From the Department of Medical Biochemistry and
Microbiology, Uppsala University, Biomedical Center, Box 575, S-751
23 Uppsala, Sweden, § Department of Cell and Molecular
Biology, Lund University, Sweden, and ¶ Department of
Biophysics, Stockholm University, Sweden
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Inter- Inter- Although I Recent observations have indicated that I For further functional studies of I Enzymes
Chymotrypsin and elastase from pig pancreas were purchased from
Boehringer-Mannheim. Thermolysin (type X) was from Sigma, hyaluronidase
from ovine testes from Calbiochem, and chondroitinase AC II Arthro from
Seikagaku Corp., Tokyo, Japan.
Purification of the Heavy Chains of Human I For the isolation of I Gel Filtration
The proteins were applied to a Superdex 200 gel (3.2/30;
Pharmacia Biotech) equilibrated with PBS at a flow rate of 50 µl/min; the absorbances at 280, 254, and 214 nm of the eluate were continuously measured. Thymidine and blue dextran were used as a markers for Vt and V0, respectively.
Calibration curves for Stokes radius and molecular mass were obtained
with thyroglobulin (8.5 nm, 668 kDa), ferritin (6.1 nm, 440 kDa),
catalase (5.2 nm, 232 kDa), aldolase (4.8 nm, 158 kDa), and bovine
serum albumin (3.55 nm, 67 kDa), all were obtained from Pharmacia Biotech.
Electron Microscopy
After thawing the protein samples, proteinase inhibitors were
added: 0.1 M 6-aminohexanoic acid, 5 mM
N-ethylmalemide, 5 mM benzamidine hydrochloride,
and 0.5 mM phenylmethanesulfonyl fluoride. The solutions
were then dialyzed at 4 °C overnight against 0.2 M
NH4HCO3, pH 7.9. Glycerol spraying and rotary
shadowing were performed as described previously (27-30). Briefly,
buffer and glycerol were added to make the final concentrations of
protein and glycerol 5 µg/ml and 400 ml/liter, respectively. The
contrast was increased by the addition of polyethylene glycol 1500 (0.01 g/liter). The samples were sprayed onto freshly cleaved mica
pieces, dried in vacuo and rotary shadowed at 9° with
carbon/platinum through electron bombardment heating in a Balzers BAF
400 D freeze etching device. The samples were observed in a Jeol 1200 EX electron microscope operated at 60 kV accelerating voltage.
Evaluation of the data from the electron micrographs was done as
described previously (27).
For the selective removal of bikunin from I Limited Proteolysis of the Heavy Chains
Chymotrypsin--
2 µl of a solution with different
concentrations of enzyme was added to 18 µl of a solution containing
heavy chain (0.4 mg/ml) in 100 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 1 mM CaCl2 to give various molar ratios of enzyme to heavy chain. After 150 min of incubation at 37 °C, the reaction was stopped by the addition of
phenylmethanesulfonyl fluoride to a final concentration of 2 mM.
Trypsin--
The reaction was performed as for chymotrypsin,
except the buffer was 100 mM Tris-HCl, pH 7.4, with 50 mM NaCl and 20 mM CaCl2.
Elastase--
The reaction was performed as for chymotrypsin,
except the buffer was 100 mM Tris-HCl, pH 7.4, and 50 mM NaCl.
Thermolysin--
The reaction was performed as for elastase but
was terminated by the addition of EDTA to a final concentration of 10 mM.
After proteolysis, the proteins were separated by electrophoresis in
15% polyacrylamide gels in Tricine buffer (31). The proteins were
stained or transferred by electrophoresis onto a nitrocellulose
membrane (Problott; Applied Biosystems; Foster City, California). The
membrane was stained, and the appropriate bands excised and subjected
to N-terminal sequencing in a 476 A amino acid sequenator (Applied Biosystems).
Circular Dichroism
Samples containing 0.1-0.3 mg/ml protein were dialyzed against
25 mM sodium phosphate buffer (pH 7.6), and the protein
concentrations were determined by quantitative amino acid analysis.
Circular dichroism spectra were obtained at room temperature on a
JASCO-720 spectropolarimeter with a 0.1 cm cell. Data points were
collected from 250 to 190 nm in 0.5 nm intervals at a rate of 50 nm/min; for each spectrum, 10 scans were performed and added. The
percentages of secondary structural elements were estimated with the
method of variable selection using a data base of 22 proteins with
known secondary structure (32, 33).
