Anders M.B. Giessing

Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr- strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m(2)A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules.

Quantitative proteomics by amino acid labeling in C. elegans.

We demonstrate labeling of Caenorhabditis elegans with heavy isotope–labeled lysine by feeding them with heavy isotope–labeled Escherichia coli. Using heavy isotope–labeled worms and quantitative proteomics methods, we identified several proteins that are regulated in response to loss or RNAi-mediated knockdown of the nuclear hormone receptor 49 in C. elegans. The combined use of quantitative proteomics and selective gene knockdown is a powerful tool for C. elegans biology.

Investigating the biomarker potential of glycoproteins using comparative glycoprofiling — application to tissue inhibitor of metalloproteinases-1

Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential are limited, calling for new methods. Here, we have investigated the cancer marker potential of the glycoprofile of tissue inhibitor of metalloproteinase-1 (TIMP-1) using a method for comparative glycoprofiling. Glycoprofiles were obtained from plasma TIMP-1 of five healthy donors and five colorectal cancer (CRC) patients showing increased amounts of TIMP-1. Furthermore, the TIMP-1 glycoprofiles of media from two colon cancer cell lines (CCC) and a prostate cancer cell line were determined as disease references. TIMP-1 was purified from IgG-depleted samples using immuno affinity and gel electrophoresis and the glycoprofiling was performed using glycopeptide enrichment and mass spectrometry. The heterogeneous glycoprofiles of TIMP-1 were found to be highly conserved among the healthy donors, proving an ideal candidate marker and showed high reproducibility of the method. Numerous CCC-specific TIMP-1 glycans were observed illustrating cancer-induced changes. Unexpectedly, quantitation revealed that the glycoprofiles of healthy donors and CRC patients varied minimally. Considering the increased CRC TIMP-1 levels and the observed CCC-specific glycans, the lack of variation indicates that the increased amount of CRC TIMP-1 is not a direct product of the cancer cells. Hence, the TIMP-1 glycoprofile holds no biomarker potential for CRC when using plasma as the sample origin. This study clearly illustrates that the technique is capable of performing individualised site-specific glycan analysis and representing a new tool for biomarker investigation of low-abundant glycoproteins.

Recognition of guanosine by dissimilar tRNA methyltransferases

Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.

1-Hydroxypyrene as a biomarker of PAH exposure in the marine polychaete Nereis diversicolor

The possibility of using the pyrene metabolite 1-hydroxypyrene as a biomarker of polycyclic aromatic hydrocarbons (PAHs) exposure was investigated by exposure of the marine polychaete Nereis diversicolor to several PAHs in the laboratory. Animals were exposed to pyrene alone and to five different PAHs - phenanthrene, anthracene, pyrene, benzo[a]pyrene and benzo[k]flouranthene. After five days of exposure the concentrations of parent PAHs and 1-hydroxypyrene were identified using three different analytical methods, high-performance liquid chromatography with fluorescence detection (HPLC/F), synchronous fluorescence spectroscopy (SFS) and gas chromatography with mass spectrometric detection (GC/MS). The SFS measurements of 1-hydroxypyrene were validated by the more sensitive method HPLC/F. The positive correlation between total PAHs and 1-hydroxypyrene concentrations in the polychaete tissues observed in experiments, suggests the feasibility of 1-hydroxypyrene as a suitable biomarker for total PAH exposure assessment. Furthermore, the possibility of employment of the simple and rapid SFS method instead of HPLC/F for biomarker analysis has been confirmed by the positive and significant correlation between results achieved by these two analytical methods.

Glycopeptide profiling of beta-2-glycoprotein I by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome.

Beta2-glycoprotein I (beta2GPI) is a five-domain protein associated with the antiphospholipid syndrome (APS), however, its normal biological function is yet to be defined. beta2GPI is N-glycosylated at several asparagine residues and the glycan moiety conjugated to residue 143 has been proposed to interact with the Gly40-Arg43 motif of beta2GPI. The Gly40-Arg43 motif has also been proposed to serve as the epitope for the anti-beta2GPI autoantibody associated with APS. We hypothesized that the structure or composition of the glycan at Asn-143 might be associated with the APS symptom by shielding or exposing the Gly40-Arg43 motif towards the anti-beta2GPI autoantibody. To test this hypothesis we used mass spectrometry (MS) for comparative glycopeptide profiling of human beta2GPI obtained from blood serum from four healthy test subjects and six APS patients. It revealed significant differences in the extent of sialylation and branching of glycans at Asn-143. Biantennary glycans were more abundant than triantennary glycans at Asn-143 in both healthy subjects and patients. In APS patient samples we observed a decrease in sialylated triantennary glycans and an increase in sialylated biantennary glycan structures, as compared to controls. These data indicate that some APS patients have beta2GPI molecules with a reduced number of negatively charged sialic acid units in the glycan structure at Asn-143. This alteration of the electrostatic properties of the glycan moiety may attenuate the intramolecular interactions with the positively charged Gly40-Arg43 motif of beta2GPI and, in turn, leads to conformational instability and exposure of the disease-related linear epitope Gly40-Arg43 to the circulating autoantibody. Thus, our study suggests a link between site-specific glycan profiles of beta2GPI and the pathology of antiphospholipid syndrome.

