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J. Biol. Chem., Vol. 280, Issue 27, 25674-25686, July 8, 2005
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-inhibitor That Act as Intermediates in the Covalent Transfer of Heavy Chains onto Hyaluronan*
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From the
Medical Research Council Immunochemistry
Unit, Department of Biochemistry, University of Oxford, South Parks Road,
Oxford OX1 3QU, United Kingdom, the Section of
Connective Tissue Biology, Department of Biomedical Engineering, Cleveland
Clinic Foundation, Cleveland, Ohio 44195,, and the¶
Department of Medical Biochemistry and
Microbiology, Uppsala University, S-751 23 Uppsala, Sweden
Received for publication, February 4, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-inhibitor (II), which is composed of three
subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate
moiety. The formation of HC·HA is likely to play an important role in
the stabilization of HA-rich extracellular matrices in the context of
inflammatory disease (e.g. arthritis) and ovulation. Here, we have
characterized the complexes formed in vitro between purified human
II and recombinant human TSG-6 (an inflammation-associated protein
implicated previously in this process) and show that these complexes
(i.e. TSG-6·HC1 and TSG-6·HC2) act as intermediates in
the formation of HC·HA. This is likely to involve two
transesterification reactions in which an ester bond linking an HC to
chondroitin sulfate in intact II is transferred first onto TSG-6 and
then onto HA. The formation of TSG-6·HC1 and TSG-6·HC2 complexes
was accompanied by the production of bikunin·HC2 and bikunin·HC1
by-products, respectively, which were observed to break down, releasing free
bikunin and HCs. Both TSG-6·HC formation and the subsequent HC transfer
are metal ion-dependent processes; these reactions have a requirement for
either Mg2+ or Mn2+ and are inhibited by
Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6
onto HA, was shown to combine with II to form new TSG-6·HC
complexes and thus be recycled. The finding that TSG-6 acts as cofactor and
catalyst in the production of HC·HA complexes has important
implications for our understanding of inflammatory and inflammation-like
processes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-inhibitor (II) family; this modification is likely to
alter both its matrix and solution properties
(3–5).
For instance, the formation of HC·HA (also known as the serum-derived
hyaluronan-associated protein·HA complex
(5)) is critical for the
stabilization of a nascent HA-rich matrix that is generated around the oocyte
of placental mammals prior to ovulation
(6–8).
The matrix expansion of the cumulus-oocyte complex (COC) is required for
successful fertilization in vivo
(9). HC·HA complexes are
also formed under inflammatory conditions, e.g. in synovial fluids of
arthritis patients (10,
11), where this modification
causes HA to become more aggregated
(4,
12,
13), which is likely to alter
its hydrodynamic size and rheological properties and makes it more resistant
to degradation by oxygen free radicals
(14).
II is an unusual proteoglycan that contains three protein chains
(HC1, HC2, and the serine protease inhibitor bikunin) held together via a
chondroitin sulfate (CS) moiety
(5). The CS chain is attached
to Ser10 of bikunin via a standard glycosaminoglycan attachment,
whereas the HCs are linked through ester bonds between carboxylate groups of
their C-terminal aspartic acid residues and the C-6 hydroxyls of internal
N-acetylgalactosamines in the CS chain
(15–17).
The HCs appear to be transferred onto HA by a transesterification reaction
because they become linked via an ester bond from their C termini to the C-6
hydroxylates of GlcNAc residues in HA
(3), i.e. analogous to
the HC ester bond to CS in II. An intact II protein is clearly
required for this process because female mice lacking the bikunin gene, which
express the HCs but are unable to assemble II (or the related
pre--inhibitor (PI), which has a different heavy chain (HC3)
linked to bikunin (18)), do
not form any HC·HA in their cumulus matrix. As a consequence, they are
infertile (6,
19). Mixing of purified HA and
II in vitro does not give rise to the formation of
HC·HA (20), indicating
that other molecules are involved
(21). Recent work has revealed
that TSG-6 (the protein product of tumor necrosis
factor-stimulated gene-6; also known as TNFIP6
(tumor necrosis factor-induced
protein-6) (22)) is
involved. TSG-6 is an inflammation-associated HA-binding protein composed
mainly of contiguous Link and CUB modules
(23–25)
and appears to have an essential role in the transfer of HCs from II
onto HA (7,
26,
27). Most importantly,
Fülöp et al.
(7) showed that
Tsg-6–/– female mice are infertile due to
their inability to form the HA-rich extracellular matrix that is essential for
cumulus expansion, a phenotype that correlates with the total absence of
HC·HA complexes in the ovaries of these animals; the administration of
murine TSG-6, either as a recombinant protein or as a transgene, rescues the
fertility of Tsg-6 null mice. In this study, HC3 (a component of
PI), in addition to HC1 and HC2, was missing from the cumulus matrix,
indicating that TSG-6 is also necessary for the transfer of HC3 onto HA
(7).
