AU7766091A - Modified heparin binding growth factors - Google Patents
Modified heparin binding growth factorsInfo
- Publication number
- AU7766091A AU7766091A AU77660/91A AU7766091A AU7766091A AU 7766091 A AU7766091 A AU 7766091A AU 77660/91 A AU77660/91 A AU 77660/91A AU 7766091 A AU7766091 A AU 7766091A AU 7766091 A AU7766091 A AU 7766091A
- Authority
- AU
- Australia
- Prior art keywords
- hbgf
- growth factor
- nts
- polypeptide
- nuclear translocation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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- IHIXIJGXTJIKRB-UHFFFAOYSA-N trisodium vanadate Chemical compound [Na+].[Na+].[Na+].[O-][V]([O-])([O-])=O IHIXIJGXTJIKRB-UHFFFAOYSA-N 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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Description
1 MODIFIED HEPARIN BINDING GROWTH FACTORS
2 BACKGROUND OF INVENTION
3 Polypeptide growth factors are modulators of cell
4 proliferation and differentiation whose biological functions
5 are mediated by the interaction of the growth factor with cell
6 surface receptors and subsequent alterations in gene
7 expression.
8 The heparin binding growth factor (hereinafter HBGF) 9. family of growth factors consists of structurally related
10 polypeptides. At the present time seven members of this
11 family have been identified and includes: two prototypic
12 members, which are HBGF-1, also known as acidic fibroblast
13 growth factor, and HBGF-2, also known as basic fibroblast
14 growth factor; three oncoprotein members int-2, also called
15 HBGF-3, hst, also called HBGF-4, and FGF-5, also called HBGF-
16 5; and, two additional members, which are FGF-6, also called
17 HBGF-6, and KGF, keratinocyte growth factor, which is also
18 called HBGF-7. Other names for each of these growth factors
19 are also known to those of skill in the art. The sequences
20 of HBGF-1 - HBGF-7 aligned with N-terminal region of HBGF-l are depicted
21 in Figure 1A.
22 The in vivo activities of the prototypic members of the
23 HBGF family include activities that influence the general
24 proliferation capacity of the majority of mesoderm-derived and
25 neuroectoderm-derived cells. These polypeptides induce
26 angiogenesis and are known to have important functions in
* 27 early development. It is also known that the mitogenic 28 activity of HBGF-1 is potentiated by heparin. All of the members of
* 29 the HBGF family appear to share an affinity for immobilized 30 heparin. Among the differences between the precursors of the
prototypic HBGF family members and the oncogenic members is the absence in the prototypic precursor proteins of a signal sequence for secretion. The HBGFs are important in angiogenesis. Angiogenesis, which involves the organized migration, proliferation, and differentiation of the endothelial cells, is initiated by the endothelial cell in response to angiogenic stimuli and can be separated into three distinct events: cell migration, cell proliferation and differentiation, whereby the cells organize into a tubular structure. These events are mediated in vitro, and most likely in vivo, by signal proteins. HBGFs induce the migration of and proliferation of endothelial cells. Endothelial cells, which line the luminal surfaces of the vascular walls . are, thus, an important component in the development of new capillaries and blood vessels, which occurs during developmental periods, such as during development of the vascular system, and as part of the pathophysiology of a variety of disease states, such as psoriasis, arthritis, chronic inflammatory conditions, diabetic retinopathy, and tumor development.
It is known that protein import into the cell nucleus requires specific binding of nuclear proteins to the nuclear pore complex. Nuclear translocation sequences have been identified in polypeptide iaitogens, such as IL-1 and in the two homodimer forms of platelet-derived growth factor, PDGF-A and PDGF- B , which are potent polypeptide mitogens for many mesenchymal cell types. In addition, the PDGF-B translation product has been found in the nucleus of virus-transformed cells (Yeh et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84: 2317) Nuclear import of proteins occurs by binding of the polypeptide to the nuclear pore complex followed by a slower, energy-dependent, translocation into the nucleus. Specific
1 amino acid sequences containing basic a ino acids have been
* 2 identified as the responsible sequences (see, e.g. Dang et al.
3 (1989) J. Biol. Chem. 264: 18019-18023, Dang et al. (1988)
" 4 Mol. Cell. Biol. 8: 4049-4058). These sequences have been
5 called nuclear targeting sequences and also nuclear
6 translocation sequences. Hereinafter they are referred to as
7 NTS.
8 By examining an array of nuclear sequences it was
9 possible to set forth a consensus NTS sequence that consists
10 of an amino-terminal proline or glycine followed by at least
11 three basic residues in a array of seven to nine amino acids
12 (see e.σ.. Dang et al., supra. and Table I, infra.) .
13 Immunohistochemical staining within neural cells of HBGF-
14 2 demonstrated nuclear localization of this polypeptide in
15 neural tissue (see, Bouche et al. Proc. Natl Acad. Sci. U.S.A.
