Wild-type hGH Research Articles

Prolonged, Controllable Protein Production of cDNA-Encoded hGH Maintained at Therapeutic Serum Levels Following One Systemic Administration of a Non-Viral, Non-Integrating, DNA-and Liposome-Based in vivo Gene Therapy Platform in Immunocompetent Mice

Abstract Recombinant human growth hormone (rhGH), the mainstay of hGH replacement therapy, is injected daily for years to enable children to achieve normal stature. Daily rhGH injections are required because its serum-T1/2 is <20 minutes. Human trials testing T1/2-extending rhGH proteins are promising, but even weekly rhGH administration poses potential short and long-term toxicity risks, including exacerbation of diabetes or hypertension and potential for oncogenesis. Frequent hGH injections produce non-physiologic IGF-I profiles, (peak serum IGF-I frequently supra-normal, trough IGF-I concentrations at baseline). Therefore, a safe, effective, single administration hGH approach that maintains hGH and IGF-I serum concentrations within their respective therapeutic ranges for approximately one year could offer significant advantages. Accordingly, DNARx tested HEDGESTM, it’s non-viral, non-integrating, DNA-and liposome-based systemic, in vivo gene therapy approach to produce long-term hGH serum levels within the therapeutic range (1 -10 ng/ml) in immunocompetent mice. Previously, one systemic HEDGES-human granulocyte colony stimulating factor (hG-CSF, serum T1/2<3 hours) cDNA administration produced durable therapeutic hG-CSF serum levels (Science advances, 2019, 5, 2019). Importantly, simply by modifying selected aspects of HEDGES’s DNA vector and liposomal components, the duration of serum hG-CSF protein production was controllable over a broad temporal range. Long-term assessment of serum hGH in immunocompetent mice was assessed by injecting either one HEDGES administration or one administration followed by one re-dose of DNA vectors expressing either A) wildtype hGH cDNA (HEDGES-1) or B) wildtype hGH fused to selected serum protein T1/2-extending DNA sequences (HEDGES-2), with these results: 1) One HEDGES-1 administration produced slowly-increasing, but sub-therapeutic hGH serum levels from day 7 to day 99 after injection. From day 99 on, serum hGH levels remained within the therapeutic range for > 230 days; 2) Concurrently, hGH-induced elevation of endogenous mouse serum IGF-I levels remained between 1 to 3 fold above baseline. 3) One HEDGES-1 re-injection was administered 35 days after initial injection, a time when hGH serum levels remained sub-therapeutic. By day 7, one re-injection further elevated cDNA-encoded hGH serum levels into the therapeutic range, maintaining them for >175 days; 4) One HEDGES-2 administration produced therapeutic hGH serum levels by day 7 that were maintained for >250 days. These studies demonstrate the feasibility of achieving very long-term therapeutic levels of hGH and IGF-I following 1-2 injections. DNARx is now focused on selectively modifying its HEDGES hGH cDNA vectors and liposomal components to produce hGH serum levels within the therapeutic range for the equivalent of one human year following administration

A novel peptide antagonist of the human growth hormone receptor

Excess circulating human growth hormone (hGH) in vivo is linked to metabolic and growth disorders such as cancer, diabetes, and acromegaly. Consequently, there is considerable interest in developing antagonists of hGH action. Here, we present the design, synthesis, and characterization of a 16-residue peptide (site 1-binding helix [S1H]) that inhibits hGH-mediated STAT5 phosphorylation in cultured cells. S1H was designed as a direct sequence mimetic of the site 1 mini-helix (residues 36–51) of wild-type hGH and acts by inhibiting the interaction of hGH with the human growth hormone receptor (hGHR). In vitro studies indicated that S1H is stable in human serum and can adopt an α-helix in solution. Our results also show that S1H mitigates phosphorylation of STAT5 in cells co-treated with hGH, reducing intracellular STAT5 phosphorylation levels to those observed in untreated controls. Furthermore, S1H was found to attenuate the activity of the hGHR and the human prolactin receptor, suggesting that this peptide acts as an antagonist of both lactogenic and somatotrophic hGH actions. Finally, we used alanine scanning to determine how discrete amino acids within the S1H sequence contribute to its structural organization and biological activity. We observed a strong correlation between helical propensity and inhibitory effect, indicating that S1H-mediated antagonism of the hGHR is largely dependent on the ability for S1H to adopt an α-helix. Taken together, these results show that S1H not only acts as a novel peptide-based antagonist of the hGHR but can also be applied as a chemical tool to study the molecular nature of hGH–hGHR interactions.