The expression of TSG-6 is up-regulated during COC expansion in the mouse
and rat
(27–33),
where the protein has been shown to co-localize with HA and II in the
cumulus matrix (30,
31). Western blot analyses
revealed that TSG-6 is present as a free protein (
35 kDa) and as a
species of 120 kDa that is immunoreactive with both anti-TSG-6 and
anti-II antibodies (30,
31). Characterization of this
120-kDa band by mass spectrometry demonstrated that it contains TSG-6,
HC1, and HC2, but not bikunin. On the basis of their molecular masses
(i.e. TSG-6 is 35 kDa, and each HC is 80–85 kDa), the
120-kDa band is thought likely to comprise a mixture of TSG-6·HC1
and TSG-6·HC2 complexes
(31). These complexes are not
sensitive to chondroitinase, indicating that the CS chain of II is not
involved in their linkage, but they are cleaved by mild NaOH treatment,
consistent with the presence of ester bonds. Therefore, the TSG-6·HC
complexes may act as intermediates in the formation of HC·HA, as has
been suggested (7). However,
prior to the present study, this had not been investigated directly. Species
of 120 kDa can also be formed when human recombinant TSG-6 and human
purified II are incubated together in vitro
(27,
34,
35), and
TSG-6·II complexes of this size have been detected in the
synovial fluids of arthritis patients
(36). However, the composition
of the 120-kDa species formed in vitro from human components,
which was reported to contain TSG-6, HC2, and bikunin held together via a
chondroitinase-sensitive linkage
(34), indicates that it might
represent a different type of complex (with TSG-6 replacing an HC on the CS
chain) compared with that formed during ovulation in the mouse.
In this study, we have characterized the complexes formed in vitro
between purified human II and recombinant human TSG-6 as
TSG-6·HC1 and TSG-6·HC2 and have shown that they act as
intermediates in the formation of HC·HA. This is accompanied by the
production of bikunin·HC1 and bikunin·HC2 by-products, which
break down to generate free bikunin and HCs. Both TSG-6·HC complex
formation and subsequent HC transfer are metal ion-dependent processes, having
a requirement for either Mg2+ or Mn2+. TSG-6, which is
released upon the transfer of HCs from TSG-6 onto HA, was shown to combine
with II to generate new TSG-6·HC complexes and thus acts as a
true catalyst for the formation of HC·HA.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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I was purified
from human serum (37), and its
concentration was determined as described previously
(38).
Formation of TSG-6·II Complexes under
Standard Conditions—In the standard assay, recombinant full-length
TSG-6 (80 µg/ml final concentration; 2.7 µM based on a
molecular mass of 30 kDa) was incubated with II (320 µg/ml final
concentration; 1.8 µM based on a molecular mass of 180 kDa) in
20 mM HEPES-HCl (pH 7.5), 150 mM NaCl, and 5
mM MgCl2 in a total volume of 25 µl for 2 h at 4
°C (i.e. on ice). The effects of protein concentration,
temperature, ionic strength, pH, and metal ions on complex formation were
investigated by varying these parameters individually while keeping all other
conditions constant. Unless otherwise stated, 7.5 µl of each sample was
analyzed on 10% (w/v) Tris/Tricine/SDS-polyacrylamide gels following reduction
with 5% (v/v) 
-mercaptoethanol in SDS protein sample buffer (5 min at
100 °C), and gels were stained with Coomassie Blue.
Effect of TSG-6 and II Protein Concentrations on
TSG-6·II Complex Formation—The
amount of TSG-6 in the assay was varied from 1 to 8 µg (40–320
µg/ml final concentration) while keeping the amount of II constant
at 8 µg (320 µg/ml final concentration). Alternatively, between 8 and 32
µg (320–1280 µg/ml final concentration) of II was used in
the presence of 1 µg of TSG-6 (40 µg/ml final concentration). As a
control, TSG-6 (1 µg) or II (8 µg) was incubated alone under
standard assay conditions. These samples were analyzed by SDS-PAGE and by
Western blotting using a rabbit anti-human polyclonal antibody raised against
TSG-6 (39) as described
(35).
Effect of Temperature, pH, and Ionic Strength on
TSG-6·II Complex
Formation—TSG-6·II complex formation was compared at
4 and 37 °C; the reactions were carried out for 0.5, 2, 5, 15, 30, 60, and
120 min. Assays were performed at pH values ranging from 4.0 to 8.0. Sodium
acetate buffer was used for pH 4.0 and 5.0; MES-HCl buffer for pH 6.0 and 6.5;
and HEPES-HCl for pH 7.0, 7.5, and 8.0 (all at a 20 mM final
concentration as in the standard assay described above). To investigate the
effect of ionic strength on TSG-6·II complex formation, the
assay was run in 20 mM HEPES-HCl (pH 7.5) in the presence of 50,
100, 150, 200, 250, or 300 mM NaCl. In these experiments, control
samples of TSG-6 or II alone were incubated at the appropriate
temperature for 2 h.
Effect of Divalent Metal Ions on
TSG-6·II Complex Formation—The
requirement for divalent metal ions during formation of the
TSG-6·II complex was tested by incubating TSG-6 and II
with 1 mM EDTA in the absence or presence of 5 mM
MgCl2, CaCl2, CoCl2, or MnCl2.
Additionally, CaCl2 and CoCl2 were co-incubated with
MgCl2 in the absence or presence of EDTA (i.e. 5
mM MgCl2 with 1 mM CaCl2 or
CoCl2; 5 mM MgCl2 with 5 mM
CaCl2 or CoCl2; and 1 mM MgCl2 and
1 mM EDTA with 5 mM CoCl2). Complex formation
was also investigated at a range of MgCl2 concentrations
(i.e. 0, 0.1, 0.5, 1.0, 5.0, 10.0, and 20.0 mM).
The effect of metal ions on TSG-6·II complex formation was
also examined using serum as the source of II. Mouse serum (20 µl;
Rockland Immunochemicals) was incubated with 2 µg of human recombinant
TSG-6 in the presence or absence of 2 mM EDTA and 5 mM
CaCl2, MgCl2, or CoCl2 in a 25-µl reaction
volume at 37 °C for 2 h. Samples (6 µl) were run on 4–20% precast
gels (Invitrogen) and analyzed by Western blotting as described previously
(31) using a rabbit anti-human
polyclonal antibody raised against TSG-6
(39).