16 84: 6770 et seq.) Because, as discussed above, nuclear
17 translocation is known to possess a structural basis involving
18 basic, amino acid residues and because NTS are known to occur
19 in polypeptide mitogens, the question arises whether there is
20 any connection between the presence of an NTS and the ability
21 of a mitogen to induce DNA synthesis and/or cell
22 proliferation. Thus, structures of the HBGF family for
23 sequences similar should be examined for the presence of the
24 consensus sequences described for polypeptides translocated
25 to the nucleus (see. Dang et al., supra.) and also to
26 ascertain the effects such sequences have on the biological
27 activity of this family of polypeptides.
28 Because of the importance of the HBGFs in the etiology
29 of many pathological states, there is also a need to develop
30 modified HBGFs that have a therapeutic use in the treatment
31 of such diseases.
SUMMARY OF THE INVENTION
It is one object of this invention to provide chimeric polypeptides, HBGF-NUR, wherein HBGF-N is a member of the heparin binding growth factor (HBGF) family and R is a nuclear translocation sequence that is derived from a polypeptide other than HBGF-NUR and wherein HBGF-NUR possesses all of the biological activities of the corresponding member of the HBGF family.
It is another object of this invention to provide a mitogenic polypeptide growth factor in which the nuclear translocation sequence (NTS) has been modified, whereby nuclear translocation of said factor is inhibited.
It is another object of this invention to provide DNA molecules that encode chimeric polypeptides, HBGF-NUR, wherein HBGF-N is a member of the heparin binding growth factor (HBGF) family and R is a nuclear translocation sequence that is derived from a polypeptide other than HBGF-NUR and wherein HBGF-NUR possesses all of the biological activities of the corresponding member of the HBGF family.
It is another object of this invention to provide DNA molecules that encode mitogenic polypeptide growth factors in which the nuclear translocation sequence (NTS) has been modified, whereby nuclear translocation of said factor is inhibited.
It is another object of this invention to provide a method for transforming cells, comprising transfecting said cells with a DNA molecule that encodes a mitogenic polypeptide growth factor in which the nuclear translocation sequence
(NTS) has been modified, whereby nuclear translocation of said factor is inhibited.
It is another object of this invention to provide a method of inhibiting DNA synthesis and cell proliferation, comprising inhibiting nuclear translocation of a mitogenic growth factor.
It is another object of this invention to provide a method of treating a disease characterized by the pathological proliferation of cells, comprising inhibiting nuclear translocation of the mitogenic growth factor responsible for said proliferation.
In accordance with this invention there are provided mutant HBGF polypeptides in which the NTS has been rendered inoperative by a deletional or insertional mutation. This invention also provides chimeric HBGF polypeptides in which the wild-type NTS is replaced with a heterologous NTS. The chimeric polypeptides prepared in accordance with this invention are exhibit substantially all of the biological activities of the wild-type protein.
This invention provides a means for severing the biological activities of growth factors, such as the HBGF, so that mutant growth factors may be prepared that possess a subset of the biological properties of the wild-type growth factor. This invention also provides growth factors that may be used both in vitro and in vivo as antagonists of the wild- type growth factor and, thus, have use as therapeutic agents for the treatment of diseases characterized by pathological cell proliferation. This invention also provides means for inhibiting nuclear translocation and for inhibiting DNA synthesis and cell proliferation.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated by reference.
BRIEF DESCRIPTION OF THE FIGURES Figure 1. Structure of the Amino-Terminal Domain of the HBGF Family Members and HBGF-1 Mutants. (A) The amino acid sequence of residues 1 to 39 is shown (1) and the amino acid sequence of the remaining HBGF family members in this region are shown (2) . Sequence identity is shown using dots and the putative nuclear translocation signal , sequence for each polypeptide underlined. (B) The amino acid sequence of residues 1 to 39 of the HBGF-1 precursor, HBGF-13 (line 1) , the amino sequence of the amino-terminal chimera, HBGF-lUα2, containing the aligned amino-terminal domain of HBGF-2 (as in A ligated to the amino- terminus of HBGF-1U (line 5) . t Figure 2. Expression of the Amino-Terminal Deletion Mutants and Chimeras of HBGF-1. (A) Heparin Affinity: The BL21(DE3)pLysS cells containing the synthetic constructs of either HBGF-lα, HBGF-1U, or HBGF-1U2 in the pET-3c vector were cultured in 2 ml of LB medium containing 50 μg/ml of ampicillin and 40 μg/ml of chloramphenicol. At late log phase of the growth, the expression of the proteins was induced by 0.5 mM
isopropylthiogalactoside for 2 hours at 37° C. The cells were collected by centrifugation, and suspended in 1 ml of 25 mM Tris-HCl, lOmM EDTA, 50mM glucose pH 7.2. The cells were freeze-thawed twice, sonicated and allowed to lyse using their endogenous lysozyme. The supernatant was adjusted to 0.1 M NaCl, mixed with 50 μl of heparin-Sepharose beads, and incubated at 4° C for 2 hours on a rotating platform. The heparin-Sepharose beads were centrifuged, washed 3 times each with 1 ml of lOmM Tris, pH 7.2 containing 1 mM EDTA (TE) and were sequentially washed with TE containing 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.9, 1.2, 2.0 M NaCl. Eluate samples (50 μl) were blotted onto a nitrocellulose membrane, and the HBGF-1 polypeptides were visualized using a rabbit anti-HBGF-1 antibody in combination with alkaline phosphatase conjugated goat anti-rabbit IgG (Promega) . (B) Purification of the HBGF-1 Mutants: The proteins were expressed as described in A and lysates from a 1 liter culture was adjusted to 0.5 M NaCl in TE. The lysate was incubated with 5 ml of heparin-Sepharose and after washing with 0.5 M NaCl in TE, the beads were packed into columns, washed extensively with 0.7 M NaCl in TE and the HBGF-1 polypeptides eluted from the columns with 1.2 M NaCl in TE. The proteins were further purified on a Vydac C4 reverse phase HPLC as described (Burgess et al. (1985) J. Biol. Chem. 260: 11389) . The HPLC-purified proteins were analyzed Jjy SDS-15% polyacrylamide gel, stained with Coomassie blue. Amino acid analysis and amino-terminal microsequencing (25 cycles) were performed with each recombinant polypeptide and the sequence of each mutant verified (data not shown) .
