Identification and Expression in Mouse of Two Heparan Sulfate
Glucosaminyl N-Deacetylase/N-Sulfotransferase
Genes*
Marion
Kusche-Gullberg
§,
Inger
Eriksson¶,
Dagmar
Sandbäck
Pikas
, and
Lena
Kjellén¶
From the
Department of Medical Biochemistry and
Microbiology, University of Uppsala, S 751 23 Uppsala, and the
¶ Department of Veterinary Medical Chemistry, Swedish University
of Agricultural Sciences, S750 07 Uppsala, Sweden
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ABSTRACT |
The biosynthesis of heparan sulfate/heparin is a
complex process that requires the coordinate action of a number of
different enzymes. In close connection with polymerization of the
polysaccharide chain, the modification reactions are initiated by
N-deacetylation followed by N-sulfation of
N-acetylglucosamine units. These two reactions are carried
out by a single protein. Proteins with such dual activities were first
purified and cloned from rat liver and mouse mastocytoma. The mouse
mastocytoma enzyme is encoded by an ~;14-kilobase (kb)
mRNA, whereas the rat liver transcript contains ~;18
kb. In the present study, the primary structure of the enzyme encoded
by the mouse 8-kb transcript is described. It is demonstrated that both
the 4-and 8-kb transcripts have a wide tissue distribution and that
they are encoded by separate genes. Characterization of the gene
encoding the 4-kb transcript demonstrates that it spans a region of
about 8 kb and that it contains at least 14 exons. The similarity of
this gene and the previously characterized human gene for the 8-kb
transcript is discussed.
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INTRODUCTION |
Heparan sulfate (HS)1
proteoglycans occur on cell surfaces and in the extracellular matrix of
loose connective tissue and in basement membranes. In connective
tissue-type mast cells, the polysaccharide is stored intracellularly in
granules. The mast cell polysaccharide is traditionally referred to as
heparin but is composed of the same structural units as HS. HS has been
implicated in cell adhesion processes, cytokine action, regulation of
enzyme activities and in the maintenance of the permeability of
basement membranes (reviewed in Refs. 1-3). The polysaccharide chain
carries many negative charges that enables it to interact
electrostatically with a wide variety of molecules. However, some of
the interactions are highly specific (4-6).
During biosynthesis, the HS-chains are polymerized by sequential
addition of glucuronic acid and N-acetylglucosamine units. Concomitantly, the polymer is modified through a series of reactions that include N-deacetylation and N-sulfation of
glucosamine residues, epimerization of glucuronic acid to iduronic
acid, and finally O-sulfation at various positions (3, 6,
7). The extent of these reactions varies, giving rise to enormous
structural heterogeneity. Compared with HS, heparin is more highly
sulfated and has a higher content of N-sulfated glucosamine
and iduronic acid (3). The first modifying enzyme, a combined
N-deacetylase/N-sulfotransferase has a prime
regulatory role in determining the overall structure and charge density
of HS as N-deacetylation followed by N-sulfation are required for all subsequent modifications (1).
Proteins, with features typical of Golgi proteins, expressing
N-deacetylase/N-sulfotransferase activities have
been purified and cloned from a mouse mastocytoma (8-10) and from rat
liver (11-14). Recently, the cDNA sequence of a human counterpart
to the rat liver transcript (15) as well as the predicted structure of
the corresponding gene was published (16).
The enzyme purified from the mouse mastocytoma is encoded by an ~4-kb
mRNA while the rat liver transcript contains ~8 kb. The major
part of the protein sequences have ~70% identity. However, the most
N-terminal 80 amino acids show a low level of structural homology, with
approximately 30% identical amino acids. In addition, both the 5' and
the 3' noncoding regions seem unrelated (9, 10, 14). In this study, we
demonstrate that the two transcripts are widely distributed in mouse
tissues and that they are encoded by different genes. The structure of
the gene encoding the 4-kb transcript is reported and shown to share
many features with the previously characterized human gene for the 8-kb
transcript.
