Inhibin, activin and follistatin bind preferentially to the transformed ...

65
Inhibin, activin and follistatin bind preferentially to the transformed
species of α 2 -macroglobulin
D J Phillips, J R McFarlane, MTWHearn 1 and D M de Kretser
Institute of Reproduction and Development, and 1 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia
(Requests for offprints should be addressed to D J Phillips, Institute of Reproduction and Development, Level 3, Block E, Monash Medical Centre,
Clayton Road, Clayton, Victoria 3168, Australia)
Abstract
α 2 -Macroglobulin (α 2 -M), a major serum glycoprotein,
has been implicated as a low-affinity binding protein for
inhibin and activin. In serum, α 2 -M exists as two major
species, a native form that is abundant and stable, and a
transformed (‘fast’) species that is rapidly cleared from the
circulation via α 2 -M receptors. In this study inhibin,
activin and their major binding protein follistatin were
investigated for their ability to bind to the native or
transformed species of purified human α 2 -M. Using native
PAGE and size exclusion chromatography, radiolabelled
inhibin, activin and follistatin bound to the transformed
α 2 -M. Inhibin and follistatin did not bind significantly to
native α 2 -M, whereas activin was able to bind to the
native species but with a lower capacity compared with
that to transformed α 2 -M. Under reducing conditions,
binding of these hormones to α 2 -M was abolished. These
findings implicate α 2 -M as a mechanism whereby inhibin,
activin and follistatin may be removed from the circulation
through α 2 -M receptors, but also whereby activin can be
maintained in the circulation through its ability to bind to
native α 2 -M.
Journal of Endocrinology (1997) 155, 65–71
Introduction
α 2 -Macroglobulin (α 2 -M) is a major serum glycoprotein
that has a multiplicity of functions including proteinase
inhibition, immune regulation and cytokine binding (for
review, see Chu & Pizzo 1994). This 725 kDa protein
exists in at least two major forms, a native or so-called
‘slow’ species, which is present in high concentrations in
serum and can entrap proteinases, and the transformed
or electrophoretically ‘fast’ species, which appears after
trapping of a proteinase or reaction with primary amines
(Van Leuven et al. 1981). A number of growth factors have
been shown to bind to α 2 -M at a site distinct from that
where proteinases are entrapped, including members of
the transforming growth factor-β (TGFβ) superfamily
(Chu & Pizzo 1994). Importantly, some polypeptides have
been shown to bind significantly only to the native form
of α 2 -M (e.g. carboxypeptidase A (Valnickova et al.
1996)), to both forms (e.g. TGFβ2 (Crookston et al.
1994)), and some to only the transformed species (e.g.
growth hormone (Kratzsch et al. 1995)). The form of α 2 -M
to which factors can bind has significant implications for
their physiology, as complexes of cytokines with native
α 2 -M remain stable in the circulation (Philip &
O’Connor-McCourt 1991, Crookston et al. 1993),
whereas factors binding to the transformed α 2 -M are
rapidly removed from the circulation via α 2 -M receptors
(Kristensen et al. 1990, Strickland et al. 1990, Misra et al.
1994).
Inhibin and activin are members of the TGFβ superfamily
with diverse physiological roles within the body
(dePaolo et al. 1991, Woodruff & Mather 1995). Follistatin
is a polypeptide that is structurally distinct from inhibin or
activin and is able to bind these cysteine knot proteins
with high affinity (Nakamura et al. 1990, Kogawa et al.
1991, Sugino et al. 1993, Schneyer et al. 1994). Investigations
into high molecular mass binding proteins of
inhibin and activin have implicated α 2 -M (Schneyer et al.
1992, Krummen et al. 1993, Vaughan & Vale 1993), but
further characterizations of these interactions were not
achieved in these earlier studies. More recently, Niemuller
et al. (1995) demonstrated that activin A is able to bind to
both the native and transformed α 2 -M with a reported
affinity of 51060 and 19030 nM respectively. These
data suggest that α 2 -M has a dual function of both
maintaining activin A in the circulation or within tissues
and facilitating the clearance of activin A depending on the
form of α 2 -M present. Whether follistatin binds to α 2 -M,
and the different species of α 2 -M that bind inhibin, have
not been previously documented.
