Understanding of the principal pathways of steroid hormone biosynthesis was established over two decades ago through advances in steroid radioisotopic and chromatographic techniques. When the enzymes of individual pathways could be examined in more detail, the dissection of the complex pattern of enzyme activities began. At many points, separate pathways employ precisely the same enzyme for equivalent catalytic steps, e.g. for 21-hydroxylase, 11 β-hydroxylase, aromatase and several dehydrogenases (Orth et al. 1992). A further economy was found for 17α-hydroxylase and 17,20-lyase activities, which co-purify with the same P450c17 polypeptide. This enzyme was later cloned and expressed in tissue culture cells, revealing that, contrary to the enzyme in rat, human and cattle, 17α-hydroxylase cannot convert 17α-hydroxyprogesterone to androstenedione (Bradshaw et al. 1987, Fevold et al. 1989). Further complexity emerged with the existence of multiple tissue-specific forms of 5α-reductase (Wilson et al. 1993), and 3β-, 11β- and 17β-hydroxysteroid dehydrogenases, most of which
R G Sutcliffe, A J Russell, C R W Edwards, and A M Wallace
A J Russell, A M Wallace, M G Forest, M D C Donaldson, C R W Edwards, and R G Sutcliffe
A 5-year-old XY pseudohermaphrodite was found to have a defect of steroid biosynthesis consistent with a partial deficiency of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD). Circulating concentrations of Δ5 steroids and Δ5 urinary steroid metabolites were elevated and remained elevated after orchidectomy. There was no evidence of salt loss, plasma renin being within normal limits, and no detectable glucocorticoid abnormality. The coding sequences of the genes for 3β-HSD types I and II were amplified by PCR and screened for mutations by denaturing gradient gel electrophoresis (DGGE) and manual and automatic DNA sequencing. A mutation in the gene for 3β-HSD type II was observed at codon 173 (CTA→CGA), leading in the affected patient to a homozygous substitution in which the leucine at residue 173 was altered to an arginine (L173R). The propositus's 2-year-old XX sister was also homozygous for L173R and showed the biochemical characteristics of partial 3β-HSD deficiency without clinical symptoms or signs. The mutation segregated as an autosomal recessive. Three related heterozygous adult females showed evidence of a small over-production of Δ5 steroids and steroid metabolites and a variable reduction in ovarian function. Concentrations of Δ5 steroids and steroid metabolites in the heterozygous father of the propositus were within the normal range.
These data are discussed in relation to the endocrine causes of pseudohermaphroditism and hirsutism. Evidence for tight linkage between the genes for 3β-HSD types I and II was obtained using a microsatellite polymorphism in the third intron of the gene for 3β-HSD type II and synonymous and non-synonymous mutations and polymorphisms in the gene for 3β-HSD type I. The latter polymorphisms were located 88 bp apart at the 3′ end of the type I coding sequence and could be physically resolved as haplotypes using DGGE. The application of DGGE to the analysis of mutations in members of a multigene family is discussed.
G K Shooter, B Magee, M A Soos, G L Francis, K Siddle, and J C Wallace
Insulin-like growth factor-I (IGF-I) analogues were produced with the aim of identifying IGF-I residues that contribute to the specificity of binding to the type 1 IGF receptor as opposed to the insulin receptor. Receptor binding properties of a series of A- and B-domain analogues were compared using rat L6 myoblasts, soluble human IGF type 1 receptors and soluble human insulin receptor isoforms HIR-A (−Ex11) and HIR-B (+Ex11). IGF-I analogues, [Leu8] IGF-I and [Phe59] IGF-I, were shown to exhibit respectively, a 28- and 17-fold decrease in affinity for the HIR-A with only a 6- and 5-fold decrease in affinity for the human IGF type 1 receptor. In contrast, the analogue [His4] IGF-I was equipotent to IGF-I in binding to the soluble type 1 IGF receptor while showing 7-fold and 4-fold increases in HIR-A and HIR-B binding respectively. Furthermore, [Leu62] IGF-I was 8-fold less potent than IGF-I in soluble IGF type 1 receptor binding but only showed a 2-fold decrease in HIR-A and HIR-B binding. Our study supports the conclusion that the co-evolution of the IGF-I and insulin receptor/ligand systems has resulted in subtle structural differences in the A- and B-regions of each ligand important for defining receptor binding specificity.
