(Received for publication, January 31, 1995; and in revised form, September 1, 1995)
From the
We showed previously that the synthesis of heparan sulfate on
betaglycan occurs at a Ser-Gly dipeptide flanked by a cluster of acidic
residues and an adjacent tryptophan (Zhang, L., and Esko, J. D.(1994) J. Biol. Chem. 269, 19295-19299). A survey of the
protein data base revealed that most heparan sulfate proteoglycans
contain repetitive (Ser-Gly) segments (n
2) and a nearby cluster of acidic residues. To study the role
of these amino acid sequences in controlling heparan sulfate synthesis,
we have examined the assembly of glycosaminoglycans on Chinese hamster
ovary (CHO) cell syndecan-1. The glycosylation sites were mapped by
making chimeric proteoglycans containing segments of CHO syndecan-1
cDNA fused to Protein A. Two sites near the transmembrane domain
(-EGS
GEQ- and -ETS
GEN-) were used solely
for chondroitin sulfate synthesis, whereas three sites near the N
terminus (-DGS
GDDSDNFS
GS
GTG-)
supported both heparan sulfate and chondroitin sulfate synthesis. The
strongest sites for heparan sulfate synthesis consisted of the repeat
unit, -S
GS
G-. An unusual coupling phenomenon
occurred across the adjacent SG dipeptides, leading to a greater
proportion of heparan sulfate than predicted by the behavior of each
site acting independently. The clusters of acidic residues adjacent to
the heparan sulfate sites play important roles as well. These sequence
motifs suggest a set of rules for predicting whether heparan sulfate
assembles at glycosylation sites in proteoglycan core proteins.
Chondroitin sulfate and heparan sulfate proteoglycans contain
glycosaminoglycan (GAG) ()chains attached to specific serine
residues of core proteins. Studies of hybrid proteoglycans (
)such as betaglycan, ryudocan, and syndecan-1 showed that
two types of GAG attachment sites
exist(1, 2, 3, 4) . One type carries
heparan sulfate or chondroitin sulfate chains, whereas the other type
bears only chondroitin sulfate. In the proteoglycan, betaglycan, a
specific amino acid sequence drives heparan sulfate
synthesis(2) . This site consists of a Ser-Gly dipeptide, a
nearby cluster of acidic residues, and an adjacent tryptophan that
augments the proportion of heparan sulfate made(2) . These
structural elements may enhance the interaction of glycosylated core
protein intermediates with a key
-GlcNAc transferase that
initiates the formation of heparan sulfate(5, 6) .
Almost all cloned heparan sulfate proteoglycans contain a cluster of
acidic residues near one or more putative heparan sulfate attachment
sites, but only a few contain nearby tryptophan. To study whether other
amino acid sequences surrounding GAG attachment sites might enhance
heparan sulfate assembly, we have examined the assembly of GAGs on
syndecan-1, a hybrid proteoglycan containing both heparan sulfate and
chondroitin sulfate(7) . Kokenyesi and Bernfield (4) showed recently that the N-terminal half of mouse
syndecan-1 contains heparan sulfate and chondroitin sulfate chains,
whereas the C-terminal half contains only chondroitin sulfate. Five
conserved Ser-Gly dipeptides may act as the sites for GAG addition, but
the identity of the sites that prime heparan sulfate was not
determined. Our study reveals that most of the heparan sulfate occurs
at an attachment site containing (Ser-Gly) through an
unusual coupling mechanism. This sequence motif occurs frequently in
heparan sulfate proteoglycans and always includes a cluster of acidic
amino acids flanking the site. We also show that the biosynthetic
capacity of the cell to make heparan sulfate and the amount of core
protein expression affect the proportion of heparan sulfate assembled.
