From the Institute of Physiological Chemistry and
Pathobiochemistry, University of Münster, D-48149 Münster,
Germany and the
Shriners Hospital for Children, McGill
University, Montreal, Quebec H3G 1A6, Canada
Received for publication, October 12, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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Biglycan is a member of the small leucine-rich
proteoglycan family. Its core protein comprises two
chondroitin/dermatan sulfate attachment sites on serine 42 and serine
47, respectively, which are the fifth and tenth amino acid residues,
respectively, after removal of the prepro peptide. Because the
regulation of glycosaminoglycan chain assembly is not fully
understood and because of the in vivo existence of
monoglycanated biglycan, mutant core proteins were stably expressed in
human 293 and Chinese hamster ovary cells in which i) either one or
both serine residues were converted into alanine or threonine residues,
ii) the number of acidic amino acids N-terminal of the respective
serine residues was altered, and iii) a hexapeptide was inserted
between the mutated site 1 and the unaltered site 2. Labeling
experiments with [35S]sulfate and
[35S]methionine indicated that serine 42 was almost fully
used as the glycosaminoglycan attachment site regardless of whether
site 2 was available or not for chain assembly. In contrast,
substitution of site 2 was greatly influenced by the presence or
absence of serine 42, although additional mutations demonstrated a
direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was
also no assembly of the linkage region. These results indicate that
xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.
The biosynthesis of proteoglycans is a complex process in
which, in the case of chondroitin and heparan sulfate
proteoglycans, the assembly of the glycosaminoglycan side chains
begins with the transfer of xylose from UDP-xylose to the hydroxyl
group of selected serine residues of the core protein (1). A
recognition consensus sequence for the attachment of xylose residues
may consist of two acidic amino acids closely followed by the
tetrapeptide Ser-Gly-Xaa-Gly, where Xaa is any amino acid (2-4).
However, a sequence comparison of several proteoglycan core
proteins indicated that the second glycine residue was present in only
19% of the cases, and there was also only an ~50% abundance of
acidic residues among the three amino acids preceding the
glycosaminoglycan attachment site (5). Furthermore, a Gly-Ser-Ala
sequence instead of the Xaa-Ser-Gly sequence is found at the
chondroitin sulfate attachment site of chicken type IX collagen (6),
and threonine substitutions of serine residues can serve as acceptors
for chain assembly, albeit at low efficiency (7). The situation is
additionally complicated by the fact that established attachment sites
are not necessarily used regularly, giving rise to so-called part-time proteoglycans. This has been considered as a result of the possibility that the attachment site is not always recognized by xylosyltransferase and that there may be competition for substrate between
xylosyltransferase and glycoprotein
N-acetylgalactosaminyltransferase (8). Furthermore, in the
glycosaminoglycan-free form of the part-time proteoglycan thrombomodulin, the respective serine residue is substituted with the
linkage region tetrasaccharide GlcA Considering the complexity of glycosaminoglycan chain assembly, we have
focused on the biosynthesis of biglycan. Biglycan belongs to the
family of small leucine-rich proteoglycans (for recent reviews see
Refs. 10-12) and carries most often two chondroitin/dermatan sulfate
chains that are linked to serines 42 and 47 of the full-length human
core protein sequence (13). The sequence around these serine residues
(marked by an asterisk) is DEEAS*GADTS*GVLD. However, usage of the two
sites is normally not complete (14), and up to 30% of the core protein
may be substituted with a single chain only (15). Biglycan contains
a prepro sequence of 38 amino acids. The propeptide whose function is
not fully known is not necessarily removed prior to or after secretion
(16). Additional proteolytic processing may take place after removal of
the propeptide, accounting for nonglycanated forms of the molecule (17,
18). The presence of only two glycosaminoglycan attachment sites that
are located in close proximity made biglycan an ideal candidate to
study glycosaminoglycan chain assembly in a still simple, yet somewhat
more complex system than in the case of the monoglycanated small
proteoglycan decorin, where glycosaminoglycan biosynthesis has
been studied in great detail (2, 7). It will be shown that the
attachment of the two chains is regulated differently.