Figure 3. Biological Activity of the HBGF-1 Mutants. (A) Binding Competition of HBGF-lα. HBGF-lα was labeled with 125I using immobilized lactoperoxidase and purified by heparin-Sepharose as described (Friesel et al.
(1989) Mol. Cell. Biol. 9: 1857). Confluent murine lung capillary endothelial cells (LE-II) were first serum starved for 4 hour in DMEM containing 0.5% FBS. The cells were then washed and incubated with Hank's balanced salt solution containing 5 units/ml heparin, 1% BSA in 20 mM HEPES (pH 7.2) (Binding buffer) at room temperature for 20 minutes. The cultures were then transferred onto ice and the cells exposed to (1251)- HBGF-lα (2 ng/ml) with the indicated concentrations of the competitor HBGF-1 or BSA (control) in the binding buffer. After incubation at 4° C for 2 hours on a rocking platform, the cells were washed three times with the binding buffer and solubilized with 0.2 N NaOH. The radioactivity of the samples was quantitated in a gamma counter. Non-specific bi •ndi•ng (the amount of (1251)-HBGF-lα bound i•n the presence of 5 g/ml of HBGF-lα) was subtracted from every value and did not exceed 20% of the total binding. All the values were determined by at least duplicate samples. (B) DNA Synthesis by LE-II Cells. LE-II cells were brought to confluency in 48-well plates in DMEM containing 10% FBS, then starved two days with DMEM containing 0.5% charcoal- absorbed FBS (starvation medium) . The test samples were added to the media at the indicated concentrations and the cells were cultured at 37° C. After 18 hr, [3H]thymidine was added to a final concentration of 1 μCi/ml. After 4 hours, the cells were washed with PBS, treated with 10% TCA, solubilized with 0.2 N NaOH, neutralized with 0.2 N HCl, and the radioactivity quantitated by liquid scintillation. All values were determined in triplicate. (C) DNA Synthesis in 3T3 Cells. N1H 3T3 2.2 cells were brought to confluency in 48-well plates in DMEM containing 10% calf serum, then starved for two days. The DNA synthesis assay was performed as described for the LE-II cells, except that the samples were added either without or with 5 units/ml of heparin (+HP) . All values were determined in triplicate.
(D) Human ENdothelial Cell Growth. Confluent human umbilical vein endothelial (HUVE) cells (passage 16) were trypsin-treated, placed into 6-well places (coated with 5 μg human fibronectin as described (see, e.g.. Burgess et al. supra.) with M199 containing 10% FBS at the density of 2.5xl03 cells/cm2, and allowed to attach at 37° C for 9 hours. The test samples were then added to the indicated final concentration with 5 units/ml of heparin (day 0) with medium changes on days 2 and 4. On day 5, the cells were detached with trypsin and viable cell numbers quantitated using a hemocytometer. All values were determined by duplicate samples except the HBGF-1-free control where 6 samples were quantitated.
