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J Biol Chem, Vol. 274, Issue 26, 18667-18674, June 25, 1999
,From the Laboratory for Glycobiology and Developmental Genetics, Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, B-3000 Leuven, Belgium
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We have identified a cDNA that encodes a
variant form of murine syndecan-1. The variant cDNA lacks the
sequence corresponding to the first 132 nucleotides of the third exon
of the syndecan-1 gene. The corresponding message is rare. The
alternative splice respects the reading frame and deletes 44 amino
acids from the protein, joining the
S45GS47GT sequence to a variant immediate
downstream context. This sequence context initiates with alanine
instead of glycine as residue 50, reducing the number of
SGXG sequence motifs in the protein from two to one.
Expression of this variant syndecan-1 in Madin-Darby canine kidney or
MOLT-4 cells yielded a recombinant proteoglycan with a reduced number
and clustering of the heparan sulfate chains. Both the conversions of
Ala50 and of Lys53 into glycine enhanced the
heparan sulfate substitution of the variant protein. These findings
support the concept that serine-glycine dipeptide signals for
glycosaminoglycan/heparan sulfate synthesis depend on sequence context
(Zhang, L., David, G., and Esko, J. D. (1995) J. Biol.
Chem. 270, 27127-27135) and imply that alternative splicing
mechanisms may in part control the molecular polymorphism of syndecan-1
and, therefore, the efficiency and versatility of this protein in its
co-receptor functions.
Syndecan-1 is a transmembrane heparan sulfate proteoglycan that is
subject to strong developmental regulations (1). The protein is
expressed in spatiotemporal- and cell type-specific patterns and shows,
in addition, tissue- and cell-specific differences in glycosylation.
This variable glycosylation pertains to the number, size, and fine
structure of the glycosaminoglycan chains that substitute the protein
(glycanation) and yields a highly polymorphic proteoglycan (2, 3). The
mechanisms that regulate this variation are not fully understood, but
the differences in glycanation can change the binding properties of the
proteoglycan (3, 4). Since heparan sulfate proteoglycans, and the
syndecans in particular, are thought to function as co-receptors for
various ligands that control cell shape, adhesion, proliferation, and differentiation, these differences in glycanation could be
developmentally significant (1).
Analysis of the glycanation of syndecan-1 from mouse mammary epithelial
cells has indicated that substitution with heparan sulfate is limited
to serines in the N-terminal half of the protein (5). Studies, in
Chinese hamster ovary cells, of recombinant chimeric proteoglycans that
contained various segments of syndecan-1, and mutations of these
segments have identified three serines, occurring in the sequence
DGS35GDDSDNFS45GS47GTG, as the
sites that prime heparan sulfate synthesis in this protein (6). The
strongest sites for heparan sulfate synthesis in these experiments
consisted of the repeat unit S45GS47G, and
evidence was obtained for a coupling phenomenon across adjacent Ser-Gly
dipeptides that enhances the substitution of these repeats with heparan
sulfate. The same studies demonstrated that the synthesis of heparan
sulfate on these serines also depended on adjacent clusters of acidic
residues and identified the sequence (Ser-Gly)n (n
We now report a variant cDNA for murine syndecan-1 that likely
results from the use of an alternative splice acceptor site. The
alternative splicing process skips part of the third exon but respects
the reading frame and, upon translation, is predicted to bring the
S45GS47GT sequence in a variant immediate
downstream sequence context. Expression of this syndecan-1 cDNA in
MDCK1 and MOLT-4 cells
indicates that, in comparison to the more common or "standard" form
of this protein, the variant protein is less substituted with heparan
sulfate and that the clustering of the heparan sulfate chains is
disrupted in this proteoglycan. Conversion of the
S45GS47GTA sequence in the splice
variant into S45GS47GTG enhances the
glycosaminoglycan substitution of this protein and restores the
clustering of the heparan sulfate chains in the proteoglycan. These
findings support the concept that the strengths of Ser-Gly dipeptides
as signals for heparan sulfate synthesis depend on sequence context and
imply that alternative splicing mechanisms may in part control the
molecular polymorphism of syndecan-1 and the efficiency and versatility
of this protein in its co-receptor functions.
Isolation of a Variant Syndecan-1
cDNA--
Poly(A)+ RNA was isolated from whole
14-day-old NMRI mouse embryos, annealed to oligo(dT), and used as
template for the synthesis of cDNA by Moloney murine leukemia virus
reverse transcriptase (10). Sequences coding for mouse syndecan-1 were
amplified from this cDNA by PCR. The PCR mixture contained 2 units
of Taq DNA polymerase, 1× Taq buffer (16.6 mM ammonium sulfate, 67 mM Tris/HCl, pH 8.8, 6.7 mM MgCl2, 10 mM
Expression of the Variant Syndecan-1 mRNA--
The
expression of the variant mouse syndecan-1 message was analyzed by a
ribonuclease protection assay kit (Ambion Inc., Austin, TX).
[32P]UTP-labeled antisense RNA complementary to the
standard mouse syndecan-1 message was synthesized with SP6 RNA
polymerase. The template was a SalI-linearized pGEM3Z
plasmid that contained a 427-bp PstI-SphI
fragment from the MoSyn-1/6 insert (spanning from residue 288 to 714 of
the sequence published by Saunders et al. (11)). The
antisense probe (of a predicted size of 446 nt) was hybridized to 10 µg of total RNA isolated from various mouse tissues, digested with
RNase, and sized in a sequencing gel. A 314-nt antisense RNA probe
complementary to the variant message was synthesized from a pGEM3Z
plasmid that contained a 295-bp PstI-SphI
fragment from the MoSyn-1/7 insert.