Effect of Chondroitinase and NaOH Treatment on
TSG-6·II Complex
Stability—TSG-6·II complexes were formed under
standard conditions (i.e. 2 h at 4 °C; see above). Aliquots of
the reaction mixture (7.5 µl) or of II incubated in the absence of
TSG-6 were then diluted with an equal volume of water. Either 1 µl of
chondroitinase ABC lyase (10 milliunits; Seikagaku Corp.) or 1.5 µl of 1
M NaOH (0.1 M final concentration) was then added,
followed by incubation for 2 h at 37 °C or for 10 min at room temperature,
respectively; 1.5 µl of 1 M HCl was added to the NaOH-treated
samples. To these (and a chondroitinase-only control (10 milliunits in 15
µl of H2O) and an untreated TSG-6·II complex (7.5
µl + 7.5 µl of H2O), both incubated for 2 h at 37 °C)
were added 15 µl of 2x SDS protein sample buffer, followed by
SDS-PAGE (with the whole sample loaded) or Western blotting (with one-third of
the sample loaded) as described above.
Characterization of TSG-6·II Complexes
by N-terminal Sequencing— Complexes were formed under standard
conditions and run on 10% (w/v) Tris/Tricine/SDS-polyacrylamide gels with or
without chondroitinase (10 milliunits) or NaOH (0.1 M final
concentration) treatment essentially as described above, except that the
"untreated" sample was not incubated at 37 °C for 2 h, and in
all cases, twice as much protein was loaded per lane. The gels were
electroblotted onto Hybond-P membrane (Amersham Biosciences) in 10
mM CAPS (pH 11) and 5% (v/v) methanol at 100 V for 2 h. The
membranes were stained with Coomassie Blue for 75 s, destained in 50% (v/v)
methanol for 10 min, and air-dried. Bands were excised (with reference to an
identical control gel) and subjected to protein sequencing on an Applied
Biosystems Procise 494A protein sequencer using standard "pulsed liquid
for polyvinylidene difluoride-bound peptides" sequencing cycles. Bands
that did not yield any visible sequence were excised from an identical gel and
subjected to in-gel digestion with trypsin, followed by mass spectrometric
analysis as described previously
(31).
Formation and Characterization of II·HA
Complexes—TSG-6·II complexes were formed under
standard conditions (2 h at 4 °C), except that HA was included in the
reaction; 1 µg of medical grade low molecular mass polymeric HA (p-HA;
120 kDa; Genzyme Corp.) or 1 µg of HA 14-mer (HA14; 2673
Da) prepared as described
(40)) was added to the assay,
and samples were analyzed by Tris/Tricine/SDS-PAGE and protein sequencing as
described above. For sequence analysis of the p-HA sample, three identical
lanes were loaded (7.5 µl of reaction mixture), followed by transfer onto
Hybond-P (in 10 mM CAPS (pH 11) for 3 h at 100 V) and
staining/destaining as described above. Equivalent bands (at the interface of
the stacking and resolving gels) were excised from the three lanes and
combined for sequencing.
Samples containing p-HA (or II alone) were also treated with
Streptomyces hyaluronidase (Seikagaku Corp.) prior to SDS-PAGE
analysis. The reaction mixture (7.5 µl) was diluted with an equal volume of
water to which 1 µl of enzyme (10 milliunits) was added, followed by
incubation at 37 °C for 2 h. II treated with NaOH as described
above was included as a control.
Effect of Divalent Metal Ions on II·HA
Complex Formation—TSG-6·II complexes were preformed
under essentially standard conditions in the presence of 0.109 mM
MgCl2 for 2 h at 4 °C, followed by the addition of 1
mM EDTA and incubation for an additional 30 min. Divalent metal
ions (MgCl2, MnCl2, CaCl2, and
CoCl2) and 1 µg of HA14 were then added (20
mM HEPES-HCl (pH 7.5), 150 mM NaCl, 0.1 mM
MgCl2, 1 mM EDTA, 5 mM M2+, and 40
µg/ml HA14 (final concentrations); 25-µl final volume) and
incubated for 2 h at 4 °C. Control experiments were also performed in the
absence of HA, EDTA, or metal ions. Alternatively, TSG-6·II
complexes were preformed in the presence of 5.4 mM MgCl2
(5 mM final concentration), and then CoCl2 was added (1
or 5 mM final concentration) and incubated for 30 min at 4 °C
before adding 1 µg of HA14 and incubating for 2 h at 4 °C.
In another experiment, TSG-6, II, and HA14 were incubated
together for 2 h at 4 °C in the absence and presence of 5 mM
metal ion (MgCl2, MnCl2, CaCl2, or
CoCl2), but without the inclusion of EDTA; co-incubation of 5
mM MgCl2, MnCl2, or CaCl2 with 5
mM CoCl2 was also performed. Gel samples were prepared
from 7.5-µl reaction volumes as described above.
The effect of metal ions on the formation of II·HA complexes
was also examined using serum as the source of II. Mouse serum (5
µl), 5 µg of high molecular mass HA (Healon GV, Pharmacia-Upjohn), and
250 ng of human recombinant TSG-6 were incubated for 24 h at 37 °C in the
presence or absence of 2 mM EDTA and 5 mM
CaCl2, MgCl2, or CoCl2 in 50 µl of
phosphate-buffered saline. Aliquots of each reaction mixture (10 µl) were
treated with 200 milliunits of Streptomyces hyaluronidase for 1 h at
37 °C. Hyaluronidase-digested and untreated samples were run on
4–20% precast gels and analyzed by Western blotting as described
previously (31) using
anti-II polyclonal antibody (Dako Corp.).