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EXPERIMENTAL PROCEDURES |
Southern Blot Analysis--
Isolated genomic DNA from adult
mouse liver was digested with XbaI, HindIII,
EcoRI, and BglII. Thirty-five µg of genomic DNA was used for each digest. The samples were fractionated by 1% agarose
gel electrophoresis in 0.9 M Tris, 0.9 M boric
acid, 0.020 M EDTA. After electrophoresis, the gel was
soaked in 0.4 M NaOH for 30 min before transfer to nylon
membrane (Hybond-N+, Amersham Pharmacia Biotech). The
membranes were hybridized overnight at 65 °C with
32P-labeled probes in 5× SSC (SSC is 0.15 M
NaCl, 0.015 M sodium citrate buffer, pH 7.0), 5×
Denhardt's solution, 2% SDS containing 100 µg/ml salmon sperm DNA,
and washed in 0.1× SSC, 0.5% SDS at 65 °C. Four different cDNA
probes, labeled with [
-32P]dCTP (NEN Life Science
Products) in the random primed DNA labeling kit (Boehringer Mannheim),
were used for hybridization. Probes A and C recognize the coding and
3'-untranslated region, respectively, of the 4-kb transcript, whereas
probes B and D hybridize with the coding and 3'-untranslated region of
the 8-kb transcript. Probe A is a HinfI cDNA fragment of
the 4-kb mouse transcript (nt 1412-1900 in Fig. 1 of Ref. 10). Probe B
was generated by RT-PCR of rat liver RNA using the same conditions as
described for the 504-bp PCR-product (10), with a sense and an
antisense primer corresponding to nt 1639-1660 and 2025-2004,
respectively, in the rat liver cDNA-sequence (14). To the 5'-end of
the primers was added a dCCGAATTC extension to create an
EcoRI site in the PCR-product. Probe C is a 356-bp
cDNA-fragment, corresponding to the 3'noncoding end of the 4 kb
mouse mastocytoma transcript (nt 2951-3307; Ref. 10). Probe D is a
HinfI fragment of the 504-bp PCR product generated by RT-PCR
of rat liver RNA (10), corresponding to nt 3396-3679 in Ref. 14.
RNA Purification--
Hepatocytes were isolated after
collagenase perfusion of a rat liver and purified by density gradient
centrifugation in Percoll (17). Total RNA was isolated from these cells
and from mouse mastocytoma (18) and mouse liver tissue using the
LiCl/urea/SDS method (19).
Northern Blot Analysis--
A single filter containing
poly(A)-selected RNA from several adult mouse tissues
(CLONTECH) was hybridized at 65 °C in 5× SSPE
(1× SSPE is 0.15 M NaCl, 10 mM
NaH2PO4, 1 mM EDTA, pH 7.4), 10×
Denhardt's solution, 2% SDS, 100 µg/ml salmon sperm DNA, at three
occasions with probes labeled with [
-32P]dCTP (NEN
Life Science Products) as described above. The probes used were a
356-bp cDNA-fragment, identical to the 356-bp cDNA fragment
(Probe C) used in Southern blotting (see above), a full-length cDNA
corresponding to an mRNA encoding a protease specific for connective tissue-type mast cells, kindly provided by Dr. Lars Hellman,
University of Uppsala (MMCP-4; Ref. 20), and a 2-kb cDNA
recognizing
-actin mRNA (CLONTECH).
A similar filter was hybridized, as described above, to a 216-bp
fragment that had been amplified from mouse liver RNA by RT-PCR using
primers corresponding to nt 2103-2123 (sense) and nt 2318-2298
(antisense), respectively, in the nucleotide sequence of the 8 kb rat
liver transcript, (14). The identity of the amplified product was
established by nucleotide sequence analysis, using
[
-35S]dATP and the Sequenase kit (U. S. Biochemical
Corp.).