Expression of Recombinant Biglycan in Human 293, Chinese Hamster
Ovary, and COS-7 Cells--
Total RNA was isolated from human MG-63
osteosarcoma cells, which are known to produce high quantities of
biglycan. It was subjected to reverse transcription with AMV
reverse transcriptase and an oligo(dT)15 primer. Then the
complete biglycan coding sequence was amplified by the Expand High
Fidelity polymerase chain reaction system (Roche Diagnostics;
polymerase chain reaction conditions: denaturation for 1 min at
94 °C, annealing for 1 min at 58 °C, and elongation for 2 min at
72 °C) using the forward primer 5'-GTTGGAATTCGAGTAGCTGCTTTCGGTCC-3' and the reverse and complement primer
5'-ATAAGAATGCGGCCGCCAAGGCTGGAAATG-3'. The primers introduce an
EcoRI and a NotI restriction site, respectively, near the 5' and 3' ends of the amplified sequence. Upon
EcoRI/NotI digestion, the purified amplification
product was ligated into the EcoRI/NotI-digested
pcDNA3.1 (+) vector (Invitrogen). The plasmid was isolated after
transformation of competent Escherichia coli TOP
10F'.
Biglycan mutations were introduced by using the QuikChange
site-directed mutagenesis kit (Stratagene) and HPSF quality
oligonucleotides (MWG Biotec) as primers. The method uses forward
and reverse primers that carry the base exchange in the center of their
sequence and anneal to identical positions of the plasmid. High
fidelity Pfu Turbo DNA polymerase was used for
amplification. The polymerase chain reaction conditions were as
follows: denaturation for 30 s at 94 °C, annealing for 1 min at
55 °C, and elongation for 14 min at 68 °C. After 30 polymerase chain reaction cycles, the methylated template was
degraded by DpnI, and XL1-Blue supercompetent cells were
used for transformation without prior ligation. The forward primers
used to introduce mutations of the wild-type sequence were as follows:
BGN S42A, 5'-GATGAGGAAGCTGCGGGCGCTGACAC-3'; BGN S42T,
5'-CGATGAGGAAGCTACGGGCGCTGACAC-3'; BGN S47A,
5'-CGCTGACACCGCAGGCGTCCTGGAC-3'. The latter primer was also
used to mutate BGN S42A for the generation of the double mutant BGN
S42A/ S47A. The primer pair
5-AAGGTGCCCAAGGGAGATTTCAGCGGGCTCC-3' and
5-GGAGCCCGCTGAAATCTCCCTTGGGCACCTT-3' served for the
construction of the triple mutant BGN S42A/S47A/ V178D. With
the use of the BGN S42A construct as template, BGN S42A/A44D was made
by using 5'-GAAGCTGCGGGCGATGACACCTCAGG-3' as
forward primer. The BGN S47A construct served as template to generate
BGN E39Q/S47A with the forward primer
5-'CATGATGAACGATCAGGAAGCTTCGGGCG-3'. Sequencing of all
constructs verified the introduction of the desired point mutations and the absence of mutations in the other parts of the cDNA sequence. Finally, a hexapeptide, IGPQVP, which is similar to
the hexapeptide following the Ser-Gly attachment site in decorin (IGPEVP), was placed between Gly43 and
Ala44 of the S42A biglycan mutant. In the first step 5'-
and 3'-portions of the desired sequence were generated by using the
primers corresponding to the vector sequences mentioned above, the
reverse and complement primer
5'-GGGCACCTGGGGGCCGATGCCCGCAGCTTCCTCATC-3' (5' portion) and
the forward primer 5'-ATCGGCCCCCAGGTGCCCGCTGACACCTCAGGCGTCC-3' (3'
portion), respectively. The amplified products were purified by agarose
gel electrophoresis and subjected to a second polymerase chain reaction
by using the two primers designed from the multicloning site. The
cDNA thus obtained was ligated into the pcDNA3.1 (+) vector
after EcoRI/XbaI digestion. The mutants used are
summarized in Fig. 1.
Cultured human 293 kidney cells were transfected with the unmodified
pcDNA3.1 (+) vector or with this vector containing one of the above
mentioned constructs by using LipofectAMINE 2000 (Life Technologies,
Inc.) according to the instructions of the manufacturer. The cells were
selected for neomycine resistance by adding 750 µg/ml G418 (Life
Technologies, Inc.). The expression of human decorin cDNA in 293 cells has been described before (19). Wild-type BGN, BGN S42A, and BGN
S47A were also used to transfect Chinese hamster ovary cells and COS-7
cells by an analogous procedure. COS cells were used for experiments 2 days after transfection.
