WO1992003478A1 - Metal ion mediated receptor binding of polypeptide hormones - Google Patents
Metal ion mediated receptor binding of polypeptide hormones Download PDFInfo
- Publication number
- WO1992003478A1 WO1992003478A1 PCT/US1991/005856 US9105856W WO9203478A1 WO 1992003478 A1 WO1992003478 A1 WO 1992003478A1 US 9105856 W US9105856 W US 9105856W WO 9203478 A1 WO9203478 A1 WO 9203478A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- histidine
- growth hormone
- hormone
- receptor
- human
- Prior art date
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Classifications
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
-
- 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
- C07K14/575—Hormones
- C07K14/57554—Prolactin
-
- 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
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Metal ions such as zinc have been shown to be useful in the prolonged parenteral release of somatotropins in an oil formulation (EP 177,478, published 04.10.84; EP 343,696, published 29.11.89).
- Similiar slow release formulations of bovine growth hormone complexed with metal ion in an oil vehicle have been shown (EP 216,485, published 01.04.87).
- Metal ions have been used to recover somatotropin from dilute aqueous solutions by forming a precipitate (EP 277,043, published 03.08.88).
- Prolactin has been examined as a regulatory hormone for zinc uptake by the prostate gland (Leake et al., J. of Endocrinology 102(1), p73-76, 1984).
- Zinc deficiency has been associated with a tendency to hyperprolactinemia (Koppelman, Medical Hypotheses, 25(2), p65-68, 1988). A review of the zinc requirement in humans can be found in Prasad (Special Topics in Endocrinology and Metabolism, vol 7, p45-76, 1985).
- hGH Human growth hormone
- hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants or growth hormone (Nichol, C. S., et al. (1986) Endocrine Reviews 7. 169). hGH is unusual among these in that it exhibits broad species specificity and binds monomerically to either the cloned
- somatogenic Leung, D. W., et al. [1987] Nature 330. 537) or prolactin receptor (Boutin, J. M., et al. [1988] Ce;.53, 69).
- the cloned gene for hGH has been expressed in a secreted form in Eschericha coli (Chang, C. N., et al. [1987] Gene 55. 189) and its DNA and amino acid sequence has been reported (Goeddel, et al. [1979] Nature 281. 544; Gray, et al. [1985] Gene 39. 247).
- the three-dimensional structure of hGH is not available.
- porcine growth hormone pGH
- pGH porcine growth hormone
- hGH Human growth hormone
- GHBP growth hormone binding protein
- GHBP in humans has also been described (Baumann G, Stolar MW, Amburn K, Barsano CP, DeVries BC, J Clin Endocrinol Metab (1986) 62:134- 141.; Herington AC, Ymer S, Stevenson J, J Clin Invest (1986) 77:1817-1823). DNA encoding human GHBP is described in PCT publication number WO 88/09818, published 15 December 1988. SUMMARY OF THE INVENTION
- Novel methods are disclosed for modulating the action of polypeptide hormones on mammalian cells, organs or whole mammals.
- Polypeptide hormone action is controlled by effecting the binding specificity of the polypeptide hormone for distinct receptors.
- the specificity for the receptor is mediated by the ability of a metal ion to bind as part of the hormone-receptor complex and thus to further determine receptor binding specificity.
- Soluble variants of the hormone receptor may be used to modulate the action or serum half-life of the polypeptide hormone.
- An example of such a polypeptide hormone system is human growth hormone (hGH) wherein receptor specificity is modulated by the metal cofactor zinc.
- hGH preferentially binds to human growth hormone receptor or growth hormone binding protein; under high zinc conditions, hGH preferentially binds to human prolactin receptor or soluble prolactin receptor variants. This is the first indication that a metal ion can mediate a direct interaction between a polypeptide hormone and an extracellular receptor or binding protein.
- Novel human polypeptide hormone variants and hormone binding protein v riants having therapeutic utility are disclosed: the variants may have a metal ion binding site deleted or inserted.
- the variants having a metal ion binding site deleted are those hormone variants having the ability to preferentially bind to specific receptors as a function of the absence of a zinc binding site.
- human growth hormone variants have histidine 21 of native human growth hormone replaced by an amino acid other than histidine glutamate, aspartate or cysteine, more specifically, the human growth hormone variant wherein histidine 21 is replaced by alanine.
- human growth hormone variants may replace histidine 18 and glutamate 174 of native human growth hormone with an amino acid other than histidine, glutamate, aspartate or cysteine, more specifically it is replaced by alanine.
- Another human polypeptide hormone variant is human placental lactogen variant wherein histidine 18 , histidine 21 or glutamate 174 of native human placental lactogen is replaced by an amino acid other than histidine, glutamate, aspartate or cysteine, more specifically with alanine.
- a mammalian growth hormone variant excluding human growth hormone, wherein the amino acid corresponding to human growth hormone amino acid histidine 18 , histidine 21 or glutamate 174 is replaced by an amino acid other than histidine, glutamate, aspartate or cysteine, more specifically with alanine.
- Other hormone variants are human growth hormone variants comprising arginine 64 and aspartate 171 substituted by alanine; and human growth hormone variants comprising lysine 168 and glutmate 174 substituted by alanine; human growth hormone variants comprising lysine 172 and glutmate 174 substituted by alanine.
- DNA sequences encoding the human growth hormone variants specifically those DNA sequences wherein said variant contains alanine in place of histidine 18 , histidine 21 and glutamate 174 .
- an expression host transformed with a DNA sequence selected from the group consisting of a DNA sequence encoding a growth hormone variant wherein histidine 21 of human growth hormone is replaced by an amino acid other than glutamate, aspartate or cysteine and a DNA sequence encoding a growth hormone variant wherein said variant contains alanine in place of histidine 18 , histidine 21 and glutamate 174 .
- a method of modifying a mammalian polypeptide hormone-receptor complex containing a metal ion binding site wherein the presence of a metal ion in the metal ion binding site determines the hormone's affinity for the mammalian hormone receptor comprising replacing a histidine, glutamate, aspartate or cysteine amino acid in a mammalian polypeptide hormone or receptor that chelates the metal ion to the mammalian polypeptide hormone-receptor complex, with another amino acid to prepare a variant hormone or receptor that is reduced in its ability to chelate the metal ion.
- the metal ion may be zinc, iron, nickel, copper, magnesium, manganese, cobalt, calcium or selenium, most preferably zinc.
- the mammalian polypeptide hormone-receptor complex containing a metal ion binding site wherein the presence of a metal ion in the metal ion binding site determines the hormone's affinity for the mammalian hormone receptor comprising replacing
- polypeptide hormone may be growth hormone or placental lactogen.
- the hormone receptor may be growth hormone receptor, prolactin receptor, placental lactogen receptor or a serum binding protein with similar receptor properties, for example, growth hormone binding protein.
- a method of stimulating a lactogenic response in a non- human mammal comprising administering to the mammal a therapeutically effective amount of a mammalian growth hormone wherein said mammalian hormone amino acid sequence contains amino acids corresponding to human growth hormone amino acids histidine 18 , histidine 21 and glutamate ⁇ , and maintaining a physiological zinc ion concentration required for said mammalian growth hormone to bind to prolactin receptor wherein a lactogenic response is elicited, preferably the total physiological zinc ion concentration is maintained between about 0.5 and 100.0 ⁇ molar.
- Also described is a method of stimulating a lactogenic response in a human comprising administering to the human a therapeutically effective amount of human growth hormone while maintaining a physiological zinc ion concentration required for said human growth hormone to bind to prolactin receptor wherein a lactogenic response is elicited, preferably the total physiological zinc ion concentration is maintained between about 0.50 and 100.0 ⁇ molar.