J M Carr, P A Grant, G L Francis, J A Owens, J C Wallace, and P E Walton
Three different molecular mass forms of IGF-binding proteins (IGFBPs) were purified from ovine plasma by IGF-I affinity chromatography and reverse-phase HPLC: a 46 kDa doublet and 29 kDa and 24 kDa forms. Amino-terminal sequence analysis confirmed that these proteins were ovine (o)IGFBP-3 (46 kDa) and two molecular size variants of oIGFBP-4. oIGFBP-3 and the 29 kDa form of oIGFBP-4 were shown to be N-glycosylated. Isoelectric points were determined to be at approximately pH 6 for oIGFBP-3 and at pH 7 and pH 7·5 for the 29 and 24 kDa forms of oIGFBP-4 respectively. The two different molecular mass variants of oIGFBP-4 had similar IGF-binding properties. Compared with human IGFBP-3 and oIGFBP-3, the two variants of oIGFBP-4 exhibited lower relative binding to amino-terminally modified IGF-I analogues in a competitive IGF-binding assay. The full protein sequence of oIGFBP-4, as deduced from the cDNA sequence, showed a high degree of identity with rat (90%), human (96%) and bovine (98%) IGFBP-4. The cDNA sequence also showed homology over regions of the 3′ non-coding sequence, particularly in comparison with bovine IGFBP-4 (96%). Northern analysis of mRNA for oIGFBP-4 indicated a 26 kb major transcript and two minor transcripts of approximately 21 and 1·8 kb. oIGFBP-4 mRNA transcripts were detected in adult ewe liver>kidney>lung>>heart and also in several fetal tissues, thus suggesting tissue-specific and developmental regulation. The availability of purified oIGFBP-4 and oIGFBP-3 as well as DNA probes for oIGFBP-4 will enable further study of the properties and functions of these proteins, as well as the establishment of specific assays for these IGFBPs.
B B Mendonça, A J Russell, M Vasconcelos-Leite, IJ P Arnhold, W Bloise, B L Wajchenberg, W Nicolau, R G Sutcliffe, and A M Wallace
A mutation (A82T) is described in the coding sequence of the gene for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) type II that is associated with variable clinical consequences. Four homozygotes are described, all of which showed elevated levels of Δ5 steroids consistent with 3ß-HSD deficiency. Two males from a consanguineous family were found to be homozygous for A82T and were affected with pseudohermaphroditism. They differed in their degree of mild salt loss. In the same family a female was found to be homozygous for A82T, but was clinically normal and had no history of premature pubarche or of abnormal menstrual cycles. However, in an apparently unrelated family, the A82T mutation was found in a female affected with premature pubarche. This is the first report of a proven mutation in 3ß-HSD type II associated with premature pubarche.
F E Carrick, M G Hinds, K A McNeil, J C Wallace, B E Forbes, and R S Norton
The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx1–279IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly19 and Asp20 of IGF-I (and the corresponding residues in IGF-II) – which are located in a reverse turn linking N-domain and C-domain interactive surfaces – are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.
G. L. Francis, M. Ross, F. J. Ballard, S. J. Milner, C. Senn, K. A. McNeil, J. C. Wallace, R. King, and J. R. E. Wells
An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1–11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1–11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1–11)-Val-Asn-[Arg3]-IGF-I, where Glu3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1–3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]IGF-I-des(1–3)IGF-I>Long [Gly3]-IGF-I>Long IGF-I>IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.
Jacqueline M Wallace, John S Milne, Raymond P Aitken, Dale A Redmer, Lawrence P Reynolds, Justin S Luther, Graham W Horgan, and Clare L Adam
Low birthweight is a risk factor for neonatal mortality and adverse metabolic health, both of which are associated with inadequate prenatal adipose tissue development. In the present study, we investigated the impact of maternal undernutrition on the expression of genes that regulate fetal perirenal adipose tissue (PAT) development and function at gestation days 89 and 130 (term=145 days). Singleton fetuses were taken from adolescent ewes that were either fed control (C) intake to maintain adiposity throughout pregnancy or were undernourished (UN) to maintain conception weight but deplete maternal reserves (n=7/group). Fetal weight was independent of maternal intake at day 89, but by day 130, fetuses from UN dams were 17% lighter and had lower PAT mass that contained fewer unilocular adipocytes. Relative PAT expression of IGF1, IGF2, IGF2R and peroxisome proliferator-activated receptor gamma (PPARG) mRNA was lower in UN than in controls, predominantly at day 89. Independent of maternal nutrition, PAT gene expression of PPARG, glycerol-3-phosphate dehydrogenase, hormone sensitive lipase, leptin, uncoupling protein 1 and prolactin receptor increased, whereas IGF1, IGF2, IGF1R and IGF2R decreased between days 89 and 130. Fatty acid synthase and lipoprotein lipase (LPL) mRNAs were not influenced by nutrition or stage of pregnancy. Females had greater LPL and leptin mRNA than males, and LPL, leptin and PPARG mRNAs were decreased in UN at day 89 in females only. PAT gene expression correlations with PAT mass were stronger at day 89 than they were at day 130. These data suggest that the key genes that regulate adipose tissue development and function are active beginning in mid-gestation, at which point they are sensitive to maternal undernutrition: this leads to reduced fetal adiposity by late pregnancy.