Requirement of TSG-6·II Complexes as
Precursors in the Formation of
II·HA—TSG-6·II
complexes were preformed under essentially standard conditions (2 h at 4
°C) and then incubated at 4 °C for a range of times (0, 2, 4, and 22
h) before the addition of 1 µg of HA14 (1 µl) or 1 µl of
water as a control and incubation for an additional 2 h. In a separate
experiment, II(8 µg) and HA14 (1 µg) were incubated
for 2, 4, or 22 h under standard conditions with various concentrations of
TSG-6 (0, 0.04, 0.2, and 2 µg) at 4 or 37 °C. Samples were analyzed by
SDS-PAGE as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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I Complexes in
Vitro—We have shown previously that incubation of recombinant human
full-length TSG-6 (expressed in Drosophila Schneider-2 cells and
purified to homogeneity) with human II (purified from serum) leads to
the production of a stable complex of 120 kDa recognized by an anti-TSG-6
antibody (35). Analysis here
of essentially identical TSG-6/II incubation mixtures by SDS-PAGE and
Coomassie Blue staining revealed that three novel protein species were formed
during this reaction: i.e. a diffuse 130-kDa band (labeled
A in Fig. 1) and an
120-kDa doublet (labeled B/C). Of these, only the 120-kDa
doublet was found to be immunoreactive with the anti-TSG-6 antiserum
(Supplemental Fig. S1), indicating that bands B and C correspond to the
TSG-6·II complex. This was subsequently confirmed by protein
sequence analysis (see below).
Fig. 1 shows that these
three species formed at both 4 and 37 °C and that bands A and C were
clearly visible after as little as 30 s. Maximal amounts of the B/C doublet
were formed after 15 min at 37 °C and after 60 min at 4 °C.
At the higher temperature, an additional band of 80 kDa (labeled
I in Fig. 1)
accumulated over time. This species ran at an identical position compared with
HC1, and N-terminal sequencing revealed that it contained both HC1 and HC2 at
an 5:1 ratio (data not shown). Band I could therefore result from
breakdown of the TSG-6·II complex or of other HC-containing
species (see below). In this regard, an 80-kDa band (albeit much fainter)
and a band of the same apparent molecular mass as species A were also seen
when II was incubated alone for 2 h at 37 °C, indicating that there
was some breakdown of II at this temperature. Additionally, at 37
°C, a high molecular mass doublet (near the top of the gel) also
accumulated over time during the incubation of TSG-6 with II, but
significant amounts of this were not formed at 4 °C; this doublet was
recently identified as a high molecular mass species of II containing
additional HCs (41).
Therefore, in subsequent experiments, incubations were generally carried out
for 2 h at 4 °C to maximize II·TSG-6 complex formation while
minimizing any production of high molecular II species or of
degradative reactions.
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I Complex Formation—The
effect of varying the protein concentration on the formation of the
TSG-6·II complex (i.e. B/C doublet) was investigated as
described under "Experimental Procedures." Similar amounts of the
B/C doublet were formed when 8 µg of II was incubated with between 2
and 8 µg of TSG-6, whereas less was seen under the other conditions tested
(i.e. 1 µg of TSG-6 in the presence of 8–32 µg of
II) (Supplemental Fig. S1). Assays conducted at different protein
concentrations (e.g. 2 or 4 µg of TSG-6 with 16 µg of
II) (data not shown) did not lead to significantly increased levels of
the B/C doublet. Therefore, 2 µg of TSG-6 and 8 µg of II
(i.e. the lowest concentrations that gave close to maximal complex
formation) were used as the standard experimental conditions so as to minimize
the amount of protein used in each assay.
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I were incubated at
pH 4.0 or 5.0 (under otherwise standard conditions). There also appeared to be
somewhat less complex formation at pH 6.0 compared with pH 6.5–8.0,
which all led to similar levels of the B/C doublet. Experiments at pH 7.5 in
which the ionic strength was varied revealed that there was a reduction in the
amount of TSG-6·II generated in 50 mM NaCl, whereas
similar amounts of complex were formed between 100 and 300 mM NaCl
(Fig. 2). Therefore, 150
mM NaCl and pH 7.5 (i.e. physiological serum conditions),
which supported optimal complex formation, were used as the standard. Preliminary experiments demonstrated that, in the absence of added metal ion, neither band A nor the B/C doublet was formed (Supplemental Fig. S2). However, these species were seen at similar levels when MgCl2 was included in the reaction mixture at a wide range of concentrations (0.1–20 mM). MgCl2 at the nominal concentration of 5 mM was chosen as the standard.
The above experiments therefore allowed us to determine the conditions
under which optimal amounts of TSG-6·II complex could be formed.
Under the standard conditions chosen (i.e. 80 µg/ml TSG-6 and 320
µg/ml II in 20 mM HEPES-HCl (pH 7.5), 150 mM
NaCl, and 5 mM MgCl2), which are close to physiological,
II was at a concentration similar to that found in normal human serum
(i.e. 400–500 µg/ml
(42)).
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I Complex Formation—As
described above, TSG-6·II complexes could be formed in the
presence of MgCl2, but did not form in the absence of added metal
ion (Supplemental Fig. S2). Consistent with this, there was no complex
formation in 0.1–50 mM EDTA (data not shown). Therefore, the
effects of other divalent cations were investigated; TSG-6 and II were
incubated under standard conditions with 5 mM M2+
(i.e. MgCl2, CaCl2, CoCl2, or
MnCl2) and 1 mM EDTA, which was added to chelate any
metal ion impurities. Fig. 3
shows that complex formation occurred with MgCl2 or
MnCl2, but the B/C doublet was not seen in the presence of the
other metal ions (e.g. Ca2+). It should be noted that
experiments conducted in the absence of any EDTA also demonstrated that either
Mg2+ or Mn2+ (but not Ca2+ or
Co2+) ions could support complex formation (data not shown).
