Native Human Growth Hormone Research Articles

Creutzfeldt-Jakob disease with unusually extensive neuropathology in a child treated with native human growth hormone

We report a case of iatrogenic Creutzfeldt-Jakob disease(iCJD) in a child with a neonatal growth hormone (GH) deficiency that was treated with native human growth hormone (hGH) between the ages of 9 months and 7 years. Three years after the end of treatment a progressive neurological syndrome consistent with Creutzfeldt-Jakob disease (CJD) developed, leading to death within a year, at age 11. Neuropathological examination showed an unusual widespread form of CJD, notably characterized by (i) involvement of the cerebellar white matter, (ii) cortico-spinal degeneration and (iii) ballooned neurons. A transitional form of the disease between common iatrogenic and panencephalopathic CJD is suggested.

Optimized clinical performance of growth hormone with an expanded genetic code

The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.

Immunogenicity, toxicology, pharmacokinetics and pharmacodynamics of growth hormone ligand–receptor fusions

A fundamental concern for all new biological therapeutics is the possibility of inducing an immune response. We have recently demonstrated that an LR-fusion (ligand-receptor fusion) of growth hormone generates a potent long-acting agonist; however, the immunogenicity and toxicity of these molecules have not been tested. To address these issues, we have designed molecules with low potential as immunogens and undertaken immunogenicity and toxicology studies in Macaca fascicularis and pharmacokinetic and pharmacodynamic studies in rats. Two variants of the LR-fusion, one with a flexible linker (GH-LRv2) and the other without (GH-LRv3), were tested. Comparison was made with native human GH (growth hormone). GH-LRv2 and GH-LRv3 demonstrated similar pharmacokinetics in rats, showing reduced clearance compared with native GH and potent agonist activity with respect to body weight gain in a hypophysectomized rat model. In M. fascicularis, a low level of antibodies to GH-LRv2 was found in one sample, but there was no other evidence of any immunogenic response to the other fusion protein. There were no toxic effects and specifically no changes in histology at injection sites after two repeated administrations. The pharmacokinetic profiles in monkeys confirmed long half-lives for both GH-LRv2 and GH-LRv3 representing exceptionally delayed clearance over rhGH (recombinant human GH). The results suggest that repeated administration of a GH LR-fusion is safe, non-toxic, and the pharmacokinetic profile suggests that two to three weekly administrations is a potential therapeutic regimen for humans.

Transglutaminase‐mediated methods for site‐selective modification of human growth hormone

Two methods for the site-selective modification of native human growth hormone (hGH) using microbial transglutaminase were developed. In the first method, 1,3-bisaminoxypropane was attached to hGH, providing a direct incorporation of reactive aminoxy groups for further modification. The reaction was shown to be selective for Gln(141), with minor modification at Gln(40). In the second method, modified glutamine substrates were developed for attachment to Lys(145) in hGH. A series of glutamine-substrates were screened, and it was shown that microbial transglutaminase was selective towards substitutions on the glutamine core structure. Products from both methods could be transformed to site selectively mono-PEGylated hGH-derivatives in good isolated yield.

A native RNA secondary structure controls alternative splice-site selection and generates two human growth hormone isoforms.

Consensus sequences at the splice donor, splice acceptor, and lariat branch point regions are necessary but insufficient determinants of splice-site selection in nuclear precursor mRNAs. Sequences outside of these regions can have a significant effect on the utilization of splice sites. Although the mode of action of such sequences is undefined in most cases, higher order RNA structures have been suggested as a potential contributor to splice-site selection. During a detailed analysis of the splicing patterns of the human growth hormone transcript, we located 2 bases in the vicinity of the exon 3 major splice-acceptor site (B) which facilitate the utilization of a competing downstream acceptor (B'). The effects of a series of site-specific mutations on the splicing pattern demonstrate that these 2 bases function by stabilizing a specific stem-loop structure in the native transcript. This defined secondary structure selectively encompasses the upstream B splice-acceptor site together with its lariat branch point region. Increasing the predicted stability of this stem by point mutations results in a corresponding shift in splicing towards the alternative B' splice-acceptor site. These results indicate that a specific secondary structure within the native human growth hormone transcript controls the relative utilization of two competing splice-acceptor sites with the consequent generation of two functionally distinct hormone isoforms.

