Lipolytic and antilipolytic effects of human growth hormone, its 20-kilodalton variant, a reduced and carboxymethylated derivative, and human placental lactogen on chicken adipose tissue in vitro

The lipolytic and antilipolytic effects of human growth hormone (22K-hGH), its 20-kilodalton variant (20K-hGH), a reduced and S-carboxymethylated derivative (RCM-hGH), and human placental lactogen were examined using chicken adipose tissue explants in vitro. Lipolysis, as determined by glycerol release, was stimulated by 22K-hGH (biosynthetic and pituitary derived), 20K-hGH (pituitary derived), and RCM-hGH (modified biosynthetic). These growth hormone preparations also exhibited similar antilipolytic activity (i.e., transient inhibition of glucagon-induced lipolysis). However, unlike human growth hormone, human placental lactogen neither stimulated lipolysis nor inhibited glucagon-stimulated lipolysis. Some augmentation of glucagon-stimulated lipolysis was observed in the presence of human placental lactogen. These results indicate that the disulfide bridges (Cys53----Cys165; Cys182----Cys189) and amino acid residues 32-46 of hGH are not required for lipolytic or antilipolytic activities of human growth hormone on chicken adipose tissue.     

Use of calcium dependence as a means to study the interaction between growth hormones and their binding proteins in rabbit liver.

The affinity of 22,000-Mr human growth hormone (22 K-hGH) for GH binding proteins in rabbit liver is increased approx. 19-fold by 25 mM-Ca2+. In contrast, ovine growth hormone (oGH) binding is Ca2+-independent up to 10 mM, and decreased by greater Ca2+ concentrations. The 20,000-Mr hGH variant (20K-hGH), lacking residues 32-46, exhibits intermediate behaviour. Without Ca2+ there is a residual 40% of maximum specific binding to liver microsomes, and this increases to 65% with liver cytosolic GH binding proteins. In contrast with 22K-hGH, Scatchard analysis of 20K-hGH binding to liver microsomes produces curvilinear plots in the presence of 25 mM-Ca2+. From these results and inhibition studies with monoclonal antibodies to the GH binding proteins, it is concluded that deletion of the region 32-46 from 22K-hGH has eliminated one component of high-affinity Ca2+-potentiable binding. The Ca2+-mediated increase in Ka for the 22K-hGH-binding protein interaction is consistent with convergence of unit negative charges on the hormone and binding protein towards an intercalated Ca2+ ion. A positive charge in the critical region of nonprimate GHs would render their interactions Ca2+-independent and of lower Ka compared with 22K-hGH. A likely candidate for the negatively charged interactive residue is glutamate-33, since it is unique to human GH and is replaced by a positively charged arginine in non-primate GHs. Its absence in 20K-hGH could explain the altered calcium-dependence of 20K-hGH binding to what is probably the type 2 binding protein [Barnard & Waters (1986) Biochem. J. 237, 885-892]. The Ca2+-dependence of 20K-hGH binding to a subset of GH binding proteins provides both a verification and a mechanistic basis for the proposal [Hughes, Tokuhiro, Simpson & Friesen (1983) Endocrinology (Baltimore) 113, 1904-1906] that 20K-hGH binds with high affinity to only a subset of binding proteins in rabbit liver membranes.     

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.     

The 20-kD human growth hormone reduces body fat by increasing lipolysis and decreasing lipoprotein lipase activity

AIM: The aim of this study was to estimate the lipolytic activity of the human growth hormone variant, 20-kD human growth hormone (20K-hGH). METHODS: Obese KV-A(y) mice were given daily subcutaneous injections of 20K-hGH (0.25, 0.5, 1.0 mg/kg), 22K-hGH (0.25 mg/kg) or saline as a control for 2 weeks. Body composition (fat, water and protein), lipolysis and lipoprotein lipase (LPL) activity were measured 24 h after the final injection. RESULTS: Both growth hormone isoforms significantly reduced relative fat pad and whole body lipids. In addition, 20K-hGH produced an inhibition of LPL activity in adipose tissue and stimulated lipolysis in adipocytes. CONCLUSION: These data strongly suggest that inhibition of LPL activity in adipose tissue and stimulation of lipolysis in adipocytes by 20K-hGH treatment reduce adipose tissue mass, resulting in body fat reduction.     

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.     

