title: cDNA-directed expression of two allelic variants of cytochrome P450 2C11 using COS1 and SF21 insect cells.
abstract: Cytochromes P450 (CYP) are ubiquitous enzymes which metabolize multiple endogenous and exogenous substrates. CYP2C11 is the most abundant among the P450s present in untreated rat liver (approximately 50% of total P450) and is known for metabolizing testosterone mainly in positions 2 alpha and 16 alpha. In Gunn rats, the specific enzymatic activities of CYP2C11 are decreased by 90%, although the protein is present at levels similar to that of the Wistar reference strain. In order to explain this difference, we cloned and expressed CYP2C11 Wistar and Gunn cDNAs in two heterologous systems. Three mutations were identified in Gunn CYP2C11 form: Val4 --> Ala in the transmembrane region, Asn116 --> Ser in the substrate recognition site SRS-1, and Phe187 --> Leu in the (E --> F) interhelical region when compared to the spatial structure of CYP101. Sf21 insect cells displayed high yields of expressed CYP2C11 proteins (up to 2 nmol CYP2C11/mg microsomal proteins) which are more than 60-fold those observed in COS1 cells and 4-fold those present in hepatic microsomes. Testosterone-hydroxylating activities from CYP2C11 Gunn expressed in insect cells were decreased by 40% when compared to CYP2C11 Wistar, reflecting with a lesser extent the decrease we have previously reported in liver microsomes. The high level of active protein obtained with the baculovirus/insect cell expression system will be useful to analyze activities of site-directed mutants of CYP2C11 in order to elucidate the respective influence of the identified mutations on the enzymatic activities.
authors.affiliation: INSERM U75, Université René Descartes, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Biagini
authors.forename: C
authors.initials: C
authors.lastname: Celier
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D014644:Genetic Variation; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D017354:Point Mutation; D051381:Rats; D011911:Rats, Gunn; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D018411:Spodoptera; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase