Proteomic analysis of Artemisia annua--towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin

Abstract

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328-331) and other genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria.

Results: The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin.

Conclusions: The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin's biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703.

Fig. 1

Functional classification (GO terms) of the Masoct-identified proteins from the trichome-enriched sample material, using the UniProtKB database (taxonomoy: viridiplantae). Mascot search results were submitted to Percolator with an ‘expect cut-off’ threshold of 0.05

Fig. 2

Functional classification (GO terms) of the Mascot-identified proteins from the trichome-enriched and trichome-depleted sample material, using the Artemis contig database: a for protein identifications of Tables 3 and 4 with higher abundance in trichome-enriched sample material; b for protein identifications of Tables 5 and 6 with lower abundance in trichome-enriched sample material. Mascot search results were submitted to Percolator with an ‘expect cut-off’ threshold of 0.05 and filtered using a minimum number of significant sequences of 2

Fig. 3

The biosynthesis of artemisinin, as it is currently best understood, depicted in three phases. Enzymes in red were identified through Mascot searches of the MS data using the taxonomy A. annua while enzymes in blue were identified using the taxonomy viridiplantae. For the latter, if needed, additional homology searching was applied, using BLASTp with ‘Artemisia’ as organism (E value < 10⁻⁴⁷). AACT – acetoacetyl-CoA thiolase; HMGS – (S)-3-hydroxy-3-methylglutaryl-CoA synthase; HMGR – (S)-3-hydroxy-3-methylglutaryl-CoA reductase; MVAK – mevalonate kinase; MVAPK – mevalonate-5-phosphate kinase; MPD - mevalonate-5-pyrophosphate decarboxylase; FPS – farnesyl pyrophosphate synthase; ADS – amorpha-4,11-diene synthase; CYP71AV1 - amorpha-4,11-diene 12-hydroxylase; DBR2 – artemisinic aldehyde Δ^11,13 reductase; ALDH1 – aldehyde dehydrogenase 1