Construction of the Expression Vectors and Transfectant Cell
Lines--
To provide the cDNAs with Kozak sequences, the segments
spanning from the PstI to the StyI restriction
sites of the MoSyn-1/7 and MoSyn-1/6 inserts were exchanged for the
corresponding segments of a full-length cDNA coding for human
syndecan-1, cloned in pBluescript SK Characterization of the Syn-1s and Syn-1v
Proteoglycan--
Transfectant cells were extracted with Triton X-100
buffer, and proteoglycan was purified from the extracts by absorption on DEAE-Trisacryl M (13). Recombinant syndecan-1 eluted from DEAE was
analyzed by SDS-polyacrylamide gradient or 4% Nusieve agarose-gel
electrophoresis and Western blotting, directly or after separation from
endogenous proteoglycan by immunopurification on mAb 281-2 (PharMingen,
San Diego, CA). Before electrophoresis, the proteoglycan samples were
treated with heparitinase, chondroitinase ABC, both enzymes, or left
untreated (13). The blots were stained with the mouse
syndecan-1-specific mAb 281-2 (14), mAb 2E9 (specific for the
cytoplasmic domains of the syndecans 1 and 3) (12), or with the
Immunofluorescence Cytometry--
Transfectant MOLT-4 cells were
analyzed for cell-surface expression of syndecan-1 protein and heparan
sulfate by flow immunocytometry, using the antibodies 281-2, 10E4, and
3G10 as described before (16). The analyses were performed on a FACSort
(Becton Dickinson, Mountain View, CA), and data were analyzed with the
program Lysis II.
Cell Binding Assay--
Cell binding to type I collagen from rat
tails (Roche Molecular Biochemicals, Mannheim, Germany) was performed
in polyvinyl 96-well U-bottom plates (Corning Costar Europe,
Badhoevedorp, Netherlands), using a modified version of the method of
Koda et al. (17). Collagen coats were made by overnight
incubation at 4 °C (100-300 µg of collagen in 1 ml of a 2:1 (v/v)
mixture of 10 mM acetic acid/PBS, pH 4.4, per well). After
removal of the non-absorbed collagen solution, the wells were rinsed
twice with 0.3% BSA (Sigma, Bornem, Belgium) in Dulbecco's PBS (Life
Technologies, Inc., Merelbeke, Belgium) and overcoated for 1 h at
room temperature with 1% heat-denatured BSA in PBS.
MOLT-4 cell cultures were split at a concentration of 3 × 105 cells/ml, 24 h before the cell binding assays.
Just prior to the assay, the cells were labeled with the vital
cytoplasmic fluorescent dye calcein AM (Molecular Probes Europe,
Leiden, Netherlands), according to Braut-Boucher et al.
(18). Cells, washed twice with DMEM/F12 (1:1) medium (Life
Technologies, Inc.), were suspended at 2 × 106
cells/ml in Dulbecco's PBS, 1% BSA and incubated for 30 min at 37 °C with 25 µM (from a 1 mM stock in
Me2SO) calcein AM in PBS, 0.5% BSA. Labeled cells were
added to the coated microtiter wells (100 µl) at 5 × 105 cells/ml (in PBS, 1% BSA) and allowed to settle for 30 min at room temperature. Then, the microtiter plates were centrifuged at 1000-4000 rpm for 5-15 min in a Heraeus Minifuge 2 and scanned by
a Fluorimager. The effect of heparin on cell adhesion was tested by
preincubating the collagen-coated wells for 30 min with 100 µg/ml
porcine intestinal heparin (Sigma) in PBS and adding 100 µg/ml
heparin to the medium during the adhesion assay.
Isolation of a Variant Murine Syndecan-1 cDNA--
By using
primers corresponding to the published N-terminal and C-terminal amino
acid sequences of murine syndecan-1 (11), we isolated two distinctive
reverse transcription-PCR products from 14-day total mouse embryo RNA:
the expected standard product of 933 bp (corresponding to residues
240-1172 of the published murine cDNA sequence) and a
"variant" shorter product. Sequencing indicated that the shorter
product was missing 132 bp, corresponding to residues 388-519 of the
published cDNA sequence (Fig. 1).
The boundaries of the missing sequence complied with the so-called
GT-AG rule for splice site selection, suggestive of alternative splicing of the syndecan-1 gene transcript. The putative splice variant
lacked the nucleotide sequence located just downstream of the
nucleotides encoding the S45GS47GT amino acid
sequence, which contains the two serines identified as the major
attachment sites for heparan sulfate in this protein (6). The removal
of these 132 bases respected the reading frame, joining the
S45GS47GT sequence to alanine
(Ala94 in the original sequence) instead of glycine as
residue 50, followed by the sequence GEKPEEGE. Thus, the splice reduced
the number of SGXG motifs in the protein and generated a
novel immediate downstream context for the Ser-Gly repeat, rich in
acidic amino acids but also containing a charged residue.
Expression of the Variant Mouse Syndecan-1 Message--
To assess
the relative abundance of the variant message, we performed
ribonuclease protection assays on total and poly(A)+ RNAs
from different embryonic and adult mouse tissues. By using a 446-nt
in vitro antisense transcript for the standard form of mouse
syndecan-1, we observed a major protected band with the size of a
430-nt fragment, consistent with the prediction for the standard
syndecan-1 mRNA. Alignment of the probe with the variant cDNA
predicted that protection by the alternatively spliced RNA should yield
fragments of 200 and 101 nucleotides. Fragments of this size were
present in 15-day mouse embryonic lung and adult lung RNA but remained
nearly undetectable in 15-day total mouse embryo or adult liver RNA,
even after prolonged exposures (Fig. 2).
The ratio of the higher (430) versus the lower (200 + 101) bands was at least 10:1, indicating that the alternatively spliced message represented at most a minor population of the total syndecan-1 message in these tissues and a fortiori the whole embryo.
The long exposure autoradiograms revealed several additional discrete protected bands that could not be accounted for by the currently identified syndecan-1 cDNAs. These bands were not due to
contaminating cDNA in the probe, since the probe was completely
degraded in the absence of added RNA. These extra bands might represent
additional splice variants or unspliced RNAs, but this possibility was
not further investigated.