Figure 4. The Induction of Tyrosine Phosphorylation by the HBGF-1 Mutants. (A) Competition of .—I.-HBGF-lα for Receptor Cross-linking bv HBGF-IU and HBGF-1U2. Confluent, serum-starved LE-II cells (as in Figure 3B)in 100-mm plates were pre-incubated for 20 minutes at 4° C and (125>I-HBGF-lα (2.4 x Los cpm/ng) at a concentration of 5 ng per ml was added to each plate in 3ml binding buffer (DMEM, 25 mM HEPES, 0.2% BSA pH 7.2) containing the indicated concentrations of unlabeled HBGF-IU or HBGF-1U2 as competitor. After 2 hours of incubation at 4° C with gentle rocking, the cells w.ere washed and cross-linked with 300 μM disuccini idyl suberate (DSS) in 3 ml phosphate-buffered saline (PBS) . The reaction was quenched with 2.0 M Tris-HCl, pH 7.2. The cells were washed in PBS and then solubilized in 50 mM Tris-HCl, 10 mM EDTA, 200 mM NaCl, 1% Triton X-100, and 0.1 phenylmethylsulfonyl fluoride (PMSF) , pH 7.2. Insoluble material was removed by centrifugation at 10,000 X g for 10 minutes at 4° C. Protein concentrations were determined using a bicinchoninic acid
assay (Pierce). 200 μg of cell lysate contai•ni#ng (125I)- labeled cross-linked complexes per sample were subjected to SDS polyacrylamide electrophoresis and to autoradiography. Cross-linked (125>I-HBGF-l-receptor complexes of Mr 170 and 150 Kd are indicated. The low molecular weight bands at 35 Kd may represent cross-linked dimers. Free (125>I-HBGF-1 migrates at the gel front. (B) The Induction of Tyrosine Phosphorylation by the HBGF-1 Mutants. Confluent, serum-starved NIH 3T3 2.2 cells in 100-mm plates were metabolically labeled for 3 hours at 37° C with 0.33 mCi of (32P)-orthophosphate per ml in phosphate-free buffer containing 125 mM NaCl, 25 mM HEPES, 4.8 mM KC1, 2.6 mM CaCl2, 1.2 mM MgS04, 5.6 mM glucose, 0.1% BSA, pH 7.4. The cells were exposed to diluent alone or to 0.1, 1.0 or 10 ng per ml of HBGF-1, HBGF-IU or HBGF-1U2 for 10 minutes at 37° C, washed once with cold PBS containing 50 mM NaF, 30 mM sodium pyrophosphate, 100 μM sodium orthovanadate, pH 7.4 (lysis buffer) . The cells were immediately scraped from the plates, vortexed and incubated on ice for 10 minutes. Lysates were centrifuged at 10,000 X g for 10 minutes at 4° C and the supernatants were added to 20 μl of a 50% suspension of monoclonal anti-phosphotyrosine antibody 1G2 coupled to Sepharose for 2 hour at 4° C with constant rotation. THe Sepharose beads were washed four times with lysis ^buffer and the (32P)-labeled phosphotyrosine-containing proteins were specifically eluted with 10 mM phenyl phosphate buffer containing 10 mM Tris-HCl, 50mM NaCl, 0.1% Triton X-100, 09.1% ovalbumin. Eluted phosphoproteins were analyzed by SDS-PAGE on 7.5% polyacrylamide gels and visualized by autoradiography. HBGF-1-stimulated proteins having Mr 150, 130, 90 and 40 are shown. Tyrosine phosphorylation was confirmed by phosphoamino acid analysis of the M 150, 130 and 90 Kd proteins.
Figure 5. The Induction of c-fos mRNA Transcript Expression bv the HBGF-1 Mutants. Confluent murine 3T32.2 cells (150-mm dishes) were serum starved as described above for two days. The cells were treated with lOng/ l of either HBGF-lα, 1U or 1U2, and incubated at 37° C. After 15 minutes, the cells were washed twice with PBS and total RNA prepared (Gay et al. (1989) J\_ Biol. Chem. 265: 3284-3292) . One μg of RNA from each sample was reverse transcribed using c-fos antisense primer (5'- CAAAGCAGACTTCTCATCTTC-3•) . The cDNA was then amplified by the polymerase chain reaction (PCR) (see, e.g. Rosenberg et al. (1987) Gene 56: 125) using both antisense and sense (S'-TGGCCGTCTCCAGTGCCi^ACTT-S') primers (see, e.g. Rakowicz- Szulczynska et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 84: 2317) . The amplified products were run on a 1.3% agarose gel, transferred to a nitrocellulose membrane, and visualized with a c-fos probe radiolabeled by random priming followed by autoradiography. The bands of the expected size (see, Rakowicz-Szulczynska et al. , supr .) of the amplified product (390 bp) are shown.
DESCRIPTION OF THE PREFERRED EMBODIMENTS In the invention disclosed herein mutated forms of HBGF family members that are able to bind to and recognize HBGF receptors and to initiate tyrosine phosphorylation and c-fos mRNA transcription, but that are unable to induce DNA synthesis and cell proliferation, are provided. This invention, thus provides a means to sever the various biological activities of the HBGF polypeptides.
As used herein, HBGF family of polypeptides includes, but is not limited to, the family of polypeptides that bind to immobilized heparin.
As used herein, HBGF-1/3 refers to the precursor of HBGF- 1. Similarly, HBGF-2/3 refers to the precursor peptide of HBGF-2. HBGF-lα refers to that can be derived from HBGF-1/3 by an amino-terminal truncation of the first 20 residues. Thus, the β designation refers to the precursor form of each protein and the α designation refers to the cleavage product.
As used herein, a nuclear translocation or targeting sequence (NTS) is a sequence of amino acids in a protein that are required for translocation of the protein into a cell nucleus. Examples of NTS are set forth in Table I, below, and in Figure 1A. One having skill in the art would know or would be able to readily identify other amino acid sequences that function as NTS.
As used herein, HBGF-NU, refers to a synthetic mutant of a member of the HBGF family in which the all or part of the NTS sequence has been deleted or otherwise rendered inoperative. "N" refers to the particular member of the HBGF family. t
As used herein, HBGF-IU is a synthetic mutant of HBGF-lα in which the amino-terminal sequence, NYKKPKL, has been deleted. HBGF-IU possesses the ability to bind to immobilized heparin in vitro but lacks the ability to induce DNA synthesis and cell growth either in the presence or absence of heparin.