Cloning and cDNA Analysis of Mouse Liver 8-kb
Transcript--
An RT-PCR-based strategy was employed to clone the
mouse homologue of the rat liver
N-deacetylase/N-sulfotransferase. Primers were
selected in regions corresponding to conserved sequences in the rat
(14) and the human (15) transcripts. One pair of primers (1)
corresponding to nt 1-18 (sense) and 1199-1182 (antisense),
respectively, in the rat liver cDNA-sequence (14) were used to
amplify the 5' half of the cDNA. An overlapping clone extending to
the 3'-untranslated region was obtained using two primers
(2) corresponding to nt 1147-1163 (sense) and nt 2717-2700
(antisense), respectively, in Ref. 14. Random hexamer primed cDNA
was synthesized from 1 µg of mouse liver total RNA using the GeneAmp
RNA PCR kit (Perkin-Elmer). The PCR reaction for primer pairs
1 was performed under conditions of: 1 cycle of 94 °C for
1 min, 30 cycles each of 94 °C for 15 s, 50 °C for 15 s, 72 °C for 1 min, and a final extension at 72 °C for 2 min. The
amplified 1199-bp fragment was subcloned into the pUC 119 vector and
sequenced. Conditions used to amplify the second part of the cDNA
using primer pairs 2 were: 1 cycle of 94 °C for 1 min, 35 cycles each of 94 °C for 20 s, 45 °C for 20 s, 72 °C
for 2 min plus 1 s for each cycle, and a final extension at
72 °C for 5 min. The amplified product (1571 bp) was subcloned into
the pCR II vector (Invitrogen) and sequenced. The sequence identities
between the mouse liver and rat liver 8-kb transcript are 96% for both
the 1199 and 1571-bp fragments (EBI/GenBankTM accession number
AF049894). To obtain a full-length clone, a unique NdeI site
at nt 1178 was used. The insert in the pCR II plasmid was excised with
NdeI and EcoRI and ligated into the same site in
the pUC vector containing the PCR product of primer pairs 1.
The sequence around the NdeI site in the resulting full-length clone was verified by nucleotide sequence analysis as
described above, using synthetic oligonucleotide primers. Sequences were analyzed with the aid of the Lasergene software package (DNAStar Inc., Madison).
Isolation of Genomic Clones--
A mouse
FIX II genomic
library (Stratagene, La Jolla, CA) was screened using as probes a
mixture of a 1.7-kb cDNA fragment (10), corresponding to a region
entirely within the coding region of the 4-kb transcript, and "probe
B" (see above), corresponding to 366 nt of the coding region of the
rat 8-kb transcript. The probes were labeled with
[
-32P]dCTP (NEN Life Science Products) as described
above. About 1× 106 plaques were screened, and
approximately 60 clones were plaque purified. Of those, two different
clones for the 4-kb transcript and 4 different clones for the 8-kb
transcript were obtained. The genomic clones were further analyzed by
restriction mapping and Southern blotting using as probes specific
cDNA oligonucleotide primers (17 nt) that had previously been used
for nucleotide sequence analysis of the mouse mastocytoma 4-kb cDNA
(10). Genomic DNA fragments obtained from the phage DNA after digestion
with SacI and/or BamHI were subcloned into the
plasmid vector pUC 119 for characterization. The gene encoding the 4-kb
transcript was further characterized, using other restriction fragments
of the
clones that were subcloned into pUC 119 and sequenced. The
size of each intron was determined by nucleotide sequence analysis.
RNA Expression Analysis Using RT-PCR--
One µg of total RNA
from each tissue in a total volume of 20 µl was used for the
generation of single-stranded cDNA in the GeneAmp RNA PCR kit.