A portion of
affinity-purified syndecan was resuspended in 0.5 M NaOH
containing 1 M sodium borohydride and incubated at 4 °C
for 24 h to -eliminate the chains. The base was neutralized by
adding 10-µl aliquots of 10 M acetic acid until bubble
formation ceased. Radiolabeled syndecan-1 and the released GAG chains
were analyzed by gel filtration HPLC (TSK G4000SW, 30 cm
7.5 mm
inner diameter, Pharmacia). Samples were eluted with 0.5 M NaCl in 0.1 M KH
PO
(pH 6.0)
containing 0.2% Zwittergent 3-12 at a flow rate of 0.5 ml/min,
and radioactivity in the effluent was determined by in-line liquid
scintillation spectrometry using Ultima Gold XR scintillant (Packard
Instrument Company, Downers Grove, IL). GAGs were digested with 20
milliunits of chondroitinase ABC (Seikagaku) in 50 mM Tris-HCl
and 50 mM sodium acetate buffer (pH 8.0) containing protease
inhibitors. Complete digestion of chondroitin sulfate by chondroitinase
ABC was assured by monitoring the extent of conversion of the carrier
to disaccharides (1 mg = 11.4 absorbance units at 232 nm).
Nitrous acid-catalyzed deaminative cleavage of heparan sulfate was
performed according to the low pH method of Shively and
Conrad(12) .
The numbers before the colon in the following list of constructs refer to a segment of amino acids from CHO syndecan-1 (mutations in parentheses), and the information after the colon refers to the set of oligonucleotide primers and templates used to amplify the desired sequence. 26/64:N26/C64; 26/64(S37A):N26/(N34/C64); 26/64(S45T):N26/C(N42B/C64); 26/64(S47T):N26/C(N42C/C64); 26/64(S45A,S47A):N26/C(N42A/C64); 26/64(S37A,S45T):N26/C(N42B/C64), and the plasmid containing 26/64(S37A) was used as a template; 26/64(S37A,S47T):N26/C(N42C/C64), and the plasmid containing 26/64(S37A) was used as a template; 187/214:N187/C214; 49/204:N49/C204; 207/240:N207/C240; 32/53:N32A/C53; 32/53(EDQD/QNQN):N32B/C53; 32/53(DDSD/DDSN): N32A/C(N39/C53); 32/53(EDQD/QNQN, DDSD/DDSN):N32B/C(N39/C53).
The plasmid, pC17, containing the full-length fibroglycan cDNA sequence (M81687, a gift from Dr. John Gallagher, Manchester, United Kingdom) was used as a template for PCR of the fibroglycan fragment (DMYLDSSSIEEASGLYPIDDDDYSSASGSGAYEDKGSPDLTTSQ).
The wild-type betaglycan construct, SPGDSSGWPDGYEDLE, and the mutant, SPGDSSGAPDGYEDLE, were made using primers described previously(2) . Overlapping PCR primers were designed to generate the following betaglycan fragments (M77809).
Overlapping PCR primers were designed for the following peptide sequences.
PCR fragments were extracted with phenol/chloroform, precipitated with ethanol, digested with EcoRI, purified by agarose gel electrophoresis, and transferred to DEAE paper (Schleicher & Schuell). After elution, the fragments were ligated into a derivative of the eukaryotic expression vector pPROTA (16) that had been treated with EcoRI and calf intestinal alkaline phosphatase. All constructs were sequenced to confirm their identity.
Stable transfectants were created by transfecting a monolayer of wild-type CHO cells (50% confluence) with 16 µg of pPROTA containing CHO syndecan-1 cDNA coding for amino acid residues 26-240. The vector pMAMNEO was included (2 µg) along with 20 µg of Transfectam reagent using conditions recommended by the manufacturer (Promega Corp., Madison, WI). Two days later, the cells were harvested with trypsin, and a serial dilution (1:3, v/v) series was prepared in a 96-well plate. Strains resistant to G418 (400 µg/ml, corrected; Life Sciences Technologies, Inc.) were selected. The medium was changed every 4 days for 2 weeks, and individual colonies were picked, expanded, and evaluated for expression of chimeras as described above.
Another
set of samples was treated for 24 h at 4 °C with 100 µl of 0.5 M NaOH containing 1 M NaBH to
-eliminate the GAG chains. [
S]GAGs were
isolated by anion exchange chromatography as described(17) .