Site-specific PEGylation of Human Growth Hormone by Mutated Sortase A

Human growth hormone(hGH), a classic therapeutic protein, which promotes growth and wound healing, is released from the pituitary gland. As a protein drug, its short half-life is its main barrier to therapeutic efficacy. Various strategies have been designed to prolong its serum half-life, the most common of which is the conjugation with polyethylene glycol(PEG), as this has been shown to significantly extend protein’s serum half-life. However, PEGylation often results in random conjugation, which can lead to impaired protein function and hinder purification, characterization and evaluation of the PEGylated protein. Therefore, site specific PEGylation is a promising direction for PEG-protein conjugation. Here we took advantages of the mutated sortase A(7M) enzyme, which can enzymatically ligate the universal α-amino acids to a C-terminal tagged protein. This then allows specific modification of the C-terminal of hGH with PEG. This site-specific bound PEG-hGH has similar efficacy, receptor binding and cell proliferation as wild-type hGH; however, pharmacokinetic analysis demonstrates that its serum half-life is almost 24 times that of wild-type hGH. Herein, we provided a promising advancement in the development of site specific PEGylated therapeutic proteins.

Preclinical characterization of eleven new Cys-PEGylated hGH mutants

We synthesized and tested for biological activity eleven new PEGylated hGH derivatives. To this aim we used different strategies. A first group of PEGylable Cys derivatives was prepared by mutating into Cys single specific aminoacid residues located either in the connecting loop between helices 1 and 2 (Ile36 and Phe44) or in the connecting loop between helices 3 and 4 (Ile138 and Phe146). A second group of mutants was synthetized by inserting new Cys close to the aforementioned positions. Other PEGylable mutants were prepared by inserting a Gly4–Cys chain either at the N- or at the C-terminus of the hGH molecule. Finally, a Cys residue of the second hGH disulfide bond (Cys182–Cys189) was made available for PEGylation by mutating its companion Cys to open up the disulfide bridge. All these mutants were tested for their ability to affect Nb2 cell proliferation in vitro and showed different effects. Two mutations (Phe44Cys and +Cys47) severely impaired and two (Ile138Cys and Phe146Cys) increased hGH bioactivity. The mutation Cys189Ser was functionally silent whereas the remaining mutations caused a 15–50% decrease in activity. 20kDa-pegylation caused a dramatic decrease in bioactivity in all mutants. The r-hGH-(Ile138Cys)–Cys138–PEG derivative showed the highest activity (15% of wild-type unpegylated hGH) and was selected for pharmacockinetic studies in vivo. It showed a fivefold longer half-life and was significantly more effective than wild-type hGH in causing weight gain when given subcutaneously twice a week to hypophysectomized rats.In conclusion, even when it is directed to residues supposed to have a marginal role in the activity of this hormone, 20kDa pegylation has detrimental effect on hGH bioactivity. These effects may be counterbalanced by the increase in half-life as it happens in pegylated Ile138Cys–hGH that could represent a promising new long-acting hGH derivative.Focal points:•Bedside: Current therapy of GH deficiency still has the important limitation of being delivered by daily subcutaneous injections and this reduces the compliance of the small pediatric patients. There is, therefore interest in developing long-acting GH derivative such as the new ones that we present here.•Benchside: By investigating the effect of Cys substitutions at aminoacidic positions that had not been investigated in previous studies, we obtained evidence that Ile138 and Phe146 have a role in GH bioactivity in vitro.•Industry: The mono-pegylated hGH mutant described in this work represents a promising candidate for human clinical pharmacology studies as long-acting derivative of h-GH.•Community: By decreasing the frequency of GH injections, long acting GH could decrease the costs of GH treatment and increase medication adherence.

Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen-Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation.

Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering.

Toward gene therapy for growth hormone deficiency via salivary gland expression of growth hormone

Salivary glands are useful targets for gene therapeutics. After gene transfer into salivary glands, regulated secretory pathway proteins, such as human growth hormone, are secreted into saliva, whereas constitutive secretory pathway proteins, such as erythropoietin, are secreted into the bloodstream. Secretion of human growth hormone (hGH) into the saliva is not therapeutically useful. In this study, we attempted to redirect the secretion of transgenic hGH from the saliva to the serum by site-directed mutagenesis. We tested hGH mutants first in vitro with AtT20 cells, a model endocrine cell line that exhibits polarized secretion of regulated secretory pathway proteins. Selected mutants were further studied in vivo using adenoviral-mediated gene transfer to rat submandibular glands. We identified two mutants with differences in secretion behavior compared to wild-type hGH. One mutant, ΔN1-6 , was detected in the serum of transduced rats, demonstrating that expression of this mutant in the salivary gland resulted in its secretion through the constitutive secretory pathway. This study demonstrates that mutagenesis of therapeutic proteins normally destined for the regulated secretory pathway may result in their secretion via the constitutive secretory pathway into the circulation for potential therapeutic benefit.

The role of human growth hormone's C-terminal disulfide bridge

ObjectiveHuman growth hormone (hGH), as well as the other members of the same polypeptide hormone family, have a four-helix bundle structure linked by two disulfide bridges, C53–C165 and C182–C189 in hGH. The C-terminal disulfide bridge of growth hormone is evolutionally conserved but its role is unknown. Our aim was to determine its importance for GH structure and/or function. DesignWe disrupted the highly conserved C-terminal disulfide bridge of hGH by substituting one or both of its cysteines by alanines. Mutant and wild type hGH genes were expressed in human embryonic kidney (HEK)-293 cells and the hGH analogs were characterized in vitro regarding biological activity, stability and binding to GH receptor (GHR) as well as GH binding protein (GHBP). ResultsDisrupting the hGH C-terminal disulfide bridge significantly reduces binding affinity to GHR and GHBP. If one of the cysteines is removed, the stability of the molecule is reduced but this feature is reversed when both cysteines are absent. However, despite decreased binding affinity and stability, biological activity is only modestly decreased when the disulfide bridge is removed. ConclusionsOur study reveals the importance of the C-terminal disulfide bridge of GH for receptor binding and the detrimental effect of its unpaired cysteines on stability as well as, to a lesser extent, biological activity. This improved knowledge of structure–function relationships helps better understand the biology of GH and related molecules. This could have an impact on diagnosis and treatment of patients with growth disorders.

ATS fusion human growth hormone has anti‐ageing effects on old mice

Protein aggregation is a major stability problem of therapeutic proteins. Previous studies showed that the ATS (acidic tail of synuclein)‐hGH (human growth hormone) fusion protein was more stable and soluble than wild type hGH. However, bioactivity of the protein was not confirmed in in vivo experiment. We executed a comparative study for hGH and ATS‐hGH in old mice. As shown in preliminary data, we verified that hGH and ATS‐hGH had bio‐activity in cell line experiment. Then, we executed in vivo animal studies, 30 μg/100 μl of hGH and ATS‐hGH were subcutaneously injected to 8 month female mice twice per a week for 14 weeks. As a result, mouse had no change in body weight. However, fat content of abdominal region and triglyceride levels in blood were more significantly reduced in hGH and ATS‐hGH group than those of control group. Most of all, fat content of abdominal region of ATS‐hGH was more reduced than hGH treated group. Also, creatinine was induced, but, urea nitrogen was not increased in blood of hGH and ATS‐hGH group. These results suggest that hGH and ATS‐hGH increased muscle capacity. In addition, immunohistochemistry result through PCNA stanning showed that proliferation of liver cell was increased in hGH and ATS‐hGH group. These result suggest that bioefficacy of ATS‐hGH is better than hGH, particulary effect in reducing the abdominal fat and in increasing the muscle content of old female mice.