In Northern blot experiments, cDNA probes for the standard and the
alternatively spliced forms of mouse syndecan-1 and also a 131-bp probe
corresponding to the fragment that was missing in the splice variant,
all recognized both the 2.6- and 3.4-kb messages for this proteoglycan,
in a similar 3:1 relative abundance as described previously (not shown).
Variant Mouse Syndecan-1 Protein as a Primer for HS
Synthesis--
Prior studies have identified the serine in the
tetrapeptide sequence SGXG as an efficient in
vitro xylosyltransferase acceptor and acidic amino acids at the
N-terminal side of this SGXG sequence as enhancers of the recognition
and substitution of the serine by xylosyltransferase (7). Consistently,
acidic residues appear to be part of the consensus structure for
attachment of glycosaminoglycans to proteins (7), whereas clusters of
acidic residues adjacent to coupled Ser-Gly dipeptides appear to be
part of the consensus structures for heparan sulfate glycosaminoglycan
attachment (6). The variant syndecan-1 cDNA coded for
S45GS47GTA instead of
S45GS47GTG (one instead of two partially
overlapping SGXG tetrapeptide sequences), flanked by a more
acidic downstream context, suggesting that the splice potentially
influenced the number, type, or structure of the glycosaminoglycan
(chondroitin or heparan sulfate) chains that were bound to the core
protein. We have analyzed this possibility by constructing Syn-1s and
Syn-1v pRc/RSV expression vectors encoding, respectively, the standard
and variant (alternatively spliced) forms of murine syndecan-1 (see
"Experimental Procedures") and expressing both these proteins in
MDCK cells. Figs. 3 and
4 show results of Western blotting
experiments with these cells.
Non-transfectant MDCK cells expressed a syndecan-1-related
proteoglycan, as indicated by the mouse monoclonal antibody 2E9, which
is specific for the evolutionarily highly conserved (and structurally
very similar) cytoplasmic domains of the syndecans-1 and -3. This
syndecan carried some chondroitin sulfate but mostly heparan sulfate.
It yielded a core of ~74 kDa after heparitinase treatment and
slightly higher yields of this protein after combined heparitinase and
chondroitinase ABC digestions, behaving as a heparan sulfate or heparan
sulfate-rich hybrid proteoglycan (Fig. 3). This canine syndecan was not
detected by the rat anti-mouse syndecan-1 antibody 281-2, which is
directed against the evolutionarily less conserved ectodomain of this
protein (not shown), but most likely represents syndecan-1 (syndecan-3
is not expressed in these cells).
MDCK cells transfected with Syn-1s cDNA (encoding the standard form
of murine syndecan-1) produced both 281-2-reactive (Fig. 4) and
2E9-reactive proteoglycans (Fig. 3). The properties of the
281-2-reactive (transfectant) and the 2E9-reactive (transfectant and
endogenous) proteoglycan species were quite similar (although not
completely identical) and reminiscent of the properties of the
2E9-reactive endogenous syndecan expressed by non-transfectant cells.
Chondroitinase ABC reduced the amount of syndecan trapped at the
interface of the stacking and separating gels but yielded no discrete
immunoreactive bands in the separating gels, indicating that these
chondroitinase-digested syndecans were still polydisperse. The effect
of the chondroitinase ABC treatment, however, was more marked on the
281-2-reactive (transfectant) forms than on the 2E9-reactive
(transfectant and endogenous) forms. Heparitinase-treated syndecans
still migrated as a smear or stagger of bands, with small yields of
~74-kDa core protein. Clearly, in relative terms, the heparitinase
digestion of the transfectant syndecan (281-2-reactive) yielded less
~74-kDa core protein than the heparitinase digestion of the
endogenous syndecan (2E9-reactive) in the non-transfectant cells.
Combined heparitinase and chondroitinase ABC digestions yielded single
2E9- or 281-2-reactive core proteins of ~74 kDa. These results
indicated that transfectant syndecan-1s was primarily a heparan
sulfate/chondroitin sulfate hybrid proteoglycan with a core protein of
~74 kDa.
MDCK cells transfected with Syn-1v (the variant murine syndecan-1)
cDNA also produced 2E9 and 281-2-reactive proteoglycan, but the
properties of the 281-2-reactive (Fig. 4) and the 2E9-reactive (Fig. 3)
syndecans were dissimilar and different from the syndecans expressed in
non-transfectant cells or in cells transfected with Syn-1s cDNA.
The 281-2-reactive (transfectant) proteoglycan in Syn-1v cells entered
the separating gels to a larger extent than the 2E9-reactive
(transfectant + endogenous syndecan-1) proteoglycan in the same cells,
the 2E9 or 281-2-reactive proteoglycan in the cells transfected with
Syn-1s, or the 2E9-reactive proteoglycan (endogenous syndecan-1) in
non-transfectants. After treatment with only chondroitinase ABC, a
substantial fraction of the 2E9-reactive syndecan-1 and an even larger
fraction of the 281-2-reactive syndecan-1 migrated as a broad band of
~70-120 kDa and small amounts as a discrete ~56-kDa band. Single
heparitinase digestion yielded a broad band of ~65-100 kDa and a
sharp band of ~56 kDa that reacted with the mAbs 2E9 and 281-2, but
the 56-kDa band was more conspicuous in the 281-2-staining than in the
2E9-staining reaction. Combined heparitinase and chondroitinase ABC
digestions yielded two sharp bands of ~74 and ~56 kDa that reacted
with mAb 2E9, and only one band of ~56 kDa that reacted with mAb
281-2. Single enzyme digestions produced also more important band
shifts and resulted in more quantitative transfers of immunostainable
proteoglycan than observed for the endogenous syndecan-1 in
non-transfectants and for transfectant syndecan-1 in cells transfected
with Syn-1s. These results indicated that transfectant syndecan-1v
represented a mixture of heparan sulfate, chondroitin sulfate, and
heparan sulfate/chondroitin sulfate hybrid proteoglycans all built on a
core protein of ~56 kDa.
