Biosynthesis of salicylic acid in fungus elicited Catharanthus roseus cells.

Feeding experiments using [1-(13)C]-d-glucose to Catharanthus roseus (L.) G.Don cell suspension cultures followed by elicitation with Pythium aphanidermatum extract were performed in order to study the salicylic acid (SA) biosynthetic pathway and that of 2,3-dihydroxybenzoic acid (2,3-DHBA) as a comparison. A strongly labeled C-7 and a symmetrical partitioning of the label between C-2 and C-6 would occur if SA was synthesized from phenylalanine. In case of the isochorismate pathway, a relatively lower incorporation at C-7 and a non-symmetrical incorporation at C-2 and C-6 would be obtained. Relatively, high- and non-symmetrical enrichment ratios at C-2 and C-6, and a lower enrichment ratio at C-7 were observed in both SA and 2,3-DHBA detected by (13)C NMR inverse gated spectrometry leading to the conclusion that the isochorismate pathway is responsible for the biosynthesis of both compounds. However, different enrichment ratios of the labeled carbons in SA and 2,3-DHBA indicate the use of different isochorismate pools, which means that their biosynthesis is separated in time and/or space.

Considerations for proteolytic labeling-optimization of 18O incorporation and prohibition of back-exchange.

Proteolytic (18)O labeling is a very powerful tool for differential analysis applied to proteome studies. However, it is a relatively new technique and the optimization of the labeling process still needs some attention. We found that the two-step post-proteolytic labeling should be favored over the conventional digestion of proteins in H(2) (18)O, since the former allows for higher sample concentrations and thus more favorable kinetics. It was demonstrated that the inhibitory effect of urea on (18)O incorporation could be compensated by the use of higher sample concentrations. Furthermore, it was shown that heat-deactivation of trypsin prevents (18)O/(16)O back-exchange. In addition, no non-specific hydrolysis of the peptides could be observed as a result of the heating. Heat inactivation of trypsin opens the way for the use of capillary electrophoresis as a separation technique in proteolytic labeling studies, as it abolishes the need for use of detrimental additives. Analysis of a labeled protein digest by capillary isoelectric focusing/mass spectrometry showed the applicability of the method. No back-exchange was observed across the entire electropherogram.

Optimization of protein identification from digests as analyzed by capillary isoelectric focusing-mass spectrometry.

Capillary isoelectric focusing (CIEF) is a high-resolution separation technique for the analysis of peptides and protein digests. When coupled to ion trap-mass spectrometry (CIEF-MS) the unique separation mechanism is combined with a highly efficient detection system. In an earlier report, we described aspects of separation and interfacing in connection to the analysis of a digest of set of standard proteins. Now, we report on different aspects of the process of protein identification. Sequest software parameters were optimized by using a standard protein digest. These settings were used for the analysis of periplasmic proteins from Escherichia coli. Since in CIEF peptides are focused according to their pI values, the mobilization time of a particular peptide is dependent on its pI value. Based on this relation, the identification of some peptides was facilitated. Furthermore, the Sequest settings that were used could be evaluated. In total, 159 proteins were identified in a single run.

Proteome analysis of the medicinal plant Catharanthus roseus.

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.

Capillary isoelectric focusing-mass spectrometry for shotgun approach in proteomics.

We report on capillary isoelectric focusing-mass spectrometry (CIEF-MS) of complex peptide mixtures in the absence of carrier ampholytes. Furthermore, the use of low concentrations of carrier ampholytes as mere spacers is investigated. Carrier ampholytes are complex mixtures of amphoteric compounds with high buffering capacity. Since all peptides are amphoteric compounds by themselves, the use of carrier ampholytes may be superfluous to establish a stable pH gradient in CIEF analysis of protein digests. Our research showed that when carrier ampholytes are omitted, the analyte ions are not focused at their isoelectric point. The analytes are charged, leading to electrophoretic mobility uncharacteristic for CIEF. The method was tested for a five-protein-mixture at 0.02 mg/mL per protein and 0.05 mg/mL per protein. At the lower concentration, the analytes were stacked during the focusing process in only a limited length of the capillary. Therefore, the higher concentration led to better separation efficiency. It was found that at low concentration (0.20%) the carrier ampholytes could work as spacers. Though it led to sensitivity losses of 15-45%, this was compensated by the higher separation efficiencies seen. The method was evaluated with an eight-protein-mixture, of which all could be identified after performing MS/MS.

The Catharanthus alkaloids: pharmacognosy and biotechnology.