Samples labeled with TRAN
S-LABEL were dissolved in 1 ml of
25 mM Tris-HCl buffer (pH 7.0) containing 6 M urea,
0.5% Triton X-100, and 0.25 M NaCl. An aliquot was counted by
liquid scintillation spectrometry.
Figure 1:
Gel filtration chromatography of CHO
syndecan-1. Nearly confluent monolayers of cells were incubated for 12
h in sulfate-deficient medium containing 100 µCi/ml of SO
. Syndecan-1 was purified from Triton
X-100-extracted proteoglycans by affinity chromatography (see
``Experimental Procedures''). One portion was treated with
alkali to
-eliminate the GAG chains. Samples were analyzed by gel
filtration HPLC before and after treatment with nitrous acid or
chondroitinase ABC (see ``Experimental Procedures''). A, syndecan-1 proteoglycan. B, syndecan-1 GAG chains.
, intact syndecan-1 or GAG chains;
, nitrous acid-treated
material;
, chondroitinase ABC-treated
material.
There are five possible GAG attachment sites in mouse, human, and rat syndecan-1 based on the presence of Ser-Gly dipeptides(7) . The same five sites were present in CHO syndecan-1, which was cloned by PCR using the mouse cDNA sequence to design appropriate primers (Fig. 2A; (13) ). The CHO syndecan-1 amino acid sequence showed 94, 87, 86, and 77% homology to the Syrian golden hamster, rat, mouse, and human sequences, respectively (Fig. 2B). More importantly, the amino acid sequences near the putative GAG attachment sites are highly conserved among all the different species(7) .
Figure 2:
Sequence of CHO syndecan-1 and comparison
with sequences from other species. A, nucleotide sequence (upper line) and predicted amino acid sequence (lower
line) of CHO syndecan-1 cDNA. CHO syndecan-1 was amplified from
the CHO-K1 quick-clone cDNA library by PCR. The sequence of the PCR
primers, indicated in lowercase letters, was derived from
mouse syndecan-1(13) . The underlined sequences define
the fragments for generating the chimeras shown in the tables
(GenBank accession number L38991). B, comparison
of predicted protein sequence of syndecan-1 from CHO, Syrian golden
hamster (SGH, M29967)(36) , mouse (Mur,
X15487)(13) , rat (Rat, M81785)(37) , and
human (Hum, X60306)(10) . -, identical amino acids; *,
a gap to improve alignment; boldface letters, potential GAG
attachment sites.
Kokenyesi and Bernfield (4) reported that both heparan
sulfate and chondroitin sulfate chains were present on the N-terminal
half of murine syndecan-1, but only chondroitin sulfate was found on
the C-terminal half. Of the five potential GAG attachment sites, three
reside in the N-terminal half and two in the C-terminal half. We showed
previously that the synthesis of heparan sulfate on betaglycan requires
a cluster of acidic residues located near a specific Ser-Gly attachment
site(2) . Two clusters of acidic amino acids border the three
Ser-Gly sites on the N-terminal part of the syndecan-1
(-EDQDGSGDDSDNFS
GS
GTG-, Fig. 2B), consistent with the idea that one or more of
these sites might support heparan sulfate assembly.
To examine how
GAGs assemble at the individual sites, we prepared chimeras composed of
CHO syndecan-1 segments fused to the IgG binding domain of
Staphylococcal Protein A with a signal peptide from the secreted
metalloprotease, transin, attached to the N terminus(16) . The
chimeras were introduced into wild-type CHO cells by transient
transfection, and the attachment of GAG chains was assessed by SO
incorporation into secreted chimeras
(``Experimental Procedures''). The activity of each Ser-Gly
site was measured independently by mutating other potential sites in
the constructs (Table 1). All three Ser-Gly dipeptides toward the
N terminus
(-EDQDGS
GDDSDNFS
GS
GTG-) were
capable of priming heparan sulfate chains, but their relative ability
varied from 9% at Ser
to 23% at Ser
. In
contrast, the two sites near the transmembrane domain
(-PTGEGS
GEQDFTFETS
GENTA-) primed only
chondroitin sulfate. A sixth Ser-Gly dipeptide exists in both CHO and
Syrian golden hamster syndecan-1 (Ser
), but not in mouse,
rat, and human syndecan-1. Expression of a 156-amino acid chimera
containing residues 49-204 did not result in incorporation of
SO
into GAGs (Table 1), suggesting that
Ser
does not act as a glycosylation site.
