(Received for publication, July 25, 1994; and in revised form, November 17, 1994)
From the
To determine its active site, growth inhibitory factor (GIF), a
central nervous system-specific metallothionein-like protein, was
digested with trypsin followed by Staphylococcus aureus protease V8 digestion. Of 5 peptide fragments separated from
trypsin-digested GIF by reverse-phase high pressure liquid
chromatography and gel filtration, only GIF1-26 or longer
peptides showed growth inhibitory activity on cortical neurons in
culture. A shorter peptide, GIF5-23, which was obtained by
further digestion of GIF1-26 with V8 protease, also showed growth
inhibitory activity. However, a synthetic peptide corresponding to
GIF5-23 did not show growth inhibitory activity. Metal-free
GIF1-26 prepared by acid treatment showed a similar level of
growth inhibitory activity to that of metal-containing GIF1-26,
indicating that metal in the peptide does not affect the activity.
Treatment of metal-free GIF1-26 with -mercaptoethanol
resulted in the loss of activity. The CD spectrum of
-mercaptoethanol-treated metal-free GIF1-26 was different
from that of nontreated metal-free GIF1-26. These results
indicate that the N-terminal portion of GIF is required for growth
inhibitory activity and that folding of the peptide via S-metal
bonding is critical for biological activity.
Alzheimer's disease (AD) ()is characterized by
progressive loss of neurons and the accumulation of senile plaques and
neurofibrillary tangles. Concurrent with these degenerative changes,
abortive somatodendritic sprouting occurs in the neocortex,
hippocampus, and basal
forebrain(1, 2, 3, 4, 5, 6, 7, 8, 9) .
The hypothesis that a lack of neurotrophic factors might be responsible
for neuronal loss (10) has not explained both neuronal loss
and abortive sprouting responses in AD brain. Indeed, contrary to the
hypothesis, AD brain extract shows higher neurotrophic activities on
cerebral cortical neurons in vitro than normal brain extract,
and this increased neurotrophic activity is due to the loss of growth
inhibitory activities that normally suppress the neurotrophic
activities(11, 12) . One of the growth inhibitory
molecules that are abundant in normal brain but deficient in AD brain
has been identified as a 68-amino acid metallothionein-like protein,
growth inhibitory factor (GIF)(13) . GIF and GIF mRNA are
detectable only in the central nervous system and not in other tissues
including the peripheral nervous system(13, 14) . GIF
is mainly localized in a subpopulation of astrocytes in the gray
matter, which appears to be closely associated with neuronal perikarya
and dendrites, where GIF may suppress abnormal sprouting of neurons
under normal conditions. Down-regulation of GIF, which occurs in
astrocytes in areas of numerous tangles and extensive neuronal loss in
the gray matter of AD cerebrum(13, 14) , may reflect a
compensatory response of astrocytes to neuronal degeneration (15) and may play a role in accelerating the death of cortical
neurons. Despite amino acid sequence homology between GIF and
metallothionein (MT), MT does not inhibit the increased neurotrophic
activities in AD brain extract on cultured cortical
neurons(13) . To investigate why GIF but not MT shows growth
inhibitory activity on cultured cortical neurons, we have attempted to
identify the active site of GIF.
Figure 1:
Separation of trypsin-digested
fragments of GIF (top) and V8 protease-digested fragments of
TG3 (bottom) on a size-exclusion column (GFA-30, 0.75
50 cm) (Asahi Chemical Industry).
Figure 2:
Growth inhibitory activities of
trypsin-digested fragments of native GIF on cortical neurons. Cortical
neurons from neonatal rat brains were cultured for 5 days in
MEM-N2-pyruvate medium containing a mixture of AD brain extract and
various concentrations of the GIF fragments. Cultured cells were fixed
and labeled with a MAP2 monoclonal antibody, and bound antibody was
quantified by ELISA. For percent inhibition, see ``Experimental
Procedures.'' , native GIF;
, TG 1;
, TG2;
, TG3.
