WO1991018924A1 - Mammalian adipogenic factors - Google Patents
Mammalian adipogenic factors Download PDFInfo
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- WO1991018924A1 WO1991018924A1 PCT/US1991/003868 US9103868W WO9118924A1 WO 1991018924 A1 WO1991018924 A1 WO 1991018924A1 US 9103868 W US9103868 W US 9103868W WO 9118924 A1 WO9118924 A1 WO 9118924A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention in the field of cell biology, physiology and medicine relates to purified mammalian adipogenic factors, genetic constructs thereof, antibodies thereto, and methods of using such factors to determine susceptibility to obesity and for evaluating efficacy of anti-obesity drugs.
- Adipose differentiation of adipogenic cell lines is under the control of factors called adipogenic factors which either trigger or stimulate the process of differen ⁇ tiation.
- the isolation and complete identification of adipogenic factors is important as, 1) they are responsible for turning on the differentiation program; 2) reports in the literature have disclosed that abnormal levels of circulating adipogenic factors exist in the blood of obese patients (Lau, D.C. . et al., 1984 Proc. 7th International Congress Endocrinology Excerpta Medica, p. 866) .
- Adipo- genie factors have been found in fetal bovine serum and in human serum and plasma.
- Crude fetuin preparations have been characterized as having an adipogenic activity that is heat sensitive and acid (pH 1) sensitive; the activity was apparently attributed to the fetuin within the preparation.
- the factor purified from human plasma is a 45 kDa protein.
- M. L. Weir and R. E. Scott, Am. J. Phvsiol. (1982), vol. 125, pp.546-554. It induces loss of proliferative potential of 3T3-T proadipocytes.
- aproliferin is different from the adipogenic factors we have discovered. Addition ⁇ ally, an active fraction has been isolated from fetal calf serum [P. Grimaldi et al (1982) EMBO J.
- the present invention is directed to novel mamma ⁇ lian, including human, adipogenic factors. These adipo ⁇ genic factors which appear to play an important role in the generation of fat cells in mammals, have are useful in a method for determining the susceptibility to obesity in a subject. The adipogenic factors are also useful for evalu ⁇ ating the efficacy of anti-obesity drugs.
- the invention is directed first to a mammalian adipogenic factor having an apparent molecular weight of about 150 to 230 kDa which is isolatable from liver cells and has adipogenic activity substantially greater than that of naturally occurring liver cells or hepatocytes in cul ⁇ ture on a per milligram protein basis.
- the adipogenic activity of this factor is susceptible to destruction by treatment with pronase, with high temperatures of about 100°C, with a pH of about 2.5, and with 0.2 M 2-mercapto- ethanol.
- the factor is of human origin and has specific adipogenic activity at least about 625 times that of the conditioned medium obtained from confluent cultures of HepG2 hepatocarcinoma cells.
- a preferred source of this adipogenic factor is a liver cell line or conditioned medium from such a line.
- the invention is also directed to a mammalian adipogenic factor having an apparent molecular weight of about 660 kDa which is isolatable from serum and has adipo ⁇ genic activity substantially greater than that of serum on a per milligram protein basis. Its adipogenic activity is susceptible to destruction by treatment with pronase, high temperature of about 100°C, and resistant to treatment with 0.2 M 2-mercaptoethanol at 25°C for 6 hours at pH 2.5. In a preferred embodiment, this factor has a specific adipo- genie activity at least 10 times that of serum and of crude fetuin.
- the invention is further directed to a mammalian adipogenic factor having an apparent molecular weight of about 230 kDa which is isolatable from serum and has adipo ⁇ genic activity substantially greater than that of serum on a per milligram protein basis. Its adipogenic activity is susceptible to destruction by treatment with pronase, with a pH of about 2.5 and about 11.0, and is susceptible to partial destruction by treatment with 0.2 M 2-mercapto- ethanol at 25°C for 6 hours. In a preferred embodiment, this factor has a specific activity at least 250 times that of serum and of crude fetuin.
- the invention is also directed to a mammalian adipogenic factor isolatable from serum and having an apparent molecular wei ⁇ nt of about 50 to 69 kDa, the factor being different from the 69 kDa glycoprotein known as "pure fetuin.”
- the adipogenic activity of this factor is sub ⁇ stantially greater than that of serum on a per milligram protein basis.
- this factor has a specific adipogenic activity at least 2 times that of serum or crude fetuin.
- the present invention also involves a method for determining the susceptibility of a subject to obesity which comprises removing a sample of a biological fluid or tissue from the subject and measuring the amount of one or more of the above-described adipogenic factors present in the fluid or tissue.
- the amount of the adipogenic factor is proportional to the susceptibility of the subject to obesity.
- the invention also includes a method for evalu- ating the efficacy of an anti-obesity drug which comprises contacting the drug being evaluated with an adipogenic cell in vitro and measuring the amount of one or more of the above-described adipogenic factors produced by the cell.
