Inhibition of Lactogenic Activities of Ovine Prolactin and Human Growth Hormone (hGH) by a Novel Form of a Modified Recombinant hGH*

Abstract

A recombinant analog of human GH (hGH) lacking 13 amino acids at the amino-terminus (Met14hGH) inhibited the hGH- or ovine PRL (oPRL)-stimulated proliferation of Nb2 lymphoma cells and bovine PRL-stimulated fat synthesis and alpha-lactalbumin secretion in explants from bovine lactating mammary gland. The inhibition was competitive in nature, and in Nb2 cells could be abolished by an excess of hGH or oPRL. Inhibition of oPRL-stimulated proliferation of Nb2 cells by Met14hGH could also be specifically abolished by anti-hGH monoclonal antibodies. Met14hGH had no growth-stimulating activity in Nb2 cells and was not cytotoxic. It also did not affect glucose uptake by the mammary gland explants. Met14hGH competed with [125I]hGH for binding to intact Nb2 cells, IM-9 lymphocytes, solubilized microsomal fraction from lactating bovine mammary gland, and microsomal fraction from the liver of female virgin rats, but its affinity for those receptors was 2 orders of magnitude lower than the affinity of hGH. Since Met14hGH used in most experiments contained about 25% impurities and degradation products, a small amount of it was further purified by immunoaffinity chromatography. Two purified fractions, one consisting of a single 20K protein and the other accompanied by a small amount of 25K protein, were obtained. Both fractions exhibited increased inhibition of hGH- or oPRL-stimulated proliferation of Nb2 cells, thus indicating that the inhibitory activity results from the intact Met14hGH molecule. To the best of our knowledge, this is the first report describing the inhibition of lactogenic hormone activities by a modified hGH.

Selective Modification of Recombinant Bovine Placental Lactogen by Site-directed Mutagenesis at Its C Terminus

“…Information obtained from mutated analogues of hGH indicates that not only the N-terminal domain (12,13,17), but also the C-terminal domain and the non-helical sequence intervening between the first and the second ␣-helix participate in receptor binding (18). Publications concerning mutation of this domain in hGH (18 -20), implicate its importance in the binding to somatogenic receptors as well to lactogenic receptors (21).…”

“…Assuming structural similarity to porcine GH (11) these mutations were aimed to remove amino acids beyond or at the beginning of the putative first ␣-helix. Results of these studies (9, 10) indicated that similarly to hGH (12-16), bPL can be selectively modified so that particular biological activities are changed while others remain relatively unaffected.Information obtained from mutated analogues of hGH indicates that not only the N-terminal domain (12,13,17), but also the C-terminal domain and the non-helical sequence intervening between the first and the second ␣-helix participate in receptor binding (18). Publications concerning mutation of this domain in hGH (18 -20), implicate its importance in the binding to somatogenic receptors as well to lactogenic receptors (21).…”

“…Assuming structural similarity to porcine GH (11) these mutations were aimed to remove amino acids beyond or at the beginning of the putative first ␣-helix. Results of these studies (9,10) indicated that similarly to hGH (12)(13)(14)(15)(16), bPL can be selectively modified so that particular biological activities are changed while others remain relatively unaffected.…”

Selective Modification of Human Growth Hormone by Site-Directed Mutagenesis: Implications for the Diversity of Biological Actions

“…Far ultraviolet circular dichroism spectra of both analogues and human GH were identical, indicating no major change in the tertiary structure [22], A reasonable hypothesis based on these results there fore indicates that either the 7 N-terminal residues are involved in the binding to somatogen receptors or that they are important for proper orientation and/or interac tion between the First and the fourth a-helix. It has pre viously been shown that truncation of 13 N-terminal ami no acids [11,12] or replacement of 23 amino acids by the corresponding sequence of bovine GH [13] drastically reduces the affinity for both lactogen and somatogen receptors. These changes probably induced disruption of the secondary structure, as estimated by the lowered con tent of a-helix [Binder and Gertler, unpubl.…”

“…Using this methodology, a recombinant ana logue of human GH was prepared and used to demon strate that a truncated form of human GH (des-13 human GH) inhibits the human GH or prolactin-receptor-me diated biological activity [11]. This analogue, however, was only a weak antagonist [12], The other analogue, bovine GH/human GH hybrid I, a chimeric bovine GH (amino acids MetAspGln and 1-23) and human GH (ami no acids ) also had lower affinity toward lactogenic and somatogenic receptors and exhibited weak agonistic activity in lactogen-receptor-mediated bioassays [13], These studies suggested that the binding site of human GH for lactogen and somatogen receptors include the N-terminal part of the molecule, and more specifically, the amino acids [8][9][10][11][12][13][14][15][16][17][18].…”