(Received for publication, May 22, 1995; and in revised form, July 28, 1995)
From the
We previously showed that serum-derived 85-kDa proteins (SHAPs, serum-derived hyaluronan associated proteins) are firmly bound to hyaluronan (HA) synthesized by
cultured fibroblasts. SHAPs were then identified to be the heavy chains
of inter--trypsin inhibitor (ITI) (Huang, L., Yoneda, M., and
Kimata, K.(1993) J. Biol. Chem. 268, 26725-26730). In
this study, the SHAP
HA complex was isolated from pathological
synovial fluid from human arthritis patients. The SHAP
HA complex
was digested with thermolysin, followed by CsCl gradient
centrifugation. The HA-containing fragments thus obtained were further
digested with chondroitinase AC II and subjected to TSK gel high
performance liquid chromatography (HPLC). Peptide-HA
disaccharide-containing fractions (the SHAP
HA binding regions)
were further purified by reverse phase HPLC. Major peaks were analyzed
by protein sequencing and mass spectrometry (electrospray ionization
mass spectrometry and collision induced dissociation-MS/MS). By
comparison with the reported C-terminal sequences of the human ITI
family, the peptides were found to correspond to tetrapeptides derived
from the C termini of heavy chains 1 of and 2 of inter-
-trypsin
inhibitor (HC1 and HC2), and heavy chain 3 of pre-
-trypsin
inhibitor (HC3), respectively, and a heptapeptide from HC1. Mass
spectrometric analyses suggested that the C-terminal Asp of each heavy
chain was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of HA chain. This report is the first
demonstration to give evidence for the covalent binding of proteins to
HA.
Hyaluronan (HA), ()has been found as a ubiquitous
component of the extracellular matrices of many tissues and in body
fluids, including the vitreous body, synovial fluid, lymph, and
blood(1, 2, 3, 4) . It has been
suggested that HA plays an important role in many biological processes,
such as gamete maturation, tissue morphogenesis, cell migration, and
cell proliferation (5, 6, 7, 8) . HA
is also involved in angiogenesis, wound healing, tumor invasion, and
pathophysiological responses of tissues to inflammation (9, 10, 11, 12) .
With regard to
functional importance, a large number of HA-binding proteins have been
reported, an important subset of which have highly homologous sequences
for HA binding. These include link proteins(13) ,
hyaluronectin(14) , glial HA-binding protein(15) ,
HA-binding proteoglycan such as aggrecan, PG-M/versican(16) ,
and CD44(17) . These are the proteoglycan tandem repeat
families of HA-binding proteins. CD44 is a typical example of the
family. Variant forms of CD44 generated by alternative splicing may
have individual functions such as lymphocyte homing and tumor cell
metastasis(18, 19) . Tumor necrosis factor-stimulated
gene-6, another new member of this family, is tumor necrosis factor or
interleukin-1-inducible and was recently shown to bind covalently to
inter--trypsin inhibitor (ITI)(20) .
We previously
showed that serum-derived HA-associated proteins (SHAPs) appear to bind
covalently to HA(21) , and therefore to mediate the binding of
HA to cell surface and other extracellular molecules, and might be one
of the serum factors involved in the HA metabolism in cultured
fibroblasts. Our recent study demonstrated that SHAPs are identical to
the heavy chains of ITI, and the SHAPHA complex could be formed
by incubating serum with exogenous HA under physiological
conditions(21, 22) .
ITI is a plasma protease
inhibitor consisting of three genetically different peptides, a light
chain (bikunin) and two heavy chains (HC1 and
HC2)(23, 24, 25, 26, 27, 28) .
The three peptides are covalently cross-linked by a chondroitin sulfate
chain(29, 30, 31, 32, 33, 34, 35) in
that each heavy chain is covalently bound to the chondroitin sulfate
chain derived from the light chain by a unique ester bond between the
carboxyl group of the C-terminal Asp of the peptides and C-6 hydroxyl
group of an internal GalNAc of the chondroitin sulfate
chain(34, 35) . Similar linkage structures exist in
pre--trypsin inhibitor (P
I). P
I is composed of one heavy
chain (HC3) and one light chain which are covalently linked to each
other by a chondroitin sulfate chain (30) .
In the present
study, we prepared SHAPHA complexes from human synovial fluid of
arthritic patients. We analyzed the binding region by protein
sequencing and electrospray ionization mass spectrometry (ESI-MS). We
give evidence to show that SHAP binds covalently to HA.
The
HA-containing fractions were precipitated with ethanol and then
subjected to digestion with chondroitinase AC II (67 milliunits/mg of
HA) in 50 mM sodium acetate, pH 6.0, at 37 °C overnight.
