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Conservation of Glutathione S-Transferase Mrna and Protein Sequences Similar bioRxiv preprint doi: https://doi.org/10.1101/2020.11.25.396168; this version posted November 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Conservation of glutathione S-transferase mRNA and protein sequences similar 2 to human and horse Alpha class GST A3-3 across dog, goat, and opossum species 3 4 Shawna M. Hubert a,b, Paul B. Samollow c,d, Helena Lindströme, Bengt Mannervike, Nancy H. Ing 5 a,d,f* 6 7 a Department of Animal Science, Texas A&M AgriLife Research, Texas A&M University, College 8 Station, TX 77843-2471, USA 9 b Department of Thoracic Head & Neck Medical Oncology, University of Texas M.D. Anderson 10 Cancer Center, Houston, TX 77030, USA 11 cDepartment of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biosciences, 12 Texas A&M University, College Station, TX 77843-2471, USA 13 d Faculty of Genetics, Texas A&M University, College Station, TX 77843-2128, USA 14 e Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories, SE- 15 10691, Stockholm, Sweden 16 f Faculty of Biotechnology, Texas A&M University, College Station, TX 77843-2128, USA 17 18 19 *Corresponding authors at: 20 Nancy H. Ing, Department of Animal Science, Texas A&M University, 2471 TAMU, College 21 Station, TX 77843-2471, United States. Tel.: +1 979 862 2790; Fax: +1 979 862 3399 22 Bengt Mannervik, Department of Biochemistry and Biophysics, Stockholm University, Arrhenius 23 Laboratories, SE-10691, Stockholm, Sweden. 24 E-mail addresses: 25 [email protected] (N.H. Ing) 26 [email protected] (B. Mannervik) 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.25.396168; this version posted November 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 27 [email protected] (S.M. Hubert) 28 [email protected] (P. Samollow) 29 [email protected] (H. Lindström) 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.25.396168; this version posted November 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 30 Abstract 31 Recently, the glutathione S-transferase A3-3 (GST A3-3) homodimeric enzyme was 32 identified as the most efficient enzyme that catalyzes isomerization of the precursors of 33 testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the 34 presence of GST A3-3 orthologs with equally high ketosteroid isomerase activity has not 35 been verified in other mammalian species, even though pig and cattle homologs have been 36 cloned and studied. Identifying GSTA3 genes is a challenge because of multiple GSTA gene 37 duplications (12 in the human genome), so few genomes have a corresponding GSTA3 gene 38 annotated. To improve our understanding of GSTA3 gene products and their functions across 39 diverse mammalian species, we cloned homologs of the horse and human GSTA3 mRNAs 40 from the testes of a dog, goat, and gray short-tailed opossum, with those current genomes 41 lacking GSTA3 gene annotations. The resultant novel GSTA3 mRNA and inferred protein 42 sequences had a high level of conservation with human GSTA3 mRNA and protein sequences 43 (≥ 70% and ≥ 64% identities, respectively). Sequence conservation was also apparent for the 44 13 residues of the “H-site” in the 222 amino acid GSTA3 protein that is known to interact 45 with the steroid substrates. Modeling predicted that the dog GSTA3-3 is a more active 46 ketosteroid isomerase than the goat or opossum enzymes. Our results help us understand the 47 active sites of mammalian GST A3-3 enzymes, and their inhibitors may be useful for 48 reducing steroidogenesis for medical purposes, such as fertility control or treatment of 49 steroid-dependent diseases. 50 51 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.25.396168; this version posted November 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 52 1. Introduction 53 It is widely appreciated that reproduction of animal species important to man centers 54 around sex steroid production by the gonads. While driven by peptide hormones from the 55 hypothalamic/pituitary axis, production of testosterone by the Leydig cells of the testis and 56 estradiol and progesterone by the granulosa cells and corpora lutea of the ovary are required 57 for successful production of offspring [1-3]. Testosterone, estradiol and progesterone act in 58 reproductive tissues by binding specific receptors which regulate the expression of sets of 59 genes [4,5]. 