Interestingly, co-incubation of 5 mM CoCl2 with 1
mM MgCl2, which was sufficient for optimal complex
formation (Supplemental Fig. S2), was found to completely inhibit production
of TSG-6·II (Fig.
3). Assays including both CaCl2 (1 or 5 mM)
and MgCl2 (5 mM) showed that Ca2+ ions had no
such inhibitory effect regardless of whether the experiments were done at 4 or
37 °C (data not shown).
Experiments conducted with mouse serum as the source of II showed
that TSG-6·II formed without any requirement for additional
metal ions (Supplemental Fig. S3). When EDTA (2 mM) was included in
the reaction mixture, complex formation was completely inhibited, which is
consistent with the results obtained with purified components (see above).
Addition of 5 mM MgCl2 to these assays rescued the
formation of TSG-6·II as expected. Interestingly, 5
mM CaCl2 or CoCl2 in the presence of 2
mM EDTA also led to complex formation (Supplemental Fig. S3). The
likely explanation for these results is that the addition of Ca2+
or Co2+ releases sufficient Mg2+ from the EDTA (or serum
proteins) to allow the reaction to proceed; this is consistent with the log
K1 values for the binding of these metal ions to EDTA.
Characterization of the TSG-6·II
Complex—As described above, the incubation of TSG-6 and II
under standard conditions led to the formation of three main species,
i.e. the B/C doublet of 120 kDa (band 2), which was
immunoreactive with anti-TSG-6 antibody, and the 130-kDa species A (band
1) (Fig. 4). These species were
characterized by N-terminal sequencing (Supplemental Fig. S4). This analysis
revealed that species A (band 1) contained the three protein chains of
II(i.e. bikunin, HC1, and HC2)
(Table I). However, no TSG-6
was detected, consistent with its lack of immunoreactivity with anti-TSG-6
antibody. Band 2, which was cut into upper and lower portions corresponding to
species B and C, respectively, contained TSG-6 in addition to both HCs (but no
bikunin). As shown in Table I,
the upper band contained TSG-6 and HC2 in similar amounts (as based on the
initial sequencing yields), with about half as much HC1 present. The converse
was observed for the lower band, which gave equivalent initial yields of TSG-6
and HC1, but less HC2. Therefore, from this analysis and considering the
molecular masses of TSG-6 and the three chains of II, it seems likely
that species B and C correspond to complexes of TSG-6 with HC2 and HC1,
respectively, whereas species A comprises a mixture of bikunin linked to one
or the other of the HCs; given the proximity of species B and C (Supplemental
Fig. S4), it is perhaps not surprising that both HC1 and HC2 were detected in
these bands.
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I Complex Stability—The
TSG-6·II complexes were formed under standard conditions and
then treated with either chondroitinase ABC lyase or NaOH. As shown in the
Western blot in Fig. 4
(lower panel), species B/C (i.e. the TSG-6·HC
complexes), which was detectable by an anti-TSG-6 antibody, disappeared
completely upon mild NaOH treatment, but the band was not altered by
chondroitinase. SDS-PAGE analysis showed that species A (band 1) and intact
II (either that remaining in the TSG-6/II reaction mixture or an
II control) were degraded by both chondroitinase and NaOH
(Fig. 4, upper panel).
It is therefore likely that the HCs in these bikunin·HC complexes are
linked via ester bonds to the chondroitin sulfate chain attached to bikunin,
as they are in the intact parent molecule
(15,
16). Both chondroitinase and
NaOH treatments led to the appearance of bands with apparent molecular masses
of 80 and 85 kDa. N-terminal sequencing revealed that the upper of
these (i.e. bands 6 and 8 in Fig.
4 and Supplemental Fig. S4) correspond predominantly to HC2,
whereas the lower (bands 7 and 9) are essentially HC1
(Table I), with only a small
amount (between 4 and 11%) of the alternative HC being detected in each of
these bands. These HC1 and HC2 species were also seen upon treatment of
II alone (Fig. 4). A
faint band of 160 kDa also appeared following chondroitinase digestion of
TSG-6/II mixtures (band 3) or of II alone, and this was shown by
mass spectrometry to contain HC1 and HC2 (14 and 18 peptides identified,
respectively) (data not shown), but did not contain any TSG-6 or bikunin. It
is likely that this species represents a partial degradation product of
II in which the two HCs remain linked by CS.
Although chondroitinase treatment clearly did not degrade the B/C doublet
(Fig. 4, lower panel),
it did alter the separation of these species upon SDS-PAGE. As shown in
Fig. 4 (upper panel),
after treatment, the tight doublet (band 2) ran as the more widely separated
bands 4 and 5 (with chondroitinase running between them). N-terminal sequence
analysis of these species (Table
I) showed that bands 4 and 5 correspond to complexes of TSG-6 with
HC2 and HC1, respectively (i.e. these are equivalent to the upper and
lower bands of species 2); in bands 4 and 5, there was only a relatively small
amount of the alternative HC detected (i.e. 9 and 22%, respectively),
presumably due to the slightly better separation of these bands (Supplemental
Fig. S4). The reason underlying this improved separation is not clear, but it
could result simply from the removal of higher molecular mass species
(i.e. intact II and species A) or the presence of the
chondroitinase enzyme itself, altering the mobility of species B and C, rather
than necessarily being due to removal of chondroitin sulfate from the
TSG-6·HC complexes.