- Another embodiment of the invention is an anti ⁇ body, either polyclonal or monoclonal, specific for an adipogenic factor.
- Such antibodies are useful both in isolation and purification of the factors as well as in the methods of the invention directed to evaluating anti- obesity drugs or in determining susceptibility to obesity.
- the antibodies are also useful in methods for treating obesity wherein an antibody to an adipogenic factor is administered to a subject who is susceptible to obesity based on increased levels of the adipogenic factor.
- the invention is further directed to polynucleo- tide molecules, including RNA and DNA which encode the adipogenic factors, as well as to vectors comprising the DNA encoding the adipogenic factors, and prokaryotic and eukaryotic host cells transformed or transfected, and capable of expressing, the DNA.
- adipogenic cell line, 1246 which can be maintained in defined medium, and the use of fetuin as the starting material were the keys for purification of the bovine factors.
- the use of 1246 cells has allowed the characterization of the human factor, since these cells, for proliferation, require only small amounts of the growth factors which are present in the fetuin but absent in human HepG2 conditioned medium (CM) , a preferred source of the human factor; other adipogenic cell lines (such as 3T3-L1 and Obl7) require greater amounts of the growth factors not found in conditioned medium (CM) of HepG2 cells, a human hepatocyte-like cell line (Knowles, B.B. et al. Science 209:497-499 (1980)), rendering the bioassay for the adipogenic factor in these latter cell lines more difficult to interpret.
- CM conditioned medium
- G3PDH glycerol-3-phosphate dehydrogenase
- an adipogenic factor is produced by normal rat hepatocytes in culture. That resulted in the identification of liver cells as the physiological source of the adipogenic factor in vivo, a discovery important for the subsequent discovery of the human adipogenic factor in the supernatant of the human hepatocyte-like cell line, HepG2. Additionally, a bovine adipogenic factor was isolated from fetuin, a bovine serum substitute known to stimulate proliferation and various functions in several different types of cells in vitro (D. Salomon et al. (1984) , in Cell Culture Methods for Molecular and Cell Biology. Vol 3 , D.W. Barnes et al. , Eds., Alan R. Liss Inc., New York, pp 125-153.)
- mammalian adipogenic factor refers to a molecule which has the capability of inducing adipose differentiation of adipogenic cells.
- the adipogenic fac ⁇ tors contemplated within the scope of the present invention are not limited to those which are purified from liver cells or serum, but to proteins or glycoproteins having adipogenic activity which have been chemically synthesized (by chemical and biochemical techniques) or produced using recombinant DNA technology.
- an adipogenic cell is a cell which can become an adipocyte (fat cell) .
- An adipo ⁇ genic factor is a substance which can induce or stimulate the differentiation of cells which are precursors of adipo ⁇ cytes, such as preadipocytes, to adipocytes.
- adipogenic factor is a substance which can stimulate proliferation of preadipocytes or adipocytes.
- Adipose differentiation can be measured in any of a number of ways which are known to those skilled in the art. A preferred way of measuring adipose differentiation is by the induction of the enzyme, G3PDH, as described herein. The assay can be done, without undue experimentation, by one of skill in the art.
- the enzyme glycerol-3-phosphate dehydrogenase (G3PDH) , represents a differentiation marker which is suitable for assaying the differentiation-inducing activity of the adipogenic factors of the present invention and is easy to quantitate.
- This enzyme is inducible by adipogenic agents.
- the level of G3PDH in an adipogenic cell such as, for example, in the 1246 cell line, is increased by about 3-10 fold.
- the enzyme level is induced as high as 100 fold. Induction of this enzyme is also measurable in primary cultures of epididymal fat pads. The induction of high levels of G3PDH specific enzyme activity is therefore an extremely useful bioassay during purification of an adipo ⁇ genic factor. A 2-fold increase in the G3PDH activity is considered induction.
- a preparation contains an adipogenic factor with adipogenic activity "substantially greater” than that of the naturally occurring cells or the serum, one compares the specific adipogenic activity in the preparation with the activity of a liver tissue homogenate or in the conditioned medium of a normal or transformed hepatocyte cell line.
- Specific adipogenic activity refers to the amount of activity per mg (or other weight unit) protein in the preparation.
- adipo ⁇ genic factor As alternatives to purified or recombinant adipo ⁇ genic factor, functional derivatives of the adipogenic factor may be used.
- a functional derivative is meant a “fragment,” “variant,” “analog,” or “chemical derivative” of the adipo ⁇ genic factor, which terms are defined below.
- a functional derivative retains at least a portion of the function of the adipogenic factor which permits its utility in accor ⁇ dance with the present invention.
- a “fragment” of the adipogenic factor refers to any subset of the molecule, that is, a shorter peptide.