The extent
of substitution of the chimeras with heparan sulfate was less than
expected from the study of endogenous syndecan-1, which contained
80-85% heparan sulfate (Fig. 1). The difference was not
due to variation in the extent of sulfation of heparan sulfate and
chondroitin sulfate chains, since comparable results were found when
the cells were labeled with [6-H]GlcN and chain
length did not vary significantly (Fig. 3). The difference also
was not due to poor expression of the chimera since only
20% of
material labeled with TRAN
S-LABEL was converted to high
molecular weight proteoglycan.
Figure 3:
Gel filtration chromatography of
[S]syndecan-1 chimeras. Syndecan-1 chimeras
containing the sequences
-EDQDGAGDDSDNFS
GTGTG-
and
-EDQDGAGDDSDNFS
GS
GTG-
were affinity-purified from
SO
-labeled cells
(see ``Experimental Procedures''). A portion of material was
-eliminated to liberate the [
S]GAGs from
the chimeras, and an aliquot was digested with chondroitinase ABC or
nitrous acid (see ``Experimental Procedures''). The samples
were then analyzed by gel filtration HPLC (see ``Experimental
Procedures''). A, intact chimeras. B,
-eliminated GAG chains. C, heparan sulfate and
chondroitin sulfate chains.
, chimeras with a single acceptor site (
-EDQDGAGDDSDNFS
GTGTG-64);
, chimeras with two acceptor sites (
-EDQDGAGDDSDNFS
GS
GTG-
);
,
-eliminated chains from the single site chimera;
,
-eliminated chains from the two-site chimera;
, heparan
sulfate chains from the two-site chimera;
, chondroitin sulfate
chains from the two-site chimera.
We also tested the chimeras in CHO ldlD cells, which cannot obtain UDP-GalNAc from UDP-GlcNAc due
to a deficiency in the 4`-epimerase that interconverts the nucleotide
sugars(18) . This strain makes less chondroitin sulfate when
deprived of exogenous GalNAc(19) . When the chimeras were
introduced into ldlD cells, the amount of heparan sulfate was
enhanced (up to 79%), but the relative amount made at each N-terminal
site remained about the same (Table 1, values in parentheses).
Interestingly, Ser (-TGEGS
GEQDF-), which
primed only chondroitin sulfate in wild-type cells, generated about 28%
heparan sulfate in ldlD cells. The other chondroitin sulfate
site at Ser
(-TFETS
GENTA-) remained
ineffective.
To test whether coupling was dependent on flanking amino acid sequences, a repetitive SGSG sequence was introduced into betaglycan. In betaglycan, the site that supports heparan sulfate synthesis contains Ser-Gly-Trp flanked by a cluster of acidic residues (Table 2). We showed previously that converting the Trp residue to Ala reduced the level of heparan sulfate from 54 to 21% (2) . When a second Ser-Gly dipeptide was substituted for the Ala residue, the normal level of heparan sulfate synthesis was restored (51%, Table 2). Thus, the coupling across adjacent Ser-Gly sites appeared to be independent of surrounding sequence. Separating the Ser-Gly repeats by one or more residues reduced the proportion of heparan sulfate, suggesting that the coupling depended on proximity (Table 2). Inserting a Trp residue next to the repeat Ser-Gly segment gave a slight enhancement of heparan sulfate (58 versus 51%).
