Wild-type hGH Research Articles

Immunodominant structures of human growth hormone identified by homolog-scanning mutagenesis

Homolog-scanning mutagenesis has been reported to be useful in elucidating the antigenic epitopes recognized by monoclonal antibodies and hGH binding to its receptor. However, little is known about which structures are recognized as immunodominant by murine serum antibodies. Therefore, the previously published series of hGH homologs and additional mutants of human placental lactogen (hPL), porcine growth hormone (pGH), and human prolactin (hPRL) were examined for their interaction with murine serum derived anti-hGH antibodies. As compared to wild-type hGH, nine of the nineteen segment substituted mutants tested showed a significant reduction in binding to anti-hGH sera. These disruptive substitutions mapped to 5 regions on a structural model of hGH: the length of helix 1 (residues 11–33), the loop between the first disulfide bond and helix 2 (residues 54–74), the beginning of helix 3 (residues 109–112), the carboxyl half of helix 4 (residues 167–182), and the final carboxyl terminus segment of the molecule (residues 184–191). In terms of the current structural model, three of the five immunodominant regions (the loop between residues 54–74, central portion of helix 4 to the carboxyl terminus and part of the amino terminus region of helix 1) closely overlaps the hGH receptor binding epitopes.

Evidence that the N-terminus of human growth hormone is involved in expression of its growth promoting, diabetogenic, and insulin-like activities.

Recent work with various point and deletion mutants of human GH (hGH) has suggested that the proximal N-terminal end of the hormone molecule is important for its growth promoting action. This study was conducted to examine the growth promoting, diabetogenic, and insulin-like activities of two N-terminal mutants of hGH, the deletion mutant Des-7 hGH (met8, ala11), and a chimeric mutant of bovine GH (bGH) and hGH containing the N-terminal 13 amino acids of bGH (met, ala 1-13/14-191, asp11). The CD spectra of these mutants are similar to that of wild-type hGH and they retain lactogenic activity on Nb2 lymphoma cells, whereas their ability to bind to somatogenic receptors on IM-9 lymphocytes and bovine liver membranes is markedly reduced. In this study, growth promoting activity of the mutants was assessed using the 9-day weight gain test in hypophysectomized rats. Des-7 hGH had a potency of 0.03 IU/mg protein in this assay, whereas the potency of the bGH/hGH chimera was 0.71 IU/mg. Diabetogenic activity was tested in the ob/ob mouse, using the elevation of fasting blood glucose and the worsening of glucose tolerance after a 3-day course of treatment as end-points. Both Des-7 hGH and the bGH/hGH chimera had reduced diabetogenic activity compared to that of biosynthetic wild-type hGH, consistent with their reduced growth activity. Insulin-like activity was assessed by testing the in vitro ability of the mutants to stimulate [14C] glucose oxidation by epididymal adipose tissue of hypophysectomized rats. Des-7 hGH had about 1% the activity of wild-type hGH, whereas the chimera was about 20% as active. When Des-7 hGH was added to the incubation medium along with wild-type hGH in ratios of 5, 12.5, or 25:1 (Des-7 hGH:hGH), the insulin-like action of hGH was significantly inhibited, indicating that the mutant is a modest antagonist of the insulin-like action of hGH. When the ability of Des-7 hGH to compete with [125I] hGH for binding to isolated rat adipocytes was tested, the mutant was about 10% as effective as wild-type hGH. Thus, Des-7 hGH appears to be more effective in binding to adipocyte GH receptors than in triggering an insulin-like response, perhaps accounting for its modest antagonistic activity. The results of this study suggest that the proximal N-terminal end of the hGH molecule is involved in the expression of the growth promoting, diabetogenic and insulin-like activities of GH.

Expression of an artificial gene coding for a human secretin precursor-like peptide in Escherichia coli

A gene coding for human secretin elongated at the C-terminus by Gly-Lys-Arg was chemically synthesized and fused to the downstream of an artifical gene encoding [Leu 14]human growth hormone (hGH) which had a leucine residue instead of methionine at position 14 in the wild type hGH protein. The fusion gene was efficiently expressed under the control of the Escherichia coli trp promoter including the consensus Shine-Dalgarno sequence. After the fusion protein was cleaved in two parts with cyanogen bromide, the desired secretin-Gly-Lys-Arg (SecGKR) was finally purified to homogeneity using high-performance liquid chromatography, yielding 11 mg protein from 16 g of wet cells. The biological activities of secretin-Gly-Lys (SecGK) and secretin-Gly (SecG) obtained by digestion of SecGKR with carboxypeptidase B were compared with SecGKR and the mature human secretin. The activities of SecGKR and SecGK were 25–30% higher than that of the mature human secretin amidated at the carboxy-terminal amino acid, but that of SecG was 25% lower.

