©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The N-terminal Portion of Growth Inhibitory Factor Is Sufficient for Biological Activity (*)

(Received for publication, July 25, 1994; and in revised form, November 17, 1994)

Yoko Uchida (1)(§) Yasuo Ihara (2)

From the  (1)Department of Neuropathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho Itabashiku, Tokyo 173, Japan and the (2)Department of Neuropathology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo Bunkyoku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 beta-mercaptoethanol resulted in the loss of activity. The CD spectrum of beta-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.


INTRODUCTION

Alzheimer's disease (AD) (^1)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.


EXPERIMENTAL PROCEDURES

Purification of Native GIF from Human Brains

Native GIF was purified from cerebral cortex of nondemented aged patients by a combination of DEAE-Sephacel chromatography, size-exclusion chromatography with a TSK G2000SW column, and reverse-phase HPLC with a VP-318 column according to the protocol previously reported(13) . GIF was detected by dot immunobinding. Aliquots of the fractions were spotted on Immobilon membrane (Millipore). The membrane was incubated with affinity-purified antibodies against synthetic GIF53-64 peptide overnight at room temperature, and reactive spots were visualized by the avidin-biotin peroxidase method.

Preparation and Peptide Sequencing of GIF Fragments

Purified GIF was digested with tosylphenylalanyl chloromethyl ketone-trypsin for 20 h at room temperature. The digest was separated by size-exclusion HPLC (GFA-30) and reverse-phase HPLC (VP-318) with a linear gradient of acetonitrile in 5 mM ammonium format (pH 6.8). Separated peptide fragments were further digested with Staphylococcus aureus V8 protease for 24 h at 37 °C. The generated fragments were similarly separated by HPLC. Purified fragments were cleaved with cyanogen bromide and oxidized with performic acid. N-terminal sequence analyses were carried out on a PSQ-1 Protein Sequencer (Shimadzu). Amino acid compositions were determined on a JLC-300 Amino Acid Analyzer (JEOL) after hydrolysis with 6 N HCl.

Preparation of Metal-free GIF

Metal-free GIF was prepared by acidification of 200 µg of native GIF in 0.1 N HCl, followed by gel filtration in 0.01 N HCl (16) .

Preparation of Reduced Metal-free GIF1-26

Reduced metal-free GIF1-26 was prepared from metal-free GIF or metal-free GIF1-26. Metal-free GIF was treated with trypsin (molar ratio, 50:1) for 18 h at room temperature. beta-Mercaptoethanol (final concentration, 2%) was added to the reaction mixture, and the fragment corresponding to GIF1-26 was separated by GFA-30 gel filtration. The other reduced metal-free GIF1-26 was prepared by acid treatment followed by 2% of beta-mercaptoethanol treatment of GIF1-26 obtained by trypsin digestion of native GIF.

Determination of Peptide Concentration

Peptide concentrations were determined by amino acid composition analysis after hydrolysis with HCl.

Circular Dichroism

Metal-free GIF1-26 and its derivatives were dissolved in Dulbecco's phosphate-buffered saline (-) to a final concentration of 15 µM. Circular dichroism spectra were recorded on a Jasco J-700 spectropolarimeter controlled by an NEC PC-9801FX computer.

Bioassay for Growth Inhibitory Activity

Cerebral cortex dissected from newborn rats was dissociated by incubation with 0.08% trypsin, 0.008% DNase I at 37 °C for 20 min and passed through a 62-µm nylon mesh. Cerebral cortical cells (1.85 times 10^4 cells/well) in 96-well microtiter plates coated with gelatin-polyornithine were cultured for 5 days in the presence of peptide fragments of GIF in MEM-N2-pyruvate medium containing brain extract from one AD patient (125 µg protein/ml) as a source of neurotrophic factors. Cultured cells were fixed with 4% paraformaldehyde and 90% methanol/acetic acid and labeled with a MAP2 monoclonal antibody (Amersham Corp.) by the avidin-biotin peroxidase method. Anti-MAP2 binding to the culture was determined by ELISA(12) . Growth inhibitory activity was quantified by the ability to inhibit the neurotrophic activity in AD brain extract and expressed as percentage of inhibition(13) .