OR12,67 Age-dependent control of Akt phosphorylation in muscles from IGFBP-2 transgenic mice as a potential target for the metabolic effects of IGFBP-2

proliferation assays, the dose-response effect induced by the human growth hormone mutants did not differ from the effects induced by wildtype hGH. Besides, wildtype hGH and the four hGH variants were able to activate STAT5 to the same extent. However, it was observed that production and secretion of the human growth hormone variants were significantly lower than wildtype hGH, as determined by fluorescent sandwich-type immunoassays and confirmed with Western blot analysis. Particularly low secretion was assessed for P89L and V110F, which showed only 15% and 24% of wildtype hGH concentration, respectively. Co-expression of wildtype and mutant human growth hormone furthermore suggested inhibition of wildtype hGH production by mutant hGH. These results indicate that the short stature in patients with one of the four heterozygous missense mutations might be caused by an intracellular event within the somatotropic cells in the anterior pituitary leading to reduced production and/or secretion of the hGH variants as well as of wildtype hGH.

Characterization of Human γ-glutamyl hydrolase in solution demonstrates that the enzyme is a non-dissociating homodimer

Human γ-glutamyl hydrolase (hGH) is a key enzyme in the metabolism of folic acid and in the pharmacology of many antifolate drugs. hGH catalyzes removal of the poly-γ-glutamate chains of intracellular folic acid and antifolates. hGH crystallized as a homodimer with two putative active sites. However, the quaternary structure and the number of species of the enzyme in solution have not been determined. hGH has now been characterized using analytical ultracentrifugation and dynamic light scattering. HisTag fusion proteins of wild-type hGH, rat GH, and hGH expressed as a glycosylated protein were studied. Analyses of HisTag wild-type hGH were conducted over a range of protein concentrations (1.4–200 μM), ionic strengths (0–1 M NaCl), and pH (4.5–8.5). A single species with a molecular mass consistent with a homodimer was observed. Glycosylated hGH and HisTag rat γ-glutamyl hydrolase also formed very stable homodimers. The lack of dissociation of the dimer, the large monomer–monomer interface, and the presence of catalytically essential Tyr-36 in the homodimer interface sequences suggest that homodimer formation is required for the hGH monomer to fold into an active conformation. The conservation of hGH monomer–monomer interface sequences in other mammalian and plant γ-glutamyl hydrolase molecules suggests that they also exist as stable homodimers.

Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis

Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH.

Partial Redirection of Transgenic Human Growth Hormone Secretion from Rat Salivary Glands

Regulated secretory pathway proteins, when delivered as transgenes to salivary glands, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, for example, human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. The C terminus of hGH is highly conserved and contains a domain similar to the regulated pathway sorting domain of pro-opiomelanocortin (POMC). We hypothesized that, similar to POMC, mutation of this domain would divert hGH secretion from the regulated to the constitutive pathway, which in salivary glands leads to the bloodstream. Several mutations were made in the C terminus of the hGH cDNA and tested in vitro. One biologically active mutant containing E174A and E186A substitutions, and with an included C-terminal extension, was studied in greater detail. Compared with wild-type hGH, we found that this mutant hGH accumulated in the Golgi/trans-Golgi network and showed increased basal secretion in AtT20 cells, a model endocrine cell line. Importantly, in vivo, the mutant hGH displayed a relative increase in the proportion of constitutive pathway secretion seen from rat salivary glands, with a significantly lower saliva-versus-serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by reengineering its sorting determinants.

