Amino acid substitutions in human growth hormone affect coiled-coil content and receptor binding

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has great relevance to human diseases such as acromegaly and cancer. HGH has been extensively engineered by other workers to improve binding and other properties. We used a computational screen to select substitutions at single hGH positions within the hGHR-binding site. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. We are particularly interested in E174 which belongs to the hGH zinc-binding triad, and which spans coiled-coil helices and obeys the coiled-coil heptad pattern. Surprisingly, substituting E174 with A leads to substantial increase in an experimental measure of coiled-coil content. E174A is known to increase affinity of hGH against hGHR; here we show that this is simply because the off-rate is slowed down more than the on-rate, in line with what has been found for other affinity-improving mutations. For E174Y (and mutations at other sites) the slowdown in on-rate was greater, leading to decreased affinity. The results point to a link between coiled-coiling, zinc binding, and hGHR-binding affinity in hGH, and also suggest rules for choosing affinity-increasing substitutions.

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The Human Growth Hormone Antagonist B2036 Does Not Interact with the Prolactin Receptor

Endocrinology ◽

10.1210/endo.140.8.7047 ◽

1999 ◽

Vol 140 (8) ◽

pp. 3853-3856 ◽

Cited By ~ 25

Author(s):

Vincent Goffin ◽

Sophie Bernichtein ◽

Océane Carrière ◽

William F. Bennett ◽

John J. Kopchick ◽

...

Keyword(s):

Growth Hormone ◽

Amino Acid ◽

Binding Site ◽

Human Growth Hormone ◽

Human Growth ◽

Phase Iii ◽

Single Amino Acid ◽

Luciferase Reporter ◽

Amino Acid Substitutions ◽

Receptor Binding Site

Abstract The human growth hormone (hGH) antagonist B2036 combines a single amino acid substitution impairing receptor binding site 2 (G120K) with eight additional amino acid substitutions that improve binding site 1 affinity. This hGH antagonist is being tested for treating pathologies linked to excess hGH levels. B2036-PEG is a polyethylene glycol (PEG) conjugated form of B2036 that has an increased half-life due to reduced renal clearance. It is currently in phase III trials for acromegaly. Human GH is also able to bind to the receptor of prolactin (PRLR). Since activation of PRLR can promote an array of pathological states (reproduction disorders, breast cancer), the ability of B2036-PEG to interact with the PRLR had to be determined. In this study, we compared four hGH antagonists (G120K, G120K-PEG, B2036 and B2036-PEG) in three bioassays: proliferation of rat Nb2 cells, binding to the human PRLR and activation of human PRLR-mediated signaling in a cell line stably expressing this receptor and a luciferase reporter gene. Agonistic and antagonistic properties were characterized. Our data show that B2036-PEG does not bind, activate or antagonize PRLRs, either from rat or human origin. These observations further demonstrate that the eight amino acid substitutions within binding site 1 provide binding specificity directed towards the human GH receptor.

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The minimal amino acid sequence of the insulin-potentiating fragments of human growth hormone: its mechanism of action

Diabetes ◽

10.2337/diabetes.29.10.782 ◽

1980 ◽

Vol 29 (10) ◽

pp. 782-787 ◽

Cited By ~ 6

Author(s):

F. M. Ng ◽

J. Bornstein ◽

C. E. Pullin ◽

J. O. Bromley ◽

S. L. Macaulay

Keyword(s):

Growth Hormone ◽

Amino Acid ◽

Amino Acid Sequence ◽

Human Growth Hormone ◽

Mechanism Of Action ◽

Human Growth

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Methionine oxidation in human growth hormone and human chorionic somatomammotropin. Effects on receptor binding and biological activities.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48266-x ◽

1987 ◽

Vol 262 (14) ◽

pp. 6472-6477 ◽

Cited By ~ 2

Author(s):

L C Teh ◽

L J Murphy ◽

N L Huq ◽

A S Surus ◽

H G Friesen ◽

...