After dilution to 40 µl, 1 µl of each sample was used for PCR in a
Rapidcycler (Idaho Technology), with Taq DNA polymerase
(MBI-Fermenta) and TaqStart Antibody (CLONTECH). The sense primer corresponds to nt 1301-1317 in the sequence of the
mouse mastocytoma 4-kb transcript (10), identical to nt 1593-1609 in
the sequence of the rat liver 8-kb transcript (14). Also, the antisense
primer corresponds to identical regions in the mouse 4-kb (nt
2299-2282; Ref. 10) and in the rat 8-kb transcript (nt 2594-2577;
Ref. 14). The 10 µl reactions were done in heat-sealed glass
capillaries in 50 mM Tris buffer, pH 8.3, containing 3 mM MgCl2 and 0.25 mg/ml bovine serum albumin.
After an initial hold for 2 min at 95 °C, each cycle included
denaturation at 95 °C for 0 s, annealing at 56 °C for 0 s, and extension at 72 °C for 30 s. After 20, 25, or 30 cycles,
the capillaries were emptied into Eppendorf tubes, and to the tubes
were added 10 µl of "restriction enzyme mix, " containing 4 units
of EcoRI or HindIII, 50 ng of pUC 119, 20 µg/ml
bovine serum albumin in the appropriate restriction enzyme buffer
(MBI-Fermenta). After incubation for 2 h at 37 °C and addition
of 4 µl of sample dye, 20 µl of the samples were electrophoresed in
1.5% agarose gels. Samples subjected to 30 PCR cycles and subsequently
incubated without any restriction enzyme were also analyzed. The
plasmid vector pUC 119 served as a control of complete restriction
enzyme cleavage, which was checked after ethidium bromide staining of
the gel. After blotting to Hybond-N+, the nylon membrane
was pre-hybridized at 42 °C for 30 min in ExpressHyb hybridization
solution (CLONTECH) and incubated in the same
solution at 42 °C for 1 h with a 32P-labeled
oligonucleotide IE 16 (dAACTATGGAAATGACCG). This sequence is found in
both transcripts from rat and mouse, and hybridizes with the intact rat
and mouse 1-kb products as well as the 829-nt EcoRI and the
668-nt HindIII fragment generated from the 1-kb band
corresponding to the rat and mouse 8-kb transcript, respectively. The
membrane was finally washed with 2× SSC, 0.05% SDS twice for 10 min
at 42 °C, before exposure to x-ray film.
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RESULTS |
Southern blots of genomic DNA isolated from mouse liver was
hybridized with 32P-labeled cDNA-probes from the coding
and from the 3'-untranslated regions of the 4-kb mouse and the 8-kb rat
transcript, respectively (Fig. 1). While
the coding regions of the 4-kb mouse and the 8-kb rat transcript are
fairly similar (~70% identity at the amino acid level), the
untranslated regions are unrelated (9, 10). The hybridization with
mouse DNA of the probe corresponding to the 3'-untranslated region of
the 8-kb rat transcript (Fig. 1D) clearly demonstrates that
also the mouse genome contains sequence information for this
transcript. The cDNA probe corresponding to the coding region of
the 8-kb transcript, hybridized to other genomic fragments (Fig.
1B), demonstrating the presence of restriction sites for
XbaI, HindIII, EcoRI, and
BglII in this gene. Notably, the hybridization pattern was
distinct from that obtained with the probes corresponding to the 4-kb
transcript (Fig. 1, A and C), indicating that the
two transcripts are encoded by different genes.

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Fig. 1.
Southern blot analysis of mouse genomic
DNA. Mouse genomic DNA was cut with XbaI
(X), HindIII (H), EcoRI
(E), and BglII (B). The resulting
fragments were separated on a 1% agarose gel. After blotting, the
filters were hybridized with probes recognizing the coding and
3'-untranslated region of the 4-kb transcript (panels A and
C, respectively) and with probes hybridizing with the coding
and 3'-untranslated region of the 8-kb transcript (panels B
and D).