In this study, we have investigated the interactions of
inhibin, activin and follistatin with the native and transformed
conformations of α 2 -M. The results, showing
that inhibin and follistatin bind significantly only to the
Journal of Endocrinology (1997) 155, 65–71 1997 Journal of Endocrinology Ltd Printed in Great Britain
0022–0795/97/0155–0065 $08.00/0

66
D J PHILLIPS and others · Hormone binding to transformed α 2 -macroglobulin
transformed species of α 2 -M, whereas activin A binds to
both the native and transformed species, suggest important
regulatory mechanisms for these factors in terms of their
availability at target tissues.
Materials and Methods
Purification of α 2 -M
Human α 2 -M was purified from time-expired plasma
obtained from the South Melbourne Blood Bank using the
method of Imber & Pizzo (1981), and incorporating the
modifications of Chu & Pizzo (1994). Briefly, the plasma
was filtered and the protein, remaining in solution in 4%
polyethylene glycol 8000 but precipitated by 16% polyethylene
glycol 8000, was then dialysed extensively against
distilled water to separate any of the transformed α 2 -M
present. The resulting precipitate containing the transformed
α 2 -M was removed by centrifugation (7600 g for
30 min) and the supernatant was loaded on to a zinc
chelate column. α 2 -M has been shown to have a high
affinity for metal ions, particularly copper and zinc (Porath
et al. 1975). The column was washed with phosphate
buffers (0·1 mol/l phosphate–0·8 mol/l NaCl, pH 6·5,
followed by 0·02 mol/l phosphate–0·15 mol/l NaCl,
pH 6·0) until minimal protein was eluted from the column.
In our hands, the α 2 -M is eluted from the column
with 0·05 mol/l EDTA–0·5 mol/l NaCl, pH 7·0. Fractions
from the protein peak eluted under these conditions
were subjected to native PAGE to assess the purity of the
material, and compared with a commercially available
preparation of human α 2 -M (Calbiochem, San Diego, CA,
USA). Those fractions containing the α 2 -M were pooled,
concentrated using an ultrafiltration cell, and stored at
80 C until further processing.
Further purification was achieved using size exclusion
chromatography on a Waters HPLC system. Aliquots
of stored α 2 -M material were chromatographed on a
Superose 12 column (HR 10/30; Pharmacia Biotech,
Uppsala, Sweden) in 0·05 mol/l PBS, pH 7·5, and fractions
of the correct size for α 2 -M (725 kDa) checked
using native PAGE. This purification step removes low
molecular mass contaminants and also monomer and dimer
forms of α 2 -M. Aggregated forms of α 2 -M (>725 kDa)
were difficult to remove from the preparation because
of the poor discriminative power of this column for
proteins of very high molecular mass. The activity of the
purified native α 2 -M was confirmed by reaction with
trypsin and the demonstration of a shift to the transformed
(proteinase-bound) species under native PAGE, and using
a trypsin-binding assay (Bonner et al. 1992, data not
shown).
To convert α 2 -M to the transformed species, native
α 2 -M was allowed to react with 300 mol/l methylamine
hydrochloride (Sigma, St Louis, MO, USA) in 50 mol/l
Tris, pH 8·2. After overnight incubation at room temperature,
the reaction mixture was dialysed against 20 mol/l
phosphate buffer, pH 7·4, overnight at 4 C. Transformation
of the α 2 -M was confirmed by native PAGE and
the lack of binding to trypsin in the binding assay of
Bonner et al. (1992).