- a “variant" of the adipogenic factor refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conve ⁇ niently prepared by direct chemical synthesis of the vari ⁇ ant peptide, using methods well-known in the art.
- amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide.
- Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combina ⁇ tion of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
- the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and pref ⁇ erably will not create complementary regions that could produce secondary mRNA structure (see European Patent Publication No. EP 75,444).
- these variants ordinarily are prepared by site-directed mutagenesis (as exemplified by Adelman et al.. DNA 2.:183 (1983)) of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
- the variants typically exhibit the same qualitative biological activity as the nonvariant peptide.
- an "analog" of the adipogenic factor refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
- a "chemical derivative" of the adipogenic factor contains additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifi ⁇ cations may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
- Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamido ethyl derivatives.
- Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloro-mercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa- 1,3-diazole.
- Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
- Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
- Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Deriva- tization with these agents has the effect of reversing the charge of the lysinyl residues.
- Suitable reagents for derivatizing alpha-amino-containing residues include i idoesters such as methyl picolinimidate; pyridoxal phos ⁇ phate; pyridoxal; chloroborohydride; tri- nitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
- i idoesters such as methyl picolinimidate; pyridoxal phos ⁇ phate; pyridoxal; chloroborohydride; tri- nitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
- Arginyl residues are modified by reaction with one or several conventional reagents, among them phenyl- glyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
- N-acetylimidizol and tetranitro- methane are used to form O-acetyl Carboxy1 side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N-C-N-R 1 ) such as 1 cyclohexyl-3-(2- morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3 (4 azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
- Glutaminyl and asparaginyl residues are frequent ⁇ ly deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
- Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or to other macromolecular carriers.
- Commonly used cross-linking agents include, e.g., l,l-bis(diazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobi- functional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succini idylpropionate) , and bifunctional maleimides such as bis-N-maleimido-l,8-octane.
- Derivatizing agents such as methyl-3-[ (p-azidophenyl) ] dithiopropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
- reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
- Such derivatized moieties may improve the solu- bility, absorption, biological half life, and the like.
- the moieties may alternatively eliminate or attenuate any undesirable side effect of the protein and the like.
- Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences. 16th ed. , Mack Publishing Co., Easton, PA (1980)
- a “liver cell line” includes hepatocytes derived from a liver or a cell line having hepatocyte functions, such as hepatocarcinoma cell line, as exemplified by HepG2.
- Constant medium refers to any culture medium in which cells have been incubated. A specific example is described herein. Generally, media are chosen that do not have significant deleterious effects on cell viability and the ability of the cell to produce a product which is being purified or assayed in a bioassay.
- a fraction of the HepG2 derived human adipogenic factor, or a serum-derived adipogenic factor, preferably a purified fraction is obtained and used to immunize an animal.
- a mouse is immunized with this antigen.
- the mouse is of the inbred strain, Balb/c.
- the term "antibody” refers both to monoclonal antibodies (mAbs) which are a substantially homogeneous population and to polyclonal antibodies which are heterogeneous populations. Polyclonal antibodies are derived from the sera of animals immunized with the above antigen stein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110.
- Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
- Hybridoma ⁇ upernatants are screened for the presence of antibody specific for the adipogenic factor by any of a number of immunoassays, including dot blots and standard enzyme immunoassays (EIA or ELISA) , which are well-known in the art. Once a supernatant has been identi ⁇ fied as having antibodies, it may be further screened by Western blotting to identify the size of the antigen to which the antibody binds.
- immunoassays including dot blots and standard enzyme immunoassays (EIA or ELISA) , which are well-known in the art.
- antibody is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab') 2 , which are capable of binding the antigen.
- Fab and F(ab*) 2 fragments lack the Fc fragment of intact antibody
- clear F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al.. J. Nucl. Med. 24:316-325 (1983)).
- Fab and F(ab') 2 and other used for the detection and quantitation of adipogenic factors are typically produced by proteolytic cleav ⁇ age, using enzymes such as papain (to produce Fab frag ⁇ ments) or pepsin (to produce F(ab') 2 fragments).
- Polyclonal or monoclonal antibodies can be used in an immunoaffinity column to purify the adipogenic factor by a one step procedure, using methods known in the art.
- the antibodies of the invention are useful for detecting and quantitate the adipogenic factors in an immunoassay, such as, for example, radioimmunoassay (RIA) or enzyme immunoassay (EIA) .
- RIA radioimmunoassay
- EIA enzyme immunoassay
- Such immunoassays are useful for detecting and quantitating an adipogenic factor in the serum or other biological fluid, or in a tissue sample or tissue extract, from a normal or obese subject.
- the concentration of one or more of the adipogenic factors of this invention is measured in a tissue extract or bio ⁇ logical fluid of a subject as a means for determining the susceptibility or the propensity of the subject for obesity.
- the susceptibility of a subject to obesity is said to be proportional to the level of the adipogenic factor.
- proportional as used herein is not intended to be limited to a linear or constant relationship between the level of the adipogenic factor and the suscep- tibility to obesity.
- the nature of the relationship between factor level and susceptibility or propensity to obesity may be highly complex. For example, the doubling of the concentration of an adipogenic factor is not neces ⁇ sarily indicative of a doubling in the susceptibility to obesity.
- the term "proportional" as used herein is intend ⁇ ed to indicate that an increased level of factor is related to an increased propensity to obesity at ranges of concen ⁇ tration of the factor that can be readily determined by one of skill in the art.
- Another embodiment of the invention is evaluating the efficacy of anti-obesity drug or agent by measuring the ability of the drug or agent being evaluated to inhibit the production of one or more of the adipogenic factors of this invention by a cell or cell line capable of producing such factors.
- the antibodies of the present invention are useful in the method for evaluating anti-obesity drugs in that they can be employed to determine the amount of the adipogenic factor in one of the above-mentioned immuno ⁇ assays. Alternatively, the amount of adipogenic factor produced is measured by bioassay, as described herein. The bioassay and immunoassay can be used in combination for a more precise assessment of the factor or factors present.
- One embodiment of the present invention is directed to polynucleotide molecules, particularly DNA, encoding the adi•pogenic factors.
- Another embodi•ment is directed to the preparation of the adipogenic factors using recombinant DNA techniques.
- vectors comprising the DNA, and host cells transformed or trans- fected with the DNA encoding an adipogenic factor.
- the DNA encoding the polypeptide portion of the adipogenic factors of the present invention is either
- DNA sequences encoding the adipogenic factor or a portion or a variant thereof are inserted into an appropriate vector, such as a plasmid or virus, and introduced into an appropriate host cell, either 0 prokaryotic or eukaryotic.
- an appropriate vector such as a plasmid or virus
- oligonucleotide probes can be prepared and used to isolated DNA (genomic or cDNA) encoding the pro ⁇ tein. Techniques for synthesizing such oligonucleotides are disclosed by, for example, Wu, R. , et al.. Prog. Nucl.
- oligonucleotides can be identified, each of which would be capable of encoding a portion of the adipogenic factor peptide. The probability that a particular oligo ⁇
- 35 nucleotide will, in fact, constitute the actual adipogenic factor peptide-encoding sequence can be estimated by con ⁇ sidering abnormal base pairing relationships and the fre ⁇ quency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells.
- Such "codon usage rules" are disclosed by Lathe, R. , et al. , J. Molec. Biol. 183:1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most proba ⁇ ble" nucleotide sequence capable of encoding the adipogenic factor peptide sequences is identified.
- amino acid sequences may be encoded by only a single oligonucleotide, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides.
- all of the members of this set contain oligonucleotides which are capable of encoding the adipogenic factor peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene.
- this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
- the oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the adipogenic peptide is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences.
- An oligo ⁇ nucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the adipogenic factor gene (Sambrook, J. et al.. supra) .
- a suitable oligonucleotide, or set of oligo ⁇ nucleotides, which is capable of encoding a fragment of the adipogenic factor gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identi ⁇ fied (using the above-described procedure) , synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing the adipogenic factor gene, such as, for example, HepG2.
- Single stranded oligo ⁇ nucleotide molecules complementary to the "most probable" adipogenic factor peptide encoding sequences can be syn- thesized using procedures which are well known to those of ordinary skill in the art (Belagaje, R. , et al. , J. Biol. Chem. 254:5765-5780 (1979); Maniatis, T. , et al.. In: Molecular Mechanisms in the Control of Gene Expression. Nierlich, D.P., et al. , Eds., Acad. Press, NY (1976); Wu, R. , et al. , Prog. Nucl. Acid Res. Molec. Biol.
- DNA synthesis may be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook, J. et al. (supra) and by Haymes, B.D., et al. (In: Nucleic Acid Hybridiza ⁇ tion. A Practical Approach. IRL Press, Washington, DC (1985) , which references are herein incorporated by refer ⁇ ence. Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for human aldehyde dehydrogenases (Hsu, L.C., et al. , Proc. Natl.
- a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA (from a cell capable of expressing adipogenic factor, such as HepG2) into an expression vector.
- the library is then screened for mem ⁇ bers capable of expressing a protein which binds to anti- adipogenic factor antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as adipogenic factor, or fragments thereof.
- DNA, or more pref ⁇ erably cDNA is extracted and purified from a cell which is capable of expressing adipogenic factor antigen.
- the purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNA frag ⁇ ments.
- DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of expression vectors whose members each contain a unique cloned DNA or cDNA fragment.
