WO1991018001A1

WO1991018001A1 - Trans-dominant suppressor genes for oligomeric proteins

Info

Publication number: WO1991018001A1
Application number: PCT/US1991/003467
Authority: WO
Inventors: Mark K. Mercola; Prescott L. Deininger; Charles D. Stiles
Original assignee: Dana-Farber Cancer Institute
Priority date: 1990-05-17
Filing date: 1991-05-16
Publication date: 1991-11-28
Also published as: EP0528979A4; JPH05506361A; CA2083164A1; EP0528979A1

Abstract

A method of making trans-dominant, suppressor genes encoding mutant subunits of two different categories of oligomeric proteins is disclosed. In one category, the protein is composed of subunits at least one of which is initially synthesized containing extraneous peptide material. This extraneous material is subsequently removed during normal processing by cleavage at a proteolytic cleavage site to generate the mature form of the subunit. According to the invention, single stranded DNA encoding the initial form of the indicated subunit is cloned, and the base sequence of the DNA in the region encoding the proteolytic cleavage site is modified by site directed mutagenesis so that processing of the translation product of the gene to the mature form of the subunit is prevented. In the other category, the oligomeric protein is one in which the monomers are bonded together by at least two disulfide bonds, at least one of which is essential for the catalytic activity of the protein. In the method of the invention, single stranded DNA encoding one of the subunits is cloned, and the base sequence of the DNA in the region encoding a cysteine residue essential for catalytic activity is modified by site directed mutagenesis.

Description

TRANS-DOMINANT SUPPRESSOR GENES FOR OLIGOMERIC PROTEINS The invention relates to trans-dominant suppressor genes.

Background of the Invention Naturally occurring dominant suppressor genes are known that trigger substantial phenotypic alterations in eucaryotic cells and tissues. Herskowitz (Nature 3_2 :219-222, 1987) has proposed that the natural process can be mimicked by altering a cloned gene so that it encodes a mutant product, capable of inhibiting the wild- type gene product in a cell, thus causing the cell to be deficient in the function of that gene product. Malin et al. (Cell 5_8:205-214, 1989) and Green et al. (Cell

5_8:215-223, 1989) performed mutational analyses of two HIV-l trans-activators essential for viral replication and obtained results which they attributed to effects of the sort described by Herskowitz. One common, naturally occurring di eric protein is platelet derived growth factor (PDGF) , first recognized and purified from human blood platelets. PDGF has been isolated from a wide variety of cells and tissues of numerous eucaryotic species. The two known PDGF subunits, A and B, are expressed from separate genes and are active as either homo or heterodimers. Dimerization occurs through disulfide linkage between cysteine residues on the individual subunits. The positioning of these residues is conserved between the A and B chains, and between species at least as divergent as human and Xenopus. Four of the eight cysteine residues on each subunit chain are believed to be essential for catalytic activity. The initial translation product of the PDGF A and B genes is a "preproPDGF**. A hydrophobic leader seguence (the "pre" sequence) and a substantial length of N-terminal material (the "pro" sequence) are removed by proteolytic cleavage to generate the mature form of a

PDGF subunit. Summary of the Invention

In general, the invention features two methods of making eucaryotic cell trans-dominant suppressor genes encoding a polypeptide translation product capable of suppressing the activity of a protein that requires an oligomeric state for function, the suppressor genes made by the methods, and the protein products of the respective suppressor genes. Each suppressor gene encodes a mutant subunit of the oligomeric protein that will suppress the wild type activity of the protein. In one aspect, the method includes isolating single stranded DNA encoding a subunit of the protein, the subunit being one that is initially synthesized containing extraneous peptide material which is subsequently removed by cleavage at a proteolytic cleavage site to generate the mature form of the subunit; modifying the base sequence in the single stranded DNA in the region encoding the proteolytic cleavage site, so that proteolytic cleavage of the initial translation product of the single stranded DNA to the mature form is prevented; and cloning the modified, single stranded DNA to form the trans-dominant suppressor gene. The modifying step includes addition or deletion of a codon for an amino acid essential for cleavage at the proteolytic cleavage site, or, preferably, substitution of one such essential codon by a codon for a different amino acid.