Glycanation of the Variant Syndecan-1 Heparan Sulfate
Proteoglycan--
The standard (s) and variant (v) forms of murine
syndecan-1 were then isolated from the corresponding metabolically
labeled transfectant MDCK cells, by immunopurification on mAb 2E9, and were separated from copurifying endogenous syndecan-1 by further immunopurification on the rat monoclonal (281-2) antibody specific for
the ectodomain of murine syndecan-1. Mab 2E9 was also used to isolate
syndecan-1 from non-transfectant MDCK cells.
Heparitinase and chondroitinase susceptibility tests indicated that the
35S-labeled recombinant syndecan-1v was more susceptible to
chondroitinase ABC than recombinant syndecan-1s and endogenous
syndecan-1 from non-transfectant MDCK cells (Fig.
5). Sizing of the HS chains, released by
alkali, and of the HS chain clusters, prepared by extensive protease K
digestion of the chondroitinase ABC-pretreated proteoglycans, was
performed by SDS-PAGE (Fig. 6) and by gel
filtration (not shown). The recombinant syndecan-1v yielded
protein-free HS chains (alkali release) that were slightly smaller
(average size of ~20 kDa) than the HS chains from recombinant
syndecan-1s (average size of ~22 kDa) and the chains from the
endogenous syndecan-1 isolated from non-transfectant MDCK cells
(average size of ~25 kDa). When prepared by protease K treatment,
most of the HS from the endogenous syndecan in non-transfectants cells
and most of the HS from the recombinant syndecan-1s migrated with
apparent sizes of ~68 kDa. Most of the HS from syndecan-1v, in
contrast, migrated with an apparent size of ~20 kDa,
indistinguishable from the size of the protein-free chains (obtained by
alkali) from this proteoglycan. These results indicated that most of
the endogenous and standard transfectant syndecan-1 core proteins were
substituted with at least two HS chains that occurred mostly together
in a protease K-resistant cluster, whereas the core proteins encoded by
the variant message were substituted with only one chain or with
several chains that were separated by a protease K-susceptible domain.
The immunopurified transfectant syndecans were also analyzed by Western
blotting, using the mAbs 281-2 (anti-core protein) and 3G10
(anti-
When the purified HS chains were digested with heparinase prior to gel
filtration over Sepharose CL6B, two discrete populations of
N-acetylated heparinase-resistant regions, one with a size ~9.3 kDa and a second with a size of ~3.6 kDa, were observed in all
three preparations. The ratios between these two peaks were similar in
all three samples. This indicated similar spacings between highly
sulfated (heparinase-sensitive) domains in all these chains (not
shown). The heparan sulfate chains from endogenous syndecan-1 and from
recombinant syndecans also eluted at near identical positions from
ion-exchange columns, suggesting similar average charge densities or
sulfations (not shown).
Recognition Signals for Glycosaminoglycan Attachment in Variant
Syndecan-1--
The variant splice reduces the number of
SGXG tetrapeptide sequences in the protein, but it also
brings the direct Ser-Gly repeat sequence in proximity to a variant and
somewhat unusual (see "Discussion") downstream sequence. To
determine the significance of these differences for the glycanation of
the proteoglycan, we converted the
S45GS47GTA50 sequence in
the variant into
S45GS47GTG50, restoring
the SGXG consensus for the second serine in the Ser-Gly repeat sequence. In a separate construct, we also substituted a glycine
for the lysine that interrupts the acidic downstream sequence
(GEK53PEE>GEG53PEE). The
constructs (Syn-1vA Functional Competence of the Variant Syndecan-1
Proteoglycan--
Expression of syndecan-1 in myeloma cells supports
the adhesion of these differentiated lymphoid cells to collagen (19, 20). In vitro studies, on the other hand, have indicated
that binding of the murine syndecan-1 ectodomain to fibrillar collagen requires intact proteoglycan and depends on cooperative bindings of
each glycosaminoglycan chain in the proteoglycan (21). To assess
whether the variant form of this proteoglycan supports cell adhesion to
collagen, we expressed the Syn-1s and Syn-1v cDNAs in MOLT-4 cells,
T lymphoid cells that do not express significant levels of endogenous
heparan sulfate proteoglycan. Analyses of the syndecan expressions in
these cells by Western blotting confirmed the main findings observed in
MDCK cells (Fig. 4). Single enzyme treatments yielded more important
band shifts and transfers of the immunoreactive materials for
syndecan-1v than for syndecan-1s. After combined heparitinase and
chondroitinase ABC treatments, the Western blots revealed a syndecan-1v
core protein that was smaller than the syndecan-1s core protein and
that stained less intensely with 3G10 than with 281-2 antibody. For
similar levels of cell-surface core protein (281-2 epitope),
quantitative fluorescence analyses of clonal transfectant MOLT-4 cell
populations revealed higher levels of cell-surface heparan sulfate
(10E4 epitope) and, after heparitinase, of We provide the first evidence for alternative splicing of the
transcript for syndecan-1, a member of the syndecan family of cell-surface proteoglycans. The variant splicing process joins the
splice donor site corresponding to the 3'-end of exon 2 of this gene
(8, 9) to an alternative splice acceptor site, deleting 132 nucleotides
from the sequences that correspond to the third exon for the originally
identified (11) and clearly more abundant mRNA for this proteoglycan.
The variant splice respects the reading frame, and deletes 44 amino
acids from the predicted translation product. The deletion occurs
immediately after the segment in the protein that harbors the serines
that were identified as sites for heparan sulfate attachment (6) and
approximates a region rich in acidic amino acids to these sequences. At
first sight, the variant therefore still reproduces a consensus pattern
(repetitive Ser-Gly sequences, flanked by both upstream and downstream
clusters of acidic amino acids) that can be observed in the other
vertebrate syndecans and the Drosophila syndecan (Fig.
