The divergent effect of insulin-like growth factor binding protein (IGFBP) - 1 on IGF-induced Steroidogenesis in bovine adrenocortical cells is not due to its phosphorylation status.

In previous studies we have shown that insulin-like growth factor (IGF)-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I. The steroidogenic effect of both, IGF-I and IGF-II, is mediated through the IGF-I receptor and modified by locally produced IGF binding proteins. Further studies demonstrate that bovine adrenocortical cells do synthesize IGFBP-1 to -4 and that their secretion is regulated differentially by IGFs and ACTH. Since IGFBP-1 selectively is induced 3- to 4- fold by ACTH, the aim of the following studies was to evaluate the effect of IGFBP-1 on IGF-induced cortisol secretion from bovine adrenocortical cells in primary culture. Coincubation of bovine adrenocortical cells with purified IGFBP-1 (30 nM) induced a time--and dose-dependent potentiating effect (38+/-2%) on IGF-I-stimulated (6.5 nM) cortisol-secretion, wheras the IGF-II induced steroidogenic effect was inhibited (40+/-3%) by IGFBP-1. Similar results were found when bovine adrenocortical cells were preincubated with IGFBP-1 before stimulation experiments with IGFs were performed. In order to evaluate the influence of different phosphorylation states of IGFBP-1 on the steroidogenic effect of IGF-I and IGF-II and on the affinity to IGFs, a highly phosphorylated (pIGFBP-1) and a dephosphorylated isoform (dIGFBP-1) of IGFBP-1 have been separated by anion exchange chromatography for further incubation experiments and binding studies. No different effects on IGF-I or IGF-II-induced steroidogenesis were observed when a highly phosphorylated IGFBP-1 isoform was used, compared to the dephosphorylated IGFBP-1 isoforms. In binding studies IGFBP-1 did show high affinity binding for IGF-I with a calculated association constant (K (a)) of 2,17 x 10 (9) M (-1). Similar association constants were calculated for dIGFBP-1 and pIGFBP-1 (1,93 x 10 (9) M (-1) and 2,67 x 10 (9) M (-1) respectively) and no difference in binding capacity was observed when IGF-II was used as ligand. IN CONCLUSION, these results demonstrate that in bovine adrenocortical cells IGFBP-1 time- and dose-dependently inhibits the steroidogenic effect of IGF-II and stimulates the effect of IGF-I. The observed modulatory effect of IGFBP-1 is independent of the mode of incubation and its phosphorylation status. The previously observed stronger steroidogenic potency of IGF-II in bovine adrenocortical cells therefore can not be explained by an interaction with IGFBP-1. The mechanisms by which IGFBP-1 divergently regulates the steroidogenic effect of IGF-I and IGF-II remain unclear at present and further investigation is necessary to elucidate the mechanisms by which IGFBP-1 modulates IGF action in the adult adrenal gland.

Characterization of human insulin-like growth factor-binding proteins by two-dimensional polyacrylamide gel electrophoresis and Western ligand blot analysis.

The insulin-like growth factor (IGF)-binding proteins (IGFBPs) from adult human serum, amniotic fluid, and cerebrospinal fluid were analyzed by a modified two-dimensional gel electrophoresis followed by Western ligand blotting. The samples were subjected to immobilized pH gradient isoelectric focusing in the first dimension, followed by nondenaturing SDS-PAGE in the second dimension and autoradiography after ligand blotting with [125I]IGF-I or [125I]IGF-II. The identity of the binding proteins was confirmed by immunoblotting and immunoprecipitation with specific antibodies. Using this method, all six human high affinity IGFBPs could be clearly separated from each other according to their molecular mass and isoelectric points (pI). All IGFBPs exhibited a variety of specific pI isoforms, which presumably represent posttranslational modifications. In adult human serum, glycosylated IGFBP-3 is found as a broad band of spots with molecular masses of 41 and 45 kDa and a pI in the range of 4.8-8.2. The two IGFBP-3 bands could be reduced to a single 36-kDa band after deglycosylation (pI 6-9). Furthermore, the specific spots for IGFBP-2 (33 kDa; pI 6.2-7.1) and deglycosylated IGFBP-4 (24 kDa; pI 6.3, 6.5, and 6.8) were found with their expected molecular masses. Additionally, the diffuse bands around 30 kDa, found in one-dimensional Western ligand blotting, could be clearly separated into distinct groups of specific spots representing IGFBP-1 (30 kDa; pI 4.0-4.8), IGFBP-6 (30 kDa; pI 4.8-5.8), glycosylated IGFBP-4 (29 kDa; pI 6.1 and 6.3), and IGFBP-5 (30/31 kDa; pI 6.4-8). As expected, IGFBP-6 was visible only when IGF-II was used as radioligand. In conclusion, two-dimensional gel electrophoresis followed by Western ligand blotting allows identification of all six high affinity IGFBPs with their isoforms on the basis of their characteristic molecular masses and pI, especially in the range of 30 kDa. This technique can be rapidly performed with small amounts of complex biological fluids and is a powerful tool for the detection and analysis of posttranslational modifications of IGFBPs.

The molecular basis for epitopes on the free beta-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCG beta-core fragment.