Keyword(s):

Growth Hormone ◽

Human Growth Hormone ◽

Receptor Binding ◽

Biological Activities ◽

Human Growth ◽

Methionine Oxidation ◽

Chorionic Somatomammotropin

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Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1984.247.5.e639 ◽

1984 ◽

Vol 247 (5) ◽

pp. E639-E644

Author(s):

C. M. Cameron ◽

J. L. Kostyo ◽

J. A. Rillema ◽

S. E. Gennick

Keyword(s):

Growth Hormone ◽

Amino Acid ◽

Human Growth Hormone ◽

Human Growth ◽

Mouse Mammary Gland ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Hypophysectomized Rats

The biological activity profile of reduced and S-carboxymethylated human growth hormone (RCM-hGH) was determined to establish its suitability for study of the diabetogenic property of hGH. RCM-hGH was found to have greatly attenuated in vivo growth-promoting activity in the 9-day weight-gain test in hypophysectomized rats (approximately 1%) and to have a similar low order of in vitro activity in stimulating amino acid incorporation into the protein of the isolated rat diaphragm. RCM-hGH also only had approximately 1% of the in vitro insulin-like activity of the native hormone on isolated adipose tissue from hypophysectomized rats. In contrast, RCM-hGH retained substantial in vivo diabetogenic activity in the ob/ob mouse, appearing to have approximately 50% of the activity of the native hormone. RCM-hGH was also found to retain significant, although attenuated (25%), in vitro lactogenic activity when tested for the ability to stimulate amino acid incorporation into a casein-rich protein fraction in mouse mammary gland explants. Because RCM-hGH exhibits a high degree of diabetogenic activity, although lacking significant anabolic or insulin-like activities, it will be useful as a "monovalent" probe for the study of the molecular mechanism of the diabetogenic action of GH.

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Investigation of sequon engineering for improved O-glycosylation by the human polypeptide N-acetylgalactosaminyl transferase T2 isozyme and two orthologues

Biochemical Journal ◽

10.1042/bcj20210382 ◽

2021 ◽

Vol 478 (19) ◽

pp. 3527-3537

Author(s):

Nicole K. Thompson ◽

Leif T. N. LeClaire ◽

Samantha Rodriguez Perez ◽

Warren W. Wakarchuk

Keyword(s):

Growth Hormone ◽

Amino Acid ◽

Human Growth Hormone ◽

Therapeutic Proteins ◽

Human Growth ◽

Bacterial Expression ◽

Intact Protein ◽

Human Interferon ◽

Kinetic Properties

We have been developing bacterial expression systems for human mucin-type O-glycosylation on therapeutic proteins, which is initiated by the addition of α-linked GalNAc to serine or threonine residues by enzymes in the GT-27 family of glycosyltransferases. Substrate preference across different isoforms of this enzyme is influenced by isoform-specific amino acid sequences at the site of glycosylation, which we have exploited to engineer production of Core 1 glycan structures in bacteria on human therapeutic proteins. Using RP-HPLC with a novel phenyl bonded phase to resolve intact protein glycoforms, the effect of sequon mutation on O-glycosylation initiation was examined through in vitro modification of the naturally O-glycosylated human interferon α-2b, and a sequon engineered human growth hormone. As part of the development of our glycan engineering in the bacterial expression system we are surveying various orthologues of critical enzymes to ensure complete glycosylation. Here we present an in vitro enzyme kinetic profile of three related GT-27 orthologues on natural and engineered sequons in recombinant human interferon α2b and human growth hormone where we show a significant change in kinetic properties with the amino acid changes. It was found that optimizing the protein substrate amino acid sequence using Isoform Specific O-Glycosylation Prediction (ISOGlyP, http://isoglyp.utep.edu/index.php) resulted in a measurable increase in kcat/KM, thus improving glycosylation efficiency. We showed that the Drosophila orthologue showed superior activity with our human growth hormone designed sequons compared with the human enzyme.

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Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.2984-2992.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 2984-2992

Author(s):

E P Slater ◽

O Rabenau ◽

M Karin ◽

J D Baxter ◽

M Beato

Keyword(s):

Growth Hormone ◽

Human Growth Hormone ◽

Receptor Binding ◽

Gene Promoter ◽

Human Growth ◽

Growth Hormone Gene ◽

First Intron ◽

Relative Binding ◽

Flanking Sequences ◽

Human Growth Hormone Gene

In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.

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