Metabolic Labeling and Proteoglycan Isolation--
Unlabeled
reference biglycan was purified from the culture media of 293 cells
stably transfected with wild-type human biglycan cDNA. Purification
included ammonium sulfate precipitation and anion exchange
chromatography as described (20), except that during the latter step
NaCl was replaced by KCl. Biglycan-containing fractions that were
eluted from the DEAE column with 0.6 M KCl were made 2 M with (NH4)2SO4 and
directly applied to a phenyl-Sepharose column (Sigma; 1 ml of gel/50 ml
of conditioned medium) equilibrated with 10 mM
K2SO4, pH 6.8, containing 2 M
(NH4)2SO4. The column was washed
with 3 volumes of starting buffer and then with 3 volumes each of this
buffer in which the (NH4)2SO4
concentration had been reduced to 1 M, 0.5 M,
and 0.2 M, respectively. Analytically pure biglycan (purity
>95%) was desorbed with water.
Metabolic labeling of subconfluent 293, Chinese hamster ovary, and COS
cells with [35S]sulfate (incubation period 24 h) and
[35S]methionine (incubation period 8 h) was
performed as described earlier (19). When [35S]methionine
labeling was performed in the presence of
p-nitrophenyl- Enzyme Treatment--
Protein A-Sepharose-bound immune complexes
were subjected to digestions with chondroitinases ABC and ACII (20)
and/or heparinases I and III (21) as described. Treatment with
peptide:N-glycosidase F was for 20 h at 37 °C in 60 µl of sodium phosphate buffer, pH 7.5, containing 0.3 mM
phenylmethylsulfonyl fluoride, 3.3 mM EDTA, 0.4% (v/v)
Nonidet P-40, 0.06% (w/v) SDS, 0.3% (v/v) 2-mercaptoethanol, and 500 units of enzyme (New England Biolabs). After enzyme treatment, the
protein A-Sepharose-bound material was washed twice with 10 mM Tris/HCl, pH 6.8, prior to
SDS-PAGE.1 Use of
[35S]methionine-labeled proteoglycan established the
complete removal of the glycosaminoglycan chains and the
N-linked oligosaccharides by this methodology.
Other Methods--
SDS-PAGE followed either by fluorography or
by Western blotting was performed as described previously (20, 22).
Fluorograms were evaluated with the ImageQuant 5.0 software program
(Amersham Pharmacia Biotech). Western blots were probed with antisera
against biglycan (see above), biglycan propeptide (16), and the HNK-1 carbohydrate epitope (a kind gift of Dr. M. Schachner, University of
Hamburg, Germany). Automated protein sequencing was performed on a
Procise 492-01 gas phase sequencer (PerkinElmer Life Sciences). A search for tyrosine O-sulfation was attempted after
hydrolysis of core proteins under nitrogen in 1 M NaOH for
24 h at 110 °C (23).
Properties of Recombinant Biglycan Synthesized by 293 Cells--
Within 24 h stably transfected 293 cells
synthesized about 0.5 pg of biglycan core protein per cell.
Surprisingly, however, its glycosaminoglycan chains were not completely
sensitive toward degradation by chondroitin ABC lyase. Between 5 and
15% of total [35S]sulfate incorporated into biglycan
remained in a monoglycanated form. The same observations were made with
Chinese hamster ovary cells. The label could be completely removed by
treatment with heparinases I and III (Fig.
2), indicating that a subset of biglycan, synthesized by these cells, is a hybrid proteoglycan. However, glycosaminoglycan-free core protein was not observed when biglycan was
treated with heparinases only. Experiments with
[35S]methionine-labeled biglycan corroborated these
results (data not shown). Thus, only when two glycosaminoglycan chains
were present could one of the chains be composed of heparan sulfate.
It was also investigated whether or not the secreted proteoglycan still
carries the propeptide sequence. With the use of propeptide-specific antibodies, the presence of such a sequence could be verified in
Western blots of biglycan purified from 293 cells (Fig.
3). However, amino acid sequencing
revealed that at most 15% of the total quantity of secreted core
protein contained the propeptide sequence, which comprised amino acids
Phe19-Asn37.
Different Use of Glycosaminoglycan Attachment Sites--
For
studying the structural prerequisites for glycosaminoglycan chain
attachment to serines 42 and 47, respectively, of biglycan core
protein, a series of mutations was created that is summarized in Fig.
