Isolation of four isomers of the 20,000 dalton variant of human pituitary growth hormone

A simple procedure has been developed which for the first time describes the isolation of isomers of the 20,000 dalton variant of human growth hormone (20K hGH). From a human pituitary hormone concentrate different hGH dimers (covalently and noncovalently linked) were enriched by chromatography on SP-Sephadex C-50, DEAE-Sepharose CL-6B and Sephadex G-100. Noncovalently-linked dimers were split by 6 M urea into 20K hGH and 22K hGH monomers. A complete group-separation of 20K hGH and 22K hGH monomers was achieved by chromatography on DEAE-Sepharose CL-6B at neutral pH. The 20K hGH monomer was resolved into four isomers either by preparative isoelectric focusing or by zone electrophoresis in agarose suspension at alkaline pH. The three latter techniques were all used in the presence of 6 M urea. Radioimmunoassay and radioreceptorassay indicated that the isomers obtained were true components of human growth hormone.     

Isolation of Four Isomers of the 20,000 Dalton Variant of Human Pituitary Growth Hormone

A simple procedure has been developed which for the first time describes the isolation of isomers of the 20, 000 dalton variant of human growth hormone (20K hGH). From a human pituitary hormone concentrate different hGH dimers (covalently and noncova-lently linked) were enriched by chromatography on SP-Sephadex C-50, DEAE-Sepharose CL-6B and Sephadex G-100. Noncovalently-1inked dimers were split by 6 M urea into 20K hGH and 22K hGH monomers. A complete group-separation of 20K hGH and 22K hGH monomers was achieved by chromatography on DEAE-Sepharose CL-6B at neutral pH. The 20K hGH monomer was resolved into four isomers either by preparative isoelectric focusing or by zone electrophoresis in agarose suspension at alkaline pH. The three latter techniques were all used in the presence of 6 M urea. Radioimmunoassay and radioreceptorassay indicated that the isomers obtained were true components of human growth hormone.     

Synthesis and purification of a deleted human growth hormone, hGH delta 135-146: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities

Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity. In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system. The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro. The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay. In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH. In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations. By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH. These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.     

The binding between the stem regions of human growth hormone (GH) receptor compensates for the weaker site 1 binding of 20-kDa human GH (hGH) than that of 22-kDa hGH

Despite the lower site 1 affinity of the 20-kDa human growth hormone (20K-hGH) for the hGH receptor (hGHR), 20K-hGH has the same hGHR-mediated activity as 22-kDa human GH (22K-hGH) at low hGH concentration and even higher activity at high hGH concentration. This study was performed to elucidate the reason why 20K-hGH can activate hGHR to the same level as 22K-hGH. To answer the question, we hypothesized that the binding between the stem regions of hGHR could compensate for the weaker site 1 binding of 20K-hGH than that of 22K-hGH in the sequential binding with hGHR. To demonstrate it, we prepared 15 types of alanine-substituted hGHR gene at the stem region and stably transfected them into Ba/F3 cells. Using these cells, we measured and compared the cell proliferation activities between 20K- and 22K-hGH. As a result, the activity of 20K-hGH was markedly reduced than that of 22K-hGH in three types of mutant hGHR (T147A, H150A, and Y200A). Regarding these mutants, the dissociation constant of hGH at the first and second step (KD1 and KD2) in the sequential binding with two hGHRs was predicted based on the mathematical cell proliferation model and computational simulation. Consequently, it was revealed that the reduction of the activity in 20K-hGH was attributed to the change of not KD1 but KD2. In conclusion, these findings support our hypothesis, which can account for the same potencies for activating hGHR between 20K- and 22K-hGH, although the site 1 affinity of 20K-hGH is lower than that of 22K-hGH.     

Monoclonal antibodies which preferentially bind to 22 K human growth hormone rather than its 20 K variant

We have established 13 hybridoma cell lines which secrete mouse IgG1 monoclonal antibodies (McAbs) to human growth hormone (hGH). Binding affinity and binding specificity of McAbs were analyzed by competitive radioimmunoassay. Among these McAbs, CL. B1 showed a high affinity of 9.8 x 10(8) l/mol, and all McAbs so far tested showed very weak cross-reactivity or none at all with human prolactin (hPRL) and human chorionic somatomammotropin (hCS; human placental lactogen). Analysis of binding sites of McAbs using hGH variant and fragments in both ELISA and RIA demonstrated that McAbs could be classified into two groups. All the McAbs obtained in this study bound to plasmin-digested fragment S2 (hGH 1-134 and 141-191) and fragment alpha 3 (hGH 1-134 and 147-191). However, five (such as 1D2) out of 13 McAbs bound to fragment F1 (hGH 1-134) and others (such as CL. B1) did not. The McAb CL. B1 in the latter group showed low affinity with 20 K hGH (residue 32-46 deleted in native 22 K hGH) in contrast to high affinity with hGH (22 K). This suggests that the former McAbs recognize an epitope located at the N-terminal two-third part of hGH. In contrast, the McAbs of the latter group are likely to recognize three-dimensional structure of native 22 K hGH.     

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12. Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation.

Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment. Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process. Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine. A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds.     

Cellular activities of 20K- and 22K-hGH do not necessarily correlate with their binding affinities for rat GH receptor

Even though 20K human growth hormone (20K-hGH) has 3-10% binding affinity for the rat liver and adipose tissue microsomes as compared to 22K-hGH, it was also reported that 20K-hGH has the same potency as 22K-hGH in the hypophysectomized rat weight gain assay. In order to investigate the reason why such controversial data exist, we have studied 20K- and 22K-hGH using the rat GH receptor extracellular domain (rGHR-ECD) and full-length rGHR. When we examined the complex formation of rGHR-ECD with 20K- and 22K-hGH in gel filtration assay, 20K-hGH formed no complex while 22K-hGH formed a 1:1 complex. Next, rGHR cDNA was introduced into Ba/F3 cells and CHO-K1 cells, and stable transfectants (Ba/F3-rGHR and CHO-rGHR) were established. In the proliferation of Ba/F3-rGHR cells, 20K-hGH had 10-fold lower activity than 22K-hGH, which is consistent with their affinities for rGHR. But surprisingly, in the Spi2.1 gene promoter activation in CHO-rGHR cells, 20K- and 22K-hGH had the same activity, which was found not only in stable CHO-rGHR clones but also in CHO-K1 cells transiently expressing rGHR. In conclusion, these results indicate that cellular activities of 20K- and 22K-hGH do not necessarily correlate with their binding affinities for rGHR.     

Comparison of the acute metabolic effects of 22,000-dalton and 20,000-dalton growth hormone in human subjects

The acute metabolic effects of 20,000-dalton human growth hormone (hGH20K) in man have not previously been tested. We compared changes in concentrations of free fatty acids (FFA), glucose, and insulin in nine growth hormone deficient children following injection of 22,000-dalton intact human growth hormone (hGH22K) and the smaller variant, hGH20K. There was a significant decline (37%) in the mean FFA concentration from baseline to 1/2 hour post-injection and from baseline to 1 hour post-injection (36%) in the children given hGH22K, but no such decline was seen after injection of hGH20K. No significant differences in mean insulin or glucose concentrations were noted between the two treatment groups, and glucose and insulin concentrations did not acutely change after injection of either hormone. The results of this study indicate that hGH20K has a diminished activity for suppression of FFA as compared to hGH22K. This suggests that GH residues 32-46, missing in hGH20K, constitute all or part of the region of hGH22K producing this response, or that the different primary structures of the two hormones result in tertiary structural differences and altered biological activity.     

The receptor binding properties of the 20K variant of human growth hormone explain its discrepant insulin-like and growth promoting activities

The 20K variant of native (22K) hGH is a full agonist for the growth promoting and lactogenic properties of the hormone in vivo but has been reported to have weak or absent insulin-like properties. To explore if these differences may be explained at the receptor level, we compared the ability of 22K and 20K hGH to inhibit the binding of 125I-22K hGH to receptors in isolated rat adipocytes, a target for the insulin-like effects of the hormone and in IM-9 cultured human lymphocytes, more specific for growth effects. Our data show that while 20K hGH is a potent agonist of native 22K hGH in the IM-9 lymphocyte assay, its potency in the rat adipocyte binding assay is only 3%, even when both cells are incubated together in identical conditions. Thus, the receptors for hGH appear to be different on various target cells, explaining why the 20K variant has different relative biological potencies at different sites of action.     

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions.

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.     

The effect of growth hormone on the proliferation of human Th cell clones

The effects of human growth hormone (hGH) on human Th cell clones were examined. Both 20K and 22K hGH stimulated the proliferation of Th2 and Th0 cells in the presence of mite antigen, whereas they did not stimulate the proliferation of Thl cells. Because the effect of 20K hGH was almost the same as that of 22KhGH, it was suggested that the action of hGH was not mediated through prolactin receptor but through hGH receptors. The application of growth hormone binding protein (GHBP) inhibited the cell growth of Th1 clones. In Th2 and Th0 cells GHBP inhibited the hGH-stimulated cell proliferation. However, GHBP alone did not affect the proliferation of Th2 and Th0 cells. hGH was detected in the supernatant of Th1 clones in the presence of mite antigen but it was not detected in Th2 clones. hGH was detected in one out of 4 batches of Th0 clones. These data indicated that hGH was secreted from Thl clones, and that Th0 clones possessed characteristics of both Th2 and Th0 clones.     

Biological activities of synthesized 20K and 22K hGH in Nb2 bioassay and IM-9 radioreceptor assay

We investigated the bioactivities of the recombinant DNA-derived methionyl 20K hGH (20K-Met-hGH) and methionyl hGH (22K-Met-hGH). The growth-promoting activities in Nb2 cells of 20K-Met-hGH and 22K-Met-hGH were 10.7% and 93.5% of pituitary hGH (P-hGH), respectively. In the IM-9 lymphocyte assay, the binding activities of 20K-Met-hGH and 22K-Met-hGH to hGH receptor were 29.0% and 87.1% of P-hGH, respectively. Our data demonstrate that 20K-Met-hGH may have weaker biological potency than P-hGH.     

