Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution.

Eva M Schmidt,Constanze Wiek,Helmut Hanenberg,Charles A Steward,Michael Gombert,Marcel Freund,Katharina Roellecke,Oliver T Parkinson,Christof M Kramm,Nadine Lottmann,Vladimir Yarov-Yarovoy,Allan E Rettie,Xinghui Qiu

doi:10.1371/journal.pone.0137110

Abstract

CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s) is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5–exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207). We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76%) in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution.

Highlights

Cytochrome P450s (CYPs) constitute a large superfamily of genes that have co-evolved with their hosts—higher plants, prokaryotic and eukaryotic organisms—from a single common ancestor [1, 2]
CYP4B1 was initially recognized as the P450 enzyme responsible for the activation of a naturally occurring pro-toxin, 4-ipomeanol (4-IPO), to an alkylating agent [6,7,8,9,10]. 4-IPO is produced by sweet potatoes (Ipomoea batatas) infected with the fungus Fusarium solani [11,12,13,14]
We aligned the genomic DNA of the corresponding CYP4B1 intron 5 sequences of great apes, monkeys, and other mammals obtained from the ensembl genome database

Summary

Introduction

Cytochrome P450s (CYPs) constitute a large superfamily of genes that have co-evolved with their hosts—higher plants, prokaryotic and eukaryotic organisms—from a single common ancestor [1, 2]. There are remarkably similar substrate specificities for the vast majority of P450 orthologs in mammals due to semi-conserved active-site sequences and ligand access channels [4], human CYP4B1 stands out as a functionally unusual member of this single-gene subfamily (reviewed in [5]). When livestock ingest moldy sweet potatoes, 4-IPO uptake induces selective cellular toxicity in the lungs as the primary organ affected [11, 12, 14] where 70% of all CYP4B1 transcripts are expressed [15,16,17,18]. Dose-escalation studies revealed that no lung toxicity, but some reversible dose-dependent liver toxicity occurred after intravenous administration of 4-IPO. No objective anti-tumor effects were noted, for either lung or liver cancer patients [20,21,22]

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Results

Conclusion