In another aspect, the method includes isolating single stranded DNA encoding a subunit of the protein, the subunit being one that is bonded in the oligomeric state by means of a plurality of cysteine residues, at least one of which is essential for the catalytic activity of the protein; modifying in the DNA strand the if*- codon for one of the cysteine residues essential for the catalytic activity of the protein so that another amino 5 acid is substituted for the one cysteine residue, the codon for at least one cysteine residue remaining unmodified; and cloning the modified, single stranded DNA to form the trans-dominant suppressor gene.

In preferred embodiments, the oligomeric protein 10 is a growth factor, preferably PDGF, and the subunit is preferably PDGF A. In other preferred embodiments the protein is a transcription factor.

In another aspect, the invention features a transgenic non-human animal that includes germ cells and 15 somatic cells including the trans-dominant suppressor gene introduced into the animal, or an ancestor of the animal, at an embryonic stage. In a preferred embodiment the trans-dominant suppressor gene is under the control of a tissue specific promoter. 20 The invention enables the systematic preparation, without undue experimentation, of a trans- dominant suppressor gene encoding a mutant subunit of an oligomeric protein that will suppress the wild type activity of the protein. Such suppressor genes can be 25 used to produce variant strains of transgenic laboratory or farm animals useful for observing the effects of tissue specific or systemic suppression of a particular wild type gene. Alternatively, the mutant product of the suppressor gene can be used in therapeutic applications. 30 Other features and advantages of the invention will be apparent from the following description of the £ preferred embodiments thereof, and from the claims.

Brief Description of the Drawings Fig. 1 is a representation of the murine 35 preproPDGF A chain and sites of directed mutagenesis. Fig. 2 is a representation of possible interactions between heterodimers of PDGF wild type and dominant suppressor subunits on the one hand, and PDGF receptor isoforms on the other hand. Description of the Preferred Embodiments

The invention enables the preparation of trans-dominant suppressor genes encoding mutant subunits of two different categories of oligomeric proteins. In one category, the protein is composed of subunits at least one of which is initially synthesized containing extraneous peptide material. This extraneous material sequence is subsequently removed during normal processing by cleavage at a proteolytic cleavage site to generate the mature form of the subunit. According to the invention, single stranded DNA encoding the initial form of the indicated subunit is cloned, and the base sequence of the DNA in the region encoding the proteolytic cleavage site is modified by site directed mutagenesis so that processing of the translation product of the gene to the mature form of the subunit is prevented.

In the other category, the oligomeric protein is one in which the monomers are bonded together by at least two disulfide bonds, at least one of which is essential for the catalytic activity of the protein. In the method of the invention, single stranded DNA encoding one of the subunits is cloned, and the base sequence of the DNA in the region encoding a cysteine residue essential for catalytic activity is modified by site directed mutagenesis. The codon for at least one cysteine residue remains unmodified.

The resulting translation product of the altered gene of either category will still bind to wildtype subunits; yet the resultant oligomers will be inactive. The protein product of the suppressor gene thus serves to suppress the wild type activity. Example of the formation of dominant suppressor genes as applied to platelet derived growth factor A (PDGF Al

A fragment spanning the open reading frame of the clone F9A5 (Mercola et al., Develop. Biol. 138; 114-122, 1990), which codes for the entire murine preproPDGF A chain protein, was cloned into the EcoRl site of the bluescript vector, pBSKS+ (Stratagene) . Single-stranded DNA was prepared using M13K07 (Promega Corp. , Madison, I) as the helper phage. Site- directed mutagenesis was carried out by the method of Kunkel et al., Methods in Enzymology, R. u and L. Grossman, eds. Vol. 154, pp. 367-382 (1987) and resulted in the isolation of two mutants carrying dominant suppressor genes, 1317 and 1308.