Figure 4:
Growth inhibitory activities of
GIF1-26 and its derivatives on cortical neurons. Cortical neurons
from neonatal rat brains were cultured for 5 days in MEM-N2-pyruvate
medium containing a mixture of AD brain extract and various
concentrations of the GIF fragments. Cultured cells were fixed and
labeled with a MAP2 monoclonal antibody (Amersham Corp.), and bound
antibody was quantified by ELISA. For percent inhibition, see
``Experimental Procedures.'' , native GIF;
, TG3
prepared from native GIF by digestion with trypsin;
, ApoTG3
prepared by acid treatment of TG3;
, ApoTG3M prepared by
-mercaptoethanol treatment of apoTG3; &cjs3649;, TApoG prepared
from metal-free GIF by digestion with trypsin and treated with
-mercaptoethanol.
We next examined the growth
inhibitory activity of another type of metal-free GIF1-26
prepared from metal-free GIF by trypsin digestion. Metals were removed
from native GIF by acid treatment. Metal-free GIF, which contained 0.02
zinc and 0.12 copper atoms per polypeptide chain, showed growth
inhibitory activity. A digest, TApoG, was prepared from metal-free GIF
by trypsin digestion followed by 2% -mercaptoethanol treatment and
gel filtration. Omitting
-mercaptoethanol treatment yielded a
higher content of aggregate on gel filtration. Sequence and composition
analyses revealed that TApoG had the sequence of GIF1-26.
However, TApoG showed no growth inhibitory activity (Fig. 4).
Because one difference in preparation between ApoTG3 and TApoG is
-mercaptoethanol treatment, we examined the growth inhibitory
activity of
-mercaptoethanol-treated ApoTG3. ApoTG3 was dissolved
in 2%
-mercaptoethanol, phosphate-buffered saline(-) and
then applied to a gel filtration column to remove the
-mercaptoethanol. The product, ApoTG3M, did not show growth
inhibitory activity (Fig. 4).
Figure 5:
CD spectra of metal-free GIF1-26 and
its derivatives. Spectra were measured in Dulbecco's
phosphate-buffered saline(-) at 22 °C. Solidline, ApoTG3 prepared by acid treatment of TG3; dashedline, ApoTG3M prepared by
-mercaptoethanol treatment of apoTG3; dottedline, TApoG prepared from metal-free GIF by digestion
with trypsin and treated with
-mercaptoethanol.
Here, we have shown that 1) GIF1-26 and a shorter
fragment, GIF5-23, prepared from native GIF by enzymatic
digestion inhibit the neurotrophic activities of AD brain extract in vitro; 2) synthetic GIF5-23 shows no growth
inhibitory activity; 3) a metal-free GIF1-26 prepared by acid
treatment of GIF1-26 obtained by trypsin digestion of native GIF
shows growth inhibitory activity; 4) -mercaptoethanol-treated
metal-free GIF1-26 shows no growth inhibitory activity; and 5)
the CD spectra of inactive metal-free GIF1-26 species differ from
that of active metal-free GIF1-26.
We have obtained two active
fragments, TG2 and TG3, by trypsin digestion of native GIF. The growth
inhibitory activities of these two fragments are at the same level as
that of native GIF. TG2 is a partially digested GIF lacking a part of
the C-terminal insert, and TG3 is the GIF1-26 peptide. The fact
that TG3 and native GIF exhibit the same level of growth inhibitory
activity indicates that the active site of GIF is in the amino-third of
the GIF molecule. This assumption is supported by the following
findings: 1) TG2, which lacks several amino acid residues in the
C-terminal region of GIF, showed a level of growth inhibitory activity
similar to that of native GIF; 2) inactive fractions of
trypsin-digested GIF, eluted before the retention time of intact GIF by
reverse-phase HPLC, contained the sequences CTSCK (GIF27-31), CAK
(GIF45-47), DCVCK (GIF48-52), and GGEAAEAEAEK
(GIF53-63); and 3) the -domain of MT, which contains almost
the same sequences as GIF32-39 and GIF64-68, showed no
inhibitory activity. The amino-third of GIF, compared with the
carboxyl-third of GIF, is well conserved among
species(13, 14, 17, 18, 19) .