The digested mixture was further subjected to TSK gel HPLC. Two TSK
G3000PWXL (7.8 mm 30 cm, Tosoh) columns were linearly connected
for the better separation. The columns were eluted with 0.2 M
NH
HCO
at a flow rate of 0.5 ml/min. The
absorbance was measured at 214 nm. The peptide-containing peak was
eluted just before the main HA disaccharide peak. The first peak was
collected and concentrated by lyophilization. The second HPLC using the
same columns was carried out to completely remove a trace of the HA
disaccharide product. The fractions containing the SHAP
HA binding
region were collected and lyophilized.
Further Purification of the
SHAPHA Binding Region by Reverse Phase HPLC-The
SHAP
HA binding region was further purified by reverse phase HPLC.
The column (TSK ODS-120T, 4.6
150 mm, Tosoh) was equilibrated
with 0.06% (v/v) trifluoroacetic acid in water. The peptides were
eluted by an acetonitrile gradient 0 to 13% acetonitrile in 0.06% (v/v)
trifluoroacetic acid in water. The flow rate was 0.5 ml/min, and the
absorbance was measured at 214 nm.
Protein Sequence Analyses-Automated Edman degradation was carried out in an Applied Biosystems model 476A sequencer with on-line phenylthiohydantoin analysis using an Applied Biosystems model 120A HPLC apparatus. The instruments were operated as recommended in the user bulletins and manuals distributed by the manufacturer.
Argon gas was used at 0.13 Pa as the collision gas at 20 eV for CID-MS/MS spectra.
Figure 1:
Isolation of the SHAPHA complex
from pathological synovial fluid of human arthritis patients. Guanidine
HCl extract of pathological synovial fluid was subjected to a series of
CsCl density gradient centrifugations as described under
``Experimental Procedures.'' After the third centrifugation
(initial density,
= 1.45 g/ml), the gradient
was partitioned into 16 fractions. Contents of HA and proteins in each
fraction were determined by carbazole reaction and micro-BCA method,
respectively.
SDS-PAGE and immunoblotting of the
SHAPHA complex thus prepared from human synovial fluid were
performed to confirm that the complex was identical with the one
prepared from the incubation mixture of human serum with HA as
described previously (22) (Fig. 2). Proteins in the
preparation from the synovial fluid were retained in the starting gels
of SDS-PAGE, indicating that the complex in the synovial fluid
preparation remained undissociated under both dissociative conditions
of 4 M guanidine HCl during the centrifugation and 1% (w/v)
SDS during the PAGE (Fig. 2A). This was also the case
with the SHAP
HA complex prepared from the incubation mixture of
human serum with HA as described previously(22) . Treatment of
both complex preparations with HA-degrading enzymes, such as
protease-free Streptomyces hyaluronidase or chondroitinase AC
II released the proteins that corresponded to HC1 and HC2 of ITI (two
bands with the higher mobility and the lower mobility, respectively),
judging from both the immunoreactivities and the mobilities of proteins
in the gels (Fig. 2, B and C). Treatment with
alkali (0.02 M NaOH) also caused their release from both the
SHAP
HA complex preparations (Fig. 3). Therefore, synovial
fluid of human arthritic patients contained a SHAP
HA complex that
was identical in both molecular structure and properties to the complex
produced by incubation of HA with serum. In addition, when the
preparation of the SHAP
HA complex from the pathological synovial
fluid was digested with V
-protease and then subjected to
SDS-PAGE, two major peptide bands resulted which have partial N-amino acid sequences identical with HC1 and HC2 of human
ITI, respectively (data not shown). Taken together, it is very likely
that the protein-HA complex obtained from human synovial fluid
corresponds to the SHAP
HA complex that we had described
previously (22) .
Figure 2:
Characterization of the SHAPHA
complex isolated from human pathological synovial fluid and comparison
with the SHAP
HA complex prepared from the incubation mixture of
human serum and hyaluronan. Aliquots (50 µg HA) of SHAP
HA
complex isolated from the pathological synovial fluid were precipitated
with ethanol, and incubated without HA degrading enzymes (lane
1), with Streptomyces hyaluronidase at 60 °C for 2 h (lane 2), with chondroitinase AC II at 37 °C for 2 h (lane 3). The samples were electrophoresed on SDS gel (9% gel,
under nonreducing conditions) and the gels were stained with Coomassie
Blue (A). About 1/20 of the same set of the samples were
electrotransferred to nitrocellulose membrane after SDS-PAGE, and
immunoblotted with antibodies to human ITI. The immune complexes were
visualized by enhanced chemiluminescence assay (B). Aliquots
of SHAP
HA complex prepared from the incubation mixture of human
serum with HA were treated, and immunoblotted the same way as described
above (C). Since the antibodies used in this experiment were
more reactive to HC2 of ITI than the other heavy chains, the observed
differences between the Coomassie Blue staining and immunostaining
patterns could be explained by such differences in the
immunoreactivity.
