Extraordinarily stable disulfide‐linked homodimer of human growth hormone

Abstract

Although a 22-kDa human growth hormone (hGH) is the predicted protein product of the hGH-N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS-PAGE under reducing conditions we isolated an unusually stable mercaptoethanol-resistant (MER) 45-kDa hGH. A 5-h incubation at 100°C in the presence of 2-mercaptoethanol was required to convert approximately 90% of MER-45-kDa hGH into a 22-kDa hGH. Other reductants were not as effective in splitting MER-45-kDa hGH. After fracturing MER-45-kDa hGH, the 22-kDa hGH fragments would spontaneously reassociate if the reductant was removed; however, alkylation of cysteine residues prevented their reassociation. Identical amino acid sequences for the first six N-terminal residues were obtained for MER-45-kDa hGH and its 22-kDa hGH cleavage product. Structural identity of MER-45-kDa hGH and 22-kDa hGH was demonstrated by MALDI-TOF mass spectrometry of tryptic digests. MER-45-kDa hGH did not break up upon incubation with EDTA and EGTA. The significance of this work to our understanding of the structure of hGH isoforms is that it demonstrates that MER-45-kDa hGH is not a single chain polypeptide but is instead a homodimer of 22-kDa hGH monomers. The MER-45-kDa hGH dimer is held together by interchain disulfide bonds and not by divalent metal cation bridges. Additionally, MER-45-kDa hGH's interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.Keywords: human growth hormone; isoform; dimer; mass spectrometry; preparative electrophoresis; purification; protein structure Pituitary GH is an important regulator of growth, metabolism, and development in humans and animals (Strobl and Thomas 1994;Ho et al. 1996;Okada and Kopchick 2001;Renaville et al. 2002;Waters and Kaye 2002). Growth hormones have long been known to stimulate body weight gain (Evans and Simpson 1931), increase the growth rate of animals (Brumby 1959;Machlin 1972), influence the metabolism of proteins, nucleic acids, lipids, and carbohydrates (Altszuler 1974;Cheek and Hill 1974;Goodman and Schwartz 1974;Kostyo and Nutting 1974), enhance the body's ratio of protein to fat (Brumby 1959;Machlin 1972;Muir et al. 1983;Chung et al. 1985), and influence the Reprint requests to: Luis S. Haro, Department of Biology, The University of Texas at San Antonio, 6900 N. Loop 1604 W., San Antonio, TX 78249-0609, USA; e-mail: luis.haro@utsa.edu; fax: (210) 458-5658.Abbreviations: SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; MER-45-kDa hGH, mercaptoethanol-resistant 45-kDa human growth hormone; TEMED, N, N, NЈ, EPPS,; CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid; EGTA, ethylene glycolbis(␤-aminoethylether)-N, N, NЈ, NЈ-tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; TCEP-HCl, tris(2-carboxyethyl)phosphine hydrochloride; hGH, human growth hormone; MALDI-TOF/ MS, matrix-assisted laser desorption/ionization time-of-fli...

Preparative alkaline urea gradient PAGE: Application to purification of extraordinarily‐stable disulfide‐linked homodimer of human growth hormone

“…A small amount of 22 kDa hGH monomer was observed in the analytical SDS polyacrylamide gel of electroeluted fractions containing dimeric MER-45 kDa hGH. However, it is not a component of the electroeluate but rather its presence in the analytical polyacrylamide gel is due to the conversion of dimeric MER-45 kDa hGH into the monomeric 22 kDa hGH when heated at 1007C in SDS sample buffer containing 2-mercaptoethanol as previously demonstrated [31]. Monomeric 22 kDa hGH, 24 kDa hGH, dimeric 45 kDa hGH and other higher oligomeric isoforms of hGH stemming from electroeluates of Bands 2 to 5 of Panel I are revealed in the analytical SDS polyacrylamide gel of Panel II (Lanes P thru X).…”

“…Dimeric MER-45 kDa hGH is a naturally occurring hGH isoform that was recently shown to be a homodimer of 22 kDa hGH subunits held together by unusually stable inter-chain disulfide bridges [31]. The hGH isoform was present in extracts of fresh pituitary glands [29] and was also observed in the culture media of human pituitary adenomas [42].…”

“…It is estimated that dimeric MER-45 kDa hGH comprises 2-5% of the pituitary hGH [13,14,43] and approximately 1% of serum hGH [13]. Although dimeric MER-45 kDa hGH was purified for structural analysis [31], the product was not useful for biological studies since the final step utilized preparative SDS-PAGE to produce a denatured hormone. Since in vitro and in vivo biological testing of the sample could not be carried out, a gap in our knowledge of the biological significance of dimeric MER-45 kDa hGH remained.…”

“…The fourth 45 kDa hGH isoform is a protein which does not dissociate into monomers even when treated for 5 min at 1007C in either 1% 2-mercaptoethanol or 1% DTT [29], and is thus referred to as a mercaptoethanol-resistant (MER)-45 kDa hGH. Recently, structural analysis of SDS-PAGE purified MER-45 kDa hGH revealed that this hGH isoform was an extraordinarily stable disulfide-linked homodimer of monomeric 22 kDa hGH subunits [31]. Unfortunately, due to the absence of a nondenaturing isolation procedure for biologically active dimeric MER-45 kDa hGH, its biological significance has remained unknown.…”

The Minor Capsid Protein VP11 of Thermophilic Bacteriophage P23-77 Facilitates Virus Assembly by Using Lipid-Protein Interactions

“…2A, I). Proteins forming strong disulfide bonds have been shown to remain in a multimeric state even after boiling in standard SDS sample buffer containing 1% SDS and 1% ␤-mercaptoethanol but could be dissociated with a higher reducing agent concentration (45). Therefore, a sample of VP11 was boiled in SDS sample buffer containing a high (5%) concentration of the reducing agent ␤-mercaptoethanol.…”