- a method of stimulating a somatogenic response in a human comprising administering to the human a therapeutically effective amount of a human growth hormone variant in which the zinc binding site required for human growth hormone binding to prolactin receptor has been deleted.
- Described is a method of screening for variants of a mammalian polypeptide hormone thought to contain a metal ion binding site wherein the presence of a metal ion in the metal ion binding site determines said hormone's affinity for a hormone receptor in a mammal comprising incubating a solution containing a chelating agent and a mammalian polypeptide hormone variant suspected of containing a metal ion binding site; then contacting the incubated mammalian polypeptide hormone with a hormone receptor; and finally, detecting the formation of a polypeptide hormone-receptor complex.
- This method may use a metal ion selected from the group consisting of zinc, iron, nickel, copper, magnesium, manganese, cobalt, calcium or selenium.
- the variant mammalian polypeptide hormone may be a variant of growth hormone or placental lactogen.
- the mammal may be any mammal, preferably selected from human, bovine, porcine, ovine, equine, feline, canine and rodentia.
- a mammalian prolactin binding protein variant comprising soluble prolactin binding protein.
- the soluble prolactin binding protein is human prolactin binding protein.
- One variant of the human prolactin binding protein has histidine 188 replaced by an amino acid other than histidine, glutamate, aspartate or cysteine, preferably by alanine.
- a mammalian growth hormone binding protein wherein another amino acid is inserted in place of an amino acid corresponding to asparagine 218 in human growth hormone binding protein that results in the ability to bind a metal ion, preferably zinc.
- the inserted amino acid is preferably histidine, glutamate, asparate and cysteine.
- the most prefered form is human growth hormone binding protein.
- These GHbP agents may be incorporated into a pharmaceutical formulation comprising mammalian growth hormone, mammalian growth hormone binding protein and zinc, wherein the mammalian growth hormone binding protein contains an amino acid substitution with histidine, glutamate, aspartate or cysteine creating a zinc binding site.
- a preferred formulation contains human growth hormone binding protein wherein asparagine 218 has been replaced by histidine.
- a DNA sequence encoding the human growth hormone variant of human growth hormone wherein the variant contains alanine in place of histidine 18 , histidine 21 and glutamate 174 .
- a DNA sequence encoding soluble human prolactin receptor wherein the human prolactin receptor encoded contains an amino acid substitution at histidine 188 , preferably alanine.
- a DNA sequence encoding human growth hormone binding protein wherein asparagine 218 is replaced by an amino acid selected from histidine, glutamic acid, asparatic acid and cysteine.
- histidine 21 of human growth hormone is replaced by an amino acid other than glutamate, aspartate or cysteine; 2) a DNA sequence encoding a soluble human prolactin receptor, 3) a DNA sequence encoding a soluble human prolactin receptor which contains an amino acid substitution at histidine 188 other than glutamate, aspartate or cysteine; 4) a DNA sequence encoding a soluble human prolactin receptor wherein histidine 188 is replaced by alanine; and, 5) a DNA sequence encoding a human growth hormone binding protein wherein asparagine 218 is replaced by histidine.
- Figure 1A Diagram of plasmid phPRLbp(1-211 ) which directs secretion of the hPRLbp into the periplasm of E. coli. Genes are indicated by arrows, replication origins by circles, and restriction sites used in the construction are indicated.
- FIG. 1B Coomassie blue stained SDS-PAGE (12.5 percent; ref. 27) of purified hPRLbp. Lanes 1-5 are: 1) an E. coli periplasmic fraction, 2) the (NH 4 ) 2 SO 4 precipitate, 3) the protein after hGH affinity chromatography, 4) the wash just before elution of hPRLbp, and 5) molecular weight standards (ranging from 14 to 97 kD), respectively.
- Figure 3 Equilibrium dialysis for binding of 65 Zn 2+ to the hGH 'hPRLbp complex.
- Figure 4. Proposed Zn 2+ binding site on hGH that mediates binding to the hPRLbp.
- Helical wheel projections show the amphipathic character of helix 1 and 4 with polar (shaded) and charged residues (blackened) on one face of the helix and non-polar (open) on the other.
- the positions of the putative zinc binding ligands, His18, His21, and Glu 174, which are involved in binding hGH to the hPRLbp are shown ( ⁇ ).
- the region where hGH binds to the hGHbp is defined roughly by the shaded circle.
- Residues marked by the symbols•, ⁇ , ⁇ and O represent sites where alanine mutations in hGH cause reductions of 2- to 4-fold, 4- to 10-fold, greater than 10-fold, or 4-fold increase in binding affinity for the hGHbp, respectively.
- Figure 5 The amino acid sequence of the extracellular domain of the human prolactin and human growth horman receptor as they are purified after expression in E. coli.
- the inset plot shows the data reformulated in a Scatchard plot to calculate of the K D (68 pM) between hGH and the hPRL bp.
- FIG. 7 Structural model of hGH based on a folding diagram for pGH determined from a 2.8 A resolution X-ray structure.
- Panel A shows a functional map of the hPRLbp epitope and Panel B shows that determined previously for the hGH bp.
- ⁇ , ⁇ and ⁇ represent sites where alanine substitutions cause a 2- to 4-fold, 4- to
- Panel B represents the position of E174A that causes greater than a 4-fold increase in binding affinity.
- Panel C shows sites where alanine mutants reduce binding affinity by ⁇ 10-fold for hPRLbp ( ⁇ ) or >5-fold for the hGHbp ( ⁇ ) without affecting substantially the binding to the hGHbp or hPRLbp, respectively.
- the ( ⁇ ) symbols show sites where alanine mutants disrupt binding to both receptors by > 10-fold.
- hGH-receptor-zinc interaction was experimentally determined using large amounts of the extracellular binding domain of the human prolactin receptor (hPRLbp) as a secreted protein from Escherichia coli.
- hPRLbp human prolactin receptor
- the binding affinity of hGH for the hPRLbp is increased about 8,000-fold (K D of 270 nM to 0.033 nM) by addition of 50 ⁇ M ZnCl 2 .
- polypeptide hormone's receptor binding specificity being determined by the presence of a metal ion complexed with the polypeptide hormone and receptor.
- the ability to change the binding specificity of polypeptide hormones, and therefore their physiological effects, permits the therapeutic control of hormone responses previously not possible.
- An example of this specificity is the complexing of zinc with human growth hormone and its receptors resulting in a change in relative receptor binding specificity from the human growth hormone receptor (somatogenic response in lower zinc) to the human prolactin receptor (lactogenic response in higher zinc).
- Polypeptide hormone may be any amino acid sequence produced in a first cell which binds specifically to a receptor on the same cell for autocrine hormones, or on a second cell type for non-autocrine hormones, and causes a physiological response characteristic of the receptor-bearing cell.
- polypeptide hormones include cytokines, lymphokines, neurotrophic hormones and adenohypophyseal polypeptide hormones such as growth hormone, prolactin, placental lactogen, luteinizing hormone, folliclestimulating hormone, thyrotropin, chorionic gonadotropin, corticotropin, ⁇ or ⁇ -melanocyte-stimulating hormone, ⁇ -lipotropin, ⁇ -lipotropin and the endorphins;
- hypothalmic releasing hormones such as corticotropin-releasing factor, growth hormone release-inhibiting hormone, growth hormone-releasing factor; and other polypeptides hormones such as insulin, insulin-like-growth factors I and II, and atrial natriuretic peptides A, B or C.
- Metal Ion Cofactors may be any divalent metal ion which will complex with a polypeptide hormone and/or receptor and increase or decrease affinity between hormone and receptor.
- preferred metal ions are zinc, iron, nickel, copper, magnesium, manganese, cobalt, calcium or selenium.