Computational analysis of ligand binding dynamics at the intermolecular hot spots with the aid of simulated tempering and binding free energy calculations

Equilibrium binding dynamics is studied for a panel of benzimidazole-containing compounds at the remodeled interface between human growth hormone (hGH) and the extracellular domain of its receptor (hGHbp), engineered by mutating to glycine hot spot residues T175 from the hormone and W104 from the receptor. Binding energetics is predicted in a good agreement with the experimental data for a panel of these small molecules that complement the engineered defect and restore the binding affinity of the wild-type hGH–hGHbp complex. The results of simulated tempering ligand dynamics at the protein–protein interface reveals a diversity of ligand binding modes that is consistent with the structural orientation of the benzimidazole ring which closely mimics the position of the mutated W104 hot spot residue in the wild-type hGH–hGHbp complex. This structural positioning of the benzimidazole core motif is shown to be a critical feature of the low-energy ligand conformations binding in the engineered cavity. The binding free energy analysis provides a plausible rationale behind the experimental dissociation constants and suggests a key role of ligand–protein van der Waals interactions in restoring binding affinity.

The Functional Binding Epitope of a High Affinity Variant of Human Growth Hormone Mapped by Shotgun Alanine-scanning Mutagenesis: Insights into the Mechanisms Responsible for Improved Affinity

A high-affinity variant of human growth hormone (hGH v) contains 15 mutations within site 1 and binds to the hGH receptor (hGHR) ∼400-fold tighter than does wild-type (wt) hGH (hGH wt). We used shotgun scanning combinatorial mutagenesis to dissect the energetic contributions of individual residues within the hGH v binding epitope and placed them in context with previously determined structural information. In all, the effects of alanine substitutions were determined for 35 hGH v residues that are directly contained in or closely border the binding interface. We found that the distribution of binding energy in the functional epitope of hGH v differs significantly from that of hGH wt. The residues that contributed the majority of the binding energy in the wt interaction (the so-called binding “hot spot”) remain important, but their contributions are attenuated in the hGH v interaction, and additional binding energy is acquired from residues on the periphery of the original hotspot. Many interactions that inhibited the binding of hGH wt are replaced by interactions that make positive contributions to the binding of hGH v. These changes produce an expanded and diffused hot spot in which improved affinity results from numerous small contributions distributed broadly over the interface. The mutagenesis results are consistent with previous structural studies, which revealed widespread structural differences between the wt and variant hormone–receptor interfaces. Thus, it appears that the improved binding affinity of hGH v site 1 was not achieved through minor adjustments to the wt interface, but rather, results from a wholesale reconfiguration of many of the original binding elements.

Growth hormone binding affinity for its receptor surpasses the requirements for cellular activity.

The human growth hormone (hGH)-receptor interaction was used to study the relationship between hormone-receptor affinity and bioactivity. hGH has two nonequivalent sites, called site 1 and site 2, that bind two molecules of receptor in a sequential fashion. We produced both site 1 and site 2 high-affinity hGH variants either by combining alanine mutants previously found to improve affinity at site 1 or by random mutagenesis of residues in site 2 followed by phage display and receptor binding selections. The two high-affinity variants, as well as one which combined them, were used in cell proliferation assays with FDC-P1 cells expressing the hGH receptor. Interestingly, none of these variants produced a change in the EC50 for cell proliferation or the levels of JAK2 tyrosine kinase phosphorylation. Next we studied the effect of a reduction in site 1 affinity on cell proliferation. A systematic series of hGH mutants were produced in which affinity for site 1 was reduced from 5- to 500-fold. Surprisingly, the EC50 for cell proliferation was unaffected until affinity was reduced about 30-fold from wild-type hGH. Thus, native hGH-receptor affinity is much higher than it needs to be for maximal JAK2 phosphorylation or cell proliferation. These studies begin to define basic functional tolerances for receptor activation that need to be considered in the design of hGH mimics.

Differential in vivo activities of bovine growth hormone analogues.