Model of TSG-6·HC Complex Formation—The
protein sequence analysis of the TSG-6/II reaction products and
characterization of the species formed following treatment with chondroitinase
or NaOH described above have allowed us to generate a model for
TSG-6·II complex formation. As shown in
Fig. 5, incubation of TSG-6 and
II in the presence of metal ions (Mg2+ or Mn2+)
leads to the formation of either a TSG-6·HC1 or TSG-6·HC2
complex (i.e. species C or B, respectively). This is likely to occur
via a transesterification reaction in which the ester bond linking one or the
other HC to CS is transferred onto TSG-6. Consistent with this, the
TSG-6·HC complexes are degraded by mild NaOH treatment (to yield free
HCs), whereas they are refractory to digestion with chondroitin ABC lyase. Via
this mechanism, formation of TSG-6·HC1 would leave HC2 still attached
to bikunin via the CS chain (i.e. bikunin·HC2) as a
by-product, whereas if the TSG-6·HC2 complex were formed, then
bikunin·HC1 would be left over. In this regard, species A (band 1 in
Fig. 4) is likely to represent
a mixture of the bikunin·HC1 and bikunin·HC2 by-products because
sequencing revealed that this species contained a similar amount of bikunin
(2.19 pmol) compared with the combined HCs (2.35 pmol) and that it was
susceptible to cleavage with chondroitinase. In this model, there would be no
cleavage of the CS chain upon transfer of an HC onto TSG-6 and consequently no
CS "stub" left attached to either of the TSG-6·HC
complexes. Overall, this model is similar to that proposed recently by
Sanggaard et al.
(41).
TSG-6 Mediates the Transfer of HCs onto HA—Recently, it was
found that covalent HC·HA complexes do not form in the COCs of
Tsg-6 null mice (7),
indicating that TSG-6 is required for the covalent transfer of HCs from
II onto HA. In addition, HC transfer onto HA has been observed in
vitro using mouse serum and human recombinant TSG-6
(7,
8). We investigated whether a
mixture containing only purified recombinant TSG-6, purified human II,
and medical grade HA is sufficient to form HC·HA complexes in
vitro. As shown in Fig. 6,
when 1 µg of p-HA was included in the standard assay (i.e.
containing TSG-6 and II), an intense additional species (band 11) at
the top of the gel was visible; no such band appeared when II and HA
were incubated together in the absence of TSG-6 (data not shown). N-terminal
sequencing of equivalent reactions (transferred onto Hybond-P membranes)
revealed that band 11 contained both HCs in approximately equal amounts
(Table I). Furthermore,
treatment of such reaction mixtures with Streptomyces hyaluronidase
(an enzyme specific for HA) released bands that ran at the same positions as
the free HCs (Fig. 6, right
panel). Hyaluronidase treatment of II alone under identical
reaction conditions did not lead to its degradation. Therefore, these data
indicate that complexes between the HCs and HA can form in vitro in
the presence of TSG-6. To investigate this further, a defined oligosaccharide
of HA (a 14-mer) was used instead of p-HA, and this led to the appearance of a
species of 85 kDa (band 12 in Fig.
6), which N-terminal sequencing showed to contain mainly HC1, but
also a small amount of HC2 (Table
I). Mass spectrometric analysis confirmed that both species were
present, perhaps with more HC2 than indicated by sequence analysis (36 and 12
peptides of HC1 and HC2 identified, respectively) (data not shown). Given the
different mobilities of HC1 and HC2 upon SDS-PAGE, it is surprising that both
HC·HA14 complexes should run at identical positions.
However, when a longer HA oligomer (i.e. HA 32-mer) was used, this
also gave rise to a single novel species of 90 kDa (i.e. a
higher apparent molecular mass than that of either HC1 or HC2). Mass
spectrometry of this band identified 36 and 15 peptides of HC1 and HC2,
respectively (data not shown), whereas amino acid sequencing indicated that
there was much more HC1 (93%) than HC2 (7%). Importantly, the finding that the
size of the HA oligomer used has a significant effect on the apparent
molecular mass of the released HCs is consistent with the formation of stable
complexes between the HCs and HA. It therefore seems likely that both HC1 and
HC2 can become covalently linked to HA oligosaccharides and that this assay
can be used to visualize the TSG-6-mediated transfer of HCs onto HA. In this
regard, co-incubation of different w/w ratios of HA14 and p-HA with
a TSG-6/II mixture revealed that this oligosaccharide is likely to be
as good a substrate for HC transfer as the 120-kDa HA preparation
(Fig. 7), and therefore,
HA14 was used in all subsequent "transfer" assays.
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|
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I, and HA14 were incubated in the presence of EDTA
under otherwise standard conditions, neither TSG-6·HC (species B/C) nor
HC·HA14 was formed (data not shown). Therefore, to test the
metal ion dependence of HC transfer onto HA, it was necessary to preform
TSG-6·HC complexes, which was done in the presence of 0.109
mM MgCl2, prior to the subsequent addition of 1
mM EDTA, followed by the addition of HA14 in the absence
and presence of various metal ions (see "Experimental
Procedures"). In control experiments in which HA14 was added
to preformed TSG-6·HC complexes in the absence of EDTA (i.e.
