Defining 17α-hydroxylase in zebrafish: expression pattern of two paralog genes (zCyp17a1; zCyp17a2) and comparative in vitro and in silico analysis
S. Parajes1, A. Griffin1, I. Miguel Escalada1, I. Rose1, A. Taylor1, L. Sacco1, J. Grötzinger2, W. Arlt1, F. Müeller1 & N. Krone1
Background: Zebrafish is emerging as a comprehensive model system in endocrinology. Zebrafish synthesise steroid hormones in the interrenal (counterpart of the mammalian adrenal), gonad and brain. Data on steroidogenic pathways is patchy and steroidogenic enzymes in zebrafish have not been well characterised. Human 17α-hydroxylase (hCYP17A1) facilitates two conversion steps, 17α-hydroxylase and 17,20-lyase reactions in the adrenal and gonad. Two zCyp17a (zCyp17a1; zCyp17a2) enzymes exist in zebrafish.
Aim: To characterise the expression pattern and the catalytic activity of zebrafish Cyp17a enzymes.
Methods: zCyp17a expression was determined by RT-PCR from whole embryos and adult tissues. Functional assays were performed using transiently transfected COS7 cells expressing zCyp17a or hCYP17A1. 17α-hydroxylase activity was assessed by the conversion of pregnenolone into 17-hydroxypregenenolone and progesterone into 17α-hydroxyprogesterone. The 17,20-lyase activity was measured by the conversion of 17α-hydroxypregenenolone into DHEA and 17α-hydroxyprogesterone into androstenedione.
Results: zCyp17a1 and zCyp17a2 expression was observed from fecundation. In adult fish, both enzymes are expressed in the interrenal, gonad and brain. zCyp17a1 and zCyp17a2 17α-hydroxylated substrates 1.53 and 611 times more efficiently than human CYP17A1. Furthermore, zCyp17a1 showed a preference for the Δ4-pathway, while zCyp17a2 converted Δ5-steroids more efficiently. In contrast to hCYP17A1, zCyp17a1 efficiently synthesised both, DHEA and androstenedione. zCyp17a2 completely lacks 17,20-lyase activity. We conducted comparative in silico analysis between two newly developed three-dimensional zCyp17a models and our human CYP17A1 model. Residue divergence within the substrate and redox interaction domains may explain our in vitro findings.
Conclusions: Herein, we demonstrated that both zCyp17a enzymes facilitate 17α-hydroxylation more efficiently than hCYP17A1. Catalytic efficiency is substrate specific. zCyp17a2 cannot synthesise androgen precursors. Expression data suggest a role of zCyp17a enzymes already during early embryologic development. Importantly, our data provides novel insights into zebrafish steroidogenesis and will help to establish a novel cutting edge tool for in vivo studies of steroidogenic disorders.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.