10), and in several other heparan sulfate proteoglycans (6).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2) and a nearby cluster of acidic residues ((D/E)n
(n 3) within 6 residues) as a structural motif that is
shared by many of the heparan sulfate proteoglycans (6). This motif
incorporates a consensus peptide structure (acidic amino acids closely
followed by the tetrapeptide SGXG) identified in prior
studies as an efficient acceptor for xylosyltransferase, the enzyme
that initiates the assembly of glycosaminoglycan chains (7). In
syndecan-1 this acceptor motif is realized twice in the sequence
S45GS47GTG, which is encoded by the junction of
the second and third exons (TCT GGC TCT GGC ACA (G/G)T) of the
syndecan-1 gene (8, 9) and the upstream sequence DDSDNF that
immediately flanks the Ser-Gly repeat.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 6.7 µM EDTA, 170 µg/ml of BSA),
10% Me2SO, 1 ng of cDNA, 1.5 mM dNTP, and
1.4 µM primer. The sense and antisense primers (Fig. 1)
were 42-mers identical, respectively, to the murine syndecan cDNA
sequence (from residue 240 to 281) and to the complement of this
sequence (from residue 1131 to 1172), as reported by Saunders et
al. (11). After 40 thermal cycles (1 min denaturation at 94 °C,
1 min annealing at 55 °C, and 1 min extension at 68 °C), the
amplification products were analyzed in 1.1% agarose gels and detected
by ethidium bromide staining. The amplification products were
electro-eluted, ethanol-precipitated, phosphorylated, and ligated in
the SmaI restriction site of pGEM-3Z (Promega Corp.,
Madison, WI). After propagation in XL1-Blue cells, several independent
recombinant plasmids were analyzed. Variant insert of only ~800 bp
(MoSyn-1/7) and insert of the expected standard size of 933 bp
(MoSyn-1/6) were further characterized by restriction site mapping and
completely sequenced by the dideoxy chain termination method (12),
using supercoiled plasmid, a modified T7 DNA polymerase (Amersham
Pharmacia Biotech, Uppsala, Sweden), T7 and SP6 primers, and both dGTP
and C7-deaza-dGTP.
. The segments of
these chimeric constructs that spanned from the (blunted)
KpnI site in the multiple cloning site of the pBS vector to
the XbaI restriction site in the human cDNA were then cloned in pRc/RSV plasmids (Invitrogen, Leek, The Netherlands), using
the blunted BstXI and the XbaI sites of this
vector. Sequencing verified that the resulting Syn-1s and Syn-1v
pRc/RSV plasmids were coding for peptides that corresponded,
respectively, to the predicted products of the MoSyn-1/6 (standard) and
MoSyn-1/7 (variant) cDNAs. The primers 5'-C TCT GGA TCT GGA ACA GGA
GGA GAG AAG CC and 5'-CA GCC GGA GAA GGA CCA GAA GAA GGA GAG CCT GTG C
and the corresponding complementary primers, in combination with
T3 and T7 primers, were used in a two-step ligation PCR procedure, to convert the
SGSGTA50GEK53PEEGEPV
sequence in the variant syndecan-1 protein into, respectively, SGSGTG50GEKPEEGEPV and
SGSGTAGEG53PEEGEPV (where boldface indicates the
substitution). The Syn-1s and Syn-1v expression plasmids were
linearized with BglII and introduced in MDCK or MOLT-4 cells
by electroporation. For the transfections, the cells were prewashed
with Ca2+/Mg2+-free PBS, incubated for 10 min
at 4 °C (107 cells in 1 ml) with 30 µg of linearized
plasmid, and electroporated at 240 V and 960 microfarads (Gene Pulser,
Bio-Rad). Selection was started 48 h later, using 500 µg/ml
G418. Stable transfectant syndecan-expressing MDCK clones were
identified by immunoblotting. Syndecan-1 expressing MOLT-4 cells were
isolated by panning on anti-syndecan-1 antibody (mAb281-2) or
anti-heparan sulfate antibody (mAb 10E4) and characterized by
immunoblotting, quantitative immunocytofluorometry, and adhesion tests.
HS-specific mAb 3G10 (15).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence of the murine syndecan-1
variant. Nucleotide sequence (upper line) of the
variant syndecan-1 cDNA (uppercase) and standard
syndecan-1 cDNA (upper + lowercase), and
predicted syndecan-1v (upright) and syndecan-1s
(upright + italics) amino acid sequences
(lower line). The nucleotide sequences corresponding to the
primers used in the reverse transcription-PCR are indicated in
bold. The nucleotide sequences corresponding to the probes
used in the ribonuclease protection assay (see Fig. 2) are
underlined. The serine residues in syndecan-1s that support
the synthesis of heparan sulfate (6) are highlighted by
shading. Acidic residues in syndecan-1s and syndecan-1v that
occur upstream or downstream of these serines within a distance of six
residues are indicated in bold. The GenBankTM
accession number for the variant cDNA sequence is AF134897.
View larger version (53K):
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Fig. 2.
Expression of variant syndecan-1 RNA in
murine tissues. Ribonuclease protection assay for syndecan-1
transcripts in RNA extracts from day 15 mouse embryonic lung
(lanes 1 and 6), adult liver (lane 2),
adult lung (lane 3), total mouse embryo (lane 4),
and embryonic liver (lane 5). In lane 1, the
labeled antisense RNA probe (314 nt) was synthesized from the syn-1v
template. Alignments predict that this lane should contain protected
fragments of 298 (syn-1v), 200, and 101 nt (syn-1s). It is to be
expected, however, that the RNase will have difficulties cleaving the
labeled strand in the asyn-1v/syn-1s RNA heteroduplex. The 298-nt band,
therefore, cannot be considered as evidence for the occurrence of
syn-1v message, but the 200- and 101-nt bands provide accurate size
markers, useful for the other lanes. In the other lanes, the antisense
probe (446 nt) was synthesized from the syn-1s template. In that case
the unpaired loop in the asyn-1s/syn-1v RNA heteroduplex forms part of
the labeled strand, and the probe is easily degraded by RNase, yielding
protected fragments of 200 and 101 nt. The 200-nt fragment should
accurately reflect the abundance of the variant message, while
theoretically the 101-nt fragment could also result from unspliced RNA.