RESULTS

N-terminal Portion of GIF Contains the Active Site

We attempted to identify the active site of GIF by digesting native GIF with trypsin. Endoprotease Asp-N, which was expected to cleave GIF into three different peptide fragments (see Fig. 1), did not cleave native GIF. 8 peptide fragments were expected to be released by trypsin digestion, but only five peptide peaks were found in the reverse-phase HPLC profile. No strong inhibitory activity was detected in the fractions corresponding to the first four peaks, although fractions corresponding to two of these peaks showed 10-20% of maximal inhibitory activity. The peptide fragments contained in the fractions corresponding to the first four peaks were shown by sequencing to be GGEAAEAEAEK (GIF53-63), CAK (GIF45-47), DCVCK (GIF48-52), and CTSCK (GIF27-31). The fifth peak fraction, which was eluted at the same time as intact GIF, was further fractionated on a size-exclusion HPLC column. Peptide peaks labeled TG1, TG2, and TG3 were detected as shown in Fig. 1, top. Sequence and amino acid composition analyses showed that TG1 was intact GIF, that TG2 was shorter than GIF with 2 Glu, 2 Ala, 2 Gly, and 1 Lys residue missing, and that TG3 had the peptide sequence of GIF1-26. All three peptides showed levels of growth inhibitory activity similar to that of intact GIF (Fig. 2). TG3 was further digested with S. aureus protease V8, and the digest was separated on a size-exclusion column. Peptide peaks labeled VG1, VG2, and VG3 were detected as shown in Fig. 1, bottom. In reverse-phase HPLC, VG1 and VG2 were eluted at the same time as intact GIF; however, two peaks were obtained from VG3: a major peak eluting before intact GIF and a minor peak eluting at the same time as intact GIF. Sequence and composition analyses revealed that VG1 was undigested TG3, VG2 had the peptide sequence of GIF5-23, the major fraction obtained from VG3 had the sequence of GIF1-4, and the minor fraction obtained from VG3 was GIF5-23. All VG peptides except GIF1-4 showed growth inhibitory activity; however, VG2 needed slightly higher concentration for the half-maximal activity than that of native GIF ( Fig. 2and 3). The metal contents of the GIF fragments were determined by atomic absorption spectroscopy. TG3 (GIF1-26) contained 2.07 copper and 1.00 zinc atoms per peptide chain. VG2 (GIF5-23) contained 1.05 copper and 0.35 zinc atoms per peptide chain.


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 times 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.'' bullet, native GIF; circle, TG 1; , TG2; , TG3.



A Synthetic GIF5-23 Peptide Shows No Growth Inhibitory Activity

Because GIF5-23 obtained by digestion of TG3 showed growth inhibitory activity, we examined the possible growth inhibitory activity of synthetic GIF5-23. The growth inhibitory activity of nonsynthetic GIF5-23 was half-maximal at a peptide concentration of 200 nM. However, synthetic GIF5-23 did not inhibit neurotrophic activity at peptide concentrations of up to 1 µM.

Reduced Metal-free GIF1-26 Shows No Growth Inhibitory Activity

To determine why synthetic GIF5-23 shows no growth inhibitory activity, we first investigated whether metals are necessary for GIF fragments to exhibit growth inhibitory activity by using GIF1-26 (TG3) obtained by trypsin digestion of native GIF. Metals were removed from TG3 by acid treatment. Atomic absorption spectroscopy revealed that metal-free TG3 (ApoTG3) contained 0.02 zinc and 0.10 copper atoms per polypeptide chain. ApoTG3 showed growth inhibitory activity similar to that of TG3 (Fig. 4), indicating that metals themselves are not essential for growth inhibitory activity.