For mutant 1317, the modification involved was a change in the proteolytic cleavage site for processing the prepro form of the murine PDGF A chain to the mature form. Referring to Fig. 1, the tetra basic amino acid sequence -ArgArgLysArg- (12) was selected as the target sequence for the protease that converts proPDGF A to PDGF A on the basis of observations that the amino terminus of mature PDGF A begins with the amino acid -Ser- (14) immediately to the carboxyl side of this tetra basic residue and that a string of three to five basic amino acids is found in a comparable position within the reading frames of other growth factors that undergo similar processing, notably PDGF B, TGF-βl, and TGF-β2. Site directed mutagenesis was used to alter the sequence -ArgLysArg- (16) at this putative proteolytic cleavage site to - SerAsnGly- (18) . Changing the amino acid sequence in this drastic manner served to prevent removal of the propeptide.

For mutant 1308, the modification involved was a change in the codon for one of four essential cysteine residues in the PDGF A chain. Again referring to the Fig. 1, site directed mutagenesis was used to alter the codon TGC (20) for the third cysteine residue in the chain to AGC, to code for serine (22) . Characterization of products of mutant genes

In order to analyze the proteins encoded by the mutant genes, the two mutant cDNAs and the wild- type PDGF A cDNA were cloned into the pMT2 expression vector, and each of these constructs was transfected into COS cells. The COS cell cultures were incubated with 35S-cysteine to label the translation products, and the conditioned media were analyzed by immunoprecipitation with PDGF A chain-specific antibodies. The wild-type PDGF A cDNA produces a species that is consistent with previous determinations of the molecular weight of PDGF A (16 kd) .

Co-transfection of COS cells with equal amounts of the wild-type and 1317 mutant constructs produces a major band at 38 kd on non-reducing gels, showing that a 1317/wild-type heterodimer is formed in which the wild-type protein is properly processed. Co-transfection of wild type with the 1308 construct did not result in any obvious structural changes. However, the abundance of the wild-type protein was decreased substantially. A series of transfections carried out with a fixed amount of the wild-type construct along with increasing amounts of the 1308 mutant showed that increasing amounts of the mutant resulted in a steady decrease in the amount of the wild-type protein expressed in the conditioned media. The mutant and the mutant/wild-type dimers appear to be subject to rapid degradation.

The biological inactivity of the protein products of the mutant genes was demonstrated using a bioassay for PDGF. Transfection of 2.5 μg of the mouse wild-type PDGF A, assayed on Balb/c-3T3 cells, resulted in 266 ng/ml of PDGF in the conditioned media. Transfection of 17.5 μg of either of the mutant constructs resulted in less than 1 ng/ml of activity. This level of expression is not significantly above the background level produced from COS cells transfected by the pMT2 expression vector alone. As a demonstration of the impact of the mutant PDGF products on the biological activity of wild-type PDGF, COS cells were co-transfected with 2.5 μg of the wild-type PDGF A chain construct along with increasing amounts of mutant constructs. Total DNA was adjusted to a constant 20 μg per transfection by addition of the pMT2 vector to minimize variations in transfection efficiency. Transfections with a 3- or 7-fold excess of 1317 DNA relative to wild-type DNA resulted in approximately 5- and 10-fold decreases in activity, respectively. Transfections with the wild- type gene and the 1308 mutant show a similar decrease in PDGF activity. Thus, the processing mutant, 1317, results in formation of a mutant/wild-type heterodimer that is efficiently secreted, but is biologically inactive, while the 1308 mutant appears to destablize the heterodimer, causing its degradation prior to release from the cells. Use

The uses of dominant suppressor genes fall into two distinct categories. Within the first category, the genes themselves would be used to produce variant strains of transgenic laboratory or farm animals. Within the second category, the genes would be used to manufacture hetero oligomeric proteins containing a suppressor subunit in association with a wild type subunit. These hetero oligomeric proteins would function as competitive inhibitors of the natural protein; thus, they could have useful therapeutic applications. l. Example of using a dominant suppressor gene to create a transgenic laboratory animal which will serve as a disease model for medical research. The amino acid sequence of the two genes which encode PDGF (PDGF A and PDGF B) has been stringently preserved from humans down through lower vertebrates. The A gene of PDGF is expressed at extremely early times in the development of mammals and amphibians. For these reasons, it is believed that PDGF regulates important processes in normal physiology; however, the true functions of PDGF in vivo have not been established. Using dominant suppressor technology, a permanent strain of a mammal such as a laboratory mouse can be created which is unable to produce PDGF. Such a mouse can be used to study the functions of PDGF in vivo and to discover new drugs which might enhance or suppress those functions as needed.

