse tissues. A, a mouse
multiple tissue Northern blot was hybridized with bovine C5-epimerase
(upper panel) and -actin (lower panel)
cDNA probes labeled with [32P]dCTP. B,
C5-epimerase activity was measured in lysates of the mouse tissues
indicated (MCT, mastocytoma) and expressed in terms of
3H2O released from 5-3H-labeled
GlcUA residues (see "Experimental Procedures").
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Fig. 5.
Nucleotide sequence of C5-epimerase cDNA
from mouse liver and predicted amino acid sequence. The predicted
amino acid sequence is shown below the nucleotide sequence.
The numbers on the right indicate the coding
nucleotide residues and the amino acid residues (boldface
italic) in the respective sequence. The 5'-untranslated region is
shown in lowercase letters. The predicted transmembrane
region is underlined.
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Fig. 6.
Alignment of C5-epimerase amino acid
sequences. Deduced polypeptide structures based on cDNAs from
mouse liver, mouse mast cells (MC), human brain, and bovine
lung are aligned. The numbers indicate the amino acid
residues of the mouse proteins. Identical residues are boxed
and shaded. The dashes indicate missing amino
acids.
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Fig. 7.
SDS-PAGE of purified epimerase samples.
The recombinant mouse enzyme expressed in insect cells was prepared as
described under "Experimental Procedures," and bovine liver enzyme
was purified as described (16). About 100 ng of each preparation was
analyzed by SDS-PAGE and stained with silver. Lane 1, purified bovine enzyme; lane 2, recombinant mouse enzyme.
The protein molecular weight standards are indicated.
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Fig. 8.
Conversion of GlcUA to IdoUA by recombinant
mouse C5-epimerase. Metabolically C1-3H-labeled K5
polysaccharide was N-deacetylated and N-sulfated
and then incubated without (A) or with (B)
purified mouse recombinant enzyme. The incubation products were treated
with HNO2 and reduced with NaBH4, and the
resultant hexuronic acid (unspecified)-aManR disaccharides
were recovered and separated by paper chromatography. The migration
positions of GlcUA-aManR (GM) and
IdoUA-aManR (IM) disaccharide standards are
indicated by arrows.
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Fig. 9.
Anion-exchange chromatography of mast cell
polysaccharides. Cultured mast cells were metabolically labeled
with [35S]sulfate, and polysaccharides were isolated as
described under "Experimental Procedures." Samples were mixed with
unlabeled chondroitin sulfate and commercial heparin and analyzed by
chromatography on DEAE-Sephacel (start of gradient 0.05-1.5
M NaCl, indicated by arrow below C).
Effluent fractions were analyzed for radioactivity (filled
circles) or for hexuronic acid (shown in A only) by the
carbazole reaction (open circles). The peak elution
positions of standard chondroitin sulfate (CS; ~1
sulfate/disaccharide unit, thus not "oversulfated") and heparin
(Hep) are indicated by arrowheads in
A. Samples were untreated (A); digested with
chondroitinase ABC (B); or reacted with nitrous acid
(C).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Schematic representation of the three
C5-epimerase preparations studied. A, the
purified bovine liver enzyme (two peptides); B, the
previously cloned bovine epimerase (11); C, the
recombinant mouse full-length epimerase.
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ACKNOWLEDGEMENTS |
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We thank Lena Nylund for technical assistance and Lena Kjellén for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Swedish Medical Research Council Grant 2309, the European Commission Grants BIO4-CT95-0026 and QLK3-CT-1999-00536, and Polysackaridforskning AB (Uppsala, Sweden).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 GenBankTM/EMBL Data Bank with accession number(s) AF330049 and AF003927.
To whom correspondence should be addressed: Dept. of Medical
Biochemistry and Microbiology, University of Uppsala, the Biomedical Center, Box 582, SE-751 23 Uppsala, Sweden. Tel.: 46-18-4714241; Fax:
46-18-4714209; E-mail: jin-ping.li@imbim.uu.se.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M011783200
2 The ~22-kDa fragment was observed in the initial purification of the bovine liver enzyme but was not identified due to contamination with peptides from a leaky concanavalin A column (11).
3 J.-P. Li, unpublished information.
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ABBREVIATIONS |
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The abbreviations used are: HS, heparan sulfate; GlcUA, D-glucuronic acid; IdoUA, L-iduronic acid; GAG, glycosaminoglycan; FISH, fluorescence in situ hybridization; DAPI, 4',6-diamidino-2-phenylindole; aManR, 2,5-anhydromannitol (formed by reduction of terminal 2,5-anhydromannose residues with NaBH4); PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair; FCS, fetal calf serum; bp, base pair; ORF, open reading frame; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; NDST, N-deacetylase/N-sulfotransferase.
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