1. In a first series of experiments the serine residues were replaced
by alanine and used for studying biglycan biosynthesis in human 293 cells. Complementary experiments were performed with Chinese hamster
ovary and COS cells. The consequences for glycosaminoglycan chain
attachment turned out to be different depending on which one of the two
serine residues was mutated. In the S47A mutant biglycan, the core
protein became substituted with a single glycosaminoglycan chain,
which was to be expected (shown for 293 cells in Fig.
4). In contrast, in the S42A mutant about
80% of the core protein was secreted into the culture medium without
being substituted with a glycosaminoglycan chain. This percentage did
not differ between the three cell types under study. The
glycosaminoglycan chain-bearing species exhibited a somewhat slower and
a less heterogeneous migration pattern during SDS-PAGE than did the
monoglycanated proteoglycan resulting from the S47A mutation. Such a
migration behavior is best explained by assuming that the
glycosaminoglycan chain of the S42A mutant is somewhat larger and less
heterogeneous in size than that of the other mutant. As an alternative
to alanine, serine-42 was also replaced by threonine. In this mutant,
too, the majority of serine 47 residues remained unused (Fig.
5). In the double mutant S42A/S47A, an
alternative use of other potential glycosaminoglycan attachment sites
could not be observed. As will be discussed below, the
glycosaminoglycan-free, but still N-glycanated, core
proteins were nevertheless substituted with [35S]sulfate
residues.
The proposed consensus sequence for glycosaminoglycan chain attachment
is fitted better for use of serine 42 than of serine 47 (Fig. 1).
Further constructs were, therefore, generated to optimize the use of
the latter site by introducing an additional acidic residue and to make
the first site less efficient by removal of an acid residue. Serine 42 was always fully substituted by a glycosaminoglycan chain regardless of
whether or not a preceding acidic residue had been exchanged for a
neutral one. Use of serine 47, however, could remarkably be stimulated,
although complete substitution was never approached (Figs. 5 and
6). The results of three or more separate
incubations are summarized in Table I.
From the data described so far it appeared that use of serine 47 was markedly dependent on a substitution of serine 42. To study this
point further, a hexapeptide was inserted in the S42A mutant in front
of the serine 47 residue. This novel mutant protein, too, was secreted
normally. However, [35S]sulfate incorporation into a
chondroitin ABC lyase-sensitive glycosaminoglycan chain was just above
the limit of detection (Fig. 7), whereas
the secretion of the glycosaminoglycan-free core protein remained
unaltered. Similarly, attempts to create a new glycosaminoglycan
attachment site at serine 180 in the S42A/S47A double mutant by
inserting aspartate in position 178 were unsuccessful (result not
shown). This negative result should be seen in the context that of all
unused Ser-Gly sites, serine 180 should have the greatest distance from
N-glycosylation sites and, in analogy to the proposed
horseshoe structure of decorin (24), should be located at the
unrestricted convex site of the molecule.
Absence of Linkage Region Saccharides on Serine 47 in the S42A
Mutant--
The majority of serine 47 residues were not substituted
with a full-length glycosaminoglycan chain in the S42A mutant. The substitution of this residue with a linkage region tetrasaccharide, however, would have escaped detection. To study this possibility, 293 cells harboring the S42A mutation were, therefore, incubated with a
high quantity of [1-3H]galactose for 24 h. The
medium was then passed over a DEAE-Trisacryl M column, and unbound
biglycan core protein was recovered by immunoprecipitation. The
immune complex was exhaustively digested with endoglycosidase F to
remove all asparagine-bound oligosaccharides. No 3H
radioactivity remained associated with the immune complex. As a control
for a proteoglycan with a single glycosaminoglycan chain, 293 cells
transfected stably with human decorin cDNA were used and labeled
under identical conditions. In this case, the whole medium was
subjected to decorin immunoprecipitation followed by digestion
of the immune complex with chondroitin ABC and ACII lyases and then
with endoglycosidase F. The immune complex still contained 1920 cpm of
3H radioactivity. It was therefore concluded that serine 47 was either substituted with a full-length glycosaminoglycan chain or
was devoid of a galactose-containing linkage region oligosaccharide. This conclusion was further supported by the observation of a slightly
different migration behavior during SDS-PAGE of the unsubstituted S42A
core protein and the S42A core protein obtained by chondroitin ABC
lyase treatment.