A stable transfectant expressing a
chimera with all five sites (residues 26-240) was analyzed (Table 3). Unlike the behavior of the transient transfectants,
the stably transfected cell line converted all of the chimera to high
molecular weight proteoglycan (data not shown) and produced about
3-fold more total S-proteoglycan than nontransfected
control cells (Table 3). The proportion of heparan sulfate was
reduced in the chimera (55%) compared with the proteoglycans found in
nontransfected parental cells (72%). Interestingly, the endogenous CHO
cell proteoglycans also contained less heparan sulfate in the stable
transfectant (54-62%). These findings suggested that the reduced
level of heparan sulfate may have been due in part to enhanced core
protein expression. Similar effects have been observed when full-length
syndecan and betaglycan constructs were expressed in
cells(1, 4) .
To gain insight into the mechanism
that results in a higher proportion of heparan sulfate at adjacent
attachment sites, we analyzed the GAG substitution pattern of
syndecan-1 chimeras containing one site (-EDQDGAGDDSDNFS
GTGTG-
)
and two sites (
-EDQDGAGDDSDNFS
GS
GTG-
).
As expected, the two-site chimera migrated with a greater hydrodynamic
volume during gel filtration than the chimera containing one site (Fig. 3A).
-elimination shifted the
[
S]GAG to a more included position, but the
mixture of GAG chains on both chimeras had essentially the same elution
position (Fig. 3B). Analysis of the heparan sulfate and
chondroitin sulfate chains on the two-site chimeras showed that they
were comparable in size as well (Fig. 3C).
The
two-site chimera presumably consisted of glycoforms containing one or
two GAG chains, with various combinations of heparan sulfate and
chondroitin sulfate chains. Analysis of S-labeled material
by SDS-PAGE revealed smeared bands characteristic of proteoglycans (Fig. 4, lane 3). The material of faster mobility
migrated in the same position as a chimera containing only one
attachment site (compare lane 3 with lanes 1 and 2), suggesting that it represented material with only one GAG
chain. The smear above this region presumably contained
S-chimeras with two chains, although some overlap occurred
with molecules containing one chain. Using the divisions shown in Fig. 4, we found that
50% of the total
S-counts were in chimeras containing two chains. This
material represented about one-third of the chimeras, given that the
size of the chondroitin sulfate and heparan sulfate chains did not vary
significantly (Fig. 3C). The degree of sulfation of
heparan sulfate and chondroitin sulfate differs somewhat (0.8 sulfate
groups/disaccharide versus 1
sulfate/disaccharide)(20) , but this effect only moderately
underestimates the extent of substitution by heparan sulfate.
Figure 4:
Electrophoresis of
[S]syndecan-1 chimeras. Chimeras were purified
from
SO
-labeled cells by IgG affinity
chromatography and dissolved in SDS-PAGE protein sample buffer. The
samples were analyzed on SDS-polyacrylamide (5-16%) gradient
gels, and the dried gel was imaged for data collection (see
``Experimental Procedures''). The positions of chimeras
containing one and two GAG chains are indicated to the right of the gel, and the protein molecular weight standards
are indicated to the left of the gel. Each lane
contained the following insert derived from syndecan-1: lane
1,
-EDQDGAGDDSDNFS45GTGTG-
; lane 2,
-EDQDGAGDDSDNFTGS
GTG-
; lane 3,
-EDQDGAGDDSDNFS
GS
GTG-
; lane 4,
-EDQDGAGDDSDNFS
GS
GTG-
after treatment with chondroitinase ABC; lane 5,
-EDQDGAGDDSDNFS
GS
GTG-
after treatment with heparin lyase III; lane 6,
-EDQDGAGDDSDNFS
GS
GTG-
after treatment with both chondroitinase ABC and heparin lyase III.
Equal portions of the samples were analyzed to show the reduction in
signal due to enzymatic digestion.