It is reasonable to conclude that this portion is essential for growth
inhibitory activity.
The active fragment GIF5-23 was obtained
by V8 protease digestion of GIF1-26. GIF1-4 purified from
the VG3 fraction did not exhibit GIF activity. Endoprotease Asp-N,
which was expected to cleave GIF before Asp, did not
produce a smaller fragment than GIF5-23. The smallest active
fragment obtained from native GIF by enzymatic digestion was
GIF5-23. The growth inhibitory activity of GIF5-23 was
slightly lower than that of native GIF but was much higher than that of
recombinant GIF(14) .
The seven metal atoms in GIF, as in
MT(20, 21, 22) , may be liganded to the
cysteinyl thiolate and may form two distinct metal clusters,
ZnCu
Cys
(GIF 1-31) and
Zn
Cu
Cys
(GIF32-68). The
bioactive N-terminal cluster domain of GIF, compared with the inactive
-domain of MT, has unique sequences: a Thr insert at position 5
and (Cys-Pro)
sequence between positions 6-9. Because
the presence of (X-Pro)
sequences is known to
results in a stiff ``elbow-hinged'' peptide
chain(23) , The MDPETCPCP sequence in GIF is predicted to
contain additional
-turns(19) , and a tightly folded
structure may be formed in this region.
Growth inhibitory activity of GIF peptides seemed to depend on the number of metal atoms in the peptides. GIF1-26 obtained by trypsin digestion of native GIF and containing 3 atom metals per polypeptide showed the highest activity, nonsynthetic GIF5-23 containing 1.5 atom metals per polypeptide showed lower activity, and synthetic GIF5-23 showed no activity. However, metals themselves are not essential for GIF activity. Metal-free GIF1-26 showed similar growth inhibitory activity to that of metal-containing GIF1-26. In metalloenzymes, metals, especially zinc atoms that are coordinated to 4 Cys residues, are known to stabilize the structure of proteins(24) . It is likely that metal ions in GIF1-26 stabilize the folded structure in the N-terminal domain of GIF.
Metal-free GIF1-26 could sustain a
complete globular structure due to the presence of Pro-rich turns and
intramolecular disulfide bonds formed by oxidation of Cys residues
closely located in a core structure after the removal of
metals(25) . Treatment of metal-free GIF1-26 with
-mercaptoethanol resulted in the loss of growth inhibitory
activity, indicating that cleavage of disulfide bonds in metal-free
GIF1-26 might loosen a core structure of GIF1-26 and that
free SH moieties might undergo different disulfide pairing in the
peptide. In apothionein after oxidation of the SH groups, the CD bands
between 250 and 280 nm are known to be due to disulfide
transitions(26) . The difference in CD spectra of this region
between metal-free GIF1-26 and
-mercaptoethanol-treated
metal-free GIF1-26 suggests that the interchange of disulfide
bonds occurs in metal-free GIF1-26 after
-mercaptoethanol
treatment. The CD spectrum is sensitive to the secondary structure of
the polypeptide chain itself and to the conformation of the amino acids
involved in S-S or S-metal binding (26) . It is
reasonable to consider that different protein folding of metal-free
GIF1-26 and disulfide-interchanged metal-free GIF1-26 may
lead to the presence or absence of growth inhibitory activity.
Thus, a folded structure formed with both a Pro-rich polypeptide chain and S-metal bonds in the amino-third portion of GIF may be necessary for growth inhibitory activity on cortical neurons in culture. This would explain the facts that recombinant GIF exhibited low growth inhibitory activity (14) and synthetic peptide GIF5-23 or metallothionein (13) showed no growth inhibitory activity.