Hormone–α 2 -M incubations
The hormones tested for binding to α 2 -M were bovine
inhibin purified from follicular fluid, human recombinant
activin A, bovine follistatin purified from follicular fluid,
and human recombinant follistatin 288 kindly provided by
the National Hormone and Pituitary Program (Baltimore,
MD, USA). Iodinations were carried out using the
chloramine T procedure for the inhibin as detailed
by McLachlan et al. (1986). For the activin and the two
follistatin preparations, iodination was achieved with
Iodogen (Pierce, Rockford, IL, USA) as described previously
(Matteri et al. 1987). The activin tracer was
further purified by gel filtration using an Ultragel AcA54
column (902·5 cm; IBF Biotechnics, Villeneuve-la-
Garenne, France). In the experiments described, an
aliquot of radiolabelled hormone (100 000 c.p.m.) was
incubated with approximately 10 µg of either native or
transformed α 2 -M at 37 C, giving a concentration of
α 2 -M in the reaction mixture of 0·4 g/l. For PAGE
analysis, the incubation time was 3 h, whereas for size
exclusion HPLC separations, an overnight incubation was
employed.
Figure 1 Native PAGE showing (a) Coomassie blue-stained and
(b) the corresponding autoradiograph of a 5% gel, with bands
corresponding to native (N) and transformed (‘fast’; F) α 2 -M.
Inhibin, activin, bovine follistatin (bFS) and follistatin 288 (FS288)
bound significantly in those lanes containing transformed α 2 -M.
Note the characteristic shift in running position on the gel for the
transformed α 2 -M compared with the native species.
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Hormone binding to transformed α 2 -macroglobulin · D J PHILLIPS and others 67
Figure 2 Size exclusion chromatography of incubations of α 2 -M with radiolabelled activin,
with profiles for activin tracer only (broken line), activin and native α 2 -M () and activin
and transformed α 2 -M (). The elution position for α 2 -M is approximately fraction 13 and
that of the unbound tracer fraction 23.
PAGE
PAGE was performed using the Minigel apparatus
(Hoeffer Scientific Instruments, San Francisco, CA,
USA), using 5% acrylamide gels for native proteins (no
dissociating agents) as previously described (Van Leuven
et al. 1981). Gels were routinely run at 20 mA for
approximately 2 h. The transformed (‘fast’) α 2 -M has a
greater mobility under these conditions and thus can be
discriminated from the native form. For PAGE in the
presence of SDS, samples were prepared in buffer containing
1% SDS and were run on SDS-containing 5%
polyacrylamide gels. Under reducing conditions, samples
were prepared with 1% SDS and β-mercaptoethanol,
incubated at 90 C for 2 min, and run on SDS-containing
7·5% polyacrylamide gels. After electrophoresis, the gels
were stained with Coomassie blue, destained, dried and,
where appropriate, exposed to Biomax MR film (Kodak,
Rochester, NY, USA). Radioactivity detected in the
autoradiographs represents only hormone bound to α 2 -
macroglobulin. Gels containing 5–7·5% acrylamide resolve
proteins of sizes greater than 75 kDa, whereas proteins
smaller than 75 kDa, including the unbound hormones
utilized in these studies, pass through the resolving gel and
are not recoverable.
Size exclusion chromatography
Size exclusion chromatography of incubation mixtures
containing radiolabelled hormones and α 2 -M were performed
using a BioSep Sec 2000 column (Phenomenex,
Torrence, CA, USA) equilibrated with buffer, pH 6·8,
containing 0·1 mol/l Na 2 SO 4 , 0·1 mol/l NaH 2 PO 4 and
0·05% BSA. The column had been calibrated with
markers of known molecular mass using the equivalent
buffer without BSA. For each hormone, column runs
were with incubation mixtures containing radiolabelled
hormone only, hormone and native α 2 -M, and hormone
and transformed α 2 -M. Fractions were collected at 30 s
intervals at a flow rate of 1 ml/min, and the radioactivity
in each fraction was determined. To focus on the binding
of radiolabelled hormone to α 2 -M and to discount unlabelled
tracer, the radioactivity in runs containing α 2 -M
was routinely expressed as a percentage of c.p.m. values
measured in the equivalent fraction of the run containing
labelled hormone only.
Results
Using both native PAGE and size exclusion chromatography,
it was demonstrated that inhibin, activin
and follistatin preferentially bound to the transformed
conformation of α 2 -M. After native PAGE, specific bands
of radioactivity were detected in those lanes containing
transformed α 2 -M, but not in those containing native
α 2 -M (Fig. 1). Some minor signal was present in all lanes
above the position of tetrameric α 2 -M, corresponding to
aggregated α 2 -M complexes that had not been removed
during the purification process.