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The expression of the 8-kb transcript in mouse liver was established
using RT-PCR followed by nucleotide sequence analysis of the isolated
clones. The nucleotide sequence of the coding region (GenBankTM
accession number, AF049894) was highly homologous to that of the
corresponding region in the rat 8-kb transcript (96% identity). On the
protein level, only a few amino acids differed between the two species
(Fig. 2). According to Northern blotting of different mouse tissues, the 8-kb transcript was widely distributed, with lung, liver, and kidney containing the highest amounts (Fig. 3).

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Fig. 2.
Alignment of the
N-deacetylase/N-sulfotransferase amino acid
sequences. Deduced amino acid sequence of mouse liver
(ML) N-deacetylase/N-sulfotransferase
8-kb transcript and alignment with mouse mastocytoma (MM)
4-kb and rat liver (RL) 8-kb transcript homologues. The
numbers indicate the amino acid residues of the proteins.
All residues in the sequence corresponding to the 8-kb mouse liver
transcript (ML 8 kb), as well as identical residues in
homologs, are boxed. Positions where amino acids are
identical in two of the sequences are shaded. Gaps
introduced to optimize alignment, using the Clustal algorithm with
PAM250 residue weight table (Lasergene software, DNAStar), are shown as
dashes ( ).
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Fig. 3.
Distribution of the 8-kb
N-deacetylase/N-sulfotransferase mRNA in
adult mouse tissues. A mouse multiple tissue Northern blot was
hybridized with a 216-bp 32P-labeled probe recognizing the
coding region of the 8 kb transcript (A) and a 2-kb cDNA
recognizing -actin (B). The filter was washed at 50 °C
in 0.1× SSC, 0.1% SDS. Sizes of RNA-markers in kilobases are
indicated.
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Isolation of Two Mouse N-Deacetylase/N-Sulfotransferase
Genes--
To isolate genomic clones containing the
N-deacetylase/N-sulfotransferase genes, two
cDNA probes corresponding to parts of the coding region of the
mastocytoma and the rat liver cDNA transcripts, receptively (see
"Experimental Procedures"), were mixed and used for screening of a
129/sv mouse genomic DNA library. Several positives clones were
isolated and subsequently identified by Southern hybridization with
cDNA fragments and oligonucleotides specific for the 4 and the 8-kb
transcript, respectively (see "Experimental Procedures"). Two
clones, 2:3 and 4:3 in Fig.
4, were derived from the gene encoding
the 4-kb transcript, but these clones did not cover the 5'-part of the
gene. An ~400-bp SacI fragment from the most 5'-terminal region of the 4:3 genomic clone was therefore used as a
probe in another screening of the library. This approach yielded two more clones (8:2 and 10:1). Together with clones
2:3 and 4:3, approximately 30 kb of genomic
sequence were now covered (Fig. 4).

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Fig. 4.
Schematic representation of the organization
of the mouse gene encoding the 4-kb
N-deacetylase/N-sulfotransferase
transcript. The four genomic clones used in determining the
sequence of the N-deacetylase/N-sulfotransferase
gene are shown (top). The clones covered together
approximately 30 kb extending in the 3' direction (- - -). Below is a
schematic view of the gene where exons are represented by
boxes while horizontal lines depict introns. The
portions of the exons that contain protein coding sequences are
filled. The box representing the left-most exon
is open to indicate that the 5'-boundary of this exon has
not been localized.
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Four of the clones identified in the initial screening were fragments
of the gene encoding the 8 kb transcript. Characterization of these
clones are in progress and will be reported in a separate communication.
Structure of the Gene Encoding the 4-kb Transcript--
A crude
map of the gene was constructed based on hybridization of restriction
fragments with oligonucleotides, previously used as specific primers in
nucleotide sequence analysis of cDNA corresponding to the mouse
mastocytoma 4-kb transcript (10). Genomic fragments generated by
digestion of the clones with SacI and/or BamHI
were subcloned into pUC 119. The exon-intron organization and the size
of exons and introns were determined by nucleotide sequence analyses of
these fragments. The data available suggest that the mouse 4-kb
N-deacetylase/N-sulfotransferase gene consists of
14 exons distributed within 8 kb of genomic DNA. However, it cannot be
excluded that additional introns may exist in the extreme 5'- and
3'-ends of the gene. The exons contain between 88 and 1322 nucleotides,
and all of the donor and acceptor splicing sites agree to the consensus
sequences for eukaryotic genes (21). These data are summarized in Fig.
