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Haplotype Diversity of NADPH-Cytochrome P450 Reductase Gene of Ophiocordyceps sinensis and the Effect on Fungal Infection in Host Insects.

ABSTRACT: Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is used as a traditional Chinese medicine. However, it is not clear how O. sinensis infects its host. The encoding gene haplotype diversity and predicted function of the nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) related to the fungal pathogenicity was analyzed for 219 individuals from 47 O. sinensis populations. Two NADPH CPR genes of O. sinensis were detected and their dominant haplotypes were widely distributed throughout the entire distribution range in Western China. Only 5.43% of all O. sinensis individuals possessed the specific private haplotypes of NADPH CPR-1 and CPR-2 genes. Bioinformatic analyses predicted that the phosphorylation sites, motifs, and domains of NADPH CPR of O. sinensis were different between those encoding by the dominant and private gene haplotypes. The one-to-one match fungus-host correspondence of the same individual suggested that the widely distributed O. sinensis with the dominant NADPH CPR gene haplotypes may strongly infect almost all host insects through a random infection by oral or respiratory pores. Conversely, O. sinensis with the specific private NADPH CPR gene haplotypes is likely to infect only a few corresponding host insects by breaching the cuticle, due to the changed NADPH CPR structure and function.

SUBMITTER: Xu Z

PROVIDER: S-EPMC7409138 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity.

Project description:The caterpillar fungus Ophiocordyceps sinensis is one of the most valuable medicinal fungi in the world, and it requires host insects in family Hepialidae (Lepidoptera) to complete its life cycle. However, the genetic diversity and phylogeographic structures of the host insects remain to be explored. We analyzed the genetic diversity and temporal and spatial distribution patterns of genetic variation of the host insects throughout the O. sinensis distribution. Abundant haplotype and nucleotide diversity mainly existed in the areas of Nyingchi, ShangriLa, and around the edge of the Qinghai-Tibet Plateau, where are considered as the diversity center or micro-refuges of the host insects of O. sinensis. However, there was little genetic variation among host insects from 72.1% of all populations, indicating that the host species composition might be relatively simple in large-scale O. sinensis populations. All host insects are monophyletic except for those from four O. sinensis populations around Qinghai Lake. Significant phylogeographic structure (NST>GST, P<0.05) was revealed for the monophyletic host insects, and the three major phylogenetic groups corresponded with specific geographical areas. The divergence of most host insects was estimated to have occurred at ca. 3.7 Ma, shortly before the rapid uplift of the QTP. The geographical distribution and star-like network of the haplotypes implied that most host insects were derived from the relicts of a once-widespread host that subsequently became fragmented. Neutrality tests, mismatch distribution analysis, and expansion time estimation confirmed that most host insects presented recent demographic expansions that began ca. 0.118 Ma in the late Pleistocene. Therefore, the genetic diversity and distribution of the present-day insects should be attributed to effects of the Qinghai-Tibet Plateau uplift and glacial advance/retreat cycles during the Quaternary ice age. These results provide valuable information to guide the protection and sustainable use of these host insects as well as O. sinensis.

Dataset Information

Haplotype Diversity of NADPH-Cytochrome P450 Reductase Gene of Ophiocordyceps sinensis and the Effect on Fungal Infection in Host Insects.

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