To produce a PDGF deficient mouse, a PDGF dominant suppressor gene, such as one of the PDGF A mutant cDNAs described above, can be placed downstream of a strong promoter which functions well in mice, such as the SV40 promoter, the cytomegalovirus promoter, or any of several retrovirus LTRs. Other usual features of a transgenic mouse vector (splice donor/acceptor sites and a polyadenylation signal) can also be incorporated into this promoter-driven PDGF A suppressor. Using procedures which are now widely in practice, such as microinjection, a permanent strain of transgenic mice which expresses the PDGF suppressor protein in virtually all tissues can be created.

Expression of the mutant protein would be of little or no consequence in tissues which do not manufacture PDGF. In mouse tissues which normally produce PDGF, the dominant suppressor protein should inactivate the wild type PDGF A and/or B chains. (It has been found that the 1317 mutation will suppress wild type PDGF A activity while the 1308 mutation will suppress both wild type PDGF A and PDGF B activity.) If PDGF has essential functions at early times in vertebrate embryogenesis, tissue specific promoters such as platelet-factor 4 can be used which would only be expressed during adult life.

2. Example of using a transdominant suppressor genes to manufacture heterodimeric proteins which are competitive inhibitors of the wild type protein

Localized overproduction of PDGF within cells which also express the PDGF receptor has been implicated as the root cause of atherosclerotic plaques and of connective tissue tumors such as glioma and sarcoma. In normal cells monomeric PDGF receptor subunits alpha (α) and beta (β) are in dynamic equilibrium with three receptor isoforms - α:α, cc. R , and β:B. In successful PDGF:receptor interaction, each half of a PDGF dimer can occupy one-half of a receptor dimer. It is known that in cells that have been stripped of alpha receptor units, so as to leave only beta subunits, PDGF A:B heterodi ers are not mitogenic; however, an excess of A:B heterodimer can inhibit the mitogenic response to a nominal dose of a B:B homodimer. Apparently, the B subunit within A:B heterodimers is able to occupy 1/2 of a beta:beta receptor dimer without activating it and thereby render it useless for a productive interaction with PDGF B:B. Dominant suppressor genes can be used to manufacture useful therapeutic reagents that would have the same effect. Referring to Fig. 2, the proPDGF A subunit produced by mutant 1317 is too bulky to occupy the α receptor site. Yet this subunit is able to dimerize with wild type PDGF (32) . This mutant heterodimer (32) should compete with either PDGF A:A or A:B, to inhibit the mitogenic response. A therapeutic reagent containing a proPDGF A:PDGF A heterodimer as described above can be administered by routine methods (e.g., intravenously) in pharmaceutically acceptable carrier substances, to a level of between 1-50 ng/ml (most preferably 1-10 ng/ml) final concentration.

Dominant suppressor genes themselves, targeted into specific human or animal cells, can be used for therapeutic applications. Dominant suppressors which inhibit expression of PDGF can be another means of treating coronary artery disease, brain tumors, and other hyper-proliferative disorders of connective tissue such as rheumatoid arthritis. Other embodiments are within the following claims.