Digesting a sample with chondroitinase ABC and heparin lyase III
prior to electrophoresis removed all of the upper band material (lane 6). Chondroitinase ABC (lane 4) removed all of
the proteoglycans from the upper smear that contained chondroitin
sulfate chains, leaving behind only those molecules containing two
heparan sulfate chains. This resistant material represented about 42%
of the counts in the upper smear or about 14% (one-third of 42%) of the
total S-proteoglycans. Treating samples with heparin lyase
III (lane 5) removed all of the glycoforms containing heparan
sulfate, and only those molecules containing two chondroitin sulfate
chains remained. The resistant material represented about 32% of the
counts in the upper smear or about 11% (one-third of 32%) of the total
S-proteoglycans. By subtraction, hybrid chimeras
containing one chondroitin sulfate and one heparan sulfate chain
represented 26% of the upper smear counts (100 - (42 + 32)),
or
9% of the total proteoglycans. These findings are summarized in Table 4.
The extent of substitution by heparan sulfate in the
two-chain glycoforms (14% of total chimeric proteoglycan) was greater
than one would predict based on the independent behavior of each site.
Constructs containing only Ser or Ser
produced 15 and 23% heparan sulfate proteoglycans, respectively (Table 1). If the sites acted independently in the chimera
containing both Ser
and Ser
, then the
proportion of chimeras containing two heparan sulfate chains should
have been
1.1% based on the probability of producing heparan
sulfate at each site (0.15
0.23) multiplied by the proportion
of chimeras containing two chains (one-third). Therefore, glycoforms
with two heparan sulfate chains accumulated more than 10-fold over the
predicted value (14 versus 1.1%). The proportion of the other
glycoforms was less than predicted (11 versus 22% for
proteoglycans containing two chondroitin sulfate chains (one-third of
0.85
0.77] and 9 versus 10% for hybrids
containing one heparan sulfate and one chondroitin sulfate chain).
Previous studies of betaglycan showed that a cluster of acidic residues downstream from the site that supported heparan sulfate synthesis dramatically affected GAG composition(2) . The sites supporting heparan sulfate synthesis in syndecan-1 also have nearby clusters of acidic residues (Fig. 2). To examine if these clusters played a similar role in syndecan-1, a chimera containing residues 32-53 and all three N-terminal sites was prepared. Mutating the Asp and Glu residues in the clusters to Asn and Gln, respectively, reduced the proportion of heparan sulfate from 38% in the control construct to 8 and 13%, depending on the cluster (Table 5). Mutating both clusters diminished heparan sulfate synthesis to background (4%). Thus, the clusters of acidic residues are important elements in both syndecan-1 and betaglycan.
This report describes the use of protein chimeras to probe the assembly of heparan sulfate chains on proteoglycans. The results suggest a set of rules for predicting whether heparan sulfate will assemble at glycosylation sites. These rules include features of the core protein, the cellular capacity to produce individual GAGs, and the relative abundance of core proteins. Together, they support a model for GAG chain assembly in which specific core protein determinants interact with a key biosynthetic enzyme involved in heparan sulfate biosynthesis.
Inspection of various cloned
proteoglycans reveals that a cluster of acidic residues always flanks
sites thought to contain heparan sulfate chains, although the position
and exact composition of the cluster varies (Table 6). These
clusters occur less frequently in chondroitin sulfate proteoglycans.
-Glycan, decorin, invariant chain, and HI-30 contain clusters of
acidic residues, but the natural proteoglycans contain only chondroitin
sulfate chains at the indicated
sites(1, 21, 22, 23) . Decorin and
the betaglycan chimeras behaved similarly (Table 6). These
findings suggest that other elements work along with the cluster of
acidic residues to drive heparan sulfate synthesis. Thus, clusters of
acidic residues appear to be necessary but not sufficient determinants
for heparan sulfate assembly.
Earlier studies of -D-xylosides in CHO cells showed
that priming of chondroitin sulfate most likely occurs by
default(24, 25) . Simple xylosides containing alkyl
chains as an aglycone prime chondroitin sulfate but not heparan
sulfate. Compounds containing fused aromatic rings, which may simulate
the structure of the indole side chain of tryptophan, drive heparan
sulfate as well as chondroitin sulfate synthesis(25) . Thus,
aromatic
-D-xylosides may mimic the hydrophobic patch
found near heparan sulfate attachment sites. Synthesis of heparan
sulfate by aromatic
-D-xylosides requires a relatively
high concentration of primer (>30 µM) compared with
endogenous intermediates, possibly because they lack negative charges
imparted by the acidic residues in natural core proteins.