in reactions containing a final concentration of 0.1 mM
MgCl2), a band corresponding to HC·HA14 was seen
(Fig. 8, upper panel, lane
1), whereas in the presence of 1 mM EDTA, no
HC·HA14 was formed (lane 3). This clearly shows
that the formation of HC·HA14 complexes requires the
presence of metal ions and is inhibited by EDTA. As shown in
Fig. 8 (upper panel),
when HA14 was added in the presence of MgCl2,
MnCl2, or CaCl2, the HC·HA14 complex
formed. In a separate experiment in which TSG-6, II, and
HA14 were all incubated together under standard conditions with 5
mM MgCl2, MnCl2, CaCl2, or
CoCl2 in the absence of EDTA, the HC·HA14 complex
(and the TSG-6·HC complex) formed only in the presence of
Mg2+ or Mn2+ ions
(Fig. 8, lower right
panel). This demonstrates that Mg2+ or Mn2+ can
support both TSG-6·HC complex formation and HC transfer, whereas
Ca2+ or Co2+ alone does not give rise to either of these
products. Therefore, in the experiment shown in
Fig. 8 (upper panel),
it is likely that transfer (i.e. formation of
HC·HA14) occurred in the presence of Ca2+ due to
its displacement of Mg2+ ions from the EDTA rather than having a
direct effect on the reaction; this is not surprising given the much larger
log K1 for the binding of Ca2+ to EDTA compared
with Mg2+. However, the possibility that Ca2+, although
not supporting the formation of the TSG-6·HC complex
(Fig. 3), could be involved in
subsequent HC transfer cannot be entirely ruled out.
|
I,
and HA14 with 5 mM MgCl2 or MnCl2
in the presence of an equimolar concentration of CoCl2 did not lead
to the formation of HC·HA14 or TSG-6·HC complexes
(data not shown). These data indicate that Co2+ ions are potent
inhibitors of TSG-6-mediated transfer of II HCs onto HA, even in the
presence of Mg2+, presumably due to their tighter binding to the
metal ion center involved in this reaction.
The requirement for divalent metal ions during HC transfer was also
examined using mouse serum as the source of II and high molecular mass
HA by Western blotting with anti-II antibody to visualize the species
formed with or without treatment with Streptomyces hyaluronidase
(Fig. 9). This showed that, in
the absence of EDTA or additional divalent cations (i.e. with just
the metal ions present in serum), the only species detected was either a high
molecular mass smear corresponding to HC·HA complexes of polydispersed
molecular masses (Fig.
9A) or a free HC (Fig.
9B) depending on whether the samples were untreated or
hyaluronidase-digested, respectively. Furthermore, there were no bands
corresponding to II or PI, indicating that all of these proteins
had been converted (in a TSG-6-mediated manner) into an HC·HA complex,
as shown previously (7).
Conversely, the presence of EDTA completely inhibited HC·HA complex
formation, and intense bands for II and PI were present
(Fig. 9), indicating that the
TSG-6-mediated transfer of HCs onto HA from both II and PI is
metal ion-dependent. The inclusion of either Ca2+ or
Mg2+ led to the formation of HC·HA complexes with the
concomitant amounts of II and PI being greatly diminished.
However, the presence of Co2+ ions caused a significant inhibition
of II-dependent transfer (a strong band for II was seen), but
did not inhibit the consumption of PI. Consequently, HC·HA
complexes were still detected. This demonstrates that the mechanisms
underlying TSG-6-mediated formation of HC·HA complexes from II
and PI are distinct. In this regard, experiments in which purified
human PI was incubated with recombinant human TSG-6 (with and without
HA) under a range of conditions in our in vitro assay did not lead to
formation of TSG-6·HC3 complexes or the transfer of HC3 onto HA (data
not shown), indicating that, in this case, TSG-6 alone is not sufficient for
the formation of HC3·HA and that another factor (e.g. in
serum) is also required.
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|
I under otherwise
standard conditions, very low amounts of TSG-6·HC complexes were
visible. Again, the bikunin·HC complexes were present in normal amounts
(Fig. 7). These data
demonstrate that the TSG-6·HC complexes are consumed upon the formation
of HC·HA, i.e. they are likely to act as intermediates in the
transfer of HCs onto HA. To investigate this further and to determine the
stability of the TSG-6·HC complexes, these were formed under standard
conditions and then incubated for a range of times (i.e. 0, 2, 4, and
22 h) before the addition of HA14 (or water) and an additional
incubation of 2 h. As shown in Fig.
10, in the absence of HA, there was no reduction in the amount of
TSG-6·HC complex (B/C doublet) even after the maximal time of
incubation (26 h in total); in fact, species B/C was more intense than at the
shorter time points. This shows that the TSG-6·HC complexes are stable
over relatively long time scales. However, a band of 80 kDa (labeled
I in Fig. 10) became
more intense over time, and this is likely to be the same species as the
80-kDa band seen in reactions mixtures of TSG-6 and II incubated
at 37 °C (Fig. 1, lower
panel). Given the stability of the TSG-6·HC complexes, it seems
probable that this species results from the breakdown of II and the
bikunin·HC by-products. Upon sequencing, this band, which was also
present in the HA-containing samples (Fig.
10), was found to contain HC1 and HC2 at an 2:1 ratio (data
not shown). The molecular mass of this band and the fact that it was formed in
both the absence and presence of HA indicate that it is likely to correspond
to free HCs, although it is not clear why HC1 and HC2 should migrate together;
in both cases, the expected N-terminal sequence was present. When HA14 was added to preformed TSG-6·HC complexes, transfer took place even after they had been incubated at 4 °C for a total of 26 h (i.e. the 22-h time point in Fig. 10); the HC·HA14 complex was seen at all incubation times, along with a decrease in the intensity of the B/C doublet compared with reactions containing no oligosaccharide. In this regard, N-terminal sequencing revealed that the HC·HA14 band contained both HC1 and HC2 (94 and 6%, respectively), which is consistent with earlier results (Fig. 6 and Table I).