- the metal ions may be any physiological acceptable salt, such as chloride, phosphate, acetate, nitrate and sulfate.
- Variant Polypeptide Sequence Notation defines the actual amino substitutions in the mutant polypeptides of the present invention, as illustrated in Table 2.
- substitutions are indicated by a letter representing the original amino acid, a number indicating the amino acid position in the polypeptide, and second letter indicating the substituted amino acid.. Therefore, each substitution is represented by a letter followed by a number which is followed by a letter.
- H18 A in Table 2 the first letter and number (H18) corresponds to the amino acid histidine at position 18 in the unmodified hGH. The last letter corresponds to the amino acid which is substituted at the position (A for alanine).
- a determination of whether a metal ion is required for a given polypeptide hormone to bind to a given receptor may be made through the use of a physiological concentration of a given metal ion and a general di- and tri-valent metal chelating agent, such as EDTA, or a transition metal chelating agent such as 1,10 phenanthroline. Under conditions of 140 mM NaCl, 20 mM Tris (pH 7.5) at 25°C.
- the metal ion at levels ranging from 5 to 1000 ⁇ M, and below a concentration that would cause metal oxides or metal beffer complexes to precipitate is incubated with the polypeptide hormone (10-100pM) and the potential receptor (10-1000pM).
- a zinc ion is required for hGH to bind to prolactin receptor
- the level of binding of the hGH to the prolactin receptor is determined.
- a chelating agent such as EDTA
- EDTA is added to the incubation mixture and the extent of hGH binding with the prolactin receptor determined by subtracting the value for plus EDTA from the value plus zinc. The difference in binding is a measure of the zinc ion requirement for prolactin receptor binding.
- Zn 2+ ⁇ hGH-hPRLbp complex was analyzed.
- the total concentration of zinc in serum varies from 5 to 20 ⁇ M in the adult population (C. Lentner ed. in Scientific Tables, Eighth Ed., (Ciba-Geigy Ltd., Ardsley, N.Y., 1981), Vol. 3, pp. 79-88; R. Berfenstam, Acta Paediat. (Uppsala) 41, suppl 82 (1952)) and about 95 percent is complexed with proteins, mostly to serum albumin (Thorlacius-Ussing, Neuroendocrinol. 45, 233 (1987); M. C. S. Koppelman, V. Greenwood, J. Sohn and P. Denster, J. Clin.
- the free Zn 2+ concentration in serum would be expected to range from about 0.1 to 5.0 ⁇ M or more preferably about 0.25 to 1 ⁇ M. This varies around the K D (0.4 ⁇ 0.2 ⁇ M) for Zn 2+ binding to the hGH ⁇ hPRLbp complex indicating that natural fluctuations in total zinc concentration can modulate the interaction between hGH and the hPRLbp complex.
- the dissociation constants (determined by Scatchard analysis) for binding of [ 125 I]hGH to hPRLbp in the presence of 5 mM CaCl 2 or 20 ⁇ M CuSO 4 are 21 ( ⁇ 5 nM) and 11 ( ⁇ 3) nM, respectively. These affinities are 300- to 600-fold weaker than for the zinc mediated complex (0.03 nM, Table 1). Thus, only zinc is capable of supporting strong binding between hGH and hPRLbp.
- other metal ions may function analogously in other polypeptide hormone-metal ion-receptor complexes.
- Zinc deficiency is often associated with alcoholism, pregnancy, some gastrointestinal disorders, severe bums, chronic renal failure, genetic disorders (acrodermatitis enteropathica and sickle cell anemia), and malnutrition. Moderate zinc deficiency leads to growth retardation (A. W. Root, G. Duckett, M. Sweetland, and E. O. Reiter, J. Nutr. 109, 958 (1979); G. Oner, B. Bhaumick, and R. M. Bala, Endocrinology 114, 1860 (1984); S. Kurtogu, T. E. Patiroglu and S. E. Karakas, Tokai J.
- Zinc is a crucial component of the large class of zinc finger proteins (notably the steroid hormone receptors) that are important regulators of transcription (A. Klug and D. Rhodes, Trends in Biochem. Sci. 12, 464 (1987); R. M. Evans and S. M. Hollenberg, Cell 52, 1 (1988); J. M. Berg, Cell 57, 1065 (1989)). Insulin is stored in complex with zinc in pancreatic cell secretory granules (J. C. Hutton, Experientia 40, 1091 (1984); G. Gold and G. M. Grodsky, Experientia 40, 1105 (1984)). Our studies extend the involvement of zinc in hormone action by showing that it mediates directly the interaction between a polypeptide hormone and an extracellular receptor.
- Any source of receptor may be used in the determination assay, including receptors bound to a cell surface, partially purified receptors or receptors produced by recombinant means.
- the formation of a polypeptide hormone-metal ion-receptor complex may be detected by commonly used assay procedures such as radionucleotides, enzyme immunoassays, or precipitation.
- the general methods as described in Example 1 may be adapted for determining metal ion requirements for any specific polypeptide hormone and receptor.
- hPRLbp extracellular binding domain
- E. coli secretion system G. Fuh et al, J. Biol. Chem. 265, 3111 (1990)
- hPRLbp extracellular domain of the hPRL receptor
- Fig. 1 A The hPRLbp was purified to near homogeneity from periplasmic extracts from E.
- the purified hPRLbp gave a single band of expected molecular weight (25 kD) on reduced SDS-PAGE.
- the purified hPRLbp had an amino-terminal sequence, Gln-Leu-Pro-Pro-Gly-Lys-Pro-Glu-Ile-Phe-Lys, indicative of proper cleavage of the signal peptide.
- binding of hGH to hPRLbp is about 8,000-fold stronger in the presence of 50 ⁇ M ZnCl 2 compared to buffer containing 1 mM EDTA (Example 1 , Table 1).
- the binding constant of hPRL to the hPRLbp is essentially the same under either condition and is close to that measured previously for the full-length recombinant hPRL receptor (2-3 nM) (G. Fuh et al., J. Biol Chem. 265, 3111 (1990)).
- binding of hGH to hPRLbp in the presence of ZnCl 2 is nearly 100-fold stronger than for hPRL, and more than 10-fold stronger than the affinity of hGH for the hGHbp.
- Scatchard analysis shows a stoichiometry of one hormone to one hPRLbp, and the binding of hGH is competitive with hPRL in the presence or absence of zinc, indicating that the hormone binding sites on the hPRLbp overlap.
- zinc actually lowers the affinity of hGH to hGHbp by 4-fold, and prolactin does not bind to hGHbp in the presence or absence of ZnCl 2 .
- the two receptors have
- the IC 50 value reported for binding of hGH to the recombinant full-length hPRL receptor in cell membranes is 0.26 nM (G. Fuh et al., J. Biol Chem. 265, 3111 (1990)) and to rat liver microsomes is 2-3 nM (J. Ray et al., Mol Endocrinol. 4, 101 (1990)) compared to the K D value for the hPRLbp in the presence of 50 ⁇ M zinc of 0.03 nM.
- the discrepancies may reflect real differences in receptor affinities. However, earlier studies did not control the level of zinc in the binding assays.
- Zinc binds to a single site at the interface between hGH and hPRLbp.
- His18 and His21 are on adjacent turns of helix 1 and are positioned near Glu 174 on helix 4. All three face in the same direction and form a plausible site for binding of Zn 2+ .
- replacing either His18, His21 or Glul74 with alanine reduces the hormone affinity for the hPRLbp by about 100-fold relative to wildtype hGH (Example 1, Table.2).
- 1 mM EDTA there is almost no difference in binding affinity between these mutants and wild-type hGH.