The secretion leader of Mucor pusillus rennin which possesses an artificial Lys-Arg sequence directs the secretion of mature human growth hormone by Saccharomyces cerevisiae

The prepro-peptide of fungal aspartic proteinase, Mucor pusillus rennin, is useful as a secretion leader for efficient secretion of human growth hormone (HGH) from Saccharomyces cerevisiae. For secretion by yeast cells of HGH with the same NH2 terminus as native HGH, an artificial Lys-Arg linker, which is one of the potential KEX2 recognition sequences, was introduced at the junction between the M. pusillus rennin secretion leader and mature HGH. The HGH directed by this construction was the same size as native HGH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid sequencing of its NH2 terminus revealed that the secretion leader peptide was removed correctly at the COOH-terminal side of the Lys-Arg linker. On the other hand, when the same plasmid was expressed in a kex2 mutant strain, unprocessed HGH of a higher molecular weight was secreted, indicating that no proteolytic cleavage at the Lys-Arg site occurred. These results clearly showed that the leader peptide with the Lys-Arg linker was recognized and specifically cleaved by the yeast KEX2 protease. The mature HGH purified from yeast culture medium was indistinguishable from native HGH in biological activity, determined by the adipocyte conversion assay, and in secondary structure, determined by circular dichroism spectroscopy.

Influence of age on responsiveness to diabetogenic action of growth hormone.

Responsiveness to the growth-promoting action of growth hormone (GH) develops gradually during post-natal life, and as the organism ages, sensitivity to the hormone declines. Our study was undertaken to determine whether there is a similar age-related pattern of sensitivity to the diabetogenic action of purified native human GH (hGH) in the obese (ob/ob) mouse. The ob/ob mouse was used because adults of this strain respond to chronic GH treatment with increases in fasting plasma insulin and blood glucose concentrations and with glucose intolerance. When hGH was given subcutaneously to adult (4-mo-old) female mice at doses of 5, 10, or 25 micrograms/day for 3 days, a significant increase in fasting blood glucose concentration and an impairment in glucose tolerance were produced by the 10-micrograms/day dose. Larger effects were obtained with the 25-micrograms/day dose. A threefold increase in fasting plasma insulin concentration was also produced with this dose of the hormone. By contrast, 25 micrograms/day of hGH had no effect on fasting plasma insulin, blood glucose, or glucose tolerance in 1-mo-old ob/ob mice. When a dose of 100 micrograms/day of hGH was given to 1-mo-old animals, a significant increase in fasting plasma insulin concentration occurred, but again there were no effects on fasting blood glucose or glucose tolerance. Mice 1.5 mo old showed marginal changes in fasting blood glucose and glucose tolerance when given hGH at doses of 25 or 100 micrograms/day. Mice 9 or 12 mo old exhibited responsiveness similar to that of 4-mo-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the antigenicities of native human growth hormone (hGH) and three forms of recombinant hGHs using monoclonal antibodies

A panel of hybridomas producing antibodies specific for human growth hormone (hGH) were prepared by using a recombinant hGH [methionylsomatotropin (r-hGH)] as an immunogen. Thirteen representative monoclonal antibodies which showed different reactivity patterns were used to analyze the antigenicities of four different forms of hGHs by RIA inhibition studies. Native hGH and r-hGH showed almost the same antigenicities with these monoclonal antibodies. A Cys-substituted recombinant hGH (r-hGH-165) retained the epitopes recognized by 11 monoclonals but not those recognized by two monoclonals. All except one of the monoclonals showed little or no reactivity with a recombinant hGH fragment (r-hGH-AB). On the basis of these results, the differences in the structures and antigenicities of the recombinant hGH proteins were discussed.