In rodents, bovine (b) growth hormone (GH) binds only to GH receptors, while human (h) GH binds to both GH and PRL receptors. The phenotypic consequences of expression of bGH and hGH in transgenic mice are different and, in some cases, opposite. In the present study, site-directed in vitro mutagenesis of the bGH gene was used systematically to eliminate its differences from hGH at one, two, three or four suspected of conferring lactogenic activity: D11, H18, S57 and T60, respectively (corresponding to sites 12, 19, 57 and 60 of the bGH molecule). The resulting bGH analogues were expressed in cell lines and in transgenic mice. All of the seven bGH analogues produced retained their ability to bind to GH receptors and exhibited somatogenic activity in vitro and in vivo. However, none of them were able to bind to PRL receptors or to elicit detectable lactogenic response in vitro. Transgenic animals expressing any of the generated analogues were characterized by gigantism and splanchnomegaly. The effects of expression of each of the double, triple or quadruple mutants on the seminal vesicle weight resembled the effects of wild-type hGH and differed from the effects of expression of wild-type bGH. There were differences between the effects of the expression of different bGH analogues on plasma PRL levels and on the PRL response to pharmacological blockade of catecholamine synthesis. Plasma LH levels in ovariectomized females were suppressed by several of the analogues tested, an effect not seen in animals expressing wild-type bGH or hGH. Dopamine turnover in the median eminence of male mice was also altered in animals expressing different bGH analogues but not in those expressing wild-type bGH or hGH. In ovariectomized females, the effects of different bGH analogs on the turnover of dopamine and norepinephrine in the median eminence included changes resembling those detected in animals expressing hGH, as well as alterations differing from the effects of both bGH and hGH. The results indicate that biological actions of these bGH analogues cannot be characterized simply in terms of enhanced or reduced somatogenic or lactogenic activity and raise a possibility that different sites, domains or features of tri-dimensional structure of GH are involved in its actions on different cellular targets.

Mitogenic and receptor activities of human growth hormone 108ndash;129

We have selectively synthesized a number of peptides encompassing the region of helix 3 of growth hormone (GH). These peptides and native human (h) GH have been evaluated for mitogenic and receptor activities in 3T3-F442A preadipocytes. In this system, wild type hGH is anti-mitogenic. In contrast, hGH 108-129 stimulated DNA synthesis while other GH-derived peptides were ineffective. hGH (L) 108-129 had an EC50 of about 0.2 nM and was maximally effective at about 0.5 nM in stimulating [3H]thymidine incorporation in 3T3-F442A cells. hGH (L) 108-129 was mitogenically as active as insulin-like growth factor-I and more active than insulin. It was less effective than transforming growth factor-beta. By cell cycle analysis, hGH (L) 108-129 increased the proportion of cells in S/G2/M phases to 28%. hGH, when coincubated with hGH (L) 108-129, blocked the mitogenic response of the peptide. A monoclonal antibody to the GH receptor significantly reduced binding of 125I-hGH to its receptor but had no effect on binding of 125I-hGH (L) 108-129. Affinity cross-linking of 125I-hGH to its receptor was not duplicated with 125I-hGH (L) 108-129. No other GH peptides or insulin competed for binding of 125I-hGH 108-129. Scatchard analysis indicated a Kd of 5.2 nM with 5.6 x 10(5) binding sites/cell for hGH (L) 108-129. These studies indicate that hGH (L) 108-129, a sequence encompassing helix 3 of hGH, acts by binding to a site other than the GH receptor and evokes high mitogenic responses.