- Other alanine variants that disrupt binding of hGH to hPRLbp produce the same reduction in binding whether in the presence of zinc or EDTA.
- the model for the zinc binding site on hGH may account for the weak or undetectable binding of non-primate growth hormones to prolactin receptors.
- Sequence alignments C. S. Nicoll, G. L. Mayer, S. M. Russel, Endocrine Rev. 7, 169 (1986)
- 19 non-primate GH's contain His21 and Glu174, but instead of His18 they contain Glnl8.
- 17 of 19 non-primate GH's contain a histidine at position 19 (hGH contains Argl9).
- Hisl9 can not coordinate zinc along with His21 because they are on opposite sides of helix 1 (Fig. 4).
- other differences between non-primate and primate GH's may contribute to the huge differences in binding affinities between these two subgroups of GH's for prolactin receptors.
- hGH-V a natural variant of hGH, known as hGH-V, binds more tightly to somatogenic than lactogenic receptors (J. Ray et al, J. Biol Chem. 265, 7939 (1990)). This homolog differs by only 13 residues out of 191 from hGH (P. H. Seeburg, DNA 1, 239 (1982)). Remarkably, instead of His18 and His21, hGH-V contains Arg18 and Tyr21. Our studies suggest that hGH-V will not bind Zn 2+ in association with the hPRL receptor, and that this is a major reason for its weaker binding.
- the zinc binding site is positioned on the edge of the epitope identified for binding to the hGH receptor (Fig. 4).
- the model suggests zinc reduces binding of hGH to the hGHbp (Example 1, Table 1) by sterically interfering with binding of hGHbp.
- binding to the hGHbp is enhanced about 4-fold by mutation of Glu 174 to Ala .
- Glu174 hinders binding of hGH to hGHbp, it is required for zinc mediated binding to hPRLbp.
- Zinc typically coordinates four ligands in proteins (B. L. Vallee and A. Galdes, Adv. Enzymol. Rel. Areas Molec. Biol. 56, 283 (1984); A. Klug and D. Rhodes, Trends in Biochem. Sci. 12, 464 (1987); R. M. Evans and S. M. Hollenberg, Cell 52, 1 (1988); J. M. Berg, Cell 57, 1065 (1989)). Having identified three ligands from hGH and realizing that both hGH and the hPRLbp are required for tight zinc binding, we evaluated the possibility that the fourth ligand comes from the hPRLbp.
- H188A showed reduced binding affinity (Example 1, Table 4). Indeed, the binding affinity for hGH was reduced more than 2,000-fold for the HI 88 A mutant in the presence of zinc. This was below the limit of accurate measurement in the assay.
- the mutational analysis supports a model wherein zinc is bound by three ligands from hGH (His 18, His21, Glul74) and one from hPRLbp (His 188).
- the dissociation constant for the hGH ⁇ hPRLbp complex measured here (68 pM) by competitive displacement of the hGHbp from hGH is roughly the same as that measured by direct binding of hGH to the hPRLbp under comparable concentrations of ZnCl 2 (25 ⁇ M).
- Homolog-scanning mutagenesis (Cunningham, B. C, Jhurani, P., Ng, P. & Wells, J. A. Science 243, 1330-1335 (1989)) was used to further localize the epitope on hGH for the hPRLbp (Table 10).
- variants of hGH that contained segment substitutions (7 to 30 residues long) derived from a non-binding homolog, pGH, or binding competent homologs, hPRL and hPL were analyzed for binding to the hPRLbp.
- pGH 48-52
- the only exception is hPRL (22-33) which causes a > 15-fold reduction in binding affinity to the hPRLbp.
- binding to the hPRLbp is very sensitive to mutations in hGH near the central portion of helix 1 and the loop region between residues 57 and 73.
- the alanine substitutions causing greater than a 4-fold reduction in binding affinity to the hPRLbp are in the central portion of helix 1 (including residues His18, His21, and Phe25), a loop region (including Ile58, Asn63, and Ser62) and the middle of helix 4 (comprising Arg167, Lys168, Lys172, Glu174, Phe176 and Arg178). These twelve residues form a patch when mapped upon a structural model of hGH (Fig. 7A). The most disruptive alanine substitutions in helix 1 and helix 4 project in the same direction. Three of these residues (His18, His21, and Glu174) along with His188 from the hPRLbp are believed to comprise the binding site for Zn 2+ that is required for the high affinity hGH-hPRLbp complex.
- the mutational analysis show that there are significant differences between the epitopes on hGH for the hGHbp and hPRLbp (Fig. 7).
- the net charge in the epitope on hGH for the hPRLbp is +5 (defined by residues causing ⁇ 4-fold reduction in affinity (Fig 7A).
- This strongly electropositive charge cluster is surrounded by a series of important hydrophobic residues, Phe25, Ile58, Tyrl64, and Phel76.
- Zinc is not required for formation of the hGH-hGHbp complex and the hGHbp epitope (Fig. 7B) is notably less electropositive (net charge for residues causing ⁇ 4-fold disruptions is +1).
- the hPRLbp epitope is elliptically shaped compared to the more circularly-shaped hGHbp.
- Fig. 7A, 7B overlap but do not superimpose.
- Ile58, Lys172, and Phel76 are important for binding to either receptor (Fig. 7C).
- Other determinants are more important for binding to the hPRLbp (especially those involved in the Zn 2+ site) and others for selective binding of hGHbp (notably Phe10, Glu56, Asp171, and Arg64).
- IL-2 receptors Robot, R. J., Greene, W. C. & Rusk, C. M. /. Exp. Med. 160, 1126-1146 (1984); Robb, R. J. Rusk, C. M. & Neeper, M. P. Proc. natn. Acad. Sci. U.S ⁇ . 85, 5654-5658 (1988)); and ANP receptors (Chang, M. S., Lowe, D. G., Lewis, M., Hellmiss, R., Chen, E., & Goeddel, D. V., Nature 341, 68-72 (1989)).
- receptor specific hormone analogs can greatly simplify this task; for example, catecholamine analogs were used to characterize ⁇ -adrenergic receptor subtypes and link specific receptor function to particular pharmacologic responses (Lefkowitz, R. J., Studel, J. M. & Caron, M. G. A. Rev. Biochem. 52, 159-186 (1983)).
- the receptor specific variants of hGH should be key reagents for probing the role of the hGH and hPRL receptors in the complex pharmacology of hGH, and for identifying other receptors for hGH.
- Prolactin receptor binding protein lacking the transmembrane region was constructed as shown in Figure 5.
- Figure 5 shows a comparison of the mature hGHbp and the mature hPRLbp. Each of these binding proteins lacks the transmembrane region.
- Variants of the PRLbp may be constructed with additional polypeptide sequences at the amino or carboxy terminals.
- Human GHBP has been described and lacks an effective binding site for zinc ion.
- DNA encoding asparagine 218 can be mutated by conventional methods to create GHBP variants that has affinity for growth hormone (Example 14). Using such conventional methods, the DNA encoding asparagine 218 was modified to encode alanine or histidine at position 218.
- histidine 218 resulted in a 30-fold increasing in affinity for hGH in the presence of zinc.
- Such complexes of hGH and GHBP incorporating zinc ion as a binding cofactor may be used as pharmaceutical formulations for therapeutic administration.
- Variants of polypeptide hormones or their receptors and binding proteins may be modified using site-directed mutagenesis or other well-known methods to modify amino acid residues to either delete required metal ion binding sites or to insert a metal ion binding site.
- the deletion of a metal ion binding site is exemplified by the modifications to human growth hormone and the modification to the soluble prolactin receptor protein.
- the amino acid sequence of a polypeptide hormone or a hormone receptor not containing a metal ion binding site may be modified to create a variant that contains a metal ion binding site.