HBGF-IU induces tyrosine phosphorylation in murine 3T3 cells at concentrations at which it fails to stimulate DNA synthesis and cell proliferation.
As used herein, HBGF-NUR, is a chimeric protein that contains HBGF-NU and a heterologous NTS. By virtue of the heterologous NTS, the biological activities of the particular HBGFs are reconstituted. "R" refers to a NTS. For example, the biological activities of the prototype HBGFs include, but are not necessarily limited to, the abilities to bind to heparin, induce intracellular receptor-mediated tyrosine phosphorylation and the expression of the c-fos mRNA transcript, and stimulate DNA synthesis and cell proliferation.
As used herein, heterologous NTS refers to an NTS that is different from the NTS that occurs in the wild-type peptide, polypeptide, or protein. For example, the NTS may be derived from another polypeptide, it may be synthesized, or it may be derived from another region in the same polypeptide.
As used herein, HBGF-1U2 is a chimeric protein that contains HBGF-IU and the yeast histone H2 nuclear translocation signal, GKKRKSKAK, at the amino-terminal end. The biological activities of HBGF-1U2 include both the ability to bind to immobilized heparin, the ability to induce tyrosine phosphorylation, and the ability to stimulate DNA synthesis and cell proliferation.
TABLE I
Examples of Typical Nuclear Translocation Sequences and
Regions of Proteins that Share
Homology with Nuclear Translocation Sequences1,2
Protein Sequence
p53 P316-Q-P-K-K-K-P
HIV Tat G^-R-K-K-R-R-Q-R-R-R-A-P f
Nucleolin P277-G-K-R-K-K-E-M-T-K-Q-K-E-V-P
1 Dang et al. (1989) J. Biol. Chem. 264: 18019-18023.
2 Amino acid sequence set forth using the single letter amino acid code; superscript adjacent to the initial amino acid in each sequence refers to position in the protein.
Because of the importance of HBGF family members in the physiological processes, such as angiogenesis and in the pathology of certain diseases, and because of the observations that suggest that at least the HBGF prototypes are located intranuclearly, the structures of the HBGF family have been examined for sequences similar to the consensus sequences described for polypeptides translocated to the nucleus and mutations in this sequence have been prepared. Analysis of the protein sequence of HBGF-1 reveals that at the amino-terminus of HBGF-lα, which an amino-terminal truncation of the precursor HBGF-1/3, contains the sequence NYKKPKL which is similar to the NTS consensus sequence, discussed above (see, also, Table I and Figure 1A) . Further, a similar sequence is also present in HBGF-2 and, with the possible exception of HBGF-5, a similar sequence motif is present in the other HBGF family members (Figure 1A) . The biological activity of HBGF-lα is substantially the same as that of the precursor polypeptide, HBGF-1/3 in that it induces tyrosine phosphorylation , the c-fos IΓLRNA transcript and induces DNA synthesis and cell proliferation. In order to ascertain the biological functions of the NTS in the HBGF family, mutants in which the NTS is deleted or otherwise rendered inoperative, such as by a point mutation, are constructed. Such mutants in any of the family members may be constructed by any means known to those of skill in the art. For example, DNA encoding the protein can be modified as desired and then expressed in a suitable host to produce the mutant or modified polypeptide. In one embodiment of this invention an HBGF-lα deletion mutant lacking residues 1-27 of the precursor polypeptide HBGF-1/3 (Figure IB) is constructed and the biological activity of the modified HBGF-1, herein called HBGF-IU, is examined.
In this embodiment a synthetic gene that encodes HBGF-1/3 is constructed. The amino acid sequence is divided into four domains, oligonucleotides encoding the structure of each domain are synthesized, ligated and cloned into individual cassettes, and the four cassettes ligated to form the complete open-reading frames (ORF) for HBGF-10 and HBGF-lα. The putative NTS is identified and the desired deletion mutant, HBGF-IU, can be prepared by deletion of DNA encoding the NYKKPKL sequence (Figure IB) . In addition, a chimeric protein in which a heterologous NTS is added to the deletion mutant, can also be constructed. To prepare the various mutant peptides any means known to those of skill in the art can be used. For example, the synthetic gene encoding the HBGF-lα ORF can be used as a template to enzymatically generate inserts with the polymerase chain reaction (PCR) (see, e.g. Rosenberg et al. (1987) Gene 56: 125) for HBGF-IU, which has the NTS deleted, and HBGF-1U2, which is a chimeric reconstituted protein, (Figure lp) using synthetic oligonucleotide primers that are designed specifically for each mutant (see, e.g. Rosenberg, et al. supra.) . To ascertain the biological function of the NTS, the biological activities of HBGF-IU, HBGF-lα, and HBGF-1U2 can then be compared. The ability of each of the peptides to bind to the HBGF-1 receptor and initiate tyrosine phosphorylation and the c-fos mRNA transcript are measured as are the ability of each of these peptides to induce DNA synthesis and cell proliferation. The ability of DNA encoding each of the peptide can also be tested for the ability to transform cells. These activities as well as any other relevant biological activities can be tested by any assays and means known to those of skill in the art.