The Crystal Structure of Affinity-matured Human Growth Hormone at 2 Å Resolution

A variant of human growth hormone (hGH), in which 15 mutations were introduced with phage display mutagenesis to improve receptor binding affinity by 400-fold, yielded two related crystal forms diffracting to high resolution. The structure of this variant was determined in both crystal forms, one at 2·0 Å resolution and one at 2·4 Å resolution, using molecular replacement with wild-type hGH taken from the receptor complex structure as a search model. Crystallographic refinement of the 2 Å structure gave an R-value of 18·5% for data in the resolution range 8 to 2 Å. The final model consists of residues 1 to 128 and 155 to 191, with three side-chains modeled in alternative conformations, together with 77 water molecules. Comparison of the structure with wild-type hGH, shows that most of the secondary structural elements are unchanged. The exception is the first turn of the third helix in the four-helix bundle core, which is unraveled in the present variant. Analysis of the two related packing environments suggests that this change is caused by crystal packing forces. A large change in the orientation of a short segment of helix found in the connection between the first two core helices is interpreted as evidence for rigid-body variability of this helical segment. Analysis of the mutations in light of the structure of the wild-type hGH/receptor complex shows that six of the mutations are buried in the hormone, whereas the remaining nine involve residues that interact with the receptor in the complex.

Affinity Maturation of Human Growth Hormone by Monovalent Phage Display

We describe a selection procedure for construction of very high affinity variants of human growth hormone (hGH) for binding to the extra cellular domain of its receptor (called the hGHbp). Five different libraries of mutated hGH genes (each containing ∼2 × 105 protein variants) were created by randomly mutating four different codons at residues that were shown to be important for receptor binding by structural or functional criteria. Mutated proteins were displayed as single copies from their respective filamentous phagemid particles and sorted in vitro for binding to the immobilized hGHbp. Phagemid particles that bound the immobilized hGHbp were eluted and propagated. After three to seven rounds of binding enrichments, hGH variants were isolated that contained 2 to 4 mutations and exhibited three- to sixfold improvements in binding affinity.Because of the limits of DNA transfection efficiency in creating the library we could not sample thoroughly mutations at more than four codons at once. Nonetheless, the free energy effects for these mutations acted cumulatively. Thus, by combining affinity enhanced mutants from libraries independently sorted we created an hGH variant with 15 substitutions that bound ∼400-fold more tightly to the hGHbp than wild-type hGH. The affinity enhancements occurred predominantly by slowing the off-rate of the hormone (>60-fold), and partly through increasing the on-rate (up to 4-fold). Residues that were shown to be important for binding by alanine-scanning were most highly conserved after binding selection, and interestingly many of them could be further improved. Thus, we found it most effective to randomly mutate the residues that were shown to modulate affinity by alanine-scanning, and to combine the selectants from separate libraries that exhibit the highest affinities. The selection procedure and mutagenesis strategy provides a framework for affinity maturation of protein-protein complexes.

Mitogenic and receptor activities of human growth hormone 108-129.

We have selectively synthesized a number of peptides encompassing the region of helix 3 of growth hormone (GH). These peptides and native human (h) GH have been evaluated for mitogenic and receptor activities in 3T3-F442A preadipocytes. In this system, wild type hGH is anti-mitogenic. In contrast, hGH 108-129 stimulated DNA synthesis while other GH-derived peptides were ineffective. hGH (L) 108-129 had an EC50 of about 0.2 nM and was maximally effective at about 0.5 nM in stimulating [3H]thymidine incorporation in 3T3-F442A cells. hGH (L) 108-129 was mitogenically as active as insulin-like growth factor-I and more active than insulin. It was less effective than transforming growth factor-beta. By cell cycle analysis, hGH (L) 108-129 increased the proportion of cells in S/G2/M phases to 28%. hGH, when coincubated with hGH (L) 108-129, blocked the mitogenic response of the peptide. A monoclonal antibody to the GH receptor significantly reduced binding of 125I-hGH to its receptor but had no effect on binding of 125I-hGH (L) 108-129. Affinity cross-linking of 125I-hGH to its receptor was not duplicated with 125I-hGH (L) 108-129. No other GH peptides or insulin competed for binding of 125I-hGH 108-129. Scatchard analysis indicated a Kd of 5.2 nM with 5.6 x 10(5) binding sites/cell for hGH (L) 108-129. These studies indicate that hGH (L) 108-129, a sequence encompassing helix 3 of hGH, acts by binding to a site other than the GH receptor and evokes high mitogenic responses.