The only samples to yield protected fragments of this size were the
embryonic and adult lung RNAs. Lane 7 contains the 446-nt
antisense probe, without RNase digestion. Lane 8 shows the
protection by yeast RNA. The lanes without numbering contain a
sequencing reaction, used as size marker.
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Fig. 3.
Expression of syndecan-1 in wild-type and
transfectant MDCK cells. MDCK cells were transfected with
pRc/RSV/Syn-1s or pRc/RSV/Syn1-v encoding, respectively, the standard
and variant form of murine syndecan-1. Total proteoglycan extracts from
wild-type MDCK cells (SYN-1E) and from the transfectants (SYN-1S and
SYN-1V) were digested (+) or not digested ( 
) with heparitinase
(Hase) and chondroitinase ABC (Case) and were
fractionated by electrophoresis in SDS gels. The Western blots of these
gels were stained with mAb 2E9, which reacts with the evolutionarily
conserved cytoplasmic domain of syndecan-1.
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Fig. 4.
Recombinant murine syndecans expressed in
MDCK and MOLT-4 cells. MDCK and MOLT-4 cells were transfected with
pRc/RSV/Syn-1s (SYN-1S) or pRc/RSV/Syn1-v (SYN-1V). Total proteoglycan
extracts from these cells were digested (+) or not digested ( ) with
heparitinase (Hase) and chondroitinase ABC (Case)
and were fractionated by electrophoresis in SDS gels. The Western blots
of these gels were stained with mAb 281-2, directed at the ectodomain
of murine syndecan-1 (top panel). Murine syndecan-1,
isolated from the detergent extracts of the MDCK cells by
immunopurification on mAb 2E9 and mAb 281-2, and total proteoglycan
from MOLT-4 cells were treated as above. The Western blots of these
gels were stained with mAb 3G10, directed at HS, the desaturated
uronate that caps the remnant of each heparan sulfate chain after
heparitinase digestion (bottom panel). All lanes were
normalized for 281-2 epitope content. The high
Mr materials remaining after the combined
digestion of syndecan-1v in MOLT-4 cells may be due to some aggregation
of the core protein, a propensity that is common to all syndecan
proteins. This phenomenon is concentration-dependent and
was not noted in other experiments.
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Fig. 5.
Heparitinase and chondroitinase ABC
susceptibility of syndecan-1. Metabolically
35SO3-labeled endogenous canine syndecan-1 was
isolated from wild-type MDCK cells, by immunopurification on mAb 2E9.
Metabolically 35SO3-labeled recombinant murine
syndecan-1 was immunopurified from MDCK cells transfected with
pRc/RSV/Syn-1s (SYN1-S) or pRc/RSV/Syn1-v (SYN1-V), by consecutive
immunopurification on mAb 2E9 and mAb 281-1. The proteoglycans were
digested (+) or not digested ( ) with heparitinase (Hase)
and chondroitinase ABC (Case), fractionated by
electrophoresis in SDS gels, and detected by autoradiography. Note that
the sulfate labeling of the variant proteoglycan is strongly reduced
and that a large part of the residual label migrates in the separating
gel after digestion with chondroitinase ABC only.
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Fig. 6.
Size and clustering of the heparan sulfate
chains in syndecan-1. Metabolically
35SO3-labeled endogenous (E) canine
syndecan-1 was isolated from wild-type MDCK cells, by
immunopurification on mAb 2E9. Metabolically
35SO3-labeled recombinant murine syndecan-1 was
immunopurified from MDCK cells transfected with pRc/RSV/Syn-1s
(S) or pRc/RSV/Syn1-v (V), by consecutive
immunopurification on mAb 2E9 and mAb 281-1. The immunopurified
syndecans were digested with chondroitinase ABC and then treated (+)
with proteinase K (PK), alkali (Alk), or
heparitinase (Hase). The various samples were fractionated
by SDS-PAGE and detected by autoradiography.
HS) as immunoprobes (Fig. 4). Doubly heparitinase and
chondroitinase ABC-digested recombinant syndecan-1s yielded ~74-kDa
core protein that was strongly stained by both 281-2 and 3G10. After a
similar treatment, recombinant syndecan-1v yielded ~56-kDa core
protein that was strongly stained by mAb 281-2 but much more weakly by
mAb 3G10. Since the heparitinase treatment leaves only one
4,5-glucuronate (3G10 epitope) per chain in association
with the core protein, these results indicated a lower HS chain
number/protein ratio in the syndecan-1v than in the syndecan-1s proteoglycans.
G and Syn-1vKG) were used in transfection experiments, and the sizes and glycanations of the recombinant proteoglycans were analyzed as described above. Converting
Ala50 into glycine increased the apparent size of the
variant proteoglycan up to that of syndecan-1s (>200 kDa), the
recombinant standard form of syndecan-1 (Fig.
7). Moreover, syndecan-1v with an Ala Gly substitution yielded clusters of heparan sulfate chains (compare
the size of the HS moiety after alkali release and after proteinase K
digestion, in Fig. 7), and after a combined heparitinase and
chondroitinase ABC digestion, it stained more intensely for the 3G10
epitope than syndecan-1v (not shown). Changing Lys53 in
syndecan-1v into glycine had no effect on the size of the intact
variant proteoglycan (not shown) but increased the ratio of the 3G10
epitope (HS stubs) over the 281-2 epitope (protein), suggesting no
change in average chain number but a more consistent substitution with
HS (Fig. 8). These data suggest that both
the reduction of the number of SGXG tetrapeptide sequences
and the variant structure of the downstream context contribute to the markedly reduced HS glycanation of the syndecan-1v core protein.
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Fig. 7.