In further experiments, competition for glycosaminoglycan chain
assembly by exogenously added
p-nitrophenyl- Core Protein-associated [35S]Sulfate--
As seen in
Fig. 2 and subsequent figures, the core protein of biglycan could be
labeled with [35S]sulfate. Combined digestion with
chondroitin ABC lyase and chondroitin ACII lyase for complete removal
of the glycosaminoglycan chains with the exception of the linkage
region tetrasaccharide did not result in complete desulfation. Because
small proteoglycans may carry tyrosine O-sulfate residues
(25, 26), biglycan core proteins were subjected to alkaline hydrolysis.
Thereafter, all radioactivity could be removed as insoluble barium
salt, thereby excluding the presence of alkali-stable tyrosine
O-sulfate residues. However, the label could completely be
removed by treatment with endoglycosidase F, indicating that sulfate
ester may be present on one or both of the two asparagine-linked
oligosaccharides of biglycan (Fig. 9).
N-linked oligosaccharides may carry a terminal 3-sulfated
GlcA residue as part of the HNK-1 carbohydrate epitope (27). Westerns
blots with antibodies against this epitope were negative for biglycan
core protein although proteins of higher molecular weight in the
medium of transfected 293 cells gave a positive immune reaction
(result not shown). Whether or not asparagine-linked oligosaccharides
of biglycan core protein contain sulfated
N-acetyllactosamine units (28) was not investigated
further.
The main result of this study is the observation that of the
two glycosaminoglycan attachment sites of biglycan the more
C-terminally located one is used nearly completely only when the first
attachment site is available. This result should be considered in light
of the high wild-type biglycan production of transfected 293 and CHO
cells. Hence, it appears unlikely that the data obtained with mutant
core proteins simply reflect the limited capacity for glycosaminoglycan chain assembly of these cells. Furthermore, [35S]sulfate
labeling studies with
p-nitrophenyl- In a study on the glycosaminoglycan attachment sites of perlecan it has
been shown that the serine residues 65, 71, and 76 could be used
individually when the two other sites were mutated to threonine (31).
As in our model, the remaining serine residue did not serve as acceptor
site in all molecules, but a sequence-dependent change in
the efficiency of usage did not become evident. A direct comparison of
the data with respect to site effectiveness, however, is difficult
because a deletion of 110 amino acids was present in the perlecan
construct eight amino acids C-terminal of serine 76, the last
glycanated serine residue. The existence of core protein-specific
pathways may also be envisaged (32).
Direct biosynthetic labeling of the linkage region together with the
results of competition experiments with
p-nitrophenyl- Xylose addition appears to begin in the endoplasmic reticulum and to
continue in intermediate compartments, perhaps including the cis-Golgi,
where galactose is added prior to the assembly of the repetitive
disaccharide structures (34-36). For an explanation of the apparent
cooperativity between the usage of the two glycosaminoglycan attachment
sites that are located close to the N terminus of the mature core
protein, it is tempting to assume that the propeptide contributes to
optimal recognition of the first serine residue by the
xylosyltransferase. The second serine residue then becomes more easily
recognized, although an independent usage of it may occur and may
become facilitated by an appropriate introduction of an additional
acidic amino acid close to its N-terminal site. The compartment where
the propeptide sequence of biglycan and decorin is normally removed has
not yet been directly defined. The recent observation that bone
morphogenetic protein-1 (procollagen C-proteinase) processes
probiglycan (37) strongly suggests that this cleavage occurs after
secretion or immediately prior to it. Thus, a regulatory influence of
the propeptide on chain initiation is compatible with the topography of
probiglycan processing. The N terminus of the propeptide was found to
be generated by proteolysis at an unusual cleavage site between
Pro18 and Phe19 and not as often after a
small amino acid (38). The predominant form of biglycan from the
secretions of rat and bovine smooth muscle cells was generated by
hydrolysis between Ala16 and Leu17. However, an
alternative rat propeptide sequence also began with Phe19,
indicating that there might be either cell- and species-specific differences in the processing of the prepro peptide of biglycan or
further degradation of the generated propeptide by aminopeptidases (39). The functional consequences of these differences are not yet known.
