Substrate plasticity of a fungal peptide α-N-methyltransferase

This work was financially supported by the Commission for Technology and Innovation (CTI/Innosuisse Grant No. CTI 25951.2), the Swiss National Science Foundation (Grant No. 31003A_173097), Wellcome Trust (Grant No. 094476/Z/10/ Z), and BBSRC (Grant No. BB/R018189/1).The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harboring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic, and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline, depending on its sequence context, can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.Publisher PDFPeer reviewe

Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive loss of motoneurons, motor weakness and death within 1–5 years after disease onset. Therapeutic options remain limited despite a substantial number of approaches that have been tested clinically. In particular, various neurotrophic factors have been investigated. Failure in these trials has been largely ascribed to problems of insufficient dosing or inability to cross the blood–brain barrier (BBB). We have recently uncovered the neurotrophic properties of the haematopoietic protein granulocyte-colony stimulating factor (G-CSF). The protein is clinically well tolerated and crosses the intact BBB. This study examined the potential role of G-CSF in motoneuron diseases. We investigated the expression of the G-CSF receptor in motoneurons and studied effects of G-CSF in a motoneuron cell line and in the SOD1(G93A) transgenic mouse model. The neurotrophic growth factor was applied both by continuous subcutaneous delivery and CNS-targeted transgenic overexpression. This study shows that given at the stage of the disease where muscle denervation is already evident, G-CSF leads to significant improvement in motor performance, delays the onset of severe motor impairment and prolongs overall survival of SOD1(G93A)tg mice. The G-CSF receptor is expressed by motoneurons and G-CSF protects cultured motoneuronal cells from apoptosis. In ALS mice, G-CSF increased survival of motoneurons and decreased muscular denervation atrophy. We conclude that G-CSF is a novel neurotrophic factor for motoneurons that is an attractive and feasible drug candidate for the treatment of ALS

Amino acid analysis in biological fluids by GC-MS

Amino acids are intermediates in cellular metabolism and their quantitative analysis plays an important role in disease diagnostics. A gas chromatography-mass spectrometry (GC-MS) based method was developed for the quantitative analysis of free amino acids as their propyl chloroformate derivatives in biological fluids. Derivatization with propyl chloroformate could be carried out directly in the biological samples without prior protein precipitation or solid-phase extraction of the amino acids, thereby allowing for automation of the entire procedure, including addition of reagents, extraction and injection into the GC-MS. The total analysis time was 30 minutes, including sample preparation and 31 amino acids could be reliably quantified using 19 stable isotope-labeled amino acids as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) were in the range of 0.03 - 12 µM and 0.3 - 30 µM, respectively. The method was validated using certified amino acid standard and reference plasma, and its applicability to different biological fluids was shown. Intraday precision for the analysis of human urine, blood plasma, and cell culture medium was 2.0 - 8.8%, 0.9 - 8.3%, and 2.0% - 14.3%, respectively, while the inter-day precision for human urine was 1.5 - 14.1%. Using two blinded sets of urine specimens containing replicates, the GC-MS method was further validated and the results were compared with those obtained for iTRAQ® derivatization HPLC-tandem mass spectrometry and ion exchange chromatography with postcolumn ninhydrin detection of amino acids. The technical error (TE), as determined by repeated aliquot measurements of various urine specimens was calculated to prove that the method was suitable for the quantitative analysis of amino acids in large clinical and epidemiological studies. The quantitative results obtained by the three methods were compared by regression analysis and Bland-Altman plotting. The method was further expanded to fatty acids. Due to the carboxy function fatty acids can be derivatized with propyl chloroformate and included in the develpoped GC-MS method. To resolve isobaric fatty acids the GC program had to be expanded and the analysis time increased to 50 min for one GC run. LODs for the fatty acids ranged from 0.08 µM to 39 µM. To that end, the method was adapted to allow the combined analysis of the total fatty acid content of 17 fatty acids and 25 free amino acids in a single gas chromatographic run. The number of amino acids amenable to GC analysis is limited and therefore, the potential of derivatization with propyl chloroformates, followed by LC-MS/MS analysis for amino acid determination was investigated. The method was expanded to tryptophan metabolites and polyamines that are of great interest in several biological projects. The intention to use an in-house synthesized internal standard for each analyte failed as experiments showed that the created standard is not suitable for quantification purposes. Therefore, isotopes labeled analytes have to act as internal standards. Using 23 stable-isotope labeled compounds as internal standards, the method aims the quantification of 41 analytes comprising amino acids, tryptophan metabolites and polyamines. It was not possible to detect tryptophan metabolites above the LLOQ in biological samples. Preliminary experiments were performed to improve the method by evaluating choice of the extraction solvent

Advances in amino acid analysis

Amino acids are important targets for metabolic profiling. For decades, amino acid analysis has been accomplished by either cation-exchange or reversed-phase liquid chromatography coupled to UV absorbance or fluorescence detection of pre-column or post-column-derivatized amino acids. Recent years have seen great progress in the development of direct-infusion or hyphenated mass spectrometry in the analysis of free amino acids in physiological fluids, because mass spectrometry not only matches optical detection in sensitivity, but also offers superior selectivity. The advent of cryo-probes has also brought NMR spectroscopy within the detection limits required for the analysis of free amino acids. But there is still room for further improvement, including expansion of the analyte spectrum, reduction of sample preparation and analysis time, automation, and synthesis of affordable isotope standards

