Irena I. Artamonova

Destabilase from the medicinal leech is a representative of a novel family of lysozymes

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

Antibacterial Non-Glycosidase activity of Invertebrate Destabilase-Lysozyme and of its Helical amphipathic peptides

Background: Since bactericidal properties of some lysozymes are independent of their glycosidase activity, we have investigated this phenomenon for destabilase-lysozyme (DL) from medicinal leech (Hirudo medicinalis). Methods: Glycosidase activity was determined on Micrococcus luteus, non-enzymatic antibacterial activity of heat-treated DL and of synthetic peptides α1, α2 and α3 (fragments of its primary structure) on M. luteus, Escherichia coli, Bacillus brevis and Streptomyces chrysomallus. Results: Glycosidase activity disappeared after the heating of native DL at 100°C for 40 min. Antibacterial activity of heat-treated DL for M. luteus MDMSU128 and MDMSU140 expressed as minimal inhibitory concentration was 9.8·10–8 and 12·10–8 M, respectively, and to E. coli MDMSU52 11·10–8M. Antibacterial activity of synthetic peptide α1 for M. luteus MDMSU128 and for E. coli MDMSU52 was 8.3·10–5 and 4.9·10–5M, respectively. Conclusion: DL is the first invertebrate lysozyme with combined enzymatic and non-enzymatic antibacterial action.

Mining sequence annotation databanks for association patterns

MOTIVATION Millions of protein sequences currently being deposited to sequence databanks will never be annotated manually. Similarity-based annotation generated by automatic software pipelines unavoidably contains spurious assignments due to the imperfection of bioinformatics methods. Examples of such annotation errors include over- and underpredictions caused by the use of fixed recognition thresholds and incorrect annotations caused by transitivity based information transfer to unrelated proteins or transfer of errors already accumulated in databases. One of the most difficult and timely challenges in bioinformatics is the development of intelligent systems aimed at improving the quality of automatically generated annotation. A possible approach to this problem is to detect anomalies in annotation items based on association rule mining. RESULTS We present the first large-scale analysis of association rules derived from two large protein annotation databases-Swiss-Prot and PEDANT-and reveal novel, previously unknown tendencies of rule strength distributions. Most of the rules are either very strong or very weak, with rules in the medium strength range being relatively infrequent. Based on dynamics of error correction in subsequent Swiss-Prot releases and on our own manual analysis we demonstrate that exceptions from strong rules are, indeed, significantly enriched in annotation errors and can be used to automatically flag them. We identify different strength dependencies of rules derived from different fields in Swiss-Prot. A compositional breakdown of association rules generated from PEDANT in terms of their constituent items indicates that most of the errors that can be corrected are related to gene functional roles. Swiss-Prot errors are usually caused by under-annotation owing to its conservative approach, whereas automatically generated PEDANT annotation suffers from over-annotation. AVAILABILITY All data generated in this study are available for download and browsing at http://pedant.gsf.de/ARIA/index.htm.

Evolutionary Dynamics of Clustered Irregularly Interspaced Short Palindromic Repeat Systems in the Ocean Metagenome

ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from the Sorcerer II Global Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, <0.0001). We developed a catalogue of elementary CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.

PEDANT genome database: 10 years online

The PEDANT genome database provides exhaustive annotation of 468 genomes by a broad set of bioinformatics algorithms. We describe recent developments of the PEDANT Web server. The all-new Graphical User Interface (GUI) implemented in Java™ allows for more efficient navigation of the genome data, extended search capabilities, user customization and export facilities. The DNA and Protein viewers have been made highly dynamic and customizable. We also provide Web Services to access the entire body of PEDANT data programmatically. Finally, we report on the application of association rule mining for automatic detection of potential annotation errors. PEDANT is freely accessible to academic users at .

Full-sized HERV-K (HML-2) human endogenous retroviral LTR sequences on human chromosome 21: map locations and evolutionary history

One of the evolutionary mechanisms for acquisition of novel functional sequences can be domestication of exogenous retroviruses that have been integrated into the germ line. The whole genome mapping of such elements in various species could reveal differences in positions of the retroviral integration and suggest possible roles of these differences in speciation. Here, we describe the number, locations and sequence features of the human endogenous retrovirus HERV-K (HML-2) long terminal repeat (LTR) sequences on human chromosome 21. We show that their distribution along the chromosome is not only non-random but also roughly correlated with the gene density. Amplification of orthologous LTR sites from a number of primate genomes produced patterns of presence and absence for each LTR sequence and allowed determination of the phylogenetic ages and evolutionary order of appearance of individual LTRs. The identity level and phylogenetic age of the LTRs did not correlate with their map locations. Thus, despite the non-random distribution of LTRs, they have apparently been inserted randomly into the chromosome relative to each other. As evidenced in previous studies of chromosomes 19 and 22, this is a characteristic of HERV-K integration.