The Catharanthus (or Vinca) alkaloids comprise a group of about 130 terpenoid indole alkaloids. Vinblastine is now marketed for more than 40 years as an anticancer drug and became a true lead compound for drug development. Due to the pharmaceutical importance and the low content in the plant of vinblastine and the related alkaloid vincristine, Catharanthus roseus became one of the best-studied medicinal plants. Consequently it developed as a model system for biotechnological studies on plant secondary metabolism. The aim of this review is to acquaint a broader audience with the recent progress in this research and with its exciting perspectives. The pharmacognostical aspects of the Catharanthus alkaloids cover botanical (including some historical), phytochemical and analytical data. An up-to-date view on the biosynthesis of the alkaloids is given. The pharmacological aspects of these alkaloids and their semi-synthetic derivatives are only discussed briefly. The biotechnological part focuses on alternative production systems for these alkaloids, for example by in vitro culture of C. roseus cells. Subsequently it will be discussed to what extent the alkaloid biosynthetic pathway can be manipulated genetically ("metabolic engineering"), aiming at higher production levels of the alkaloids. Another approach is to produce the alkaloids (or their precursors) in other organisms such as yeast. Despite the availability of only a limited number of biosynthetic genes, the research on C. roseus has already led to a broad scientific spin-off. It is clear that many interesting results can be expected when more genes become available.

Analysis of anthraquinones in cell cultures of Cinchona 'Robusta' by HPLC with photodiode array and mass spectrometry detection.

An HPLC-PAD-ESI/MS method has been developed for the analysis of anthraquinones in cell cultures of Cinchona 'Robusta'. Using a C18 column and gradient elution with a mobile phase system containing acetonitrile, water and trifluoroacetic acid, a satisfactory separation of both anthraquinone glycosides and aglycones in a crude dichloromethane extract could be obtained. Robustaquinone B was identified as a major anthraquinone in the extracts, and another five anthraquinones were tentatively identified from spectroscopic data. A number of minor unknown compounds were detected and were distinguished from the known anthraquinones. An isocratic system for the quantitative determination of robustaquinone B has also been developed.

High-performance liquid chromatography assay for 1-deoxy-D-xylulose 5-phosphate synthase activity using fluorescence detection.

A high-performance liquid chromatography assay for activity of 1-deoxy-D-xylulose 5-phosphate synthase, an early enzyme in the recently discovered 2-C-methyl-D-erythritol-4-phosphate pathway, was developed. In this assay, the enzymatic product 1-deoxy-D-xylulose was first derivatized with a fluorescent reagent 2-anthranilic acid, followed by separation using HPLC on a Nova-Pak phenyl column with a mobile phase containing CH3CN-water-1-butylamine-tetrahydrofuran-H3PO4 (2:97:0.125:0.5:0.25, v/v). The eluate was monitored by fluorescence detection at an excitation wavelength of 320 nm and an emission wavelength of 425 nm for quantitation of the fluorescent derivative. A linear response was obtained between 5 and 200 ng of 1-deoxy-D-xylulose. This assay was successfully applied to measure the 1-deoxy-D-xylulose 5-phosphate synthase activity in a recombinant E. coli overexpressing dxs gene. It demonstrated that this assay is simple, sensitive and selective compared to the methods used at present.

Capillary electrophoretic separations of proteins using carrier ampholytes.

Capillary separations of proteins using carrier ampholytes are performed between an anolyte and a catholyte of same pH (pH 3). Depending upon the concentration of carrier ampholytes used, two different separation processes take place. At a 10% concentration, the high-resolution separation of six model proteins is achieved, which can be described as a transient capillary isoelectric focusing (cIEF) system moving isotachophoretically. The isotachophoretic (ITP) behaviour of the system is evidenced by the influence of the catholyte concentration on the separation. The separation is neither pure cIEF nor pure cITP and the migration order of the proteins results from the influence of both their isolelectric points and their mobilities.

Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus.

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly to identify possible rate limiting steps, a transgenic cell line over-expressing tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was fed with various amounts of precursors (tryptophan, tryptamine, loganin and secologanin) in different time schedules and analyzed for TIA production. When these precursors were added to this culture it was found that the optimal time for supplying the precursors was at inoculation of the cells into the production medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or secologanin; however, the secologanin feeding was less effective. Tryptamine or tryptophan alone had no effect on TIA accumulation. The over-expression of Tdc causes this cell line to produce quite large quantities of alkaloids after feeding loganin or secologanin. However, in combination with tryptophan or tryptamine, feeding of these precursors resulted in an even further increase of alkaloid accumulation and under optimal conditions line T22 accumulated around 1200 micromol l(-1) of TIAs whereas the control cultures accumulated less than 10 micromol l(-1) TIAs.