Gel Filtration
Analysis of I-inhibitor (I
I) is a 180-kDa serum
protein consisting of three polypeptides. Two of these, the heavy
chains 1 and 2 (H1 and H2), are of 75-80 kDa and have similar amino
acid sequences. The third polypeptide, bikunin, has a molecular mass of
25 kDa and contains a 7-kDa chondroitin sulfate chain that is
covalently linked to the C-terminal amino acid residues of H1 and H2.
I
I has been shown to be required for the formation of the
hyaluronan-containing extracellular matrix of certain cell types. How
I
I exerts this function is not known, but it appears that upon
interaction with cells, the heavy chains are released and become
covalently linked to hyaluronan. Our results indicate that I
I and
its heavy chains are extended molecules; thus, upon electron
microscopy, I
I appeared to consist of two globular domains connected
by a thin structure 31-nm long and the isolated heavy chains of a
globular domain and a "tail" about 15-nm long. Analysis of the
heavy chains by partial proteolysis showed that the C-terminal halves
are particularly sensitive to hydrolysis indicating that they are
loosely folded. Furthermore, electron microscopy showed that partially
degraded heavy chains lacked the extended regions. Taken together,
these results suggest that the N-terminal half of the heavy chains
forms a globular domain, whereas the other half has an extended and loosely folded structure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-inhibitor
(I
I)1 is a plasma protein
synthesized by hepatocytes. It consists of bikunin, which has a
molecular mass of about 25 kDa (1, 2), and two other polypeptides of
75-80 kDa, the heavy chains 1 and 2 (H1 and H2) (3). Bikunin carries a
7-kDa chondroitin sulfate chain (4, 5), which is linked to the
-carbons of the C-terminal amino acid residues of H1 and H2 through
two ester bonds (6). Pulse-chase experiments with isolated hepatocytes
have shown that the coupling of bikunin to the heavy chains occurs in
the Golgi complex immediately after the chondroitin sulfate chain has
been completed (7, 8). Bikunin, which occurs in plasma also in free
form (9), is a proteinase inhibitor with a wide specificity range but
with relatively low affinity to the proteolytic enzymes tested so far
(10).
I was isolated more than 30 years ago (11), its
physiological role is still unclear (for a review see Ref. 3). Recent
findings indicate that this protein is required for the maturation of
oocytes; in vitro experiments have shown that I
I stabilizes the hyaluronan-containing extracellular matrix that is
formed by the cells surrounding the oocytes (12). The observation that
the capillaries near the follicles become leaky to plasma proteins
during ovulation supports the idea that the observed effect occurs
in vivo (13). I
I has also been shown to be required for
the formation of the hyaluronan-containing structure that surrounds
fibroblasts and mesothelial cells (14). The physiological role of these
coats is unclear, but it has been suggested that they may protect
against compression (15), promote cell migration (16), and help to keep
cells separated (15). It is not clear in what way I
I stabilizes this
extracellular structure, but it appears that when the
bikunin-containing proteins interact with cells, the bikunin moiety is
displaced by hyaluronan molecules that become covalently linked to the
heavy chains (17, 18).
I may also play a role in
inflammation. Thus hyaluronan isolated from the synovial fluid of
patients with rheumatoid arthritis has been found to contain covalently
linked heavy chains (19, 20). Furthermore, upon inflammation,
fibroblasts and monocytes secrete a protein named TSG-6, which reacts
with I
I by displacing H1 and forming a covalent link with the
chondroitin sulfate of bikunin (21). Upon this reaction, the ability of
bikunin to inhibit plasmin is enhanced, and the formed complex appears
to have a strong anti-inflammatory capacity (22). The molecular details
of the interactions of I
I and TSG-6 are still unknown.
I, detailed structural
information will be helpful. The amino acid sequences of the heavy chains and bikunin are known, as are the structures of their glycans and disulfide arrangements (1, 23). In this study, we have investigated
the conformation of I
I and its isolated heavy chains with various techniques.
MATERIALS AND METHODS
I
I, a side fraction from the commercial
production of factor IX was used (kindly provided by I.-M.
Lööf; Pharmacia-Upjohn, Stockholm, Sweden). After dialysis
against phosphate-buffered saline (PBS) and removal of insoluble
protein, this material was subjected to gel filtration on Sepharose
S-400, which yielded pure I
I. For the release of the heavy chains, 2 M NaOH was added to a solution of I
I (1 mg/ml in PBS) to
give a final concentration of 0.05 M (24). After 15 min at
room temperature, Tris-HCl, pH 8.0, was added to yield a final
concentration of 0.25 M. Alternatively, one volume of I
I
(1 mg/ml in PBS) was mixed with two volumes of 0.4 M sodium
acetate, pH 6.0, followed by the addition of chondroitinase and
phenylmethanesulfonyl fluoride to yield the final concentrations 20 milliunits/ml and 0.2 mM, respectively (25). The mixture was incubated for 20 h at 37 °C. The sample was then dialyzed against 20 mM sodium phosphate, pH 7.6, and applied to an
anion exchange gel (MonoQ HR 5/5; Pharmacia Biotech) equilibrated with the same buffer. The proteins were eluted at 4 °C at a linear flow
rate of 20 cm × h
1 with 100 ml of a gradient from 0 to 0.7 M NaCl in 20 mM sodium phosphate, pH 7.6 (26). The fractions were analyzed by SDS-PAGE followed by staining with
Coomassie Brilliant Blue. Unless specified otherwise, protein
concentrations were determined by UV measurements. The absorbance
coefficients for the protein moieties of I
I, H1, and H2 were
calculated from the amino acid compositions (Lasergene, DNASTAR Inc.).
The published carbohydrate contents were then used for the calculation
of the corresponding values for the whole proteins: 0.60, 0.47, and
0.72 mg
1 ml cm
1, respectively. The protein
solutions were stored at
20 °C until they were used for experiments.
I, the procedure of
Enghild et al. (24) was used: a sample containing 0.9 mg/ml I
I and 6 µg/ml hyaluronidase in PBS was incubated for 2.5 h
at 37 °C. H2 was selectively removed by elastase digestion
essentially as described by Balduyck et al. (26) using an
enzyme to protein ratio of 1:1,000 with the conditions described below
for digestion of the isolated heavy chains. Limited chymotrypsin
digestion of H1 was done as described below with an enzyme to protein
ratio of 1:50. Subsequent electrophoretic analysis of the different protein samples showed bands with the expected apparent molecular masses.
RESULTS
I, H1, and H2 by gel filtration (Fig.
1A) showed that more than 90%
of the applied material eluted as one peak. Based on their amino acid
sequences and known carbohydrate contents, the molecular masses of the
constituents of I
I (H1, H2, and bikunin) are 77, 78 (1), and 25 kDa
(1, 2), respectively, yielding a total mass of 180 kDa for the whole
protein. The gel filtration experiments showed that I
I, H1, and H2
behave like globular proteins of 350 ± 7, 100 ± 4, and
140 ± 1 kDa (Fig. 1B,
; three determinations) indicating that they have extended structures. The corresponding Stokes
radii are 6.4 ± 0.2, 3.8 ± 0.2, and 4.5 ± 0.1 nm,
respectively (Fig. 1B,
). The ratios between these radii
and those of spherical proteins of the same masses, the frictional
ratios, are 1.7, 1.4, and 1.6, respectively (34).
View larger version (13K):
[in a new window]
Fig. 1.
Characterization of I I, H1, and H2 by gel
filtration. A, the proteins were subjected to gel
chromatography and their elution times determined. Thymidine was
present in the samples and its elution position, apparent as an
absorbance peak, yielded Vt.
V0 was determined with blue dextran (Pharmacia
Biotech) in a separate experiment and the Kd values
for the proteins were calculated. B, proteins of known
Stokes radius (
) and molecular mass (
) were analyzed as in
panel A and a calibration graph was plotted. The
Kd values of I
I, H1, and H2 are indicated on the
x-axis.
Electron Microscopy
II molecules visualized by rotary shadowing (Fig.
2, A and B) showed
particles with a diameter of 11 ± 2 nm. Most of these (63%)
occurred pairwise joined by a thin strand. The average distance between
the centers of the particles measured along the strand (Fig.
3A, shaded bars)
was 42 ± 8 nm (Fig. 3B) implying that the strand was
about 31-nm long. In the middle of the strand, there was a small
globular structure the size of which was at the limit of the resolution
of the method (Fig. 2B, arrowheads). To test the
idea that this globule was bikunin, I
I was subjected to limited hyaluronidase treatment, which has previously been shown to selectively remove bikunin (24). As shown in Fig. 2C, the treated
molecules lacked the globule, 95% of the total number. To further test
the identity of the structures seen by electron microscopy, H2 was specifically released from I
I by limited elastase digestion (26). Fig. 2D shows that this treatment leads to the disappearance
of one of the large globular domains, whereas the small globule
remained (indicated by arrowheads). Isolated H1 studied with
the same technique (Fig. 2, E and F) displayed a
globular domain (diameter, 11 ± 2 nm) with a thin tail; the
average length of the molecule (Fig. 3A, open
bars) was 20 ± 5 nm (Fig. 3B), the tail being
about 15 nm. Mild proteolytic treatment of H1 (Fig. 2G)
resulted in removal of the tail, whereas the size of the globular
domain seemed unaffected. Apparently identical results were obtained
with H2 (data not shown). The extended structure of I
I and its
isolated heavy chains was also seen with negatively stained material
(data not shown).