High level expression of human transforming growth factor α gene in Escherichia coli in a fusion gene system with human growth hormone gene

An artificial gene encoding [Leu 14]human growth hormone(hGH), which had a leucine residue at position 14 instead of methionine as in the wild type hGH protein, was constructed and expressed in Escherichia coli at a high level, as well as the [Met 14]hGH gene, under the control of the trp promoter including a consensus SD sequence. A human transforming growth factor α (TGFα) gene, which had a methionine codon attached to the 5′-terminal region, was chemically synthesized and fused to the [Leu 14]hGH gene. The fusion gene was efficiently expressed in E. coli, yielding 48 mg per gram of the wet cell weight. After the fusion protein was cleaved into two parts at the single methionine site by treatment with cyanogen bromide, the desired TGFα was made to form disulfide bonds by a refolding reaction, and then purified by HPLC. The structure of the TGHα was confirmed by amino acid sequence analysis. The biological activity of the purified TGFα was assessed.

Engineering human prolactin to bind to the human growth hormone receptor

A strategy of iterative site-directed mutagenesis and binding analysis was used to incorporate the receptor-binding determinants from human growth hormone (hGH) into the nonbinding homolog, human prolactin (hPRL). The complementary DNA for hPRL was cloned, expressed in Escherichia coli, and mutated to introduce sequentially those substitutions from hGH that were predicted by alanine-scanning mutagenesis and other studies to be most critical for binding to the hGH receptor from human liver. After seven rounds of site-specific mutagenesis, a variant of hPRL was obtained containing eight mutations with an association constant for the hGH receptor that was increased more than 10,000-fold. This hPRL variant binds one-sixth as strongly as wild-type hGH, but shares only 26 percent overall sequence identity with hGH. These studies show the feasibility of recruiting receptor-binding properties from distantly related and functionally divergent hormones and show that a detailed functional database can be used to guide the design of a protein-protein interface in the absence of direct structural information.

Engineering Human Prolactin to Bind to the Human Growth Hormone Receptor

A strategy of iterative site-directed mutagenesis and binding analysis was used to incorporate the receptor-binding determinants from human growth hormone (hGH) into the nonbinding homolog, human prolactin (hPRL). The complementary DNA for hPRL was cloned, expressed in Escherichia coli, and mutated to introduce sequentially those substitutions from hGH that were predicted by alanine-scanning mutagenesis and other studies to be most critical for binding to the hGH receptor from human liver. After seven rounds of site-specific mutagenesis, a variant of hPRL was obtained containing eight mutations with an association constant for the hGH receptor that was increased more than 10,000-fold. This hPRL variant binds one-sixth as strongly as wild-type hGH, but shares only 26 percent overall sequence identity with hGH. These studies show the feasibility of recruiting receptor-binding properties from distantly related and functionally divergent hormones and show that a detailed functional database can be used to guide the design of a protein-protein interface in the absence of direct structural information.

High-Resolution Epitope Mapping of hGH-Receptor Interactions by Alanine-Scanning Mutagenesis

A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four.

468 AMINO ACID RESIDUES 93–96 OF THE HUMAN GROWTH HORMONE MOLECULE ARE IMPORTANT FOR POLYCLONAL ANTISERUM RECOGNITION

The precise regions of the human growth hormone (hGH) molecule that mediate its biologic effects and immunoreactivity are not known. To address this issue, we have introduced mutations into the coding region of a cloned hGH gene and expressed the resulting mutants in a eukaryotic expression vector. Two clones with alterations in the DNA sequences encoding amino acids 93-96 within the hGH molecule were created by the insertion of Bam HI DNA linkers into the Mst II site. The hGH derived from these mutants had characteristics indistinguishable from those of wild type hGH in the NB-2 cell lactogenic assay, the IM-9 cell radioreceptor assay, and in the binding of four anti-hGH monoclonal antibodies. However, significant differences were observed between the mutants and standard human pituitary GH or hGH produced by the normal gene in the binding of polyclonal hGH antisera. Both mutants bound to the polyclonal antibody poorly but did not displace labeled standard hormone in a proportionate manner.These results suggest that amino acids 93-96 of hGH regulate the expression of antigenic determinants that are detected by polyclonal antiserum, but these determinants are distinct from those recognized by the four monoclonal antibodies that were tested. Moreover, hGH molecules with abnormal immunologic reactivity may retain normal biologic activities in certain assays.