60 Testosterone, estradiol and progesterone are produced from cholesterol by a series of 61 enzymatic modifications [6,7]. This reaction sequence differs among species: rodents utilize 62 the △4 pathway whereas humans and other larger mammals utilize the △5 pathway [7]. The 63 biosynthetic pathway for testosterone in human and horse testes involves the alpha class 64 glutathione S-transferase A3-3 (GST A3-3) as a ketosteroid isomerase that acts as a 65 homodimer to convert △5-androstene-3,17-dione to △4-androstene-3,17-dione, the 66 immediate precursor of testosterone [8-15]. The recombinant human GST A3-3 enzyme is 67 230 times more efficient at this isomerization than hydroxy-△5-steroid dehydrogenase, 3β- 68 hydroxysteroid delta-isomerase 2 (HSD3B2), the enzyme to which this activity previously 69 had been attributed [2]. Estradiol synthesis is likewise dependent upon GSTA3-3 in humans 70 and horses because testosterone is further converted to estradiol by aromatase [7]. GST A3-3 71 also participates in the synthesis of progesterone in female mammals utilizing the △5 72 pathway by isomerizing △5-pregnene-3,20-dione to △4-pregnene-3,20-dione (i.e., 73 progesterone). 74 From a general perspective, the GST family of enzymes is recognized for having 75 active roles in detoxification. The GSTA class of genes is best characterized in humans. It is 76 represented by 12 genes and pseudogenes with almost indistinguishable sequences. These 77 arose from gene duplications and in humans are clustered in a 282 kbp region on 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.25.396168; this version posted November 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 78 chromosome 6 [16]. Because of the redundancy of GSTA genes, the GSTA3 genes are 79 inexactly identified, if identified at all, in the current annotations of the genomes of most 80 mammals. This gap in knowledge limits studies of GSTA3 gene expression because standard 81 GSTA3 mRNA and protein detection reagents crossreact with other four gene products in the 82 GSTA family, which are ubiquitously expressed [11]. 83 Understanding the regulation of expression of GSTA3 gene and the activities of 84 GSTA3-3 enzyme is critical to fully comprehending the synthesis of sex steroid hormones 85 that direct reproductive biology in animals. In extracts of a steroidogenic cell line, two 86 specific inhibitors of GSTA3-3 (ethacrynic acid and tributyltin acetate) decreased 87 isomerization of delta-5 androstenedione with very low half maximal inhibitory 88 concentrations (IC50) values of 2.5 uM and 0.09 uM, respectively [17]. In addition, 89 transfection of such a cell line with two different small interfering RNAs both decreased 90 progesterone synthesis by 26%. In stallions treated with dexamethasone, a glucocorticoid 91 drug used to treat inflammation, testicular levels of GSTA3 mRNAs decreased by 50% at 12 92 hours post-injection, concurrent with a 94% reduction in serum testosterone [18,19]. In 93 stallion testes and cultured Leydig cells, dexamethasone also decreased concentrations of 94 steroidogenic factor 1 (SF1) mRNA [20]. In a human genome-wide study of the genes 95 regulated by SF1 protein, chromatin immunoprecipitation determined that the SF1 protein 96 bound to the GSTA3 promoter to up-regulate the gene [13]. Notably, the SF1 transcription 97 factor up-regulates the expression of several gene products involved in steroidogenesis [2]. 98 These data indicate that there are reagents and pathways by which GSTA3 gene expression 99 and steroidogenesis can be altered in vivo. This could be useful for reducing steroidogenesis 100 for fertility control or other medical purposes. 101 The equine GST A3-3 enzyme is structurally similar to the human enzyme and 102 matches or exceeds the very high steroid isomerase activity of the latter [15]. Remarkably, 103 the cloning of GSTA3 homologs from the gonads of domestic pigs (Sus scrofa) and cattle 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.25.396168; this version posted November 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 104 (Bos taurus) yielded enzymes that were much less active ketosteroid isomerases for reasons 105 yet to be determined. In the current work, GSTA3 mRNA was cloned from the testes of the 106 dog and goat (eutherian mammals), and gray short-tailed opossum (metatherian mammal). 107 The purposes were (1) to compare mRNA and protein sequences with the human and horse 108 counterparts to search for more highly active ketosteroid isomerases, (2) to generate GSTA3- 109 specific reagents for future studies of the regulation of GSTA3 genes, and (3) to understand 110 the scope of the GSTA3-3 function in steroidogenesis across a range of mammalian species 111 in which reproduction is important to humans.
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