Additional species of 160 and 90 kDa were visible in the samples
containing HA14. As noted above
(Fig. 6), the 160-kDa band
(labeled II) most likely corresponds to an oligomer with both HC1 and
HC2 attached. The 90-kDa band (labeled III) was shown by
N-terminal sequence analysis to also contain both HCs (with HC1 and HC2 at a
2:1 ratio) (data not shown). This species was formed only in the presence of
HA14 and is likely therefore to represent an
HC·HA14 complex, but why this should run at a slightly
higher molecular mass is not clear.
On the basis of the above data, it therefore seems likely that TSG-6·HC in the presence of HA converts into a HC·HA complex. Consistent with this, there was a clear increase in the intensity of the TSG-6 band in samples in which the formation of the HC·HA14 complex had taken place, i.e. TSG-6 was released upon the transfer of HCs onto HA.
TSG-6 Is a Catalyst for HC Transfer—The observation that
TSG-6 may be released upon the transfer of HCs from TSG-6·HC onto HA
led to the hypothesis that such molecules of TSG-6 may be able to form new
complexes with II and thus can be recycled. If this were the case, then
it would be expected that suboptimal concentrations of TSG-6 should support,
over time, the formation of significant amounts of HC·HA, given that
neither II or HA is limiting. To test this possibility, standard
amounts of II(45 pmol of 1.8 µM) and HA14
(15 µM) were incubated with various concentrations of TSG-6 for
2, 4, or 22 h. Experiments conducted at 4 °C showed that, when TSG-6 was
present at 0.27 or 0.054 µM (i.e. 10- and 50-fold lower
concentrations than in the standard assay), very little, if any,
HC·HA14 complex was formed after 2 or 4 h (data not shown);
some transfer occurred with 0.27 µM TSG-6 (but not 0.054
µM) after 22 h.
However, at 37 °C, a small amount of HC·HA14 complex
was formed after 2 h even in the presence of the lowest concentration of TSG-6
(0.054 µM); and after 4 or 22 h, a significant amount of
HC·HA14 was apparent
(Fig. 11).
Fig. 11 shows that, although
there was a relatively constant amount of HC·HA14 generated
between 2 and 22 h in reactions containing 2.7 µM TSG-6, when
TSG-6 was present at lower concentrations, the HC·HA14
complex accumulated over time, with the concomitant disappearance of
II. Importantly, in the samples containing 0.27 or 0.054
µM TSG-6, no TSG-6·HC complexes (i.e. B/C
doublet) were seen. It is clear, however, that TSG-6·HC complexes had
been formed because species A, corresponding to the bikunin·HC
by-products of this reaction (Fig.
5), were present. These data indicate that any TSG-6·HC
complexes made are converted into HC·HA14 and that the TSG-6
released is indeed recycled. If all of the II (45 pmol at 1.8
µM) was consumed, then a maximum of 45 pmol at
HC·HA14 could be generated (i.e. one molecule of
II is converted into one molecule of either HC1·HA14
or HC2·HA14), which would require 45 pmol of
TSG-6·HC1/TSG-6·HC2 complexes to be formed and converted.
Although not all of the II was consumed in any of these experiments,
Fig. 11 shows that 50% of
the II disappeared after 22 h in the presence of 0.054 µM
TSG-6 (1.3 pmol). This corresponds to the formation of >20 pmol of
HC·HA14, which is >15-fold the amount of TSG-6
present.
Therefore, the above data show that TSG-6 can be recycled. Thus, it not only acts as an essential cofactor for HC transfer, but is also a true catalyst for this reaction, as illustrated in Fig. 12.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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I led to the formation of TSG-6·HC1 and
TSG-6·HC2 complexes with the generation of the corresponding
bikunin·HC2 and bikunin·HC1 by-products in a pH-, salt
strength-, and metal ion-dependent manner. Our characterization of the
TSG-6·HC complexes and bikunin·HC by-products and the mechanism
of complex formation we have proposed (Fig.
5) (43) agree well
with a recent study (41).
Earlier work from Wisniewski et al.
(34) suggested that the
TSG-6·II complexes formed in vitro consist of
TSG-6·bikunin·HC2 and that this complex is susceptible to
degradation by chondroitinase, which does not agree with our data.
Furthermore, Sanggaard et al.
(41) demonstrated that, in the
TSG-6·HC2 complex at least, TSG-6 is linked directly to the C-terminal
aspartic acid of HC2, providing definitive evidence that TSG-6·HC
complexes are covalent in nature, as was suspected
(31). Given our previous
finding that monoclonal antibody A38, which recognizes an epitope in the TSG-6
Link module (44), can inhibit
the formation of the TSG-6·HC complexes
(27), it seems likely that
this domain is directly involved in complex formation and may be the site of
covalent attachment (as shown in Fig.
5). However, the Link module alone is unable to form a covalent
complex with II (45),
indicating that other regions of TSG-6 are also necessary for this process
(see below).
Sanggaard et al.
(41) also observed and
characterized high molecular mass species that are likely to correspond to
II with one or two additional HCs attached. These were present only in
very small amounts in our experiments conducted at 4 °C. Therefore, it
seems likely that these species result from a side reaction not required for
the formation of the TSG-6·HC1 and TSG-6·HC complexes. Free
bikunin was also visualized by a trypsin inhibition counterstaining technique
(41), whereas no band
corresponding to bikunin was seen on our SDS-polyacrylamide gels, which were
stained with Coomassie Blue. However, N-terminal sequence analysis of the
35-kDa "TSG-6" band from gels equivalent to those shown in
Fig.