- An "expression vector” is a vector which (due to the presence of appropriate transcriptional and/or transla- tional control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing a polypeptide or protein.
- Expres- sion of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expression vector is employed, then the appro ⁇ priate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Similarly, if a eukary- otic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences. Importantly, since eukaryotic DNA may contain intervening sequences, and since such sequences cannot be correctly processed in prokaryotic cells, it is preferable to employ cDNA from a cell which is capable of expressing adipogenic factor in order to produce a prokaryotic genomic expression vector library.
- Nucleic acid detection assays can be predicated on any characteristic of the nucleic acid molecule, such as its size, sequence, susceptibility to digestion by restric ⁇ tion endonucleases, etc.
- the sensitivity of such assays may be increased by altering the manner in which detection is reported or signaled to the observer.
- assay sensitivity can be increased through the use of detectably labeled reagents. A wide variety of such labels have been used for this purpose.
- Kourilsky et al. U.S.
- Patent 4,581,333 describe the use of enzyme labels to increase sensitivity in a detection assay. Radioisotopic labels are disclosed by Falkow et al. (U.S. Patent 4,358,535), and by Berninger (U.S. Patent 4,446,237). Fluorescent labels (Albarella et al.. EP 144914) , chemical labels (Sheldon III et al.. U.S. Patent 4,582,789; Albarella et al.. U.S. Patent 4,563,417), modified bases (Miyoshi et al.. EP 119448) , etc. have also been used in an effort to improve the efficiency with which detection can be observed.
- One method for overcoming the sensitivity limita ⁇ tion of nucleic acid concentration is to selectively a pli- fy the nucleic acid whose detection is desired prior to performing the assay.
- Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Patent 4,237,224), Maniatis, T. , et al. , etc.
- PCR polymerase chain reaction
- the polymerase chain reaction provides a method for selectively increasing the concentration of a particu- lar nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.
- the method can be used to amplify either single- or double-stranded DNA.
- the essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.
- a molecule of DNA or RNA possesses directionality, which is conferred through the 5'-3* linkage of the phosphate groups of the molecule. Sequences of DNA or RNA are linked together through the formation of a phosphodiester bond between the terminal 5' phosphate group of one sequence and the terminal 3 ' hydro ⁇ xyl group of a second sequence. Polymerase dependent ampli ⁇ fication of a nucleic acid molecule proceeds by the addition of a 5' nucleotide triphosphate to the 3 • hydroxyl end of a nucleic acid molecule.
- oligonucleotide probes of the PCR are selected such that they contain sequences identical to, or complementary to, sequences which flank the particular nucleic acid sequence whose amplification is desired.
- the oligonucleotide sequences of the "first" probe is selected such that it is capable of hybridizing to an oligonucleotide sequence located 3 ' to the desired sequence
- the oligonucleotide sequence of the "second” probe is selected such that it contains an oligo ⁇ nucleotide sequence identical to one present 5 • to the desired region. Both probes possess 3' hydroxy groups, and therefore can serve as primers for nucleic acid synthesis.
- the reaction conditions are cycled between those conducive to hybridiza ⁇ tion and nucleic acid polymerization, and those which result in the denaturation of duplex molecules.
- the nucleic acids of the sample are transiently heated, and then cooled, in order to dena- ture any double-stranded molecules which may be present.
- the "first" and “second” probes are then added to the sample at a concentration which greatly exceeds that of the desired nucleic acid molecule.
- the "first" probe will hybridize to the nucleic acid molecule of the sample at a position 3 * to the sequence to be amplified.
- the "second" probe will hybridize to the complementary strand of the nucleic acid molecule at a position 3 • to the sequence which is the complement of the sequence whose amplification is desired.
- the 3* ends of the "first” and (if the nucleic acid molecule was double- stranded) "second" probes will be extended.
- the extension of the "first” probe will result in the synthesis of an oligonucleotide having the exact sequence of the desired nucleic acid.
- Extension of the "second” probe will result in the synthesis of an oligonucleotide having the exact sequence of the complement of the desired nucleic acid.
- the PCR reaction is capable of exponential ampli ⁇ fication of specific nucleic acid sequences because the extension product of the "first" probe, of necessity, contains a sequence which is complementary to a sequence of the "second” probe, and thus can serve as a template for the production of an extension product of the "second” probe.
- the extension product of the "second” probe contains a sequence which is comple ⁇ mentary to a sequence of the "first” probe, and thus can serve as a template for the production of an extension product of the "first” probe.
- recombinant molecules can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by 5 application of recombinant DNA techniques. Techniques for synthesizing such molecules are disclosed by, for example, Wu, R. , et al. (Prog. Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978)). Procedures for constructing recombi ⁇ nant molecules in accordance with the above-described 0 method are disclosed Sambrook et al. (supra)
- the 3' terminus of the above-described recombi ⁇ nant molecule is preferably treated to render it unsuitable for polymerization.