Effect of restoring the number of
SGXG signal sequences in syndecan-1v.
Metabolically 35SO3-labeled recombinant murine
syndecan-1 was immunopurified from MDCK cells transfected with
pRc/RSV/Syn-1s (S), pRc/RSV/Syn-1v (V), or
pRc/RSV/Syn-1v Ala50 Gly (AG) by consecutive
immunopurification on mAb 2E9 and mAb 281-1. The immunopurified
syndecans were left untreated or digested with chondroitinase ABC
(Case) and then treated with proteinase K (PK) or
alkali (Alk). The various samples were fractionated by
SDS-PAGE and detected by Western blotting using mAb 281-2 (left
panel) or by autoradiography (right panel).
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Fig. 8.
Effect of the positive charge in the variant
downstream sequence context. Recombinant murine syndecan-1 was
immunopurified from cells transfected with pRc/RSV/Syn-1v
(V) or pRc/RSV/Syn-1v Lys53 Gly
(KG). The immunopurified syndecans were digested with
heparitinase and chondroitinase ABC, fractionated by SDS-PAGE,
transferred to Zeta-probe membranes, and stained with the mAbs 281-2 (core protein) and 3G10 (HS).
-heparan sulfate (3G10
epitope) in Syn-1s than in Syn-1v transfectants (not shown).
Mock-transfected, Syn-1s-transfected, and Syn-1v-transfected MOLT-4
cells all adhered to type I collagen. Expression of the variant form of
syndecan-1 only slightly increased the background adhesion to collagen,
and the adhesion of mock-transfected and Syn-1v- transfectants was
essentially not sensitive to the addition of heparin in the assay.
Cells expressing the standard form of syndecan-1, in contrast, showed
enhanced binding to collagen but only in the absence of heparin (Fig.
9). Comparison of the forces needed for
de-adhesion (pelleting of the cells at differing speeds of
centrifugation), consistently indicated stronger collagen adhesiveness
of the Syn-1s than the Syn-1v transfectants.
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Fig. 9.
Adhesion to collagen of syndecan-1
transfectant MOLT-4 cells. MOLT-4 cells transfected with pRc/RSV
(O), pRc/RSV/Syn-1s (S), or pRc/RSV/Syn1-v
(V) were labeled with calcein AM and left to adhere to
U-shaped wells coated with bovine serum albumin (BSA) or
type I collagen (COL) (duplicate assays). After a spin,
non-adherent cells collected in the center of the wells. The cells were
detected by fluorimaging. The syn-1s and syn-1v transfectant cells
shown expressed similar (moderate) levels of cell-surface syndecan-1
protein as detected by fluorescence-activated cell sorter. The cells
expressing syndecan-1s adhered more strongly (larger diameter of the
pellet) to collagen than the cells expressing syndecan-1v or control
transfectants. Adding heparin (+H) to the cell suspension
during the adhesion assay abolished this difference.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Sequences flanking the SG repeats in the
syndecans. Alignment of the sequences that flank the SG repeats in
the N-terminal domain of the variant form of murine syndecan-1
(S1v), in syndecan-1 (S1), (11, 23, 24),
syndecan-2 (S2) (10, 25, 26), syndecan-3 (S3)
(27, 28), and syndecan-4 (S4) (24, 29) from human
(h), mouse (m), rat (r), and chick
(c) origin, and in the syndecan from Drosophila
melanogaster (dr) (30). Acidic residues are indicated
in bold. The SG sequences are indicated by shaded
boxes.
Yet, in MDCK and in MOLT-4 cells, this variant syndecan-1 protein is
less efficient at priming the synthesis of heparan sulfate than the
full-length form of this protein. A first indication is provided by the
marked size differences of the heparan sulfate proteoglycans (after
chondroitinase ABC digestion) but not of their constituent heparan
sulfate chains. The second evidence is the clustering of heparan
sulfate chains in the standard form of the proteoglycan but not in the
variant form. The final and direct indication is the lowering of the
HS:protein epitope ratio in the variant form.
The precise structures that determine the glycanation of a protein remain enigmatic. Both heparan sulfate and chondroitin sulfate are O-linked via a galactose-galactose-xylose linkage to serine residues in the core proteins of proteoglycans. Sequence alignments indicate that the substituted serine residues are followed by a glycine residue, and peptides containing a Ser-Gly dipeptide can serve as acceptors for xylosyltransferase-catalyzed transfer of xyloside (31). However, additional signals are involved in the biosynthetic recognition of the core proteins by the xylosyltransferase, since most proteins that contain Ser-Gly sequences are not proteoglycans and not all Ser-Gly sequences of proteoglycan core proteins are substituted with glycosaminoglycan. Testing synthetic polypeptides for the ability to accept xylose has indicated that acceptor activity could be enhanced by the presence of a doublet or triplet of acidic residues preceding the Ser-Gly sequence and the occurrence of the Ser-Gly dipeptide in the sequence context of Ser-Gly-Xaa-Gly (where Xaa stands for any amino acid) (7). This consensus motif can be found in many of the known core proteins, but it is clear that it does not encompass all the sites used for glycosaminoglycan attachment. The reduced glycanation of syndecan-1v (in comparison to syndecan-1s) and the enhanced glycanation of syndecan-1v when its Ala50 is converted into glycine, is consistent with the in vitro xylosyltransferase acceptor activity studies which indicated that peptide containing the sequence Ser-Gly-Xaa-Ala retained only 10% of the activity measured for the tetrapeptide Ser-Gly-Xaa-Gly in an otherwise similar context (7).