Effects of growth hormone substitution on mental performance in adults with growth hormone deficiency: a pilot study

Five patients ages 22 to 36 years with growth hormone deficiency received substitution with native human growth hormone and biosynthetic methionyl human growth hormone, 8 IU i.m. three times weekly, for two separate 4-week periods. Before and at the end of each treatment period, five psychometric tests evaluating cognitive functions were performed. The results suggest a beneficial effect of growth hormone on certain cognitive functions. The results were similar for the two hGH preparations.

Alkaline Phosphatase Fusions: A Tag to Identify Mutations that Result in Increased Expression of Secreted Human Growth Hormone from Escherichia coli

We describe a method for the mutagenesis of a signal sequence, and the identification of mutations leading to the enhanced expression of periplasmicly secreted human growth hormone (HGH) from Escherichia coli. A plasmid was engineered, using deoxyoligonucleotide directed site specific mutagenesis, to contain two unique restriction sites flanking the signal sequence for HGH. The signal was mutagenized specifically using nitrous acid. Mutants resulting in enhanced expression of secreted HGH were detected by expressing and secreting a human growth hormone–alkaline phosphatase fusion protein possessing phosphatase activity. Such fusions show activity only when secreted. The signal sequence mutations isolated were transferred into plasmids directing the expression and secretion of native HGH and the phenotypes of enhanced expression of secreted protein were largely preserved.

Trypsin-resistant forms of human growth hormone have diabetogenic and insulin-like activities

Although diabetogenic and insulin-like activities are intrinsic properties of the growth hormone (GH) molecule, it has been frequently suggested that the hormone must be proteolytically processed for these activities to be expressed. If this is correct, then derivatives of GH having resistance to appropriate proteolytic attack might not have diabetogenic and/or insulin-like activity. The purpose of the present study was to prepare derivatives of human GH that are resistant to digestion by trypsin and to determine whether they posses diabetogenic or insulin-like activity. Three derivatives were prepared from purified native human GH in which lysine residues were modified with methyl acetimidate, citraconic anhhydride of S-ethylthioltrifluoroacetate, and one in which arginine residues were modified with camphorquinone-10-sulfonic acid. Comparisons of peptide maps of tryptic digests of these derivatives with that of unmodified human GH indicated that all four were resistant to proteolysis by trypsin. All of these trypsin-resistant forms of human GH were found to posses ssignificant growth-promoting, diabetogenic and insulin-like activities, although all activities were attenuated to some extent in each derivative. The relative potencies of the human GH derivatives in a radioimmunoassay for human GH were somewhat similar to their order of potency in the growth-promoting and diabetogenic assays. These results suggest that if proteolytic processing of the GH molecule is involved in the expression of one or more of its biological activities, such processing probably does not involve a trypsin-like proteinase.

Diabetogenic activity of native and biosynthetic human growth hormone in obese (ob/ob) mouse.

It has been repeatedly suggested that diabetogenic activity is not an intrinsic property of native pituitary growth hormone (GH) and that the diabetogenic effects produced by GH preparations are due to low-molecular-weight contaminants or degradation products of the hormone. This possibility was evaluated in this study by assessing the ability of purified native human GH (hGH) and biosynthetic methionyl-hGH to exacerbate fasting hyperglycemia and glucose intolerance in the obese (ob/ob) mouse. Native hGH that had been purified by DEAE-cellulose chromatography (A-type; 1.8 IU/mg) produced fasting hyperglycemia and glucose intolerance in the ob/ob mouse when injected subcutaneously at doses of 50 micrograms/day or greater for 3 days. It had no effect when a single subcutaneous dose of 200 micrograms was administered 24 h previously. To eliminate possible contamination with smaller peptides, the hGH was gel-filtered on a column of Sephacryl S-200 in 6 M guanidine-HCl. When injected subcutaneously into ob/ob mice at a dose of 50 micrograms/day or greater for 3 days, the guanidine-treated hGH produced glucose intolerance. Also biosynthetic methionyl-hGH produced marked fasting hyperglycemia and glucose intolerance when injected subcutaneously at doses of 50 or 100 micrograms/day for 3 days. These results support the conclusion that hGH itself is indeed diabetogenic but that chronic exposure of the organism to the hormone is required for its effects on glucose metabolism to become clearly manifest.