4 and in Table I. Exon 1 contains part of
the 5'-untranslated region, whereas the remainder of the 5'-untranslated sequence is located in exon 2, which contains the
translation start site. This exon, which is much larger than any of the
other exons, also encodes both the short N-terminal cytoplasmatic
domain and the transmembrane part of the protein in addition to a large
portion of the luminal domain of the
N-deacetylase/N-sulfotransferase. While most of
the intron junctions are of the 0 type (splicing occurs between
codons), introns 3 and 8 are type 1 introns (where splicing occurs
after the first base of the codon). Introns 9 and 12, finally, are of
type 2 (splicing occurs after the second base of the codon).
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Table I
Exon-intron organization of the mouse 4-kb
N-deacetylase/N-sulfotransferase gene
The exons are numbered starting at the 5' end of the gene. Exon
sequences are in capital letters; intron sequences are in lowercase
letters. The amino acids corresponding to the exon sequence are shown
immediately below the nucleotide sequence.
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Expression of the 4-kb N-Deacetylase/N-Sulfotransferase
Transcript--
It has generally been assumed that the 4-kb transcript
encodes the N-deacetylase/N-sulfotransferase
involved in heparin biosynthesis and thus that this transcript is
confined to mast cells. We previously noted a low expression of the
4-kb transcript in rat liver (10). Since mast cells in the liver
capsule may have been the source of this transcript, we decided to look
for the presence of the 4-kb transcript in purified hepatocytes using a
PCR approach. In this method, a primer pair is used that will amplify a
1-kb band from cDNA generated by reverse transcription of either of the 4 or 8-kb transcripts2.
(The nucleotide sequence of the 4- and 8-kb transcripts is identical in
the regions where the primers anneal.) After the PCR, the 1-kb band is
incubated with a restriction enzyme that cleaves the band derived from
the 8-kb transcript but leaves the band generated from the 4-kb
transcript intact. For RNA derived from rat tissues, EcoRI
is used, while HindIII cleaves the 1-kb band generated from the mouse 8-kb transcript (See Fig. 5
where exon 8 of the gene encoding the 4-kb transcript is compared with
the corresponding exon in the mouse gene for the 8-kb transcript.). As
shown in Fig. 6, most of the 1-kb PCR
product obtained using rat hepatocyte cDNA as template is cleaved
by EcoRI, but small amounts of the 4-kb transcript are
present in these cells, represented by the weak intact 1-kb band (Fig.
6A). Mouse liver, in contrast, appears to contain similar
amounts of the 4- and 8-kb transcripts (Fig. 6B), and mouse
mastocytoma contains almost exclusively the 4-kb transcript (Fig.
6C). Small amounts of the HindIII cleavage
product, not evident in the figure, can be detected after additional
PCR cycles (data not shown). In Fig. 6, the large difference in
expression of N-deacetylase/N-sulfotransferase
mRNA in mastocytoma and liver is also evident.

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Fig. 5.
Comparison of the nucleotide sequence of
corresponding exons in the mouse genes encoding the 4- and 8-kb
transcripts. Exon sequences are in capital letters,
while intron sequences are in lowercase letters. The
HindIII site, present in the exon from the gene encoding the
8-kb transcript but absent from exon 8 of the gene encoding the 4-kb
transcript, is underlined. y = t or
c.
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Fig. 6.