The molecular basis for antigenic determinants on the free beta-subunit of human chorionic gonadotrophin (hCG beta), its carboxyl-terminal peptide (hCG beta CTP) and the hCG beta-core fragment (hCG beta cf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCG beta cf preparation, a synthetic peptide corresponding to the hCG beta CTP (beta 109-145), overlapping synthetic peptides spanning the entire amino acid sequence of hCG beta, and a reduced and alkylated hCG beta preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a soluble-phase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCG beta, nine different epitopes (beta 1-beta 9), arranged in three spatially distinct domains, could be distinguished. Epitopes beta 1-beta 7 were located in a single large domain on both hCG beta and the hCG beta cf whereas hCG beta CTP contained two topographically distant determinants, designated beta 8 and beta 9 respectively. All but the two epitopes located on hCG beta CTP (beta 8 and beta 9) were destroyed by reducing and alkylating hCG beta, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCG beta CTP is linear. At a molecular level, amino acid residues spanning hCG beta 45-52, hCG beta 137-144 and hCG beta 113-116 contributed to the formation of epitopes beta 5, beta 8 and beta 9 respectively. We have also shown that the hCG beta cf represents the immunodominant part of the free beta-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence beta 45-52. The hCG beta CTP does not play a critical role in the immunologically important tertiary structure of hCG beta and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues beta 113-116 and beta 137-144 respectively.

Variants of human chorionic gonadotropin from pregnant women and tumor patients recognized by monoclonal antibodies.

In biological fluids, hCG and its free alpha- (hCG alpha) and beta-subunits (hCG beta), occur in multiple forms. These various forms differ at the molecular level primarily in glycosylation, but also differ in protein backbone modifications corresponding to the urinary low molecular weight fragment of the hCG beta-subunit (beta-core fragment). This microheterogeneous nature can be demonstrated by isoelectric focusing in which variants are separated into bands with different isoelectric points (pI). To determine whether such isoelectric variants differ in antigenicity and consequently might escape immunoassay detection due to overspecificity of monoclonal antibodies (MCA), urinary pregnancy hCG (NIH, CR123) and tumor hCG preparations, such as a tumor-specific acidic variant of hCG (hCGav) and the hCG beta-core fragment, were separated by isoelectric focusing in the absence or presence of 8 M urea, or by sodium docedyl sulfate-polyacrylamide gel electrophoresis and enzymatically immunostained using an MCA panel directed against 17 different hCG epitopes. MCA against 14 different epitopes accessible on holo-hCG recognized all pI variants of pregnancy holo-hCG or tumor-derived hCGav, as was true for the three MCA recognizing epitopes hidden on holo-hCG but accessible on the free subunits after hCG dissociation by urea. We conclude that each individual pI-isoform of holo-hCG and its free subunits expresses the entire set of epitopes recognized by our MCA panel. The carbohydrate moieties that form a biochemical basis for hCG heterogeneity seem to be neither of major antigenic relevance, nor are they structurally related to any particular epitope. Thus, various glycosylation forms of hCG, hCG alpha, hCG beta, and hCG beta-core in normal as well as in pathological samples should safely be detectable and measureable by immunoassays employing MCA with appropriate subunit specificity.

Cross-reactivity of a commercial chemiluminescence immunoassay for human chorionic gonadotropin with the free beta-subunit.

An enhanced chemiluminescence immunoassay for the determination of serum human chorionic gonadotropin (HCG) in specimens from oncology patients has been assessed with respect to its cross-reactivity with the free HCG beta-subunit (HCG-beta). The assay, standardized against the First International Reference Preparation 75/537, had a cross-reactivity with the free beta-subunit of 625% (molar basis). Therefore this assay achieves high sensitivity for the detection of either intact HCG or free HCG-beta in serum of patients with seminomatous or nonseminomatous testicular cancers. Results of both assays, the in-house immunoradiometric assay (+HCG-beta) and the Amerlite HCG-60 assay, showed a close correlation (R = 0.854-0.960) when serum samples from tumour patients were analyzed. Moreover, the content of free beta-subunit determined in a specific HCG-beta assay, could be quantitatively measured in the enhanced chemiluminescence immunoassay. Thus, this assay is suitable for oncology use, but also highlights the limitations of measuring HCG in serum samples.

Testicular cancer secretes intact human choriogonadotropin (hCG) and its free beta-subunit: evidence that hCG (+hCG-beta) assays are the most reliable in diagnosis and follow-up.

Human choriogonadotropin (hCG) and free hCG-beta values for 934 serum samples from patients with seminomatous or nonseminomatous testicular cancer were measured by highly specific immunoradiometric assays (IRMAS). In non-seminoma samples, hCG and hCG-beta were highly correlated (r = 0.82, P less than 0.001). Of 112 "marker-positive" seminoma samples, only 46 (41.1%) showed both increased hCG and hCG-beta. In 39 cases (34.8%) only hCG-beta and in 27 cases (24.1%) only dimer-hCG was increased. This makes the determination of hCG and hCG-beta, either by two assays or by a single hCG (+hCG-beta) assay, most reliable in these patients. For all samples, hCG (+hCG-beta) was measured by a polyclonal RIA and a monoclonal IRMA, which differed in their cross-reactivities with hCG-beta (234% and 720%, respectively). The hCG (+hCG-beta) IRMA, as a result of its higher hCG-beta cross-reactivity, was superior to the hCG (+hCG-beta) RIA in detecting slightly increased hCG-beta. Additionally, 11 widely used commercial hCG kits were tested for their hCG-beta cross-reactivities and showed values between less than 3% and 264%.