Asparagine-linked glycosylation of the scrapie and cellular prion proteins

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of Mr26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPSc yielded equimolar amounts of two proteins of Mr26K and 28K. The significance of this altered stoichiometry between the 26- and 28-kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 27-30, which is produced by limited proteolysis of PrPSc, exhibited a reduced number of charge isomers after PNGase F digestion. The molecular weight of PrP 27-30 was reduced from 27K-30K by PNGase F digestion to 20K-22K while anhydrous hydrogen fluoride or trifluoromethanesulfonic acid treatment reduced the molecular weight to 19K-21K and 20K-22K, respectively. Denatured PrP 27-30 was radioiodinated and then assessed for its binding to lectin columns. PrP 27-30 was bound to wheat germ agglutinin (WGA) or lentil lectins and eluted with N-acetylglucosamine or alpha-methyl-mannoside, respectively. Digestion of PrP 27-30 with sialidase prevented its binding to WGA but enhanced its binding to Ricinus communis lectin. These findings argue that PrP 27-30 probably possesses Asn-linked, complex oligosaccharides with terminal sialic acids, penultimate galactoses, and fucose residues attached to the innermost N-acetyl-glucosamine. Whether differences in Asn-linked oligosaccharide structure between PrPC and PrPSc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.     

Effects of 22K or 20K human growth hormone on lipolysis, leptin production in adipocytes in the presence and absence of human growth hormone binding protein.

OBJECTIVE AND METHOD: We studied the effects of human growth hormone (hGH) on leptin production and lipolysis stimulation in the presence or absence of human growth hormone binding protein (hGHBP) using 3T3- L1-hGHR adipocytes which efficiently express human growth hormone receptor. RESULTS AND CONCLUSION: It was clarified that (1) hGH decreases leptin secretion after hGH-induced lipolysis stimulation, and (2) the reduction of leptin production and lipolysis stimulation by 22K hGH was attenuated with hGHBP, whereas that by 20K hGH, which is a naturally occurring isoform of 22K hGH, was not affected with hGHBP.     

Correlation between active form and dimeric structure of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart

The active form of purified mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated by crosslinking with dimethylsuberimidate and SDS-PAGE, with or without pretreatment with the inactivating detergent Triton X-100. In the absence of detergent, crosslinked isomers of the dimeric form of 208–235 kDa were obtained. Addition of detergent led to the simultaneous loss of the dimers and the bulk of the activity. Removal of the detergent led to a partial restoration of both activity and the dimeric forms. The results suggest that the active form is a dimer, and that the detergent-dependent conversion to the largely inactive monomer is reversible. It is proposed that the mechanism of inactivation of transhydrogenase by Triton X-100 involves a disruption of essential hydrophobic interactions between the membrane-spanning regions of the monomers.     

Characterization of the dimerization process of a domain-swapped dimeric variant of human pancreatic ribonuclease

It has been previously reported that the structure of a human pancreatic ribonuclease variant, namely PM8, constitutes a dimer by the exchange of an N-terminal domain, although in an aqueous solution it is found mainly as a monomer. First, we investigated the solution conditions that favour the dimerization of this variant. At 29 degrees C in a 20% (v/v) ethanol buffer, a significant fraction of the protein is found in dimeric form without the appearance of higher oligomers. This dimer was isolated by size-exclusion chromatography and the dimerization process was studied. The dissociation constant of this dimeric form is 5 mm at 29 degrees C. Analysis of the dependence of the dimerization process on the temperature shows that unlike bovine pancreatic ribonuclease, a decrease in the temperature shifts the monomer-dimer equilibrium to the latter form. We also show that a previous dissociation of the exchangeable domain from the main protein body does not take place before the dimerization process. Our results suggest a model for the dimerization of PM8 that is different to that postulated for the dimerization of the homologous bovine pancreatic ribonuclease. In this model, an open interface is formed first and then intersubunit interactions stabilize the hinge loop in a conformation that completely displaces the equilibrium between nonswapped and swapped dimers to the latter one.     

Bioactivity of human growth hormone in urine from acromegalic patients

Bioactivity of hGH in urine from five acromegalic patients was determined by Nb2 bioassay and IM-9 receptor modulation assay (RMA). Urine samples were concentrated by immunoadsorbent chromatography, dialysis and lyophilization. The ratio of the bioactivity by Im-9 RMA and the immunoactivity by RIA was between 0.81 and 1.24 (1.01 +/- 0.19, mean +/- SD). The ratio of the bioactivity by Nb2 bioassay and the immunoactivity ranged from 0.45 to 0.60 (1.37 +/- 0.85). Gel chromatography of hGH in urine samples measured by sensitive sandwich enzyme immunoassay showed that more than 97% of hGH activity was the 22K form. Urinary hGH from acromegalic patients was bioactive in Nb2 bioassay and IM-9 RMA and the bioactivity showed a close correlation with the immunoactivity. The major immunoactive form of hGH (22K) seems to correspond to the bioactivity.     

Interleukin-22 forms dimers that are recognized by two interleukin-22R1 receptor chains

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-γ (IFN-γ). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.