Fragments of human growth hormone produced by digestion with bromelain: Chemistry and biological properties

In an effort to produce small discrete fragments of human growth hormone (GH), we examine the action of the proteolytic enzyme, bromelain, on this molecule. Purified human GH incubated for 40 min at 22°C with crude bromelain and gel-filtered on Sephadex G-100 resulted in a major digestion product, peak 2. SDS-urea gel electrophoresis in the presence of β-mercaptoethanol suggested that peak 2 was composed of two polypeptide chains. Two polypeptide fractions were isolated by the reduction and S-alkylation of peak 2 in 6 M guanidine-HCl and subsequent chromatography on Sephacryl S-200 in 6 M guanidine-HCl. These two fractions, A and B, had the same mobilities as the two components of peak 2 on SDS-urea gels. Amino-terminal analysis, tryptic peptide mapping, carboxypeptidase digestion, cyanogen bromide cleavage, and amino acid analysis of fraction A indicated that it was peptide 1–135. Amino-terminal analysis and tryptic peptide mapping of fraction B suggested the presence of a mixture of peptides 143–191, 145–191 and 146–191. Thus, peak 2 is heterogeneous and appears to be a mixture consisting of peptide 1–135 + peptide 143–191, peptide 1–135 + peptide 145–191 and peptide 1–135 + peptide 146–191, in each case the N-terminal peptide being joined to the C-terminal peptide by the disulfide bridge between residues 53 and 165. In the weight-gain test in hypophysectomized rats, two preparations of peak 2 appeared to be somewhat less active than the native human GH preparations from which they were derived. Several preparations of peak 2 showed equivalent potency in stimulating [ 14C]glucose oxidation to 14CO 2 by isolated epididymal adipose tissue of hypophysectomized rats. Also, most of the peak 2 preparations were somewhat less active than native human GH in displacing 125I-labeled human GH bound to antibodies to human GH.

Purified human growth hormone from E. coli is biologically active.

Human growth hormone (HGH), somatotropin, is a protein consisting of 191 amino acids with a molecular weight of ∼22,000. It is essential for linear growth and its application in the treatment of hypopituitary dwarfism is well established1–3. Growth hormone may also be effective in the treatment of other disorders including bone fractures, burns and bleeding ulcers4. Because growth hormone is species specific, human cadavers have previously been the only source of HGH. Moreover, the standard purification of pituitary-derived HGH does not provide a homogeneous product, but rather a mixture of native HGH and modified forms and fragments of HGH, each of which may have different physiological roles5. The expression of HGH in Escherichia coli using recombinant DNA technology6 provides an inexhaustible source of a single eukaryotic protein. The bacterially synthesized HGH, produced from E. coli, is methionyl-HGH (Met-somatotropin), the extra methionine arising from the AUG start codon inserted at the beginning of the gene. This cloned product has now been purified to homogeneity and its biological properties examined. For pituitary and bacterially synthesized HGH, specific activities as determined by radioimmunoassay are comparable.

Demonstration by Radioligand-Binding Assay of the Structural Similarity of a Partially Synthetic Growth Hormone Recombinant Molecule to Its Natural Analog and to Native Human Growth Hormone*

The recombinant resulting from the noncovalent interaction of a natural amino-terminal fragment of reduced and carbamoylmethylated human GH (residues 1-134) with a synthetic carboxyl-terminal fragment of reduced and carbamoyl-methylated human GH (residues 141-191) reacts in seven immunoassay systems and two receptor assay systems in a similar manner and with comparable potency to its naturally occurring analog, but with less avidity overall than does the native hormone. This similarity of reactivity is strong evidence for similarity of conformation among these molecules. The demonstration that a synthetic fragment can restore radioligand-binding properties to a proteolytic fragment of human GH implies that partially synthetic variant GH molecules have biological activity.