Unexpectedly it was discovered that deletion of the NTS severs the biological activities of the HBGF peptide. The HBGF-1 mutant that lacks a functional NTS is able to initiate tyrosine phosphorylation and the c-fos mRNA transcript, but, unexpectedly, is unable to initiate DNA synthesis and cell proliferation. This result is unexpected in that tyrosine phosphorylation and c-fos mRNA were believed to be the mediators of the mitogenic activity of the peptide. Unexpectedly, however it has been discovered that mitogenic activity requires that the HBGF-1 peptide be translocated to the nucleus. It has also been discovered that endothelial cells transfected with cDNA encoding HBGF-lα and HBGF-1U2 exhibit a transformed phenotype; whereas, endothelial cells that are transfected with cDNA encoding HBGF-IU do not. Thus, nuclear translocation of the HBGF growth factor is required for transformation. The ability to delete and reconstitute the mitogenic activity of HBGF- lα by manipulation of a nuclear translocation sequence without significantly altering receptor-mediated tyrosine phosphorylation and the induction of the c-fos mRNA transcript indicates that translocation of HBGF-1 to the nucleus appears to be a requisite for the stimulation of DNA synthesis at least .in vitro, if not in vivo. These results also indicate that the induction of tyrosine phosphorylation of membrane-associated and cytosolic polypeptides and the expression of the c-fos mRNA transcript are not sufficient for the initiation of DNA synthesis or endothelial cell proliferation. Thus, HBGF-1 may ultimately act as an intracellular, nuclear translocated polypeptide mitogen, which activity would not require a signal sequence.
Because the mutants of the HBGF polypeptides that lack a functional NTS can bind to HBGF receptors and initiate tyrosine phosphorylation and c-fos mRNA transcription, they are useful as an antagonist of HBGFs that function as extracellular polypeptide mitogens in vivo. The mutant HBGF polypeptides, thus, can be used as therapeutic agents for the treatment of diseases, such as cancer, arthritis, psoriasis, chronic inflammatory conditions, and diabetic retinopathy, that involve pathological proliferation of endothelial cells and other HBGF-responsive cells.
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
EXAMPLE 1 Computer-assisted analysis of the secondary structure of HBGF-lα and the desired deletion mutant HBGF-IU (Figure IB) suggested that deletion of the NYKKPKL sequence would not substantially alter the conformation of HBGF-IΛ Computation of the hydropathy index for. HBGF-lα using the SOAP Program showed the sequence indicated that the sequence NYKKPK (amino acids 21-27) has an index of -20 to -5, and that there are six similar hydrophilic regions with a similar index in the HBGF-1 molecule. Computation of the antigenicity index for HBGF-1 using the ANTIGEN Program indicated that this amino acid region has a moderate antigenicity index 0 to 1 and that there are five regions more antigenic than this area in the structure of HBGF-lα. Further, residue Cys^, which may be involved in the determination of the secondary structure of HBGF-lα is conserved among HBGF- lα, HBGF- 1U and HBGF-1U2. Thus, it appeared that deletion of the NYKKPKL sequence would result in minimal change in the conformation of HBGF-lα.
To construct the NTS deletion mutant, the synthetic gene that encoded the HBGF-lα ORF was used as a template to enzymatically generate inserts with the polymerase chain reaction (PCR) for HBGF-IU and HBGF-1U2 (see, e.g. Rosenberg et al., supra.) . The constructs were cloned into the pEτ-3c vector between the Ndel and BamHI sites. The synthetic construct of HBGF-lα in pUClδ was amplified by the polymerase chain reaction using different sense oligonucleotide primers, each containing the Ndel restriction site and 7 codons of HBGF-lα. The sense primer for HBGF-1U2 contained 9 codons encoding the histone 2B nuclear translocation sequence in addition to the Ndel restriction site and 7 codons of HBGF-lα. A common antisense oligonucleotide complimentary to the 3' region of the HBGF-lα constructs was synthesized. so that the products could be amplified prior to digestion with Bglll. Thus, HBGF-lα, HBGF- 1U, and HBGF-1U2 were amplified, digested with Ndel and Bglll, and cloned between the Ndel and BamHI sites in pετ-3c. The primers used were as follows: oligo #3125'-GGCATATGGCTAATTACAAGAAGCCC-3■ forHBGF-lα sense; Oligo #463 5'-CCGAATTCGCTAGCCATATGCTCTACTGTAGCAACGGGGGC-3' for HBGF-IU sense; Oligo #571 5'-CTAGCCATATGGGGAAGAAAAGGAAGTCCAAGGCCAAGAT GCTCTACTGTAGCAACGGGGGC-3• for HBGF-1U2 sense; and, oligo #494 5•GAACAGATCTCTCTTTAATCAGAAGA-3' for antisense common to HBGF-lα, HBGF-IU, and HBGF-1U2. Each construct in the p_2_~3c vector was used to transform the expression host, BL21(DE3) pLysS cells, and single colonies of the cells containing each vector were selected on ampicillin plates.