Towards Liquid Chromatography Time-Scale Peptide Sequencing and Characterization of Post-Translational Modifications in the Negative-Ion Mode Using Electron Detachment Dissociation Tandem Mass Spectrometry

Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational modifications (PTMs) and produces predictable and interpretable fragment ion types (a·, x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented in large-scale peptide characterization strategies. We successfully increased the EDD fragmentation efficiency (up to 9%), and demonstrate for the first time the utility of EDD-MS/MS in liquid chromatography time-scale experiments. Peptides and phosphopeptides were analyzed in both positive- and negative-ion mode using electron capture/transfer dissociation (ECD/ETD) and EDD in comparison. Using approximately 1 pmol of a BSA tryptic digest, LC-EDD-MS/MS sequenced 14 peptides (27% aa sequence coverage) and LC-ECD-MS/MS sequenced 19 peptides (39% aa sequence coverage). Seven peptides (18% aa sequence coverage) were sequenced by both EDD and ECD. The relative small overlap of identified BSA peptides demonstrates the complementarity of the two dissociation modes. Phosphopeptide mixtures from three trypsin-digested phosphoproteins were subjected to LC-EDD-MS/MS resulting in the identification of five phospho-peptides. Of those, one was not found in a previous study using a similar sample and LC-ETD-MS/MS in the positive-ion mode. In this study, the ECD fragmentation efficiency (15.7% av.) was superior to the EDD fragmentation efficiency (3.6% av.). However, given the increase in amino acid sequence coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a more comprehensive strategy of analysis in proteome research.

3‐(3‐amino‐3‐carboxypropyl)‐5,6‐Dihydrouridine is one of two novel post‐transcriptional modifications in tRNALys(UUU) from Trypanosoma brucei

tRNA is the most heavily modified of all RNA types, with typically 10–20% of the residues being post‐transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications [ 1 ], many of which are chemically similar. Furthermore, the tRNA has to be investigated with single‐nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNALys(UUU) from Trypanosoma brucei, and provide a complete overview of its post‐transcriptional modifications. The first step was MALDI‐TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass‐changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher‐order tandem MS. Phylogenetic comparison with modifications in tRNALys from other organisms was used through the entire analysis. We identified modifications on 12 nucleosides in tRNALys(UUU), where U47 exhibited a novel modification, 3‐(3‐amino‐3‐carboxypropyl)‐5,6‐dihydrouridine, based on identical chromatographic retention and MS fragmentation as the synthetic nucleoside. A37 was observed in two versions: a minor fraction with the previously described 2‐methylthio‐N6‐threonylcarbamoyl‐modification, and a major fraction with A37 being modified by a 294.0‐Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin.

Identification and characterization of the Thermus thermophilus 5-methylcytidine (m5C) methyltransferase modifying 23 S ribosomal RNA (rRNA) base C1942.

Background: The enzymes methylating C1942 and C1962 in 23 S ribosomal RNA of Thermus thermophilus remained to be identified. Results: The methyltransferase targeting C1942 was identified and characterized with respect to substrate, exact product, and structure. Conclusion: The methyltransferase has structural similarities with previously characterized methyltransferases producing 5-methylcytidine and 5-methyluridine. Significance: The structure of RNA-methylating enzymes is important in the delineation of their evolution. Methylation of cytidines at carbon-5 is a common posttranscriptional RNA modification encountered across all domains of life. Here, we characterize the modifications of C1942 and C1962 in Thermus thermophilus 23 S rRNA as 5-methylcytidines (m5C) and identify the two associated methyltransferases. The methyltransferase modifying C1942, named RlmO, has not been characterized previously. RlmO modifies naked 23 S rRNA, but not the assembled 50 S subunit or 70 S ribosomes. The x-ray crystal structure of this enzyme in complex with the S-adenosyl-l-methionine cofactor at 1.7 Å resolution confirms that RlmO is structurally related to other m5C rRNA methyltransferases. Key residues in the active site are located similar to the further distant 5-methyluridine methyltransferase RlmD, suggestive of a similar enzymatic mechanism. RlmO homologues are primarily found in mesophilic bacteria related to T. thermophilus. In accordance, we find that growth of the T. thermophilus strain with an inactivated C1942 methyltransferase gene is not compromised at non-optimal temperatures.