The present study yielded several additional results. First, the
glycosaminoglycan-free biglycan core protein is transport-competent and
becomes secreted as the fully N-glycosylated species. Its extracellular stability, however, may be decreased, considering the
finding that the conformational stability of biglycan depends to a
greater extent than in the case of decorin on the presence of
chondroitin/dermatan sulfate chains (40). Second, as expressed in 293 and CHO cells, biglycan may be substituted with a single heparan
sulfate chain, thereby representing a hybrid proteoglycan. Recombinant
biglycan synthesized by HT-1080 cells also appears as a hybrid species
because its glycosaminoglycan chains cannot completely be removed by
chondroitin ABC lyase (41). The factors that govern the substitution
with either two galactosaminoglycans or a single galactosaminoglycan
and an additional glucosaminoglycan chain and their possible cell type
specificity have not yet been defined. Some experimental variability
was also noted in the present investigation. Third, sulfation of
asparagine-bound oligosaccharides of a chondroitin/dermatan sulfate
proteoglycan was shown for the first time. This sulfate substitution
may have escaped detection in previous studies because of its low
relative amount. The functional significance and the detailed structure
of the sulfated oligosaccharides remain to be determined.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
1-3Gal
1-3Gal
1-4Xyl
(9), raising the possibility that it is the transfer of the fifth
monosaccharide (either GalNAc or GlcNAc) that represents the most
critical step in glycosaminoglycan chain biosynthesis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the glycosaminoglycan chain
attachment consensus sequence with the sequence of mature wild-type and
mutant biglycan. Mutated amino acids are indicated by bold
single letters in italics. a designates an acidic amino
acid, and x designates any amino acid. GAG,
glycosaminoglycan; KO, knockout.
-D-xyloside (Sigma), the
xyloside was already present during the 1-h preincubation period in
methionine-free medium. For labeling with [1-3H]galactose
(ICN Pharmaceuticals; specific radioactivity 18 Ci/mmol), Waymouth MAB
87/3 medium containing 4% fetal calf serum and only 1.4 mM
glucose was used. Incubation was for 24 h in the presence of 14 ml
of culture medium with 500 µCi of 3H radioactivity per
75-cm2 culture flask. At the end of the incubations, medium
was mixed with proteinase inhibitors and made 70% saturated with
(NH4)2SO4. After centrifugation,
the pellet from one 25-cm2 culture flask was dissolved in
300 µl of 0.1 M Tris/HCl buffer, pH 7.4, containing 1 M NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, and
proteinase inhibitors. The solution was first allowed to react with
protein A-Sepharose (Sigma; 1.5 mg of dry gel per 25-cm2
flask), which had been preincubated with preimmune rabbit antisera as
described (15). Biglycan was subsequently immunoprecipitated by
applying 6 mg of protein A-Sepharose per 25-cm2 flask,
which had been coated with a monospecific rabbit antiserum against a
KLH-coupled biglycan-derived peptide raised analogously as
described (13).
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Fig. 2.
Heparinase sensitivity of recombinant
biglycan. [35S]Sulfate-labeled biglycan was isolated
from 293 cells transfected with wild-type biglycan cDNA and
subjected to treatments with chondroitin ABC lyase (ABC)
and/or heparinases I and III (HSase) prior to SDS-PAGE in a
10% separation gel followed by fluorography. The migration distance of
reference proteins is indicated in the right margin.
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Fig. 3.
Identification of biglycan propeptide.
Biglycan was purified from the conditioned medium of 293 cells
transfected with wild-type biglycan cDNA. After digestion with
chondroitin ABC lyase, it was subjected to SDS-PAGE, Western blotting,
and immunochemical reaction with a propeptide-specific antibody
(lane 1) and an antibody directed against the mature core
protein (lane 2). The prosequence obtained by gas phase
sequencing is shown in the right panel.
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Fig. 4.
Glycosaminoglycan substitution of wild-type
biglycan and biglycan mutants S42A, S47A, and S42A/S47A.
[35S]Sulfate- and [35S]methionine-labeled
biglycan was isolated after biosynthetic labeling and subjected to
SDS-PAGE and fluorography. CV, control vector;
WT, wild type.
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Fig. 5.
Glycosaminoglycan substitution of biglycan
mutants S42T, S42A/A44D, and E39Q/S47A as determined by
[35S]sulfate incorporation. Secreted biglycan was
obtained by immunoprecipitation from the mutants indicated and from 293 cells transfected with wild-type (WT) biglycan or
insert-free control vector (CV) prior to SDS-PAGE and
fluorography.