All this evidence and prior findings on syndecan-1 (6) would be consistent with a mostly three-heparan sulfate chain model for the standard form of syndecan-1, one chain occurring on Ser35 and the clustered chains occurring on the (protease K-resistant) S45GS47G tetrapeptide that has been identified as the major heparan sulfate-glycanation site (6), and a mostly two-heparan sulfate chain model, one chain on Ser35 and one on Ser45 (Ser47 remaining unprimed), for the variant from of syndecan-1. The consistency of the results in MDCK and MOLT-4 cells suggests that the findings are not accidental and due to imbalances between the protein expression levels and the capacity of the glycanation machinery of the cells (16, 22). The findings do, however, not imply that the natural equivalents of syndecan-1s (or syndecan-1v) in different cell types and stages of differentiation invariably correspond to the substitution model identified here in these two cell types.
Attempts to define core protein features that designate a Ser-Gly
dipeptide for heparan sulfate rather than chondroitin sulfate attachment have revealed a coupling phenomenon between adjacent Ser-Gly
dipeptides that leads to a preferential substitution of these sites
with heparan sulfate, and sequence alignments indicate that most
heparan sulfate proteoglycan core proteins contain repetitive (Ser-Gly)n segments and a nearby cluster of acidic residues (6). This incorporates the consensus motif defined above but with Xaa
standing for serine. However, also this motif does not cover all sites
known to be substituted with heparan sulfate, and it is clear that
sequences in non-glycosaminoglycan binding regions of the core protein
(e.g. the SEA module from perlecan) can influence the
utilization of the attachment sites for heparan sulfate synthesis (32).
From the mutagenesis of fusion proteins (6) and the analysis of the
structural motifs represented in all the known heparan proteoglycans
(6), one might also suspect that the assembly of HS chains on the
variant could be influenced by the proximity of the acidic cluster
downstream of the Ser-Gly repeat. Proximal downstream acidic sequences
are common in heparan sulfate proteoglycans, but decreasing the number
of amino acids between the downstream acidic cluster and the Ser-Gly
attachment site in betaglycan, for example, progressively reduces the
priming of heparan sulfate by this protein (33). On the other hand, the
novel flanking region in variant syndecan-1 reads as AGEKPEEGE, and
scrutiny of the published heparan sulfate core protein sequences also
indicates that positively charged residues tend to be excluded from the
sequences that flank the Ser-Gly dipeptides. The follow-up mutagenesis
experiments on the splice variant, converting the lysine residue in the
AGEKPEEGE sequence to glycine, supported the suggestion that the
positive charge may suppress HS priming. The substitution increased the
HS:protein ratio after heparitinase digestion but not the size of
the intact proteoglycan, suggesting it favors extending linkage regions
initiated on Ser35 and Ser45 with HS rather
than chondroitin sulfate but does not stimulate the initiation of a
third glycosaminoglycan chain on Ser47. Substituting
glycine for Ala50 in variant syndecan-1 enhanced heparan
sulfate synthesis and restored heparan sulfate chain clustering but
still yielded a mixture of heparan sulfate and chondroitin sulfate
proteoglycan (approximately 40% chondroitin sulfate). The gain in size
noticeable for the intact proteoglycan persisted after single
heparitinase or single chondroitinase digestions (results not shown),
indicating syndecan-1vAG carried an excess of both heparan sulfate
and chondroitin sulfate chains in comparison to syndecan-1v and an
excess of chondroitin sulfate in comparison to syndecan-1s. Indirectly,
this suggests that not only the reduction of Ser-Gly-Xaa-Gly consensus
sequences but also the novel downstream sequence context in syndecan-1v negatively affects the substitution of the Ser-Gly repeat with heparan sulfate.
Although the precise structural features that determine the lesser
efficiency of the syndecan splice variant as proteoglycan remain
unresolved, the present data extend the repertoire of mechanisms that
may account for the molecular polymorphism of the heparan sulfate
proteoglycans and specifically that of syndecan-1 (2). It is generally
accepted that this polymorphism largely determines the various receptor
or co-receptor functions that have been ascribed to cell-surface
proteoglycans. In this context it had previously been shown that
syndecan-1 from mouse mammary epithelial cells binds with high affinity
to type I collagen, through the intermediate of its heparan sulfate
chains, but that the protein-free heparan sulfate chains of this
proteoglycan do not bind collagen, suggesting that binding of the
proteoglycan is based on chain cooperation (21). Although it remains
formally unproven that the relative failure of syndecan-1v to support
or assist the adhesion of MOLT-4 cells to collagen is due to its
reduced glycanation, this failure appears consistent with this binding
model. It is also consistent with the evidence that all three sites of
heparan sulfate attachment in syndecan-1 are needed for optimal
activity of this syndecan in mediating cell-cell and cell-matrix
adhesion of myeloma cells and inhibiting the invasion of these cells in
collagen gels (34). These findings impart functional importance to the
chain valency of the proteoglycans and to the mechanisms that may
determine or affect this valency, and encourage further investigations
that should establish whether alternative splicing represents a
biologically significant means of regulating the functional versatility
of the syndecans and their effects on cell behavior.
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FOOTNOTES |
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* This work was supported in part by Grant G.0181.97 of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, the Geconcerteerde Onderzoeksacties 1996-2000, the Interuniversity Network for Fundamental Research sponsored by the Belgian Government, and the Flanders Interuniversity Institute for Biotechnology.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) AF134897.
Supported by the Human Capital and Mobility Program of the
European Union Fellowship ERBCHBI-CT 92-0224. Present address: Departament de Bioquímica i Biologia Molecular, Facultat de
Veterinària, Universitat Autònoma de Barcelona,
08193-Bellaterra, Spain.
§ Research Director of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. To whom correspondence should be addressed: Center for Human Genetics, Campus Gasthuisberg, 0&N6, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-345863; Fax: 32-16-347166; E-mail: guido.david{at}med.kuleuven.ac.be.
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ABBREVIATIONS |
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The abbreviations used are: MDCK, Madin-Darby canine kidney; PCR, polymerase chain reaction; BSA, bovine serum albumin; Me2SO, dimethyl sulfoxide; mAb, monoclonal antibody; PBS, phosphate-buffered saline; bp, base pair; nt, nucleotide; PAGE, polyacrylamide gel electrophoresis; HS, heparan sulfate.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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