ECE2014 Oral Communications IGF-1 and Thyroid Basic (5 abstracts)
Background: Besson A et al. described a patient with homozygous for C53S-hGH suffering from IGHD (JCEM2005 90:2493-9). They observed reduced ability of the mutant to bind and activate GHR in vitro. C53A-hGH lacks the disulfide bond between C53 and C165, which is conserved in GH/prolactin family.
Methods: Mouse pituitary AtT-20 cells, HEK-293 and CHO-K1 cells were transfected with plasmids containing C53S, C53A or C53S/C165A cDNA. hGH from cell supernatants and lysates was characterized with special immunoassays, western blots, a GHBP binding assay and proliferation assays using BaF-B03 and Nb2 cells.
Results: Western blot analysis of the AtT20 cell lysates under non-reduced conditions demonstrated the main band of C53S-hGH at 44 kDa, which could be reduced to lower molecular mass (22 kDa, 24 kDa) bands. However, C53S-hGH in supernatants of transfected AtT-20 cells appeared as both lower molecular mass and 44 kDa bands. In contrast the supernatants and lysates from HEK-293 and CHO-K1 cells contained mainly lower molecular mass C53S-hGH. In addition, the levels of C53S-hGH in supernatants and lysates from AtT-20 cells were only 32.0% and 9.8% of wt hGH levels respectively (P<0.001). C53S-hGH dimer exhibited very low binding to GHBP. The secreted C53S-hGH showed also reduced binding to GHBP (IC50 12.0 vs 3.0 nM, P<0.001) and decreased bioactivity in BaF-B03 assay (EC50 0.167 vs 0.036 nM, P<0.05) and in NB2 assay (EC50 0.150 vs 0.037 nM, P<0.01) compared to wt-hGH. The C53A-hGH mutation yielded the same results. In contrast, the double mutant C53S/C165A was unable to form a hGH dimer.
Conclusions: The unpaired cysteine at C165 in the C53S-hGH mutant leads to formation of a non-native disulfide bond linking two hGH molecules in pituitary cells. The C53S-hGH dimer possesses very low bioactivity. The production and secretion of C53S-hGH from pituitary cells was significantly reduced.