Automated GC-MS analysis of free amino acids in biological fluids

A gas chromatography–mass spectrometry (GC–MS) method was developed for the quantitative analysis of free amino acids as their propyl chloroformate derivatives in biological fluids. Derivatization with propyl chloroformate is carried out directly in the biological samples without prior protein precipitation or solid-phase extraction of the amino acids, thereby allowing automation of the entire procedure, including addition of reagents, extraction and injection into the GC–MS. The total analysis time was 30 min and 30 amino acids could be reliably quantified using 19 stable isotope-labeled amino acids as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) were in the range of 0.03–12 μM and 0.3–30 μM, respectively. The method was validated using a certified amino acid standard and reference plasma, and its applicability to different biological fluids was shown. Intra-day precision for the analysis of human urine, blood plasma, and cell culture medium was 2.0–8.8%, 0.9–8.3%, and 2.0–14.3%, respectively, while the inter-day precision for human urine was 1.5–14.1%

Promiscuity of Omphalotin A Biosynthetic Enzymes Allows de novo Production of Non‐Natural Multiply Backbone N‐Methylated Peptide Macrocycles in Yeast

Multiple backbone N-methylation and macrocyclization improve the proteolytic stability and oral availability of therapeutic peptides. Chemical synthesis of such peptides is challenging, in particular for the generation of peptide libraries for screening purposes. Enzymatic backbone N-methylation and macrocyclization occur as part of both non-ribosomal and ribosomal peptide biosynthesis, exemplified by the fungal natural products cyclosporin A and omphalotin A, respectively. Omphalotin A, a 9fold backbone N-methylated dodecamer isolated from the agaricomycete Omphalotus olearius, can be produced in Pichia pastoris by coexpression of the ophMA and ophP genes coding for the peptide precursor protein harbouring an autocatalytic peptide α-N-methyltransferase domain, and a peptide macrocyclase, respectively. Since both OphMA and OphP were previously shown to be relatively promiscuous in terms of peptide substrates, we expressed mutant versions of ophMA, encoding OphMA variants with altered core peptide sequences, along with wildtype ophP and assessed the production of the respective peptide macrocycles by the platform by high-performance liquid chromatography, coupled with tandem mass spectrometry (HPLC–MS/MS). Our results demonstrate the successful production of fifteen non-natural omphalotin-derived macrocycles, containing polar, aromatic and charged residues, and, thus, suggest that the system may be used as biotechnological platform to generate libraries of non-natural multiply backbone N-methylated peptide macrocycles.ISSN:1439-4227ISSN:1439-763

Amino acid analysis in physiological samples by GC-MS with propyl chloroformate derivatization and iTRAQ-LC-MS/MS

Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography-quadrupole mass spectrometry (GC-qMS) analysis in single-ion monitoring mode. Derivatization is carried out directly in aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC-MS. The method delivers the quantification of 26 amino acids. The isobaric tagging for relative and absolute quantification (iTRAQ) method employs the labeling of amino acids with isobaric iTRAQ tags. The tags contain two different cleavable reporter ions, one for the sample and one for the standard, which are detected by fragmentation in a tandem mass spectrometer. Reversed-phase liquid chromatography of the labeled amino acids is performed prior to mass spectrometric analysis to separate isobaric amino acids. The commercial iTRAQ kit allows for the analysis of 42 physiological amino acids with a respective isotope-labeled standard for each of these 42 amino acids

Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols

Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid-base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid-base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells

Enzyme-mediated backbone N-methylation in ribosomally encoded peptides

Backbone N-methylation as a posttranslational modification was recently discovered in a class of ribosomally encoded peptides referred to as borosins. The founding members of the borosins are the omphalotins (A-I), backbone N-methylated, macrocyclic dodecapeptides produced by the mushroom Omphalotus olearius. Omphalotins display a strong and selective toxicity toward the plant parasitic nematode Meloidogyne incognita. The primary product omphalotin A is synthesized via a concerted action of the omphalotin precursor protein (OphMA) and the dual function prolyloligopeptidase/macrocyclase (OphP). OphMA consists of α-N-methyltransferase domain that autocatalytically methylates the core peptide fused to its C-terminus via a clasp domain. Genome mining uncovered over 50 OphMA homologs from the fungal phyla Ascomycota and Basidiomycota. However, the derived peptide natural products have not been described yet, except for lentinulins, dendrothelins and gymnopeptides produced by the basidiomycetes Lentinula edodes, Dendrothele bispora and Gymnopus fusipes, respectively. In this chapter, we describe methods used to isolate and characterize these backbone N-methylated peptides and their precursor proteins both in their original hosts and in the heterologous hosts Escherichia coli and Pichia pastoris. These methods may pave the path for both the discovery of novel borosins with interesting bioactivities. In addition, understanding of borosin biosynthetic pathways may allow setting up a biotechnological platform for the production of pharmaceutical leads for orally available peptide drugs.ISSN:0076-687

Urinary metabolite quantification employing 2D NMR spectroscopy

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy is a fairly novel method for the quantification of metabolites in biological fluids and tissue extracts. We show in this contribution that, compared to 1D 1H spectra, superior quantification of human urinary metabolites is obtained from 2D 1H−13C heteronuclear single-quantum correlation (HSQC) spectra measured at natural abundance. This was accomplished by the generation of separate calibration curves for the different 2D HSQC signals of each metabolite. Lower limits of detection were in the low to mid micromolar range and were generally the lower the greater the number of methyl groups contained in an analyte. The quantitative 2D NMR data obtained for a selected set of 17 urinary metabolites were compared to those obtained independently by means of gas chromatography−mass spectrometry and liquid chromatography−mass spectrometry of amino acids and hippurate as well as enzymatic and colorimetric measurements of creatinine. As a typical application, 2D-NMR was used for the investigation of urine from patients with inborn errors of metabolism