Size exclusion chromatography showed that radioactivity
could be detected in the eluted fractions
Journal of Endocrinology (1997) 155, 65–71

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D J PHILLIPS and others · Hormone binding to transformed α 2 -macroglobulin
Figure 3 Size exclusion chromatography showing binding of (a) inhibin, (b) activin, (c) bovine follistatin, and (d) human follistatin 288 to
native () or transformed () α 2 -M. Note that the radioactivity detected in each fraction is expressed relative to that measured in the
corresponding tracer-only fraction.
corresponding to the predicted position for α 2 -M that
preceded the peak of free labelled hormone (Fig. 2). When
profiles were expressed as a percentage of control runs
to focus on the binding of α 2 -M to the radiolabelled
hormones (Fig. 3), it was apparent that the inhibin and the
two follistatin preparations bound significantly only to the
transformed α 2 -M, as also shown by native PAGE. However,
with this more sensitive technique it was demonstrated
that activin was able to bind to both forms of α 2 -M,
with a greater capacity for the transformed species. For
activin, the percentage of tracer added that bound to α 2 -M
was 4·7 and 18% for the native and transformed conformations
respectively. The percentage of tracer added that
bound to transformed α 2 -M was 12·0, 12·7 and 15·1% for
human recombinant inhibin, bovine follistatin and human
recombinant follistatin 288 respectively.
To investigate the nature of the binding of these
hormones to α 2 -M, PAGE under SDS and reducing
conditions were performed. Under SDS non-reducing
conditions, the α 2 -M tetramer was broken down to
dimeric and monomeric forms (Fig. 4). In these subunit
states, inhibin, activin and follistatin remained bound to
transformed α 2 -M. In addition, radioactive bands for
activin were also detected for native α 2 -M under SDS
non-reducing conditions, with a lower intensity than that
for transformed α 2 -M. Trace amounts of the signal for
radiolabelled inhibin and follistatin were found in the
native α 2 -M lanes, probably because of contamination of
the native α 2 -M with a proportion of the transformed
species. When preformed radiolabelled ligand–α 2 -M complexes
were run under reducing conditions, in which
α 2 -M is present predominantly in a monomeric form,
radioactive bands were not detectable for any of the
hormones (Fig. 5). This result and the comparative result
obtained under non-reducing conditions (Fig. 4) indicate
that the ligand–α 2 -M complexes may involve a disulphide
interchange which is reducible by β-mercaptoethanol,
rather than weak hydrophobic and electrostatic
interactions between the radiolabelled hormones and
α 2 -M.
Journal of Endocrinology (1997) 155, 65–71

Hormone binding to transformed α 2 -macroglobulin · D J PHILLIPS and others 69
Figure 4 PAGE under SDS non-reducing conditions showing (a) Coomassie blue-stained
5% gel and (b) corresponding autoradiograph of radiolabelled hormones allowed to react
with α 2 -M. See the legend of Fig. 1 for abbreviations. The positions of the α 2 -M dimeric
and monomeric forms are indicated.
Figure 5 PAGE under SDS-reducing conditions showing (a) Coomassie blue-stained 7·5%
gel and (b) corresponding autoradiograph, and illustrating no binding of labelled hormones
to α 2 -M. See the legend of Fig. 1 for abbreviations. The positions of the monomeric α 2 -M
are indicated.
Discussion
In this study we have determined that inhibin and
follistatin bind significantly to the transformed α 2 -M,
whereas activin is able to bind to both species, but with a
lesser capacity for the native (slow) species. The data for
activin are consistent with those published by Niemuller
et al. (1995). Previous studies had implicated α 2 -M as
a binding protein for inhibin (Schneyer et al. 1992,
Krummen et al. 1993, Vaughan & Vale 1993), but without
differentiating which species of α 2 -M was the
predominant binder.