- Such a modification is exemplified by the modification to the human growth hormone binding protein by inserting histidine 218 in place of arginine 218 , resulting in the formation of a hormone-receptor complex containing zinc ion.
- the method of modifying a mammalian polypeptide hormone-receptor complex not containing a metal ion binding site to contain a metal ion binding site comprises determining the amino acid sequence of a polypeptide hormone or polypeptide hormone receptor not containing a metal ion binding site wherein the determined amino acid sequence has regions of homology with a known polypeptide hormone or receptor having a metal ion binding site.
- the amino acid sequence of the mammalian polypeptide hormone or hormone receptor not containing a metal ion binding site is modified to contain one or more amino acids analogous to the polypeptide hormone or hormone receptor containing a metal ion binding site. This results in the insertion of a metal ion binding site.
- the preferred metal ion binding site is zinc, although other metals may be used such as iron, nickel, copper, magnesium, manganese, cobalt, calcium and selenium.
- Selection methods for isolating the polypeptide hormone variants include any method that permits selection of those variants forming stable complexes with a metal ion, for example, phagemid isolation methods.
- mice were immunized with E. coli derived hPRLbp using standard methods and serum was collected after each boosting.
- Serum was passed over an hGH column (7) in which the hPRLbp was covalently cross-linked to the hGH by reaction with dimethylsuberimidate (10 mM final in phosphate buffered saline; PBS).
- the hGH-hPRLbp column was washed sequentially with 2 M NaCl, 8 M urea, and 3 M KSCN to remove non-covalently bound components.
- Serum was passed over an hGH column to remove any anti-hGH antibodies that may have been produced from a low level contamination by hGH during hGH affinity purification by the hPRLbp
- the flow-through was adsorbed onto the hGH-hPRLbp column, washed with 1 M NaCl, and eluted with 3 M KSCN.
- the non-blocking anti-hPRLbp antibodies were dialyzed into PBS and titered for the optimal concentration needed to precipitate hPRLbp in the assay.
- DNA sequence encoding the parent polypeptide is cloned and manipulated so that is may be expressed in a convenient host.
- DNA encoding parent polypeptides can be obtained from a genomic library, from cDNA derived from mRNA, from cells expressing the parent polypeptide or by synthetically constructing the DNA sequence (Maniatis, T., et al. [1982] in Molecular Cloning. Cold Springs Harbor Laboratory, N. Y.).
- E. coli K12 strain 294 (ATCC No. 31446) may be used as E. coli B.
- E. coli X1776 ATCC No. 31557
- R CQH c600 and c600hf1 E. coli W3110 (F_, ⁇ _, prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis and other
- enterobacteriaceae such as Salmonell typhimurium or Serratia marcesans. and various pseudomanas species.
- the preferred prokaryote is E. coli W3110 (ATCC No. 27325).
- the polypeptides When expressed in prokaryotes the polypeptides typically contain an N-terminal methionine or a formyl methionine, and are not glycosylated.
- eukaryotic organisms such as yeast cultures or cells derived from multicellular organism may be used.
- any such cell culture is workable.
- interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a repeatable procedure (Tissue Culture. Academic Press, Kruse and Patterson, editors [1973]).
- useful host cell lines ar VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7 and MDCK cell lines.
- plasmid vectors containing replication and comeol sequences which are derived from species compatible with the host cell are used in connection with these hosts.
- the vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells.
- E. coli may be transformed using pBR322, a plasmid derived from an E. coli species (Mandel, M. et al. ri9701 J. Mol. Biol. 53 . 154). Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for selection.
- a preferred vector is pBO475 (Fig 8). This vector contains origins of replication for phage and E. coli which allow it to be shuttled between such hosts thereby facilitating mutagenesis and expression.
- “Expression vector” refers to DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host.
- control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribsome finding sites, and sequences which control termination of transcription and translation.
- the vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
- "plasmid” and “vector” are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.
- operably linked when describing the relationship between two DNA or polypeptide regions, simply means that they are functionally related to each other.
- a presequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence.
- a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
- segment-substituted DNA sequences which encode for the changes in amino acid sequence defined by the analogous segment being substituted.
- segmentsubstituted polypeptides are obtained.
- recovery of the parent polypeptide or segment-modified polypeptide may be facilitated by expressing and secreting such molecules from the expression host by use of an appropriate signal sequence operably linked to the DNA sequence encoding the parent polypeptide or segment-modified polypeptide.
- Table 1 illustrates zinc dependence for binding of hGH or hPRL to their purified binding proteins (bp).
- Dissociation constants (K D ) for binding to hGHbp (0.1 nM final) were measured in assay buffer (20 mM Tris-HCl (pH 7.5), 0.1 percent w/v BSA), by competitive displacement of [ 125 I]hGH (2, 5-7).
- Binding to the hPRLbp (0.01 nM final) was measured in assay buffer containing 50 ⁇ M ZnCl 2 as described for Fig. 2.
- Table 3 illustrates the ratio of bound to free 65 Zn 2+ (0.2 ⁇ M total) at a fixed concentration of hGH mutant and hPRLbp (each 2 ⁇ M ). 65 ZnCl 2 was allowed to equilibrate in dialysis cells, and bound and free zinc concentrations were determined as described for Fig. 3.
- Figure 2 illustrates the binding of [ 125 I]hGH to the hPRLbp in the presence of 0.1 percent BSA (crystallized high grade fraction V; Sigma), 140 mM NaCl, 10 mM MgCl 2 , 20 mM Tris (pH 7.5) and variable concentrations of total ZnCl 2 .
- BSA crystallized high grade fraction V
- a fixed 1:1 ratio of hGH and hPRLbp (0.01 nM final) was incubated 16 h in the presence of the indicated concentration of ZnCl 2 and the bound [ 125 I]hGH was immunoprecipitated using affinity purified rabbit polyclonal antibodies directed against the hPRLbp.
- the zinc concentration exceeded 100 ⁇ M, some protein precipitated, thus reducing the amount of native hGH-hPRLbp complex formed.
- FIG. 3 illustrates equilibrium dialysis for binding of 65 Zn 2+ to the
- hGH-hPRLbp complex All stock solutions were made from metal-free deionized water, and reagents were of highest quality available. Plastic dialysis cells and 3500 molecular weight cutoff membranes (preboiled in 5 percent w/w NaHCO 3 and washed) were soaked in 1 mM EDTA and washed thoroughly with dialysis buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 (used to reduce non-specific binding of Zn 2+ ) dialysis membrane) and 140 nM NaCl.
- a 0.34 mM stock solution of 65 ZnCl 2 (500 ⁇ Ci/0.1 ml in 50 mM HCl; DuPont) was prepared from a 1 M ZnCl 2 , 0.05 M HCl stock solution prepared gravimetrically from anhydrous ZnCl 2 .
- One-half of the dialysis cell (0.2 ml total) contained hGH-hPRLbp (1.2 ⁇ M final) in dialysis buffer and the total zinc concentration was diluted over a range of 0.1 to 10 ⁇ M.
- 65 ZnCl 2 was added initially on the side of the cell lacking hGH and the hPRLbp. Cells were sealed and rotated slowly for 16 hr at 25oC. Aliquots (50 ⁇ l) from each half of the dialysis cell were counted, and bound and free zinc concentrations were calculated. The binding studies were performed in the absence of carrier protein to avoid adventitious binding of Zn 2+ .
- Figure 4 illustrates the proposed Zn 2+ binding site on hGH that mediates binding to the hPRLbp.
- Helical wheel projections show the amphipathic character of helix 1 and 4 with polar (shaded) and charged residues (blackened) on one face of the helix and non-polar (open) on the other.