Applying negative rule mining to improve genome annotation

BackgroundUnsupervised annotation of proteins by software pipelines suffers from very high error rates. Spurious functional assignments are usually caused by unwarranted homology-based transfer of information from existing database entries to the new target sequences. We have previously demonstrated that data mining in large sequence annotation databanks can help identify annotation items that are strongly associated with each other, and that exceptions from strong positive association rules often point to potential annotation errors. Here we investigate the applicability of negative association rule mining to revealing erroneously assigned annotation items.ResultsAlmost all exceptions from strong negative association rules are connected to at least one wrong attribute in the feature combination making up the rule. The fraction of annotation features flagged by this approach as suspicious is strongly enriched in errors and constitutes about 0.6% of the whole body of the similarity-transferred annotation in the PEDANT genome database. Positive rule mining does not identify two thirds of these errors. The approach based on exceptions from negative rules is much more specific than positive rule mining, but its coverage is significantly lower.ConclusionMining of both negative and positive association rules is a potent tool for finding significant trends in protein annotation and flagging doubtful features for further inspection.

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

The emergence of ribosomes and translation factors is central for understanding the origin of life. Recruitment of translation factors to bacterial ribosomes is mediated by the L12 stalk composed of protein L10 and several copies of protein L12, the only multi-copy protein of the ribosome. Here we predict stoichiometries of L12 stalk for >1,200 bacteria, mitochondria and chloroplasts by a computational analysis, and validate the predictions by quantitative mass spectrometry. The majority of bacteria have L12 stalks allowing for binding of four or six copies of L12, largely independent of the taxonomic group or living conditions of the bacteria, whereas some cyanobacteria have eight copies. Mitochondrial and chloroplast ribosomes can accommodate six copies of L12. The last universal common ancestor probably had six molecules of L12 molecules bound to L10. Changes of the stalk composition provide a unique possibility to trace the evolution of protein components of the ribosome.

Evolution of the Exon-Intron Structure and Alternative Splicing of the MAGE-A Family of Cancer/Testis Antigens

Cancer/testis antigens (CT-antigens) are proteins that are predominantly expressed in cancer and testis and thus are possible targets for immunotherapy. Most of them form large multigene families. The evolution of the MAGE-A family of CT-antigens is characterized by four processes: (1) gene duplications; (2) duplications of the initial exon; (3) point mutations and short insertions/deletions inactivating splicing sites or creating new sites; and (4) deletions removing sites and creating chimeric exons. All this concerns the genomic regions upstream of the coding region, creating a wide diversity of isoforms with different 5′-untranslated regions. Many of these isoforms are gene-specific and have emerged due to point mutations in alternative and constitutive splicing sites. There are also examples of chimeric mRNAs, likely produced by splicing of read-through transcripts. Since there is consistent use of homologous sites for different genes and no random, indiscriminant use of preexisting cryptic sites, it is likely that most observed isoforms are functional, and do not result from relaxed control in transformed cells.

Multiple forms of medicinal leech destabilase-lysozyme

Earlier, three genes Ds1, Ds2, and Ds3 encoding corresponding destabilase-lysozyme isoforms were identified. However only one form of the enzyme encoded by Ds3 gene coincided with the protein CNBr fragments [Mol. Gen. Genet. 253 (1996) 20]. In this work we found by ESI-TOF mass spectrometry that the enzyme preparation consists of at least three forms with molecular masses of 12677.6, 12839.7, and 12938.2Da, each of which contains seven disulfide bridges. Only one mass (12839.7Da) fits to the calculated mass for the protein encoded by Ds3 gene. Further analysis of the CNBr fragments of the enzyme showed the heterogeneity of large 5.5 kDa peptide at positions 64 (threonine or arginine) and 67 (histidine or arginine) in the wild-type amino acid sequence. One CNBr peptide, with Arg and His at positions 64 and 67, respectively, correlates in the molecular mass with the protein encoded by Ds3. In addition, we have found a new acid form of destabilase-lysozyme, P-Ac, which differs from all known destabilase-lysozyme structures by its N-terminal amino acid sequence.