Molecular Analysis of the Human 3beta-Hydroxysteroid Dehydrogenase Delta5/Delta4 Isomerase Gene Family

McBride, Martin William (1996) Molecular Analysis of the Human 3beta-Hydroxysteroid Dehydrogenase Delta5/Delta4 Isomerase Gene Family. PhD thesis, University of Glasgow.

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3-beta hydroxysteroid dehydrogenase Delta5/Delta4 isomerase (3beta-HSD) catalyses the oxidative conversion of ?4-3beta-hydroxysteroids to their corresponding Delta4-3-ketosteroid configuration, and is essential for the biosynthesis of all classes of steroid hormone; mineralocorticoids, glucocorticoids, progestins, androgens and oestrogens. In humans two homologous linked, genes, HSD3B1 an HSD3B2, encoding 3beta-HSD type I and type II enzymes, have been isolated and mapped to the human chromosomal region Ip 13.1. RNase protection assays have localised 3beta-HSD type I transcripts principally in placenta and skin, while 3beta-HSD type II transcripts are almost exclusively found in the adrenals and gonads. The two forms are 93.5% identical in primary structure and antibodies raised against purified human placental protein react immunocytochemically with 3beta-HSD in all four tissues. Such tissue specificity is shown by monoclonal antibody FDO 161G which reacts with 3beta-HSD, and was originally raised against human syncytiotrophoblast cells from the placenta. However, a further mouse monoclonal antibody, FDO 26G, raised against purified 3beta-HSD from placental villous tissue showed more restrictive tissue reactivity. Immunocytochemically, FDO 26G reacted with 3beta-HSD in villous syncytiotrophoblast and adrenals, but reacted weakly with extravillous trophoblast and Leydig cells of the testis. These experimental observations provide evidence for the expression of a further isoform of 3B-HSD exists in extravillous trophoblast and adult Leydig cells; either a post-translational modification within the region of monoclonal antibody binding (the epitope) or the expression of as yet unidentified 3beta-HSD gene that encodes a different amino acid sequence over the FDO 26G epitope. Using a combination of lac Z fusion polypeptides and synthetic peptides, the FDO 26G epitope was located to residues 354-366 at the carboxy terminal end of 3B-HSD type I, an amino sequence that is identical in the type I and type II forms of the enzyme. This epitope contains a consensus for caesin kinase II phosphorylation , with serine 359 as the candidate target of phosphorylation. This suggested that the lack of reactivity of FDO 26G in certain trophoblast cells of the placenta might be due to phosphorylation of serine 359. Peptide 354-366 was synthesised with phosphoserine at residue 359. FDO 26G reactivity to the phosphopeptide and unphosphorylated peptide was compared. FDO 26G bound the phospho-peptide at least as strongly as the unphosphorylated peptide. It was concluded that phosphorylation of serine 359 was not responsible for lack of FDO 26G reactivity in placental trophoblast populations. A number of 3beta-HSD cDNAs and genes have been cloned from a variety of vertebrate species. In mouse six homologous 3beta-HSD cDNAs have been isolated and shown to be expressed tissue- and sex- specifically. This gene family clustered on mouse chromosome 3 includes two functionally distinct groups of proteins; enzymes that catalyse the oxidative conversion of ?5-3beta-hydroxysteroids (characterised by an aspartic acid at residue 36) and enzymes that exclusively act as 3-ketosteroid reductases (characterised by a tyrosine (rat) or phenylalanine (mouse) at residue 36). Several bands of hybridisation are detected when Southern blots of human genomic DNA were probed with HSD3B1 exon specific probes suggesting that 3beta-HSD-like sequences other than HSD3B1 and HSD3B2 exist in the human genome. In addition when screening amplified segments of patient DNA for mutations in 3beta-HSD types I and II, it is relatively common to amplify novel but closely related 3beta-HSD sequences. To estimate the size of the 3beta-HSD gene family two human genomic lambdagem 11 libraries were screened with 3beta-HSD type I cDNA under non-stringent conditions. From a total of 1.4x10e6 clones, fifty-seven positive clones hybridised reproducibly through to the second stage of screening. These clones were tested as templates for PCR using primers that were known to amplify homologous members of the gene family (from mutation screening). PCR products were classified into groups depending on their mobility on denaturing gradient electrophoresis gels. Representatives of each group were sequenced. The remaining PCR-amplified products were directly sequenced. A total of seven distinct 3beta-HSD gene family members, including HSD3B1 and HSD3B2, were identified and mapped to the human chromosomal region Ip13. All of the newly identified genes contained 3beta-HSD-like exon and intron sequences indicating that these clones were not processed pseudogenes. Novel 3beta-HSD sequences were subcloned and sequenced from each of the new members of the family. The presence of frameshift and in some cases, missense mutations associated with 3beta-HSD type II deficiency suggests that these genes are not expressed as functional 36-HSD enzymes. There was no evidence of a human 3beta-HSD ketosteroid reductase enzyme.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Roger Sutcliffe
Keywords: Molecular biology, Genetics, Endocrinology
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-74769
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 13 Nov 2019 15:58
Last Modified: 13 Nov 2019 15:58

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