Expression of 4- and 8-kb transcripts in rat
hepatocytes, mouse liver, and mouse mastocytoma. After reverse
transcription of total RNA, PCR was performed with primers selected to
give a 1-kb band when cDNA corresponding to both 4- and 8-kb
transcripts from either murine or rat tissues was used as template (see
"Experimental Procedures"). After 20, 25, and 30 cycles, the
samples were incubated with EcoRI (rat hepatocyte samples)
or HindIII (mouse mastocytoma and liver samples). This
treatment selectively cleaves the 1-kb band corresponding to the 8-kb
transcript. The enzyme-treated samples, uncleaved controls, and size
markers were electrophoresed in 1.5% agarose and subsequently blotted
to nylon membrane. After hybridization with a 32P-labeled
oligonucleotide, recognizing intact mouse and rat 1-kb product as well
as the 829-nt EcoRI and 668-nt HindIII fragments,
the membrane was washed and exposed to x-ray film.
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The distribution of the 4-kb transcript in different adult mouse
tissues was also studied by Northern blotting using a probe specific
for the 4-kb transcript (Fig.
7A). All tissues examined expressed the transcript, testis, liver, and kidney containing the
highest amounts of the mRNA (Fig. 7A). The distribution
of an mRNA encoding a protease specific for connective tissue-type mast cells (MMCP-4; Ref. 20) was different, skeletal muscle containing
considerable amounts of the transcript while lower levels could be
detected in heart, spleen, and lung (Fig. 7B). From these results, it
can be concluded that expression of the 4-kb
N-deacetylase/N-sulfotransferase transcript is
not restricted to mast cells.

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Fig. 7.
Distribution of the 4-kb
N-deacetylase/N-sulfotransferase mRNA in
adult mouse tissues. A mouse multiple tissue Northern blot was
hybridized with 32P-labeled probes (see "Experimental
Procedures") recognizing the 3' noncoding region of the 4-kb
transcript (the filter was washed at 50 °C in 2× SSC, 0.05% SDS)
(A), mRNA for the mast cell-specific protease MMCP-4
(the filter was washed at 50 °C in 1× SSC, 0.05% SDS)
(B), -actin mRNA (the filter was washed at 65 °C
in 0.1 × SSC, 0.05% SDS) (C). Sizes of RNA-markers in
kilobases are indicated.
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DISCUSSION |
Southern blotting of mouse genomic DNA (Fig. 1) showed that the
two different N-deacetylase/N-sulfotransferase
enzymes, encoded by the 4 and 8-kb transcript, respectively, originate
from related but distinct genes. Nucleotide sequence analysis of
cDNA corresponding to the 8 kb mRNA (Fig. 2), established that
mice also express this transcript. The nucleotide sequence of the rat
and human transcripts have previously been published (14, 15). The
mouse protein is very similar to its rat counterpart with 99% identity on the protein level (Fig. 2). As demonstrated by Northern blotting, the 8-kb transcript is widely distributed and was detected in all
tissues analyzed (Fig. 3).
The gene encoding the human 8-kb transcript has previously been
characterized (16). Characterization of the mouse gene encoding the
4-kb transcript, which spans at least 7.4 kb and is split into 14 exons
(Fig. 4), revealed a high resemblance between the two genes. The exons
have the same size, except for exons 1, 2, and 14. In addition, the
exon-intron phases from exon 2 and downstream are of the same type
(eight type 0, two type 1 and two type 2 boundaries). The structural
similarities between these genes strongly suggest that they have
evolved from a common ancestral gene. However, some differences between
the two genes may be noted. 1) The introns of the human gene encoding
the 8-kb transcript are much larger (16). This gene was estimated to
span approximately 35 kb of genomic DNA. 2) In the murine gene encoding
the 4-kb transcript, the initiation codon is found in the large exon 2, which contains 1005 nucleotides of coding sequence, while the
corresponding region in the other gene (1008 nucleotides) is divided
between the two first
exons.3 3) In
the human gene, the splice donor site of exon 8 is a GC rather than the
expected GT.3 This rare variant was not found in the murine
gene encoding the 4-kb transcript. 4) Exon 14 in the mouse gene has 123 nucleotides of coding region, whereas the corresponding exon in the
human gene contains 117 nucleotides. While so far only two different genes encoding glucosaminyl
N-deacetylase/N-sulfotransferases have been
identified, we cannot exclude the presence of additional genes.