Figure 3:
Alkali treatment of SHAPHA complexes
from human pathological synovial fluid and from the incubation mixture
of human serum with hyaluronan. SHAP
HA complex isolated from
pathological human synovial fluid was treated with 0.02 M NaOH
for 0, 30, and 60 min (A). The complex from the incubation
mixture of human serum and hyaluronan was subjected to the same
treatment for 60 min (B). The samples after neutralization
with acetic acid were treated with (+)/without (-) Streptomyces hyaluronidase and subjected to SDS-PAGE and
subsequent immunoblotting the same way as described in Fig. 2.
Intact ITI and metabolic products of ITI
were also present in synovial fluid (data not shown). In our
purification procedures, the SHAPHA complex was recovered in the
bottom fractions after the first and second CsCl isopycnic
centrifugation, while ITI, P
I, and most of bikunin were in the
upper fractions (data not shown). Complete separation from those
proteoglycans were appraised by SDS-PAGE of the purified preparation
before and after the treatment with either Streptomyces hyaluronidase or chondroitinase AC II and subsequent
immunoblotting with anti-human ITI (Fig. 2). Since Streptomyces hyaluronidase degrades HA only while the
chondroitinase degrades both HA and chondroitin sulfate, the fact that
no distinctive difference was observed in the immunostaining pattern of
protein bands between the Streptomyces hyaluronidase- and
chondroitinase AC II-treated samples indicated that the purified
preparation from the synovial fluid was neither contaminated with ITI
nor with P
I. The slight difference in mobility of the stained
bands between the two samples could be explained by the difference in
size of the products between the two enzymes. It is known that major
digestion products of HA with Streptomyces hyaluronidase are
tetramers and hexamers while the chondroitinase AC II digestion of HA
yields dimers. In addition, there were also some differences between
the Coomassie Blue staining and immunostaining patterns (Fig. 2, A and B). These differences could be explained by the
apparent differences in the immunoreactivity of the anti-human ITI
antibodies between HC1 and HC2.
Figure 4:
TSK gel HPLC of thermolysin- and
chondroitinase AC II-digested products of SHAPHA complex.
SHAP
HA complex was treated with thermolysin and subjected to CsCl
gradient centrifugation. HA-containing fractions were pooled and
treated with chondroitinase AC II as described under
``Experimental Procedures.'' The digested mixture was loaded
onto two linearly connected TSK G3000 HPLC columns and eluted with 0.2 M NH
HCO
(A). The
peptide-containing fraction (peak I) was collected and
rechromatographed on the same columns (B). The
peptide-containing fraction (peak 1) was collected and
lyophilized for the next purification step.
The chromatography of peak II in Fig. 4A on SAX 10 column (37) indicated that
none of any disaccharide products derived from chondroitin, chondroitin
4-sulfate and chondroitin 6-sulfate other than the HA disaccharide was
detected in this fraction, judging from the elution positions of the
standard disaccharides (data not shown). The result further confirmed
no contamination with ITI and PI in the SHAP
HA complex
prepared from the human synovial fluid.
Further purification of the
SHAPHA binding regions was performed by applying the peak 1 from
the second TSK gel HPLC onto C
reverse phase HPLC. Elution
with an acetonitrile gradient as described in the experimental
procedures yielded 12 major peaks detected by the absorption at 214 nm
and numbered by numerical order (Fig. 5).
Figure 5:
Reverse phase HPLC of the TSK gel
fraction for the purification of the SHAPHA binding region. The
lyophilized peak 1 of the second TSK gel HPLC was applied onto a
C
reverse phase HPLC, eluted by an acetonitrile gradient
in 0.06% (v/v) trifluoroacetic acid as described under
``Experimental Procedures.'' The absorbance was measured at
214 nm. The separated peaks were designated as peaks 1-12 in numerical order. Each peak was collected and lyophilized,
respectively, for protein sequencing and mass spectrometry
analysis.
Figure 6:
Negative ion ESI CID-MS/MS of the Peak 6
sample. Precursor ion: (M - H) at m/z 835. The inset shows proposed fragmentations. Weak
signals from m/z 650-900 are mostly due to instrument
noise.
Figure 7:
Negative ion ESI CID-MS/MS of the Peak 7
sample. Precursor ion: (M - H) at m/z 808. The inset shows proposed fragmentations. Weak
signals from m/z 700-900 are mostly due to instrument
noise.