In vivo interaction of human growth hormone (HGH) with subcellular structures of rat liver

The interaction of circulating HGH with subcellular structures of female rat liver was studied in the intact animal. Various subcellular fractions including plasma membranes (Biochim. Biophys. Acta 154 : 540, 1968) and Golgi fractions (J. Cell. Biol. 59 : 45, 1973) were prepared from liver homogenates at different time intervals after a single injection of 125I-HGH into a peripheral vein. Labeling of plasma membranes occurred within the first minutes following injection, whereas labeling of Golgi fractions was delayed and reached a maximum by 15 minutes. Labeling of both subcellular structures was inhibited by excess (500 μg) native HGH but was unaffected by insulin. At all times studied virtually no radioactivity was found in the nuclear and mitochondrial-lysozomal fractions. The 125I-HGH eluted from the plasma membranes and from Golgi fractions appeared intact as tested by TCA precipitation and rebinding to rat liver membrane. The results suggest that, following binding to the surface of liver cells, HGH is translocated into Golgi elements under a biologically active form.

BINDING STUDIES OF GROWTH HORMDNE TO RAT LIVER CELLS

The interaction of human growth hormone (HGH) with isolated liver cells from normal rats has been studied. Cells prepared with the method described by Berry and Friend were incubated at room temperature with 125I-labeled HGH. Free and cell-bound hormone were separated by filtration or centrifugation. The binding of 125I-HGH to liver cells is a specific, time dependent, saturable and reversible process. At a hormone concentration of 5 × 10−11M, equilibrium of binding is achieved in 120 minutes. The bound labeled HGH is rapidly dissociated by addition of an excess of unlabeled hormone. The specific binding of 125I-HGH is saturable at a hormone concentration of 6 × 10−10M. 1500 molecules of HGH are specifically bound per cell. Native HGH inhibits binding of 125I-HGH to cells; half maximal inhibition occurs at 4 × 10−9M. No significant inactivation or degradation of 125I-HGH or of the specific receptors occurs. Preliminary data indicate that those receptor sites are biologically meaningful.

Isolation and biological characterization of fragments of human growth hormone produced by digestion with plasmin.

The biologically active component of plasmin digested human growth hormone consisted of residues 1-134 attached to residues 141-191 by the disulfide bond between residues 53 and 165. This large fragment of the hormone retained the in vivo capacity to stimulate weight gain and cartilage metabolism in hypophysectomized rats and exhibited the diabetogenic property of the native hormone in partially pancreatectomized, dexamethasone-treated rats. This fragment also retained the in vitro ability to stimulate protein synthesis and amino acid and sugar transport into isolated diaphragm muscle of hypophysectomized rats, Furthermore, a smaller peptide (residues 1-134) derived from the large fragment by reduction and carbamidomethylation of the disulfide bond retained high activity in in vitro systems and the in vivo capacity to stimulate cartilage metabolism provided the peptide was injected intravenously. Thus the present studies demonstrate that many of the in vivo and in vitro metabolic effects of human growth hormone on tissues of hypophysectomized rats can be elicited with a peptide comprising the aminoterminal 134 amino acid residues of native human growth hormone.

Human pituitary growth hormone: Immunochemical investigations of biologically active fragments

Guinea pig antisera to human growth hormone were tested for their ability to recognize the two biologically active fragments of the hormone produced by human plasmin digestion and a synthetic active fragment. A precipitin line was obtained with native human growth hormone, plasmin-treated human growth hormone, and its NH 2-terminal fragment (residues 1–134). In the microcomplement-fixation and radioimmunoassay experiments, the NH 2-terminal plasmin fragment (residues 1–134) showed a greater immunoreactivity than the COOH-terminal plasmin fragment (residues 141–191). This, in turn, was more active than the synthetic fragment (residues 95–136).