- Such treatment may be accomplished by blocking the terminus by chemical means, or by modifying 5 the terminal bases such that they sterically interfere with polymerase action.
- such treat ⁇ ment is accomplished by immobilizing the 3* terminus, such as by coupling it to a solid support (such as, for example, glass, plastic, latex, etc.).
- the support may be of any 0 form (i.e. a sheet, rod, sphere, ovoid, etc. Procedures for such immobilization are well known to those of ordinary skill.
- the 3' end of the recombinant molecule is covalently bound to the solid support.
- a spacer region may be used to extend the probe 5 outward from the solid support as long as (1) it will not sterically hinder any function or characteristic of the recombinant molecule, and (2) the sequence of the spacer region does not participate in the hybridization or poly ⁇ merization reactions of the assay. It is typically desir ⁇ able to immobilize several, and preferably, a large number of such recombinant molecule to the support.
- a genetic construct is produced that possesses the necessary control elements to permit appropriate transcription and translation of the nucleic acid sequence.
- a promoter is a double-stranded DNA or RNA molecule which is capable of binding RNA polymerase and promoting the transcription of an "operably linked" nucleic acid sequence.
- a "promoter sequence” is the sequence of the promoter which is found on that strand of the DNA or RNA which is transcribed by the RNA polymerase.
- a “promoter sequence complement” is a nucleic acid molecule whose sequence is the complement of a “promoter sequence.” Hence, upon extension of a primer DNA or RNA adjacent to a single-stranded “promoter sequence complement” or, of a “promoter sequence,” a double-stranded molecule is created which will contain a functional promot ⁇ er, if that extension proceeds a nucleic acid molecule which is operably linked to that strand of the double- stranded molecule which contains the "promoter sequence” (and not that strand of the molecule which contains the "promoter sequence complement”) .
- RNA polymerases exhibit a high specifici ⁇ ty for such promoters.
- the RNA polymerases of the bacte- riophages T7, T3, and SP-6 are especially well characterized, and exhibit high promoter specificity.
- the promoter sequences which are specific for each of these RNA polymerases also direct the polymerase to utilize (i.e. transcribe) only one strand of the two strands of a duplex DNA template. The selection of which strand is transcribed is determined by the orientation of the promoter sequence. This selection determines the direction of transcription since RNA is only polymerized enzymatically by the addition of a nucleotide 5' phosphate to a 3' hydroxyl terminus.
- Two sequences of a nucleic acid molecule are said to be "operably linked” when they are linked to each other in a manner which either permits both sequences to be transcribed onto the same RNA transcript, or permits an RNA transcript, begun in one sequence to be extended into the second sequence.
- two sequences such as a promoter sequence and any other "second" sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked second sequence.
- two sequences such as a promoter sequence and any other "second" sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked second sequence.
- a promoter sequence in order to function as a promoter, a promoter sequence must be present as a double-stranded molecule.
- the two strands of a functional promoter sequence are referred to as a "transcript” strand and a “complement” strand.
- the "transcript” strand is that strand of the duplex which will be transcribed by the RNA polymerase (i.e. which serves as the template for tran ⁇ scription) .
- the "complement” strand is the strand which has a sequence complementary to the "transcript” strand, and which must be present, and hybridized to the "tran ⁇ script” strand, in order for transcription to occur.
- the promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Suitable promoters are repressible, or, more preferably, constitu ⁇ tive. Examples of suitable prokaryotic promoters include promoters capable of recognizing the T4 (Malik, S. et al.. J. Biol. Chem. 263:1174-1181 (1984); Rosenberg, A.H. et al.. Gene 59:191-200 (1987); Shinedling, S. et al. , J. Molec. Biol. 195:471-480 (1987); Hu, M. et al.. Gene 42:21- 30 (1986), T3, Sp6, and T7 (Chamberlin, M.
- Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Ha er, D., et al.. J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (Mc night, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C. , et al.. Nature (London) 290:304-310 (1981)); and the yeast ga!4 gene promoter (Johnston, S.A. , et al. , Proc. Natl. Acad. Sci. (USA) 79.:6971-6975 (1982); Silver, P. . , et al..
- Strong promoters are the most preferred promoters of the present invention. Examples of such preferred promoters are those which recognize the T3, SP6 and T7 polymerase promoters; the P L promoter of bacteriophage 1; the recA promoter and the promoter of one which is capable of recognizing the T7 polymerase promoter.
- the sequences of such polymerase recognition sequences are disclosed by Watson, J.D. et al. (supra) .
- myosin 200 kDa
- beta- galactosidase 116 kDa
- phosphorylase B 97 kDa
- BSA 66 kDa
- egg albumin 43 kDa
- the invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well. the treatment of human subjects, although it is intended for veterinary uses as well.