EXAMPLE 2 The biological properties of HBGF-IU (Figure IB) , which lacks the amino-terminal sequence, NYKKPKL, were examined. HBGF-IU was able to bind immobilized heparin (Figure 2A) with an affinity similar to that described for HBGF-lα so that HBGF-IU could be purified from J _ coli translation lysates (Figure 2B) . Confirmation of the deletion within HBGF-IU was determined by amino terminal microsequencing (see, Burgess et al. (1986) Proc. Natl. Acad. Sci. U. S.A 83: 7216). The biological activity associated with HBGF-IU was examined using a (hi)dThd-incorporation assay in murine 3T3 cells (see, e.g.. Schreiber et al. (1985) Proc. Natl. Acad. Sci. U. S.A 82: 6138) and endothelial cells (see. Burgess et al. (1985) J. Biol. Chem. 260: 11389). This assay as well as a human endothelial cell proliferation assay (see, e.g.. Maciag et al. Science 225: 932) demonstrated that HBGF-IU was not able to induce either DNA synthesis or cell growth (Figure 3B,C,D) . In addition, HBGF-IU was unable to induce either DNA synthesis or cell growth when these assays were performed in the presence of heparin (Figure 3C,D) .
EXAMPLE 3 Because exogenous HBGF-1 exerts a mitogenic response by the occupancy of a high affinity receptor, because HBGF-1 receptor binding stimulates tyrosine phosphorylation of membrane bound and cytosolic polypeptides and because the HBGF-2 receptor contains an intrinsic c-fms-like intracellular tyrosine kinase domain, the ability of HBGF-IU to induce tyrosine phosphorylation in murine 3T3 cells was examined. Immunoprecipitation analysis with antiphosphotyrosine antisera demonstrated that HBGF-IU and HBGF-lα were able to induce the phosphorylation of Mr 150 Kd, 130 Kd, 90 Kd, and
40 Kd polypeptides at concentrations where HBGF-IU failed to stimulate cell proliferation and DNA synthesis .in vitro (Figure 4B) . These data suggested that, although HBGF-IU was not able to either initiate DNA synthesis or cell division, the mutant was capable of initiating the activation of tyrosine-specific phosphorylation of membrane bound (Mr 150 Kd, 130 Kd) and cytosolic (Mr 90 Kd, 40 Kd) polypeptides. To further substantiate this effect, the ability of HBGF-IU to induce the expression of the c-fos mRNA transcript, an immediate-early response gene for HBGF-lα, was examined. HBGF-IU was able to induce the expression of the c-fos mRNA transcript (Figure 5) . Thus these data suggest that the mitogenic defect within the structure of HBGF-IU may be due to the failure of the mutated polypeptide to undergo nuclear translocation.
EXAMPLE 4 To assess the possibility that HBGF-IU fails to undergo nuclear translocation, the polypeptide HBGF-1U2 was prepared as discussed in Example 1. HBGF-1U2 (shown in Figure IB) , is a chimera containing the HBGF-IU sequence and the yeast histone 2B NTS at the amino-terminus of HBGF-IU. The sequence, GKKRKSKAK, within the yeast histone 2B polypeptide is a well-known NTS (see, e.g.. Moreland et al. ^1987) Mol. Cell. Biol. 7: 3527). Like HBGF-lα and HBGF-IU, the chimeric polypeptide, HBGF-1U2 ( Figure IB) was able to bind to the HBGF-1 receptor, initiate tyrosine phosphorylation of the membrane bound and cytosolic polypeptides, and induce the expression of the c- fos mRNA transcript (Figure 5) .
Unlike HBGF-IU, however, the chimeric polypeptide was biologically active in the cell proliferation and DNA synthesis assay systems. Thus, addition of a NTS to the mutant polypeptide that lacks the NTS, reconstituted the ability of HBGF-IU to induce DNA synthesis and cell proliferation. This indicates that nuclear translocation is essential for this activity. Also, in the absence of heparin, HBGF-1U2 was as potent as the recombinant HBGF-lα polypeptide in its ability to initiate DNA synthesis in murine 3T3 cells and stimulate human endothelial cell division (Figure 3B,C,D) . In addition, the ability of heparin to potentiate the biological activity of HBGF-1 was also reconstituted in the chimeric polypeptide, HBGF-1U2, (Figure 3C,D) .