CID MS/MS analysis of (M - H) at m/z 764 in peak 6 suggested that the same linkage occurs between HC3
and the HA disaccharide (data not shown). However, its limited sample
amount prevented us from carrying out detailed structural studies.
Taken together, the analyses demonstrated linkages between the C-6
of the reducing terminal N-acetylhexosamine residue of HA
disaccharide and a carboxyl group of the C-terminal aspartic acid of
the peptides derived from the heavy chains, HC1, HC2, and HC3 of ITI
and PI.
Enghild et al.(30, 34) showed that
three different heavy chains (HC1, HC2, and HC3) were covalently linked
to bikunin by chondroitin sulfate chain in ITI and PI. We have
shown in this study that a similar linkage structure exists between
SHAPs (HC1, HC2, and HC3) and HA. Mass spectrometic analyses revealed
esterification of the carboxyl group of the C-terminal Asp with C-6
hydroxyl group of reducing terminal N-acetylhexosamine of the
unsaturated HA disaccharide. Thus, we conclude that SHAPs (heavy chains
of ITI and P
I) are covalently linked to HA by the esterification
with the C6-hydroxyl groups of N-acetylglucosamine of HA via
the C-terminal Asp.
The present study using ESI CID-MS/MS technique
did not provide information to show which of the two carboxylate groups
in the C-terminal Asp participated in the ester linkage formation. This
is because the low energy collision-induced dissociation in the
quadrupole instrument under our conditions failed to cleave C-C
bonds in the aspartic acid. We assume tentatively that the
-carboxylate is the one which covalently bonds to the C6-hydroxyl
group of the N-acetylhexosamine by an ester linkage, by
analogy to the previously reported structure for the interchain linkage
between the heavy chains and the light chain (bikunin core protein) via
a chondroitin sulfate chain originating from the light chain in ITI or
P
I(30, 34) . This suggests that SHAP
HA
complex may be formed simply by transferring the heavy chains from the
chondroitin sulfate to HA (substitution reaction of HA for the
chondroitin sulfate). However, the mechanism for the reaction has not
been determined yet.
We noted that two fractions with different
retention time in reverse phase HPLC contained peptides with the
identical amino acid sequence (for example, peaks 4 and 6 in Table 1and Table 2). Since there was no significant
difference observed between those two fractions by peptide sequencing
or by ESI CID-MS/MS, this could be due to difference of the anomeric
configuration of hydroxyl group at the reducing end ( and
)
of the HA disaccharide.
In mouse ovulation, preovulatory synthesis
of hyaluronan within the cumulus mass plays an important role for
cumulus expansion(8) . Recently, Chen et al.(38, 39) reported that the cumulus extracellular
matrix stabilizing factor in fetal bovine serum is a member of the ITI
family, and that stabilizing ability is achieved through its direct
binding to HA, which is sensitive to ionic strength and has a
dissociation constant of 1.9 10
M at pH 7.2. Therefore, the properties of the interaction of cumulus
extracellular matrix stabilizing factor with HA appear to be different
from those for the formation of SHAP
HA complex.
The tight
binding of ITI to HA in human pathological synovial fluid was firstly
reported in 1965(40) . The present results show that the
formation of a covalent linkage is involved in this binding. TSG-6, a
35-kDa glycoprotein of the proteoglycan tanden repeat HA-binding
family, also exists in pathological synovial fluid of patients with
arthritis(41) . Recently, Wisniewski et al.(20) have shown that TSG-6 forms a covalently bound
complex (120 kDa) with serum ITI. This TSG-6ITI complex was
formed rapidly even in the apparent absence of other proteins at 37
°C, but not at 4 °C. TSG-6 appeared to form a direct covalent
bond to the chondroitin 4-sulfate chain of ITI for the stability of the
TSG
ITI complex(20) . In our study, we have not detected
any peptides derived from TSG-6 in the SHAP
HA binding regions
prepared from human pathological synovial fluid. Therefore, TSG-6 may
not be involved in the formation of SHAP
HA complex in synovial
fluid.
It is interesting to note that the formation of the
SHAPHA complex from HA and ITI or P
I is accompanied by the
release of bikunin. Bikunin contains two tandem repeats of Kunitz-type
domains, and the trypsin inhibitor activity of ITI is localized in this
part(23, 42) . Bikunin is also identified in urine as
UTI (urinary trypsin inhibitor), which was shown to be a urine
proteoglycan with molecular mass ranging from 40 to 45
kDa(43, 44, 45) . Some tumor cells have UTI
receptors(46) . UTI and fragments derived from UTI by limited
proteolysis efficiently inhibit tumor cell invasion and
metastasis(46, 47, 48, 49) .
Therefore, the formation of SHAP
HA complex might be related to
some defense mechanism from proteolysis.