Monoclonal antibodies specific for different regions of human apolipoprotein A-I. Characterization of an antibody that does not bind to a genetic variant of apoA-I (Glu----136 Lys)

Three monoclonal mouse hybridoma antibodies, designated 2AI, 4AI, and 5AI, specific for human plasma apolipoprotein A-I (apoA-I) were characterized. In an enzyme-linked immunosorbent assay (ELISA) each of the antibodies reacted with purified apoA-I and with A-I in normal human serum. Immunoblotting of apoA-I subjected to isoelectric focusing revealed that the three antibodies reacted with all the charge isomorphs of apoA-I and with proapoA-I. Using a solid phase competitive displacement assay, the antigenic determinant for antibody 5AI could be localized to cyanogen bromide fragment 3 of apoA-I (residues 113-148), while the epitope for antibody 4AI resided in cyanogen bromide fragment 4. Dot blot experiments and data obtained by the competitive displacement assay revealed that antibody 2AI reacts with high affinity with CNBr fragment 2 but that it also reacts with lower affinity with fragments 1 and 4. The antibody 5AI did not bind to a genetic variant of apoA-I (Glu----136 Lys), demonstrating that the substitution of a single amino acid in human apoA-I can cause the loss of an antigenic determinant.     

The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia.

Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys). Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxyl-terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia.     

Localization of a gene for the human low-voltage EEG on 20q and genetic heterogeneity.

The localization of a gene responsible for a normal variant of the human electroencephalogram to the distal part of chromosome 20q is reported. A linkage analysis, including 17 families with 191 individuals, tested with 73 RFLPs and 22 blood and serological markers, was performed for the low-voltage electroencephalogram. This is a normal variant of the human electroencephalogram with an autosomal dominant mode of inheritance. The results present strong evidence for close linkage with the highly polymorphic marker CMM6 (D20S19) and for genetic heterogeneity.     

A state of acquired IL-10 deficiency in 0.4% of Danish blood donors

Autoantibodies against a variety of growth factors and cytokines are present in preparations of pooled normal human IgG, such as IVIg. The present study demonstrated that healthy Danish blood donors produced high concentrations of anti-IL-10 IgG antibodies that bound IL-10 with extremely high avidity. The antibodies were of IgG class, polyclonally derived and acted as competitive IL-10 inhibitors in vitro, substantially inhibiting cellular IL-10 receptor binding and neutralizing IL-10 activity in vitro. The antibodies failed to bind viral forms of IL-10 or other members of the human IL-10 family (IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, IL-29). The production of anti-IL-10 antibodies was stable from months to years, and high positive donors were likely to acquire a state of IL-10 deficiency in the circulation during this period. Anti-IL-10 antibodies were readily measurable even in highly diluted plasma samples, providing the explanation for the fact that relatively low antibody activity can be detected in normal human pooled IgG, derived from the plasma of over 1000 blood donors.     

Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra), an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.     

Effects of acid water exposure on plasma cortisol, ion balance, and immune functions in the "cobalt" variant of rainbow trout

This study was undertaken to examine physiological responses to acidification of environmental water in the "cobalt" variant of rainbow trout (Oncorhynchus mykiss), which exhibits malformation of the pituitary, by following changes in plasma levels of cortisol and electrolytes, blood pH, gill Na(+), K(+)-ATPase activity, and immune functions after exposure to acid water (pH 4.5). Resting levels of plasma cortisol and lysozyme were significantly lower in the cobalt variant than in the normal trout, whereas plasma ceruloplasmin was significantly higher in the cobalt variant, suggesting that some endocrine factors, lacking or deficient in the cobalt variant, are important for the regulation of its immune functions. Blood pH was slightly but significantly lower in the cobalt variant at rest. After exposure to acid water for 24 h, both the normal trout and cobalt variant showed a significant elevation in plasma cortisol, although the increased level in the cobalt variant was still lower than that in the normal trout transferred to neutral water. No differences were seen in blood pH, plasma electrolytes, and gill Na(+), K(+)-ATPase activity between the normal trout and the cobalt variant, indicating that the cobalt variant regulates ion balance when exposed to acid water, despite malformation of the pituitary. Although the normal trout showed a reduction in plasma lysozyme level after acid exposure, there was no significant change in the cobalt trout. Adverse effects of pituitary malformation on ion balance and immune functions may be compensated by extrapituitary factors in the cobalt variant when it is exposed to acid water.     