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.'' bullet, native GIF; , TG3 prepared from native GIF by digestion with trypsin; , ApoTG3 prepared by acid treatment of TG3; up triangle, ApoTG3M prepared by beta-mercaptoethanol treatment of apoTG3; &cjs3649;, TApoG prepared from metal-free GIF by digestion with trypsin and treated with beta-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% beta-mercaptoethanol treatment and gel filtration. Omitting beta-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 beta-mercaptoethanol treatment, we examined the growth inhibitory activity of beta-mercaptoethanol-treated ApoTG3. ApoTG3 was dissolved in 2% beta-mercaptoethanol, phosphate-buffered saline(-) and then applied to a gel filtration column to remove the beta-mercaptoethanol. The product, ApoTG3M, did not show growth inhibitory activity (Fig. 4).

Secondary and Tertiary Structures Differ between Nonreduced and Reduced Metal-free GIF1-26

The secondary structures of inactive TApoG and ApoTG3M were compared with that of active ApoTG3 using circular dichroism (CD) analysis (Fig. 5). ApoTG3, metal-free GIF1-26, exhibits a negative CD intensity below 200 nm with a negative shoulder near 230 nm but no band above 250 nm. On the other hand, the spectra of ApoTG3M and TApoG, both of which are beta-mercaptoethanol-treated metal-free GIF1-26, were different from that of ApoTG3. There was no negative shoulder near 230 nm in the spectra of ApoTG3M and TApoG. Besides a negative CD band below 200 nm, ApoTG3M had positive bands at 260 and 295 nm. TApoG had a positive band at 270 nm and negative bands at 250 and 307 nm in addition to a negative band below 200 nm. Because the spectrum below 220 nm is assigned to polypeptide bonds, the strong CD intensity below 200 nm in the spectra of all three metal-free GIF1-26 species shows that the initial conformation of the GIF1-26 polypeptide chain is a random coil. Because none of the three GIF1-26 derivatives contained aromatic amino acids or metals, the CD intensities above 220 nm must be attributed to disulfide transition. ApoTG3M and TApoG were treated with beta-mercaptoethanol, and their S-S bonds were cleaved once and reformed by auto-oxidation. In addition to the disappearance of a negative shoulder near 230 nm, the appearances of positive bands near 260-270 nm and at 295 nm and negative bands at 350 and 307 nm in beta-mercaptoethanol-treated ApoTG3M or TApoG suggest that disulfide bridges were newly formed at positions in the GIF1-26 molecule different from those in untreated ApoTG3. Newly formed disulfide bonds may change the peptide folding pattern to a different one from that of ApoTG3. Thus, the difference in CD spectra of metal-free GIF1-26 with or without beta-mercaptoethanol treatment may reflect a difference in the secondary structures of these peptides and in the conformation of the polypeptide chain involved in growth inhibitory activity.


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 beta-mercaptoethanol treatment of apoTG3; dottedline, TApoG prepared from metal-free GIF by digestion with trypsin and treated with beta-mercaptoethanol.




DISCUSSION

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) beta-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 alpha-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^18, 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, Zn(1)Cu(2)Cys(9) (GIF 1-31) and Zn(2)Cu(2)Cys (GIF32-68). The bioactive N-terminal cluster domain of GIF, compared with the inactive beta-domain of MT, has unique sequences: a Thr insert at position 5 and (Cys-Pro)(2) sequence between positions 6-9. Because the presence of (X-Pro)(n) sequences is known to results in a stiff ``elbow-hinged'' peptide chain(23) , The MDPETCPCP sequence in GIF is predicted to contain additional beta-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 beta-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 beta-mercaptoethanol-treated metal-free GIF1-26 suggests that the interchange of disulfide bonds occurs in metal-free GIF1-26 after beta-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.


FOOTNOTES

*
This work was supported by Grant-in-aid for Scientific Research on Priority Areas 4268103 from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 813-3964-3241 (ext. 3050); Fax: 813-3579-4776.

(^1)
The abbreviations used are: AD, Alzheimer's disease; GIF, growth inhibitory factor; MT, metallothionein; HPLC, high pressure liquid chromatography; MEM, minimum essential medium; MAP2, microtubule-associated protein 2; ELISA, enzyme-linked immunosorbent assay.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.