- His21, and Glu174, which are involved in binding hGH to the hPRLbp are shown ( ⁇ ).
- the region where hGH binds to the hGHbp is defined roughly by the shaded circle.
- Residues marked by the symbols•, ⁇ , ⁇ and O represent sites where alanine mutations in hGH cause reductions of 2- to 4-fold, 4- to 10-fold, greater than 10-fold, or 4-fold increase in binding affinity for the hGHbp, respectively.
- Table 4 illustrates the effect of mutations at conserved His and Cys residues in hPRLbp on binding of hGH in the presence of ZnCl 2 . Mutants of the hPRLbp were produced by site-directed mutagenesis (25), purified and assayed i ⁇ . the presence of Zn 2+ as described in Figs. 1, 2, and Table 1.
- Table 5 illustrates the effect of divalent cations at physiological total serum concentrations (19) on the complexation of [ 125 I]hGH to hPRLbp (at 60 or 5,000 pM).
- MnCl 2 -4 H 2 O, MgCl 2 -6 H 2 O were obtained from Johnson-Matthey Purotonic, Sigma, Mallinckrodt, Mallinckrodt and Johnson-Matthey Purotonic, respectively.
- Binding assays were performed as described in Fig. 2 in 0.1% BSA, 140 mM NaCl, 20 mM Tris (pH 7.5) at 25oC. The percentage of complex formed was calculated from the ratio of the amount of [ 125 I]hGH-hPRLbp complex immunoprecipitated to total [ 125 I]hGH present in the assay.
- FIG. 1A illustrates the diagram of plasmid phPRLbp(1-211) which directs secretion of the hPRLbp into the periplasm of E. coli.
- the hPRLbp gene fragment is transcribed under control of the alkaline phosphatase (phoA) promoter and secreted under direction of the stll signal sequence. Genes are indicated by arrows, replication origins by circles, and restriction sites used in the construction are indicated.
- a cDNA encoding the hPRL receptor (3) in a Bluescript plasmid (Stratagene) was purchased from Dr. Paul Kelly (Royal Victorial Hospital, McGill University, Montreal, Canada). Site-directed mutagenesis (25) using an oligonucleotide with the sequence
- 5'-AGCCACAGAGATAACGCGlCTATGTATCATTCAT-3' (Seq ID 4) was performed on this plasmid to introduce a stop codon and Mlul restriction site (indicated by asterisks and underline, respectively) after the threonine 211 codon which immediately precedes the transmembrane domain of the receptor.
- the 600 bp Bglll-Mlul fragment from this plasmid was then cloned into the Nsil-Mlul backbone of plasmid phGHbp (1-246) (Boutin, J. M. et al, Mol Endocrinol. 3, 1455 (1989)).
- a synthetic linker that spans the Nsil and Bglll sites was used to fuse the hPRLbp onto the Stll secretion signal sequence and restore the 5' end of the hPRLbp gene.
- the bottom strand of this linker has the sequence 5'- GATCTCAGGTTTTCCAGGA GGTAACTGTGCA- 3' (Seq ID 5).
- the top strand is complementary to this but 4 bp shorter on each end to match the restriction site termini. Dideoxy sequencing (26) was used to confirm the construction.
- Figure 1B illustrates Coomassie blue stained SDS-PAGE (12.5 percent) (U. K.
- hPRLbp Laemmli, Nature 227, 680 (1970)) of purified hPRLbp.
- the hPRLbp was purified essentially as described for the hGHbp except that 50 ⁇ M ZnCl 2 was added to the ammonium sulfate precipitate prior to solubilizing and loading onto the hGH affinity column.
- the column was washed with 1 M KSCN and eluted with 2 M KSCN plus 50 mM NaCl, 0.02% NaN 3 , 20 mM Tris-HCl (pH 7.5). The eluate was dialyzed into the same buffer minus KSCN and stored frozen (at -70°C).
- Lanes 1-5 are an E. coli periplasmic fraction, the (NH 4 ) 2 SO 4 precipitate, the protein after hGH affinity
- a synthetic hGH gene was made that had 18 unique restriction sites evenly distributed without altering the hGH coding sequence.
- the synthetic hGH DNA sequence was assembled by ligation of seven synthetic DNA cassettes each roughly 60 base pairs (bp) long and sharing a 10 bp DNA fragment shown form Nsil to Bg1II. The ligated fragment was purified and excised from a
- polyacrylamide gel and cloned into a similarly cut recipient vector, pB0475, which contains the alkaline phosphatase promoter and stll signal sequence (Chang, C. N., et al.
- pBO475 was constructed as follows: the f1 origin DNA from filamentous phage contained on a Dral, Rsal fragment 475bp in length was cloned into the unique PvuII site of pBr322 to make plasmid p652. Most of the tetracycline resistance gene was then deleted by restricting p652 with Nhel and Narl, filling the cohesive ends in with DNA polymerase and dNTPs and ligating the large 3850bp fragment back upon itself to create the plasmid p ⁇ 652.
- p ⁇ 652 was restricted with EcoRI, EcoRV and the 3690bp fragment was ligated for a 1300bp EcoRI, EcoRV fragment from phGH4R (Chang, C. N., et al [1987] Gene 55, 189) containing the alkaline phosphatase promoter, STII signal sequence and natural hGH gene.
- This construction was designated as pBO473.
- Synthetically derived DNA was cloned into pBO473, was restricted with Nsil, Bglll, and ligated to a 420pb Nsil, Hindlll fragment and a 1170bp hindll, Bglll fragment, both derived from synthetic DNA.
- the resulting construction pB0475 contains DNA coding for the natural polypeptide sequence of hGH but possesses many new unique restriction sites to facilitate mutagenesis and further manipulation of the hGH gene.
- the unique restriction sites in hGH sequence in pBo475 allowed insertion of mutagenic cassettes (Wells, J. A., et al. [19851 Gene 34. 315) containing DNA sequences encoding analogous segments from the analogs pGH, hPL and hPRL.
- the hGH and hGH variants were purified as follows: to 200g of cell paste, four volumes (800ml) of 10mM tris pH 8.0 was added. The mixture was place on an orbital shaker at room temperature until the pellets were thawed. The mixture was homogenized and stirred for an hour in a cold room. The mixture was centrifuged at 7000 for 15 min. The supernatant was decanted and ammonium sulfate was added to 45% saturation (277 g/l) and stirred at room temperature for one hour. After centrifugation for 30 minutes at 11,000g, the pellet was resuspended in 40ml lOmM tris pH 8.0.
- E. coli W3110. degP (Strauch, K. L., et al. [1988] PNAS USA 85. 1576) was transformed with the expression vector and grown in low phosphate media (Chang, C. N. [1987] Gene 55, 189) in a fermenter at 30oC.
- the 246 amino acid hGHbp was used to generate preliminary data.
- a slightly shorter hGHbp containing amino acids 1 through 238 was used in the examples herein. The results obtained with that receptor were indistinguishable from those obtained with the 246 amino acid hGHbp.
- hPL Human placental lactogen
- hPL The overall sequence of hPL is 85% identical to hGH. Within the three regions that broadly constitute the receptor binding epitope on hGH, hPL differs at only seven positions and contains the following substitutions: P2Q, I4V, N12H, R16Q, E56D R64M, and I179M. (In this nomenclature the residues for wild-type hGH is given in single-letter code, followed by its position in mature hGH and then the residue found in hPL; a similar nomenclature is used to describe mutants of hGH.) Single alanine substitutions have been produced in hGH at each of these seven positions.
- the alanine substitutions were found to cause 2-fold or greater reduction in binding affinity including I4A, E56A, R64A and I179A.