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Fig. 6.
Glycosaminoglycan substitution of biglycan
mutants S42T, S42A/A44D, and E39Q/S47A as determined by
[35S]methionine incorporation. The experiment was
performed analogously to that described in the legend to Fig. 5.
ABC denotes digestion with chondroitin ABC lyase.
WT, wild type.
Composition of glycosaminoglycan-containing biglycan secreted by 293 cells
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Fig. 7.
Further decline of glycosaminoglycan
substitution by hexapeptide insertion into biglycan mutant S42A.
Cultures of 293 cells expressing wild-type (WT) biglycan,
biglycan S42A, and S42A with a hexapeptide insert were incubated in
parallel with [35S]sulfate prior to SDS-PAGE of secreted
biglycan followed by fluorography. Note that wild-type biglycan was
overexposed. ABC denotes digestion with chondroitin
ABC lyase.
-D-xyloside was studied.
Neither in the S42A nor in the S47A mutant was the use of the remaining
glycosaminoglycan chain attachment site influenced by increasing doses
of the competitor. However, shorter glycosaminoglycan chains were
assembled at high xyloside concentrations (Fig.
8). These data suggest that the activity
of galactosyltransferases is not the rate-limiting step during the
formation of the linkage region. Hence, it seems likely that serine 47 was also not substituted with xylose in the glycosaminoglycan-free
species of the S42A mutant.
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Fig. 8.
Influence of
p-nitrophenyl- -D-xyloside
on biglycan biosynthesis. Cells stably transfected with wild-type
(WT) biglycan cDNA or one of the mutant constructs were
preincubated with the xyloside concentrations given in the figure
(µM) for 1 h before they were incubated further for
6 h in the presence of [35S]methionine and the same
concentrations of xyloside. Medium was then subjected to
immunoprecipitation, SDS-PAGE, and fluorography.
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Fig. 9.
Sensitivity of core protein sulfation to
endoglycosidase F treatment. Cells transfected with the double
mutant S42A/S47A were labeled with either [35S]methionine
or [35S]sulfate before biglycan was obtained by
immunoprecipitation and subjected to digestion with endoglycosidase F
(Endo F) prior to SDS-PAGE and fluorography. Note that in
this experiment some biglycan had undergone limited proteolysis of the
core protein.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-xyloside as primer
molecule also supported this conclusion. Monoglycanated biglycan has
also been extracted from normal tissue. However, it has not yet been
investigated systematically which of the two glycosaminoglycan
attachment sites were used in these cases. In bovine articular
cartilage both sites were almost completely substituted (14) whereas,
as anticipated from the present study, the second site was less
frequently used in biglycan from human bone (29). Analogous data were
obtained with chicken decorin, which resembles mammalian biglycan
because it may also carry two chondroitin/dermatan sulfate chains
linked to serine 4 and serine 16, respectively. In the monoglycanated
proteoglycan form, serine 4 was used exclusively (30). Information on
the usage of the multiple sites of other chondroitin/dermatan sulfate
proteoglycans is not available.
-D-xyloside strongly
suggested that in the S42A mutant, serine 47 was either linked with a
full-length glycosaminoglycan chain or not substituted at all with
carbohydrate. This finding indicated that
UDP-D-xylose:protein
-D-xylosyltransferase (EC 2.4.2.26) is the rate-limiting
step in glycosaminoglycan chain assembly and not, as in the case of thrombomodulin, a
-N-acetylgalactosaminyltransferase (9). Also in other aspects of glycosaminoglycan chain initiation,
thrombomodulin appears to exhibit special features, because the
attachment consensus sequence is less well preserved, and, most
strikingly, different serine residues can be used in different cells
(33).
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ACKNOWLEDGEMENT |
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The expert technical help of Barbara Liel is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 496, Project A6).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Inst. of Physiological Chemistry and Pathobiochemistry, Waldeyerstr. 15, D-48149 Münster, Germany. Tel.: 49-251-8355581; Fax: 49-251-8355596; E-mail: kresse@uni-muenster.de.
¶ Present address: Dept. of Orthopedics, University of Ulm, 89069 Ulm, Germany.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M009321200
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ABBREVIATIONS |
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The abbreviation used is: SDS-PAGE, SDS-polyacrylamide gel electrophoresis.
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