- Conditions for the culture of 1246 cells useful for the bioassay of adipogenic factors are modifications of methods described previously (Serrero and Khoo (1982) , Anal. Biochem. 120, 351-359; G. Serrero, (1985), In Vitro Cell. Dev. Biol. 21, 537-540), and are hereby incorporated by reference.
- Adipose differentiation assay On day 0, subconfluent 1246 cells were plated at a density of 1.5 x 10 4 cells per well (having a surface area of 4.5 cm 2 ) in 12-well cluster plates (Costar) in DM/F12 medium supplemented with 2% FCS. At day 1, the medium was replaced by DME/F12 supplemented with insulin (10 ⁇ g/ml) (Sigma, St. Louis, MO) , transferrin (10 ⁇ g/ml) (Sigma) , and fibroblast growth factor (5 ng/ml) (Collaborative Research, Waltham MA) .
- insulin 10 ⁇ g/ml
- transferrin 10 ⁇ g/ml
- fibroblast growth factor 5 ng/ml
- the starting material for large scale purifica- tion of the human adipogenic factor is the culture medium conditioned by the HepG2 cells.
- the HepG2 cell line is available from American Type Culture collection (ATCC HB 8065). For the isolation of this factor, see Aden, D.P. et al. (1979) Nature 282 615. HepG2 cells are cultivated in defined medium, RITC-807 medium + 10% FBS. RITC-807 medium is described in M. Kan, and I. Yamane (1982) J. Cell Phvsiol. 111. 155-162. At confluence, they are cultivated RITC-807 medium. In these conditions, the cells secrete several proteins in the culture medium including the adipo- genie factor.
- Conditioned medium from HepG2 cells was concen ⁇ trated 25-fold by ultrafiltration with a 10,000 molecular weight cut-off Filtron membrane system. Ammonium sulfate precipitation was carried out as using standard procedures which are well-known in the art.
- the protein factor pre ⁇ cipitated by 30-50% (w/v) ammonium sulfate was resuspended in phosphate buffer (20 mM, pH 7.0) and diluted. The diluted fraction was chromatographed on a heparin-sepharose column equilibrated in 20 mM sodium phosphate buffer pH 7.0. The active fraction was eluted with a gradient of NaCl between 0.35 M - 1 M NaCl.
- the adipogenic factor represents at least 30% of the total protein in the fraction.
- the human adipogenic factor isolated as described above, was analyzed by SDS-PAGE.
- a major band has a molecular weight of 230 kDa and two minor bands are of lower molecular weight.
- Figure 1 depicts a similar profile obtained from running an ammonium sulfate precipi ⁇ tate onf Sephacryl S-300.
- bovine adipogenic factors The starting point for purification of these factors is crude fetuin, prepared according to the method of Pedersen
- the crude fetuin was dialyzed against start buffer (25 mM imidazole-CH 3 COOH, pH 7.4) and then load it on a chromatofocusing polybuffer exchange column PBE 94 gel (sold by Pharmacia) that had been equilibrated with start buffer. Unbound proteins were washed out with the start buffer and collected in the "flow-through” fraction. Factor F j was present in the flow-through fraction. The column was then washed with polybuffer 96-CH 3 COOH, pH 6.0 (purchased from Pharmacia, chemical composition undisclosed) and subsequently washed with 1.0 M NaCl. Factor F j j was eluted with 1.0 M NaCl.
- F j J was subsequently loaded on a heparin- sepharose column equilibrated with 20 mM phosphate buffer pH 7.0.
- the column was washed in a stepwise manner with 20 mM phosphate buffer pH 7.0, then with 0.3 M NaCl in 20 mM phosphate buffer pH 7.0 and finally with 1 M NaCl in phos- phate buffer pH 7.0.
- FII was eluted with 1 M NaCl.
- F r t was subsequently loaded on lectin sepharose column equili ⁇ brated with 20 mM phosphate buffer, pH 7.0, containing 0.15 M NaCl.
- the active fraction was eluted with 0.5 M alpha- methyl mannoside, dialyzed against 20 mM phosphate buffer, pH 7.0, and then loaded on a Mono Q ion exchange column. Elution was performed with a NaCl gradient from 0.1 M to 0.5 M NaCl. The active fraction was chromatographed on a hydrophobic interaction phenyl sepharose column. Elution was performed with a descending gradient of NaCl.
- the Fj fraction was loaded on a heparin sepharose column equilibrated with 20 M phosphate buffer at pH 7.0.
- the active fraction was eluted with the same buffer con ⁇ taining 0.1 M NaCl.
- F l eluted with a molecular weight of 660 kDa.
- F j j had an apparent molecular weight of 230 kDa.
- Protease sensitivity was examined by incubating samples with immobi- lized pronase conjugated to agarose beads (Streptomyces griseus, Sigma) at 37°C for 6 h. Pronase was removed by centrifugation before use.