|
|
Partial Proteolysis
Flexible regions of proteins are generally more sensitive to proteolytic cleavage than the tightly folded ones (35). Partial proteolysis can therefore be used for the structural analysis of proteins. Fig. 4A shows the effect of incubating H1 with increasing amounts of different proteinases as judged by SDS-PAGE followed by staining with Coomassie Brilliant Blue. Upon treatment at a molar ratio of thermolysin to H1 of 1:1,000 (lane 2), the apparent molecular mass of the major band shifted from 79 to 74 kDa, and three groups of bands of about 60, 40, and 29 kDa appeared. The separated fragments were transferred to a membrane and the N-terminal sequences of two of these (denoted t1 and t2) were determined. The obtained sequences were the same as the N-terminal sequence of the intact protein (Fig. 4B) showing that the fragments had been formed by C-terminal truncation (Fig. 4C). Increasing the relative amount of the enzyme 10-fold (lane 4) resulted in the disappearance of t1, t2 (and other, larger fragments) and the appearance of three major bands of 27, 18, and 10 kDa (denoted t3, t4, and t5). Sequence analysis showed that t3 was an N-terminal fragment and that t4 and t5 were the C- and N-terminal halves of t3 (Fig. 4, B and C). Further increases in the enzyme concentration lead to the disappearance of t3 and a simultaneous increase of t4 and t5 (lanes 5 and 6). As judged by its apparent molecular mass on SDS-PAGE, t4 appears to extend beyond the C terminus of t3 (Fig. 4C). Whether this is actually the case or whether t4 behaves anomalously upon electrophoresis remains to be clarified.
|
Treatment of H1 with chymotrypsin (lanes 8-12) yielded fragments with apparent molecular masses close to those obtained with thermolysin (bands c1-c4). Their positions within the molecule were essentially the same as those found for thermolysin (Fig. 4, B and C). The fragments generated by treating H1 with elastase (lanes 14-18) or trypsin (not shown) were similar in size to those obtained with the two other enzymes; their N-terminal amino acid sequences were not determined.
We also used partial proteolysis to probe the structure of H2 (Fig. 5A). At a thermolysin/H2 ratio of 1:10,000, a number of bands in the range of 80-40 kDa were formed (lane 2). Some of these (collectively denoted t1) were analyzed by amino acid sequencing and found to have the N terminus of the intact polypeptide (Fig. 5, B and C). It should be noted that this degradation occurred at a proteinase concentration ten times lower than that required to achieve a similar effect on H1. When the concentration of enzyme was increased (lanes 3-5), new bands appeared (t2-t4) apparently through truncation of the C-terminal end. At the highest proteinase concentrations used (lane 5), cleavage near the N terminus also took place (band t4). Similar results were obtained with chymotrypsin (lanes 8-10) and elastase (lanes 13-15).
|
Circular Dichroism
The relative content of secondary structures in the heavy chains
was assessed by circular dichroism measurements. The spectra of the two
proteins were essentially identical (Fig.
6). The relative occurrence of different
secondary structures were obtained by fitting the spectra to those of
proteins with known secondary structure (32, 33). Three determinations
were performed yielding values varying less than 2%. The estimated
uncertainty in the procedure was less than 7% for each type of
structure. This analysis yielded the following results: 34% -helix,
11% anti-parallel
-sheets, 8% parallel
-sheets, 16% turns, and
32% unordered structures; similar compositions have been obtained for
globular proteins (36).
|
![]() |
DISCUSSION |
---|
In this study, we have found that II and its isolated heavy
chains behave as extended molecules in free solution; their Stokes radii as estimated by gel filtration are 40-70% larger than those of
globular proteins of the same mass. These results are supported by
electron microscopy, which shows that I
I consists of two globular domains connected by a thin flexible structure (Fig. 2, A
and B). With the same technique, the isolated heavy chains
seem to consist of a globular domain of the same size as those in I
I and of a thin tail whose length is about half that of the strand connecting the globular domains in the whole molecule (Fig. 2, E and F). These tails appear to be the C-terminal
ends of the polypeptides, because upon treatment of the heavy chains
with low concentrations of proteinases, the C-terminal parts of the polypeptides are degraded (Figs. 4C and 5C) and
the extended regions seen by electron microscopy removed (Fig.