The enhanced synthesis of heparan sulfate across
adjacent Ser-Gly sites deserves further study. The upstream site in
syndecan-1 (Ser) couples to the downstream sites at
Ser
and Ser
(Table 1). Shworak et
al.(3) showed that stable transfectants expressing
ryudocan contain more heparan sulfate when multiple attachment sites
were present. These findings may indicate that coupling can occur at a
distance, possibly by juxtaposing the sites through secondary and
tertiary structure. Interestingly, chimeras containing the sites in
syndecan-3 (-SDLEVPTSSGPSGDFEIQEEEETT-) and in N-syndecan (-TTTQDEPEVPVSGGPSGDFELQEE-) do not
prime much heparan sulfate (7 and 8%, respectively). Both sequences
contain a proline residue between the Ser-Gly units, which may act as
an inhibitor. The low degree of coupling between closely spaced Ser-Gly
sites in betaglycan also may have been due to an intervening proline
residue(2) , which may have altered the conformation of the
peptide (Table 3). If correct, this idea is reminiscent of the
inhibitory effect of Pro on the attachment of
Glc
Man
GlcNAc
oligosaccharides from
dolichyl-P intermediates to Asn-Xaa-Ser/Thr sites in
glycoproteins(27) . Perhaps negative regulatory sequences/amino
acids play a role in proteoglycan assembly as well.
As shown in Table 6, repetitive Ser-Gly dipeptides with a flanking cluster of
acidic residues represent a common motif in a variety of heparan
sulfate proteoglycans. Thrombomodulin also contains adjacent Ser-Gly
dipeptides, but lacks a cluster of acidic residues and therefore does
not prime heparan sulfate. Searching the Wisconsin gene bank for
sequences consisting of (Ser-Gly) and a
nearby cluster of acidic amino acids ((D/E)
) within 6 residues yielded 15 out of 16 heparan sulfate
proteoglycans cloned to date. The search also yielded proteins from
bacteria, viruses, parasites, and eukaryotic subcellular organelles.
Cell surface and secreted proteins, including agrin, a tyrosine kinase
receptor, prostatic spermine-binding protein, and sporozoite surface
antigen have a high chance of bearing heparan sulfate chains based on
their location on the surface or outside the cell. Among them, agrin
was shown recently to bear heparan sulfate chains(28) .
Interestingly, a chimera prepared from the tyrosine kinase receptor
sequence (M35196) primed heparan sulfate, suggesting that the native
protein might contain a heparan sulfate chain.
Proteoglycans vary in
composition in different tissues and cells. For example, serglycin
contains chondroitin sulfate chains when expressed in various myeloid
cells (30) and a mixture of heparin and chondroitin sulfate
chains when expressed in connective tissue mast cells. ()Syndecan-1 also varies in composition in different cells (31) and in response to growth factors (32) . No
systematic study of the biosynthetic enzymes has been undertaken in
these tissues, but it is reasonable to assume that changes in enzyme
expression could alter GAG composition. Thus, the actual complement of
chains borne by a proteoglycan depends on biosynthetic capacity as well
as permissive elements embedded in the core protein sequence.
Although the proportion of heparan sulfate declines, the actual
amount of material increases after transfection. The data presented in Table 2show that wild-type cells produced about 3.2
10
cpm of [
S]heparan sulfate (4.4
10
cpm
72%). The stable transfectant
produced 8.1
10
cpm of
[
S]heparan sulfate (1.5
10
54%). This 2-3-fold increase in heparan sulfate
synthesis is offset by a much larger increase in chondroitin sulfate
synthesis, causing the relative composition to change. The
compositional difference mimics the effect of
-D-xylosides, which depress the proportion of heparan
sulfate because the primers preferentially stimulate chondroitin
sulfate assembly(25) .