It was previously suggested that the enzyme encoded by the 4-kb
transcript, first recognized in mouse mastocytoma (8), was restricted
to connective tissue mast cells and to the biosynthesis of heparin (9,
10). However, the tissue distribution of the 4-kb
N-deacetylase/N-sulfotransferase mRNA
reported in this paper (Figs. 6 and 7), demonstrates that this
transcript is widely distributed and that it is present in cells other
than mast cells. Accordingly, the enzyme encoded by the 4-kb transcript
appears to take part both in heparin and heparan sulfate biosynthesis.
The high level of expression of the 4-kb transcript in mastocytoma
compared with its expression in e.g. liver (Fig. 6), may be
the reason why this transcript was not previously recognized in other
cells (9). As indicated by overexpression of the enzymes in
vitro (12, 22), the two enzymes may have different catalytic
properties; transfection of cDNA corresponding to the 4-kb
transcript in the human kidney cell-line 293 resulted in a dramatic
increase in N-sulfation of the heparan sulfate produced by
the cells (22), while overexpression in COS cells of cDNA for the
8-kb mRNA did not cause any significant change in the overall
N-sulfation of the polysaccharide (12). The enzyme encoded
by the 4-kb transcript thus appears to participate in the production of
HS with higher N-sulfate content. Tentatively, different
locations of the 4- and 8-kb transcripts in a certain tissue may result
in a local variation of HS structure. Such local variation of HS
structure was recently reported for kidney, using a monoclonal antibody recognizing N-unsubstituted glucosamine residues, found in
HS in the glomerular basement membrane but not in the basement
membranes of the tubules (23). These results imply that the fine
structure of heparan sulfate may be carefully regulated. Expression of
the 4- or 8-kb transcript may be one of the means for the cell to control the structure of the heparan sulfate produced, which in turn
will be important for the ability of the proteoglycan for functionally important interactions with effector molecules
such as cytokines, enzymes, and enzyme inhibitors (4, 6).
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ACKNOWLEDGEMENT |
We thank Britt-Marie Fogelholm for excellent
technical assistance.
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FOOTNOTES |
*
This work was supported by grants from the Swedish Medical
Research Council (6525 and 12074). Konung Gustav V:s 80-årsfond, Magnus Bergvalls stiftelse, Stiftelsen Lars Hiertas minne,
Polysackaridforskning AB (Uppsala, Sweden), and the European Commission
Contract No. B104-CT95-0026.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) AF049894.
§
To whom correspondence should be addressed: Dept. of Medical
Biochemistry and Microbiology, University of Uppsala, The Biomedical Center, Box 575, S-751 23, Uppsala, Sweden. Fax: 46-18-471 42 09; E-mail: Marion.Kusche{at}medkem.uu.se.
1
The abbreviations used are: HS, heparan
sulfate; bp, base pair(s); kb, kilobase pair(s); nt, nucleotide(s);
RT-PCR, reverse transcriptase-polymerase chain reaction; SSPE,
saline/sodium phosphate/EDTA.
2
The nucleotide sequence of the four different
PCR products corresponding to the 4- and 8-kb transcripts in rat and
mouse have been determined. A unique EcoRI site is found in
the 1-kb band generated from the rat 8-kb transcript, whereas the mouse
8-kb mouse transcript contains a unique HindIII site.
3
In the partly characterized murine gene encoding
the 8-kb transcript (Inger Eriksson, Marion Kusche Gullberg, Hanna
Wlad, Rikard Pehrson, and Lena Kjellén, work in progress), exons
1 and 2 are organized in the same way as in the human counterpart. Also, the sequence of the splice donor site of intron 8 (GC instead of
GT) is conserved (see Fig. 5).
 |
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