Detection of bound and free IGF-1 and IGF-2 in human plasma via biomolecular interaction analysis mass spectrometry

Insulin like growth factor (IGF)-1 and IGF-2 were assayed from human plasma via biomolecular interaction analysis mass spectrometry, utilizing antibodies as ligands for affinity retrieval. Detection of both targeted and non-targeted IGFs in the mass spectra indicated possible protein complex retrieval by the individual antibodies. A series of control experiments eliminated the possibility of analyte cross-walking between flow cells, significant antibodies cross-reactivity, and direct IGF interactions. To disrupt the putative protein complex and release its constituent proteins, plasma samples were treated with detergents. An SDS-treated plasma yielded IGF signals in a different ratio than the one observed in the mass spectra from the non-treated plasma, suggesting disruption of the protein complex, and its retrieval from non-treated plasma. Novel truncated IGF-2 variant, missing its N-terminal Alanine, was detected in all mass spectra.     

Determination and validation of off-target activities of anti-CD44 variant 6 antibodies using protein biochips and tissue microarrays

We describe a novel procedure for determination and validation of off-target activities of anti-cluster designation antigen identity 44 (CD44) variant 6 recombinant antibodies by combining two complementary technology platforms; namely UNIchip AV-400, containing a printed serial dilution of CD44 variant 6 and approximately 400 different human proteins, and TISSOMICS, enabling human tissue microarray analysis in high-throughput mode. We have analyzed the performance of two human monoclonal recombinant antibodies directed against CD44 variant 6 protein, BMS 116 and BMS 125, in a blinded study. The antibodies exhibit a clear differentiation with regard to their binding profiles in the two systems. BMS 116 shows a low degree of specificity in the normal human Food and Drug Administration (FDA) tissue panel, which was confirmed by binding to more than 206 proteins on the protein biochip. In contrast, BMS 125 gives a highly selective membranous staining on selected human epithelial tissue components with no off-target activities observed on the protein biochip. Additionally, antibody BMS 125 shows a higher sensitivity to its antigen CD44 variant 6 than antibody BMS 116 as determined by the protein biochip.     

Hepatitis B virus surface antigen binds to apolipoprotein H.

We have previously demonstrated that a plasma membrane-enriched fraction isolated from human liver is capable of binding recombinant hepatitis B surface antigen (rHBsAg) (P. Pontisso, M. A. Petit, M. Bankowski, and M. E. Peeples, J. Virol. 63:1981-1988, 1989). In this study we have separated the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used a ligand-blotting technique to identify a 46-kDa rHBsAg-binding protein. This protein could be removed from the membranes with a weakly acidic buffer, implying that it is peripherally bound. Examination of human serum revealed that the 46-kDa binding protein is a serum protein. Isolation of plasma lipoproteins revealed that the binding protein is in part associated with chylomicrons and high-density lipoproteins, both of which are targeted to the hepatocyte during the normal course of lipid metabolism. The binding protein was identified as apolipoprotein H (apo H), also known as beta 2-glycoprotein I, on the basis of copurification of the rHBsAg-binding activity with the apo H protein and the ability of cDNA-expressed apo H to bind rHBsAg. Serum-derived HBsAg also binds to apo H, indicating that binding is not unique to rHBsAg. Binding is saturable, requires only the small S protein of rHBsAg, and is inhibited by excess rHBsAg, antibodies to HBsAg, and antibodies to apo H. The binding activity of apo H is destroyed upon reduction, indicating that 1 or more of its 22 disulfide bonds are required for interaction with rHBsAg. The possibility that an interaction between hepatitis B virus particles and lipoprotein particles may facilitate entry of the virus into hepatocytes is discussed.     

Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth hormone (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa GH was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.     

Determination of insulin-like growth factor-I in normal subjects and in patients with growth hormone disorders by radioimmunoassay using biosynthetic homologous peptide

A highly sensitive and specific RIA for IGF-I has been developed using recombinant DNA-derived IGF-I of very high purity and specific antiserum to it. This assay system could detect IGF-I at as low concentrations as 20-30 ng/ml. The intra-assay and interassay coefficients of variation at various concentrations of IGF-I were 4.9 to 6.5% and 5.4 to 8.0%, respectively. The recovery rate of pure IGF-I added to plasma was 77.0 +/- 3.7%. The antiserum did not cross-react with porcine insulin, biosynthetic human insulin, hGH, hEGF, the synthetic C-domain of IGF-I or that of IGF-II, but reacted equally with an analog, Thr59-IGF-I. Plasma IGF-I was extracted by the acid-ethanol method before assay to separate IGF-I from its binding protein. When plasma IGF-I was assayed without extraction, the inhibition curves of serial dilution of plasma samples from several individuals were not parallel to the standard curve of IGF-I. The plasma concentration of IGF-I was 147 +/- 49 ng/ml (mean +/- SD) in 156 normal adults aged from 20-59 years. As reported by others, the IGF-I levels were low in cord plasma (41.8 +/- 23.5 ng/ml) and plasma of patients with GH deficiency (64.6 +/- 42.0 ng/ml), while its levels were high in normal children of pubertal ages (12-13 yr, 365 +/- 126 ng/ml) and in patients with active acromegaly (562 +/- 115 ng/ml). This RIA system is a simple and useful method for determining plasma IGF-I in normal and diseased states.     