- the alanine substitutions have a greater effect on binding than homologous substitutions from human prolactin. Therefore, the effect of some of the substitutions from hPL introduced into hGH were investigated. Whereas the I179A substitution caused a 2.7-fold reduction in affinity, the I179M caused only a slight 1.7-fold effect.
- the R64A and R64M substitutions caused identical and much larger reduction (about 20-fold) in binding affinity.
- the double mutant (E56D:R64M) in hGH was even further reduced in affinity by a total of 30-fold.
- E56D and R64M primarily determine the differences in receptor binding affinity between hGH and hPL.
- the double mutant D56E, M64R in hPL therefore substantially enhances its binding affinity for the hGH receptor.
- Additional modifications such as M179I and V4I also enhance binding of hPL to the hGH receptor.
- NNN represents the new codon at position 174 and asterisks indicate the mismatches to eliminate the Kpnl site starting at codon 178.
- Mutant codons were as follows: Gln, CAG; Asn, AAC; Ser, AGC; Lys, AAA; Arg, AGG; His, CAC; Gly, GGG ; Val, GTG; GTG; Leu, CTG.
- the plasmid pool was enriched for the mutation by restriction with Kpnl to reduce the background of wild-type sequence. All mutant sequences were confirmed by dideoxy sequence analysis (S anger, F., et al. [1977] Proc. Natl. Acad. Sci. USA 74, 5463-5467).
- Table 7 illustrates the comparative binding of hGH variants to the hPRLbp and hGHbp.
- Mutants of hGH produced by homolog-scanning mutagenesis are named according to the extremes of the segment substituted from the various hGH homologs: pGH, hPL, or hPRL. The exact description of the mutations introduced is given by the series of single mutants separated by commas. The component single mutants are designated by the single-letter code for the wild-type residue followed by its codon position in mature hGH and then the mutant residue. Mutants of hGH were produced and purified as previously described.
- Binding of the hGH mutants to the hPRLbp was measured by competitive displacement of [ 125 I]hGH as described for the hGHbp except that assays included 50 ⁇ M ZnCl 2 and 10 mM MgCl 2 .
- Affinity purified rabbit polyclonal antibodies raised against the hPRLbp were used to precipitate the hGH -hPRLbp complex.
- the relative reduction in binding affinity (K D (mut)/K D (hGH)) reported for the hGHbp was taken from S. S. Abdel-Meguid et al., Proc. Natl. Acad. Sci. U.S.A. 84, 6434 (1987).
- the change in receptor preference was calculated by dividing the ratio of the relative reduction in binding affinity for the hPRLbp by that for the hGHbp.
- WT wild-type
- SD standard deviation.
- the alanine substitutions causing greater than a 4-fold reduction in binding affinity to the hPRLbp are in the central portion of helix 1 (including residues His18, His21, and Phe25), a loop region (including Ile58, Asn63, and Ser62) and the middle of helix 4 (comprising Arg167, Lys168, Lys172, Glu 174, Phe176 and Arg178). These twelve residues form a patch when mapped upon a structural model of hGH (Fig.7A). The most disruptive alanine substitutions in helix 1 and helix 4 project in the same direction. Three of these residues, (His18, His21, and Glu 174) along with His 188 from the hPRLbp, are believed to comprise the binding site for Zn 2+ that is required for the high affinity hGH-hPRLbp complex.
- Table 9 illustrates the binding of double mutants of hGH designed to discriminate between the hGH and hPRL binding proteins (hGHbp and hPRLbp). Mutants of hGH were prepared by site-directed mutagenesis, purified (Cunningham, B. C. & Wells, J. A. Science 244, 1081-1085 (1989)), and assayed for binding to the hGHbp (Fuh, G., Mulkerrin, M. G., Bass, S., McFarland, N., Brochier, M., Bourell, J. H., Light, D. R., & Wells, J.A. /. Biol Chem. 265, 3111-3115 (1990)) or hPRLbp as described in Table 7.
- Table 10 illustrates the additive effects of mutations in hGH upon binding to the hGH or hPRL binding proteins.
- the change in the free energy of binding ( ⁇ G binding ) for the variant relative to wild-type hGH was calculated from the reduction in binding affinity according to: The values of (K D (muty/sToOiGH)
- Figure 6 illustrates the competition between hGH and hPRL binding proteins for binding to [ 125 I]hGH.
- concentrations of [ 125 I]hGH and purified hGHbp domain were fixed at 0.2 nM.
- Increasing concentrations of purified hPRLbp were added and the three components were allowed to reach equilibrium in assay buffer containing 25 ⁇ M ZnCl 2 , 20 mM Tris-HCl (pH 7.5) and 0.1 percent w/v BSA for 12 h at 25°C.
- a non-neutralizing monoclonal antibody to the hGHbp (Mab263, Bernard, R., Bundesen, P. G., Rylatt, D. B., & Waters, M. J.
- Figure 7 illustrates the structural model of hGH based on a folding diagram for pGH determined from a 2.8 A resolution X-ray structure (Abdel-Megnid, S. S., Shieh, H. S., Smith, W. W., Dayringer, H. E., Violand, B. N., & Bentle, L. A. Proc. Natn. Acad. Sci. U.S.A. 84, 6434-6437 (1987)).
- Panel A shows a functional map of the hPRLbp epitope
- Panel B shows that determined previously for the hGH bp (taken from S. S. Abdel-Meguid et al., Proc. Natl Acad.
- the symbols•, ⁇ , ⁇ and ⁇ represent sites where alanine substitutions cause a 2- to 4- fold, 4- to 10-fold, 10-fold to 80-fold, or >80-fold reductions in binding affinity, respectively, for each receptor binding domain.
- the O in the hGHbp epitope represents the position of E174A that causes greater than a 4-fold increase in binding affinity.
- Panel C shows sites where alanine mutants reduce binding affinity by > 10-fold for hPRLbp (D) or >5-fold for the hGHbp ( ⁇ ) without affecting substantially the binding to the hGHbp or hPRLbp, respectively.
- the A symbols show sites where alanine mutants disrupt binding to both receptors by > 10-fold.
- the absence of zinc causes formation of a PEG-precipitate of hPL in assays using labelled hPRL.
- prolactin receptor can compete with hPL for binding to the prolactin. The binding of hPL to the prolactin receptor has a Kd of greater than 10 nM.
- Table 12 shows the binding of hPL mutants to the hPRL receptor in the presence of 10 mM MgCl 2 plus 50 ⁇ M zinc using displacement of labelled hGH from the prolactin receptor.
- V4LD56E,M64R,M179I 48 1.5 Kd (nM) values for mutants of hPL were determined using recombinant human growth hormone binding protein (hGHbp) (Table 13).
- hGH human growth hormone
- hPRLr human prolactin receptor
- Table 14 shows a comparison of part of the amino acid sequence of growth hormone and prolactin receptors from several different species.
- the histidine at position 188 in the prolactin receptors is conserved; furthermore, no histidine is present in any of the growth hormone receptors at the corresponding position (residue 218).
- Site-directed mutagenesis of residue 188 in hPRLbp has demonstrated that it is essential for the high affinity zinc-mediated binding of hGH (Example 1, Table 4).
- mutant binding proteins were expressed in E. Coli KS330 cells and either partially purified by fractionation with 45% ammonium sulphate or extensively purified using an hGH affinity column. Binding of the mutant receptors to hGH in the presence of 50 ⁇ M ZnC-2 or lmM EDTA was measured by competitive displacement of [ 125 I] hGH by unlabelled hGH as previously described (S. A. Spencer et al. [1988], J. Biol. Chem. 263 pp.7862-7867).
- the amount of wild-type or mutant binding protein used in the assay was determined empirically by titration of the binding protein with [ 125 I] hGH: the concentration of binding protein in the assay was chosen to be that which gave approximately 20% [ 125 I] hGH bound in the preliminary titration.