2G). Biochemical analysis has shown that the C-terminal ends
of the two heavy chains in I
I are covalently linked to the
chondroitin sulfate chain of bikunin (6). In the electron micrographs
of I
I, a small globule can be seen in the middle of the strand
connecting the larger globular domains (Fig. 2B,
arrowheads). This structure appears to be bikunin, because
it is not present in molecules subjected to mild hyaluronidase
digestion (Fig. 2C), a treatment selectively releasing the
bikunin moiety (24).
It is possible that the chondroitin sulfate chain of bikunin
constitutes part of the thread-like structure between the globular domains in II. However, the finding that the sizes of the isolated heavy chains are about half of the whole molecule indicates that the
contribution of the polysaccharide is small; the fact that the bikunin
polypeptide is selectively released upon mild hyaluronidase treatment
might be because of the heavy chains being linked to the chondroitin
sulfate chain at relatively closely located sites (24). An alternative
explanation for our results is that the release of the heavy chains
from I
I was incomplete and that the tails are bikunin. However, the
observation that the heavy chains ran as homogeneous bands upon
SDS-PAGE with an apparent molecular mass of the expected value argues
against this possibility. In this study, we used two procedures for
releasing the heavy chains from I
I: short exposure to high pH and
chondroitinase digestion. The former method is the simpler to perform
but could possibly bring about conformational changes. However, we have
not been able to detect any such differences between heavy chains
prepared by either procedure.
Our results suggest that the heavy chains of II have a club-like
structure. There are numerous examples of proteins of this kind. For
example, the subunit of the cartilage oligomeric matrix protein (COMP),
which has a molecular mass of about 120 kDa, appears to consist of a
globular domain with a diameter of 5 nm and a thin flexible strand of
28 nm (37). Extended protein structures are often decorated with
carbohydrate groups, and it has been suggested that one of their
functions is to decrease the flexibility of the polypeptide chain (38).
It is interesting to note that most of the carbohydrate groups on H1
and H2 are near the C termini (see Fig.
7).
|
In one of the heavy chains (H1) there is a disulfide bridge connecting
the C- and N-terminal halves of the molecule (see Fig. 7). The
existence of this cross-link indicates that the extended part of the
molecule is formed mainly by the polypeptide segment beyond this
disulfide, which contains about 150 amino acid residues. If this part
of the polypeptide would form an -helix, it would have a length of
about 20 nm. As judged by electron microscopy, the length of the tail
is about 15 nm implying that the extended part of the heavy chains
contains little tertiary structure. Our observation that proteolytic
cleavage in this region of the protein occurs at many closely spaced
sites (Figs. 4A and 5A) is in agreement with this
conclusion. Furthermore, there is a high frequency of the
structure-breaking amino acid proline near the C termini of H1 and H2
(39). The fact that the proteinase-sensitive parts of the heavy chains
constitute approximately half the polypeptides (see Fig. 7) indicates
that there are regions other than the tails that contain flexible
structures. However, as judged by electron microscopy (Fig.
2G) and gel
filtration,2 the sizes of the
globular domains were not affected by partial proteolytic digestion.
These findings indicate that the fragments formed from the globular
domains were not released.
It is possible that the structures of II and its isolated heavy
chains were altered during the preparation for electron
microscopy. The fact that this technique did not reveal any
difference in the sizes between H1 and H2, whereas gel filtration
showed that that H1 was less extended than H2, suggests that this is
the case. Possibly, electron microscopy of unstained, vitrified samples could provide the sizes at more physiological conditions.
The experiments with partial proteolytic cleavage described in this
paper is an extension of an earlier study in which it was shown that
upon treatment of intact II with elastase, fragments of H1 and H2
were released by C-terminal cleavages (26). Similar results were also
obtained upon incubation of I
I with tumor cells (40). Whether this
cleavage process is of physiological significance remains to be seen.
It is still unclear how I
I supports the formation of the
hyaluronan-containing pericellular coat on various cell types. It has
been suggested that the heavy chains bind to the cell surface and then
become covalently linked to hyaluronan molecules through their
C-terminal ends (41). If this is the case, it would make sense for the
globular domains to be close to the cell surface and the flexible
C-terminal parts to extend out of the glycocalyx (38).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Å. Engström for help with the gel filtration experiments and T. C. Laurent for valuable suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Swedish Medical Research Council (Grants 12567 and 11196) and the Swedish Society for Medical Research.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. Tel.:
46-18-471-41-99; Fax: 46-18-471-49-75; E-mail:
Erik.Fries{at}medkem.uu.se.
2 A. M. Blom and E. Fries, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
II, inter-
-inhibitor;
H1 and H2, heavy chain 1 and 2, respectively;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered
saline;
Tricine, N-[2-hydroxy-1,1-bis
(hydroxymethyl)ethyl]glycine..
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|