EXAMPLE 5 Although the mitogenic activity of HBGF- lα and HBGF-1U2 were very similar, HBGF-1U2 was approximately 25-fold less efficient than HBGF-lα in displacing (125I)HBGF-lα from its receptor (Figure 3A) . Similar data were obtained using competitive ligand-receptor covalent cross-linking methods (Figure 4A) . HBGF-lα and HBGF-1U2 not only stimulated HBGF receptor-mediated phosphorylation and c-fos mRNA transcript expression at similar concentrations, but HBGF- 1U2 also induced these responses at concentrations that were not competitive with HBGF-lα for receptor binding (Figure 3A and 4A) . Further HBGF-IU was approximately 80 to 100-fold less efficient than HBGF-lα in competing (125I)-HBGF-lα from its receptor (Figure 3A and 4A) , but was also able to initiate tyrosine phosphorylation (Figure 4B) and c-fos expression
(Figure 5) at concentrations that were not competitive with HBGF-lα for receptor binding. (Figure 3A and 4A) . However, HBGF-IU required a 10-fold excess of polypeptide to stimulate either tyrosine phosphorylation or c-fos expression than HBGF-lα or HBGF-1U2 (Figures 4B and 5) . This difference could reflect a difference in the K„ for initiation of tyrosine phosphorylation and receptor binding and the K^ for the intranuclear mitogenic activities or the presence of a nuclear translocation sequence may in some manner override deficiencies in HBGF-1U2 receptor binding and thus may enable relatively small concentrations of the polypeptide to initiate DNA synthesis and cell growth.
EXAMPLE 6 NIH 3T3 cells and normal endothelial cells were transfected with cDNA encoding each of HBGF-lα, HBGF-IU, and HBGF-1U2. Both the 3T3 cells and the normal cells exhibited a transformed phenotype following transfection with HBGF-lα and HBGF-1U2, but did not following transfection with HBGF-IU.
Since modifications will be apparent to those of skill in the art, it is intended that this invention be limited only by the scope of the appended claims.
Claims (28)
- We claim: 1. A chimeric polypeptide, HBGF-NUR, wherein HBGF-N is a member of the heparin binding growth factor (HBGF) family and R is a nuclear translocation sequence that is derived from a polypeptide other than HBGF-NUR and wherein HBGF-NUR possesses all of the biological activities of the corresponding member of the HBGF family.
- 2. A mitogenic polypeptide growth factor in which the nuclear translocation sequence (NTS) has been modified, whereby nuclear translocation of said factor is inhibited.3. The polypeptide growth factor of claim 2 , wherein said modification is selected from the group consisting of selected from the group consisting of deletions of all or a part of said NTS and insertions of at least one amino acid into said NTS.
- 3. A polypeptide of claim 2 which is selected from the group consisting of HBGF-IU and HBGF-2U.
- 4. A polypeptide of claim 1, which is selected from the group consisting of HBGF-1U2 and HBGF-2U2, wherein said NTS is derived from yeast histone 2B. t
- 5. A DNA molecule that encodes the polypeptide of claim 1.
- 6. A DNA molecule that encodes the polypeptide of claim 2.
- 7. A DNA molecule that encodes a polypeptide of claim 3.
- 8. A DNA molecule that encodes the polypeptide of claim 4.
- 9. A method for transforming cells, comprising transfecting said cells with the DNA molecule of claim 5.
- 10. A method for transfomring cells, comprising transfecting cells with the DNA molecule of claim 8.
- 11. A method of inhibiting DNA synthesis and cell proliferation, comprising inhibiting nuclear translocation of a mitogenic growth factor.
- 12. The method of claim 11, wherein said growth factor is a member of the heparin binding growth factor family.
- 13. The method of claim 12, wherein said growth factor is HBGF-1 or HBGF-2.
- 14. The method of claim 11, wherein nuclear translocation is inhibited by mutation of the nuclear translocation sequence of said growth factor.
- 15. The method of claim 14, wherein said mutation is selected from the group consisting of deletions of all or a part of said NTS and insertions of at least one amino acid into said NTS.
- 16. The method of claim 14, wherein said mutation is a point mutation.
- 17. The method of claim 14, wherein said mutated growth factor is a heparin binding growth factor that has the nuclear translocation sequence (NTS) deleted.
- 18. The method of claim 14, wherein said growth factor is HBGF-IU or HBGF-2U.
- 19. A method of treating a disease characterized by the pathological proliferation of cells, comprising inhibiting nuclear translocation of the mitogenic growth factor responsible for said proliferation.
- 20. The method of claim 19, wherein said growth factor is a member of the heparin binding growth factor family.
- 21. The method of claim 20, wherein said growth factor is HBGF-1 or HBGF-2.
- 22. The method of claim 20, wherein nuclear translocation is inhibited by mutation of the nuclear translocation sequence (NTS) of said growth factor, whereby said translocation is prevented.
- 23. The method of claim 22, wherein said mutation is selected from the group consisting of deletions of all or a part of said NTS and insertions of at least one amino acid into said NTS.
- 24. The method of claim 22, wherein said mutation is a point mutation.
- 25. The method of claim 22, wherein said mutated growth factor is a heparin binding growth factor that has the nuclear translocation sequence (NTS) deleted.
- 26. The method of claim 25, wherein said growth factor is HBGF-IU or HBGF-2U.
- 27. The method of claim 19, wherein said disease is selected from the group consisting of psoriasis, arthritis, chronic inflammatory conditions, diabetic retinopathy, and cancer.
- 28. The method of claim 19, wherein said inhibition is effected by competitively inhibiting binding of endogenous growth factor to its cellular receptor by introducing a modified form of said growth factor that has a mutation in its nuclear translocation sequence into a host animal afflicted with said disease.
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JP (1) | JPH05507844A (en) |
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EP0525054A4 (en) | 1993-05-12 |
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