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ABSTRACT

Transgenic mice which expressed human IGF-binding protein-3 (hIGFBP-3) were generated by pronuclear injection of an hIGFBP-3 cDNA driven by the mouse metallothionein 1 promoter. Two of the seven founder mice had measurable levels of hIGFBP-3 in the circulation. The serum levels of hIGFBP-3 increased as the mice were bred to homozygosity and were further induced by supplementing the drinking water with 25 mm ZnCl₂. While the birth weight, litter size and body weight of transgenic mice were not significantly different from non-transgenic litter mates or wild-type mice derived from the same genetic background, the transgenic mice demonstrated selective organomegaly. The spleen, liver and heart of mice derived from both founders were significantly heavier compared with organs from non-transgenic mice (P<0·05, P<0·005 and P<0·01 respectively). The weights of the brain and kidney were similar in transgenic and non-transgenic mice. Expression of the transgene was detected in the kidney, small intestine and colon by Northern blot analysis.

Western ligand blotting of serum from transgenic mice did not demonstrate any change in the abundance of the IGFBPs detected by this method. When serum from transgenic mice was incubated with ¹²⁵I-labeled IGF-I and analyzed by Sephacryl S-200 chromatography under neutral conditions a significantly (P<0·05) increased amount of the radioactivity was found in the 140 kDa ternary complex compared with serum from wild-type mice. Immunoreactive hIGFBP-3 was detected in the 140 kDa ternary complex but the majority of immunoreactive hIGFBP-3 present in transgenic mouse serum eluted in later fractions indicating that it was not associated with the acid-labile subunit. These data demonstrate that modest constitutive expression of hIGFBP-3 has a selective effect on organ growth and development. The establishment of these IGFBP-3 transgenic mouse strains may provide useful models to investigate further the physiological role of IGFBP-3.

ABSTRACT

Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common α subunit and a hormone-specific β subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian β subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-β. Using site-directed mutagenesis, each of these two lysines in hCG-β was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine α subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine α subunit bound to hCG-β (Glu²) and to hCG-β (Glu¹⁰⁴), as well as with the control, i.e. the bovine α subunit bound to the hCG-β wild-type subunit. In two in-vitro assays, one a competitive binding assay with ¹²⁵I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant β subunit exhibited bioactivity, but their potencies were lower than that of the bovine α subunit bound to the hCG-β wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-β participate, either directly or indirectly, in receptor binding.

ABSTRACT

Graves' disease is an autoimmune thyroid disease characterized by the presence of pathogenic autoantibodies to the TSH receptor (TSH-R). By using polymerase chain reaction, the extracellular region of the human TSH-R cDNA has been amplified and used to prepare recombinant TSH-R (extracellular) protein fused with glutathione-S-transferase (GST). Purification of the recombinant TSH-R (extracellular)-GST fusion protein was achieved by preparative gel electrophoresis in SDS or by preparative isoelectric focusing in urea. Following removal of SDS by detergent exchange or urea by dialysis, the purified recombinant receptor preparations were assessed for binding to the hormone or to autoantibodies from Graves' disease patients. The purified recombinant receptor preparations fail to show any binding to the hormone or autoantibodies either by inhibition of binding assays or by immunoblotting. The results imply that the correct folding and/or post-translational modifications of the polypeptide chain which are not achieved in recombinant proteins produced in Escherichia coli may be important for the binding of the hormone or Graves' disease autoantibodies to the TSH-R. The recombinant receptor prepared in this manner will be useful for immunological and cellular investigations in patients with Graves' disease.

ABSTRACT

Since the cloning of the TSH receptor (TSH-R), the target autoantigen of Graves' disease, the receptor has been expressed in a variety of eukaryotic cells to obtain a functional molecule. Despite this success, the levels of receptor expression have been marginally higher than the extremely low levels found in thyroid cells, preventing any progress on the purification of the molecule. In this study, the large extracellular region of the TSH-R, without the membrane spanning segments, has been expressed in insect cells using recombinant baculovirus to generate substantial quantities of the receptor protein. A monoclonal antibody previously generated to a bacterial TSH-R fusion protein was used to characterize and monitor the expression of the truncated receptor in insect cells. Two polypeptides of 63 and 49 kDa were recognized as the components of the truncated recombinant receptor. The 63 kDa protein was shown to be the glycosylated form of the smaller, 49 kDa, component. Expression in different insect cell lines showed that an increase in expression of approximately tenfold was apparent in High Five cells when compared with Sf21 cells. Very small quantities of the truncated receptor were secreted by the three insect cell lines examined, with the majority of the molecule being retained within the cells. Immunoaffinity purification of milligram quantities of the truncated receptor was achieved using the monoclonal antibody. The availability of the purified TSH-R has allowed the establishment of an enzymelinked immunosorbent assay to measure autoantibodies in the sera of patients with Graves' disease. Although the truncated receptor interacts with autoantibodies, our results show that it does not bind TSH and differs in this respect from other glycoprotein hormone receptors.

ABSTRACT

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2^b and H-2^d) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22–35 (A10 and All, both IgG2b from H-2^b animals), amino acids 402–415 (A7, IgG2b from H-2^b animals) and amino acids 147–228 (A9, IgG1 from H-2^d animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and All was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and All, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.