- Table 15 shows the effect of 50 ⁇ M ZnCl 2 on binding of hGH to wild-type hGHbp and to hGHbp mutants N218H and N218A.
- N218H mutant the binding is not significantly different to wild-type in the absence of zinc, but in the presence of 50 ⁇ M ZnCl2 the binding is dramatically (>30-fold) tighter, presumably due to the incorporation of a zinc ligand into the interaction.
- the N218 A mutant shows the effect of removing the asparagine side-chain without introducing a zinc ligand: binding to growth hormone is approximately 2-fold weaker than wild-type in the absence of Zn 2+ and 6-fold tighter in the presence of Zn 2+ .
- GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC 3110 CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC 3160 GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC 3210 GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC 3260 CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG 3310 TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG 3360 TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC 3410 CCGGTAAGAC ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT 3460 TAGCAGAGCG AGGTATGTAG GCGG
- GCGTCAACAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT 4510 CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC 4560
- GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAAAA CAGGAAGGCA 4660 AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA 4710 TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC 4760
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- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US56893690A | 1990-08-17 | 1990-08-17 | |
US568,936 | 1990-08-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1992003478A1 true WO1992003478A1 (en) | 1992-03-05 |
Family
ID=24273373
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1991/005856 WO1992003478A1 (en) | 1990-08-17 | 1991-08-16 | Metal ion mediated receptor binding of polypeptide hormones |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0550629A1 (en) |
JP (1) | JPH06500559A (en) |
CA (1) | CA2087267A1 (en) |
WO (1) | WO1992003478A1 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0787497A3 (en) * | 1996-02-02 | 1999-04-14 | MITSUI TOATSU CHEMICALS, Inc. | Pharmaceutical preparation containing human growth hormone |
US7378506B2 (en) | 1997-07-21 | 2008-05-27 | Ohio University | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins |
EP2133427A1 (en) | 2004-01-14 | 2009-12-16 | Ohio University | Methods of producing peptides/proteins in plants and peptides/proteins produced thereby |
US7855066B1 (en) | 1999-06-28 | 2010-12-21 | Genentech, Inc. | Methods for making Apo-2 ligand using divalent metal ions |
EP2348043A1 (en) | 2001-10-02 | 2011-07-27 | Genentech, Inc. | APO-2 ligand variants and uses thereof |
EP2500032A1 (en) | 2002-06-24 | 2012-09-19 | Genentech, Inc. | APO-2 ligand/trail variants and uses thereof |
US8623812B2 (en) | 2004-04-19 | 2014-01-07 | Ohio University | Cross-linkable glycoproteins and methods of making the same |
US8871468B2 (en) | 1997-07-21 | 2014-10-28 | Ohio University | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0089666A2 (en) * | 1982-03-22 | 1983-09-28 | Genentech, Inc. | Human growth hormone variants |
WO1990004788A1 (en) * | 1988-10-28 | 1990-05-03 | Genentech, Inc. | Method for identifying active domains and amino acid residues in polypeptides and hormone variants |
-
1991
- 1991-08-16 WO PCT/US1991/005856 patent/WO1992003478A1/en not_active Application Discontinuation
- 1991-08-16 JP JP51662491A patent/JPH06500559A/en active Pending
- 1991-08-16 CA CA 2087267 patent/CA2087267A1/en not_active Abandoned
- 1991-08-16 EP EP19910917895 patent/EP0550629A1/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0089666A2 (en) * | 1982-03-22 | 1983-09-28 | Genentech, Inc. | Human growth hormone variants |
WO1990004788A1 (en) * | 1988-10-28 | 1990-05-03 | Genentech, Inc. | Method for identifying active domains and amino acid residues in polypeptides and hormone variants |
Non-Patent Citations (8)
Title |
---|
European Journal of Biochemistry, volume 153, no. 3, 1 March 1985 (Berlin, DE) T. Tokunaga et al.: "Synthesis and expression of a human growth hormone (somatotropin) gene mutated to change cysteine-165 to alanine", pages 445-449, see the whole article * |
Journal of Biological Chemistry, volume 266, no. 17, 15 June 1991 (Baltimore, US) H.B. Lowman et al.: "Mutational analysis and protein engineering of receptor-binding determinants in human placental lactogen", pages 10982-10988, see the whole article * |
Proceedings of the National Academy of Sciences of USA, volume 88, no. 10, 15 May 1991 (Washington, US) S.H. Bass et al.: "A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor", pages 4498-4502, see the whole article * |
Proceedings of the National Academy of Sciences of USA, volume 88, no. 8, 1 April 1991 (Washington, US) B.C. Cunningham et al.: "Rational design of receptor-specific variants of human growth hormone", pages 3407-3411, see the whole article * |
Science, volume 244, 2 June 1990 (Lancaster, PA, US) B.C. Cunningham et al.: "High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis", pages 1081-1085, see the whole article * |
Science, volume 247, 23 March 1990 (Lancaster, PA, US) B.C. Cunningham et al. "Engineering human prolactin to bind to the human growth hormone receptor", pages 1461-1465, see the whole article * |
Science, volume 250, 21 December 1990 (Lancaster, PA, US) B.C. Cunningham et al.: "Zinc mediation of the binding of human growth hormone to the human prolactin receptor", pages 1709-1712, see the whole article * |
Science, volume 253, no. 5019, 2 August 1991 (Washington DC, US) B.C. Cunningham et al.: "Dimerization of human growth hormone by zinc", pages 545-548, see the whole article * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0787497A3 (en) * | 1996-02-02 | 1999-04-14 | MITSUI TOATSU CHEMICALS, Inc. | Pharmaceutical preparation containing human growth hormone |
US6013773A (en) * | 1996-02-02 | 2000-01-11 | Mitsui Chemicals, Inc. | Pharmaceutical preparation containing human growth hormone |
US7378506B2 (en) | 1997-07-21 | 2008-05-27 | Ohio University | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins |
US8871468B2 (en) | 1997-07-21 | 2014-10-28 | Ohio University | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins |
US8563687B2 (en) | 1997-07-21 | 2013-10-22 | Ohio University | Synthetic genes for plant gums and other hydroxyproline rich glycoproteins |
EP2339003A2 (en) | 1999-06-28 | 2011-06-29 | Genentech, Inc. | APO-2 ligand substitutional variants |
EP2311956A1 (en) | 1999-06-28 | 2011-04-20 | Genentech, Inc. | Methods for making APO-2 ligand using divalent metal ions |
US7855066B1 (en) | 1999-06-28 | 2010-12-21 | Genentech, Inc. | Methods for making Apo-2 ligand using divalent metal ions |
EP2348043A1 (en) | 2001-10-02 | 2011-07-27 | Genentech, Inc. | APO-2 ligand variants and uses thereof |
EP2500032A1 (en) | 2002-06-24 | 2012-09-19 | Genentech, Inc. | APO-2 ligand/trail variants and uses thereof |
EP2133427A1 (en) | 2004-01-14 | 2009-12-16 | Ohio University | Methods of producing peptides/proteins in plants and peptides/proteins produced thereby |
US9006410B2 (en) | 2004-01-14 | 2015-04-14 | Ohio University | Nucleic acid for plant expression of a fusion protein comprising hydroxyproline O-glycosylation glycomodule |
US8623812B2 (en) | 2004-04-19 | 2014-01-07 | Ohio University | Cross-linkable glycoproteins and methods of making the same |
Also Published As
Publication number | Publication date |
---|---|
JPH06500559A (en) | 1994-01-20 |
EP0550629A1 (en) | 1993-07-14 |
CA2087267A1 (en) | 1992-02-18 |
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