Structural characterization of a chain termination mutant of human serum albumin

Mutant forms of human serum albumin have been detected on the basis of their abnormal electrophoretic mobility which is either faster or slower than that of normal albumin. In the present work we have studied the structure of a slow variant, referred to as albumin Ge/Ct, in order to define the cause of its genetic abnormality. The protein was isolated from the serum of a young healthy woman homozygous for the variant. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585). This fragment was isolated on a preparative scale and subjected to tryptic digestion. All tryptic peptides were purified by reverse-phase high performance liquid chromatography and characterized. Sequential analysis of three abnormal peptides revealed that albumin Ge/Ct has a shortened chain with the following COOH-terminal sequence: Leu-Val-Ala-Ala-Ser-Lys580-Leu-Pro. The presence of an additional lysine residue accounts for the electrophoretic behavior of the variant. It is likely that the variant may be caused by a single base deletion in the structural gene, a Cyt in mRNA codon 580, and the consequent shift in reading frame.     

Flow cytometric characterization of normal and variant cells with monoclonal antibodies specific for glycophorin A

Quantitative immunofluorescence measurements were performed on erythrocytes labeled with monoclonal antibodies to glycophorin A (GPA) to assess the level of binding of these antibodies to normal and variant cell types. The seven antibodies used in this study include two that bind preferentially to the M form of GPA, three that bind preferentially to the N form, and two that bind equally well to both. Flow cytometric analysis of mixtures of cells differing in M,N type showed binding specificities of greater than 100-fold for most of the antibodies, and showed that three antibodies bind cell-bound GPA with an affinity of approximately 10(9) M(-1). These data also showed that the level of expression of GPA varies by less than 10% from cell to cell and from individual to individual. Flow measurements were also done on human erythrocytes with the following variant forms of glycophorin: Mc, Mg, Mk, En(F), En(UK), Mi-I, Mi-II, Mi-III, S-s-U-, Tn+, and St(a+). Other cell types analyzed included erythrocytes from chimpanzee, rhesus, African green, and capuchin monkeys, and cells from the human erythroleukemia cell line, K562. Flow analysis with our seven antibodies showed these cell types have distinctive labeling patterns consistent with the known or inferred altered glycophorins presented on these cells. In most cases, variant alleles were expressed at normal levels. Our results support other observations that the variants En(UK) and St(a+) contain hybrid GPA-GPB proteins, and suggest that their level of expression is largely determined by the 3' end of the hybrid genes.     

A simple semiquantitative radiometric measurement of antithyroglobulin antibodies in human serum. Comparison with hemagglutination method

A simple solid-phase radiometric assay for the measurement of thyroglobulin autoantibodies (TgRA) was developed and evaluated. The assay is semiquantitative, and the results were expressed as a ratio between sample versus negative control (normal human serum). In 59 normal subjects, the mean ratio was 0.93 +/- (SD) 0.34. Thyroglobulin antibodies by radiometric assay, by hemagglutination (TgHA), as well as microsomal antibodies by hemagglutination (MCHA) were measured in 41 patients with a histopathologic diagnosis of Hashimoto's thyroiditis (n = 22), adenomatous goiter (n = 10), carcinoma (n = 5), adenoma (n = 4), and in 59 patients without histopathologic diagnosis of thyroid disease. In patients with Hashimoto's thyroiditis, TgRA, TgHA, and MCHA were positive in 54, 31, and 81% of patients, respectively. 1 patient had positive TgRA with negative MCHA levels, and 2 had negative antibody titers by all methods. Thyrotropin-stimulating hormone levels were elevated (greater than 10 microU/ml) in 17 of these patients. Our results suggest that although the TgRA method is more sensitive than TgHA for detecting thyroglobulin antibodies, its diagnostic sensitivity is not equal to that of MCHA.