- Fj was found to have a pi > 9.4, to be heat and alkaline labile, protease sensitive, and stable during treatment with 2-mercaptoethanol or acid.
- F x r was found to have a pi
- isolelectric point determinations of F j and F j j are of preparations containing some impurities, which may contribute to a greater or lesser degree to the observed pi. Furthermore, the glyco- protein nature of these factors, and the possibility that other sugars or proteoglycans are present in the fractions, may also contribute to the pi. The key point is the fact that two distinct adipogenic factors are discernible and capable of separation by chromatofocusing.
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Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US53139390A | 1990-06-01 | 1990-06-01 | |
US531,393 | 1990-06-01 |
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WO1991018924A1 true WO1991018924A1 (en) | 1991-12-12 |
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PCT/US1991/003868 WO1991018924A1 (en) | 1990-06-01 | 1991-05-31 | Mammalian adipogenic factors |
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EP (1) | EP0535038A4 (en) |
AU (1) | AU8087591A (en) |
CA (1) | CA2084062A1 (en) |
WO (1) | WO1991018924A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5281582A (en) * | 1992-02-27 | 1994-01-25 | Alliance Pharmaceuticals, Corp. | Serum growth factor |
EP0623142A1 (en) * | 1992-01-17 | 1994-11-09 | W. Alton Jones Cell Science Center | Mammalian adipogenic factors |
WO1998026067A1 (en) * | 1996-12-12 | 1998-06-18 | Incyte Pharmaceuticals, Inc. | Adipocyte-specific differentiation-related protein |
WO2002039923A2 (en) * | 2000-10-27 | 2002-05-23 | Wayne State University | Inhibition of alpha-2 hs glycoprotein (ahsg/fetuin) in obesity and insulin control of glucose homeostasis |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH02503229A (en) * | 1987-04-02 | 1990-10-04 | ザ ベス イスラエル ホスピタル アソシエイション | How to diagnose and treat obesity due to genetic causes |
-
1991
- 1991-05-31 AU AU80875/91A patent/AU8087591A/en not_active Abandoned
- 1991-05-31 EP EP19910910884 patent/EP0535038A4/en not_active Withdrawn
- 1991-05-31 CA CA002084062A patent/CA2084062A1/en not_active Abandoned
- 1991-05-31 WO PCT/US1991/003868 patent/WO1991018924A1/en not_active Application Discontinuation
Non-Patent Citations (5)
Title |
---|
Biochimica et Biophysica Acta, Vol. 846, issued 1985, GAILLARD et al., "Fetuin Modulates Growth and differentiation of Ob17 preadipose cells in Serum-Free Hormone-Supplemented Medium", pages 185-191, see entire document. * |
International Journal of Obesity, Vol. 10, No. 4, issued 1986, HAUNER et al., "Adipogenic Factors in Human Serum Promote the Adipose Conversion of 3T3-L1 Fibroblasts", pages 323-330, see Abstract No. 06058650. * |
Journal of Cellular Physiology, Vol. 144, No. 3, issued September 1990, ZAITSU et al., "Pedersen Fetuin contains three Distinct biochemical characteristics", pages 485-491, see entire document. * |
Klinische Wochenschr., Vol. 65, No. 17, issued 1987, LOFFLER et al., "Adipose Tissue Development: The Role of Precursor Cells and Adipogenic Factors", pages 812-817, see Abstract No. 06367305. * |
See also references of EP0535038A4 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0623142A1 (en) * | 1992-01-17 | 1994-11-09 | W. Alton Jones Cell Science Center | Mammalian adipogenic factors |
EP0623142A4 (en) * | 1992-01-17 | 1995-07-12 | Alton Jones Cell Science Cente | Mammalian adipogenic factors. |
US5281582A (en) * | 1992-02-27 | 1994-01-25 | Alliance Pharmaceuticals, Corp. | Serum growth factor |
WO1998026067A1 (en) * | 1996-12-12 | 1998-06-18 | Incyte Pharmaceuticals, Inc. | Adipocyte-specific differentiation-related protein |
WO2002039923A2 (en) * | 2000-10-27 | 2002-05-23 | Wayne State University | Inhibition of alpha-2 hs glycoprotein (ahsg/fetuin) in obesity and insulin control of glucose homeostasis |
WO2002039923A3 (en) * | 2000-10-27 | 2003-04-17 | Univ Wayne State | Inhibition of alpha-2 hs glycoprotein (ahsg/fetuin) in obesity and insulin control of glucose homeostasis |
Also Published As
Publication number | Publication date |
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CA2084062A1 (en) | 1991-12-02 |
EP0535038A4 (en) | 1993-12-29 |
EP0535038A1 (en) | 1993-04-07 |
AU8087591A (en) | 1991-12-31 |
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