The consequences of inhibin binding only to transformed
α 2 -M are highly relevant to the mechanism for
removal of inhibin from the circulation. Receptors for
α 2 -M, the lipoprotein receptor-related protein, are found
in many sites throughout the body (Moestrup et al. 1992).
A second α 2 -M receptor has also been recently characterized
on macrophages (Misra et al. 1994). It is conceivable
that by binding to transformed α 2 -M, inhibin (and activin
and follistatin) are being targeted to these tissues whereby
they act through the α 2 -M receptor. Nevertheless, when
radiolabelled growth factors complexed to transformed
α 2 -M are injected into mice, over 50% of normalized
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D J PHILLIPS and others · Hormone binding to transformed α 2 -macroglobulin
radioactivity is recovered from the liver (LaMarre et al.
1991, Philip & O’Connor-McCourt 1991, Crookston
et al. 1993, Niemuller et al. 1995). In the liver, α 2 -M–
proteinase complexes bind to receptors on hepatocytes,
leading to endocytosis and degradation of the ligand
(Gliemann & Davidsen 1986). It is unclear whether
α 2 -M–growth factor complexes targeted to hepatocytes
undergo a similar process of degradation, although recent
evidence suggests that cytokines bound to transformed
α 2 -M may have intracellular actions after being internalized
through the α 2 -M receptor (Borth 1992, Stouffer et al.
1993).
Activin has been shown in this study and previously
(Niemuller et al. 1995) to bind to both native and transformed
α 2 -M. Although activin binds with a lower capacity
to the native conformation, this form is predominant
in the circulation and is present in high abundance. As
hypothesized by others (Schneyer et al. 1992, Krummen
et al. 1993, Vaughan & Vale 1993, Niemuller et al. 1995),
native α 2 -M may play a role as a low-affinity carrier for
activin in the circulation or within tissues where follistatin,
a higher-affinity binding protein, is not abundant. The
affinity of native α 2 -M for activin has been estimated as
510 nM (Niemuller et al. 1995), whereas follistatin has an
affinity for activin in the range 0·01–0·09 nM (Nakamura
et al. 1990, Kogawa et al. 1991, Sugino et al. 1993,
Schneyer et al. 1994). Therefore consideration needs to be
made at the tissue and circulatory level not only of the
affinity of the binding proteins present, but also their
relative abundance. We are currently exploring whether
activin, when bound to α 2 -M, can subsequently be
released under conditions that favour binding to follistatin.
It is of interest that follistatin, a binding protein for
inhibin and activin, was capable of binding to transformed
α 2 -M. We examined two preparations of follistatin,
that purified from bovine follicular fluid and human
recombinant follistatin 288; both were able to bind
α 2 -M with apparently similar capacities. This binding
phenomenon may represent a mechanism whereby excess
follistatin is removed from the circulation or follistatin
is targeted to tissues expressing the α 2 -M receptor. An
intriguing question is whether complexes of activin and
follistatin are able to bind to α 2 -M. This process would be
feasible if the binding domains for α 2 -M on activin and
follistatin are not masked after formation of activin–
follistatin complexes. Although the binding domain for
α 2 -M on activin is almost certainly in an analogous position
to that for TGFβ (Qian et al. 1992), it is unclear if this site
is involved in binding to follistatin. On the other hand, a
binding site for α 2 -M on the follistatin molecule has yet to
be determined, although the regions responsible for binding
to activin have been determined (Inouye et al. 1991,
Sumitomo et al. 1995). It is important for these issues to
be resolved, particularly the implications of whether
follistatin-bound activin can or cannot be cleared via an
α 2 -M binding process.
In summary, inhibin, activin and follistatin were found
to bind to the transformed species of α 2 -M, with activin
also binding to a lesser extent to the native form. The
complexes were reversible, and as such provide potentially
important mechanisms whereby these hormones can be
distributed to various target tissues.
Acknowledgements
These studies were performed with the support of the
National Health and Medical Research Council of
Australia. We thank the National Hormone and Pituitary
Program for the provision of the human recombinant
follistatin 288.
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Received 25 July 1996
Revised manuscript received 3 February 1997
Accepted 21 April 1997
Journal of Endocrinology (1997) 155, 65–71