For example, murine trans-dominant PDGF A suppressors can be suppressors of murine PDGF B chain activity. The murine PDGF A dominant suppressors can also be used effectively in a wide range of species because the cysteines are highly conserved. This possibility has been confirmed by blocking the biological activity of the Xenopus laevis A chain using these mutants. When the open-reading frame from pXOl, a Xenopus A chain cDNA clone, is expressed in COS cells along with mutants 1317 and 1308, the mouse PDGF mutants are both effective at suppressing Xenopus PDGF. Other growth factors which are appropriate for the application of this technology include members of the transforming growth factor beta (TGF-β) superfamily and colony stimulating factor 1 (CSF-1) . All members of the TGF-β superfamily and CSF-1 are growth factors which function in a dimeric state like PDGF. Dominant suppressor mutants of TGF-βl and TGF- β2 have been constructed by disrupting basic amino acids near the propeptide processing region in a manner described for the PDGF A 1317 mutation. TGF- βl contains the following amino acid sequence at the processing site:

-SerArgHisArgArgAla-. This sequence was changed to -SerGlnHisSerGlyAla-. TGF-β2 contains the following sequence at its processing site: -ArgArgLysLysArgAla- . This sequence was changed to -ArgGlnAsnGlnGlyAla- In both cases, the modification was effective in suppressing the corresponding growth factor.

In a similar manner, dominant suppressor genes can be prepared that will inhibit the activity of any oligomeric protein in which either, one subunit is initially synthesized containing a subsequently cleaved extraneous sequence (e.g., a "pro" sequence or an internal sequence) , or the monomers of the protein are bonded together by at least two disulfide bonds, at least one of which is essential for the catalytic activity of the protein.

Any conventional method of site directed mutagenesis, including amino acid substitution, addition or deletion, that will not disrupt the reading frame of the resulting cDNA, is appropriate for implementing dominant suppressor gene technology. What is claimed is:

Claims

Claims 1. A method of making a eucaryotic cell trans-dominant suppressor gene encoding a polypeptide translation product capable of suppressing the activity of a protein that requires an oligomeric state for function, said method comprising the steps of: isolating single stranded DNA encoding a subunit of said protein, said subunit being one which is initially synthesized containing extraneous peptide material which is subsequently removed by cleavage at a proteolytic cleavage site to generate a mature form; modifying the base sequence in said single stranded DNA in the region encoding said proteolytic cleavage site, whereby proteolytic cleavage of the initial translation product of said single stranded DNA to said mature form is prevented; and cloning said modified, single stranded DNA to form said trans-dominant suppressor gene.

2. The method of claim l wherein said modifying step comprises altering the codon for an amino acid essential for cleavage at said proteolytic cleavage site, whereby a different amino acid is substituted for said essential amino acid in said initial translation product of said single stranded DNA.

3. The method of claim 1 wherein said modifying step comprises deleting the codon for an amino acid essential for cleavage at said proteolytic cleavage site.

4. The method of claim 1 wherein said modifying step comprises adding a codon for an amino acid at said cleavage site.

5. A method of making a eucaryotic cell trans-dominant suppressor gene encoding a polypeptide translation product capable of suppressing the activity of a protein that requires an oligomeric state for function, said method comprising the steps of: isolating single stranded DNA encoding a subunit of said protein, said subunit being bonded in said oligomeric state by means of a plurality of cysteine residues, at least one of which is essential for the catalytic activity of said protein; modifying in said DNA strand the codon for one of said cysteine residues essential for the activity of said protein by substituting a codon for another amino acid in place of the codon for said one cysteine residue, the codon for at least one other cysteine residue remaining unmodified; and cloning said modified, single stranded DNA to form said trans-dominant suppressor gene.

6. The method of claim 1 or claim 5 wherein said protein is a growth factor.

7. The method of claim 6 wherein said growth factor is PDGF.

8. The method of claim 7 wherein said subunit is PDGF A.

9. The method of claim 1 or claim 5 wherein said protein is a transcription factor.

10. The eucaryotic cell trans-dominant suppressor gene made by the method of claim 1 or claim 5.

11. The protein translation product of the trans-dominant suppressor gene of claim 10.

12. A transgenic non-human animal comprising germ cells and somatic cells comprising the trans- dominant suppressor gene of claim 10 introduced into the animal, or an ancestor of the animal, at an embryonic stage.

13. The transgenic animal of claim 12 wherein said trans-dominant suppressor gene is under the control of a tissue specific promoter.