Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2-which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.     

Functional and evolutionary analyses on expressed intronless genes in the mouse genome

Using computational approaches we have identified 2017 expressed intronless genes in the mouse genome. Evolutionary analysis reveals that 56 intronless genes are conserved among the three domains of life--bacteria, archea and eukaryotes. These highly conserved intronless genes were found to be involved in essential housekeeping functions. About 80% of expressed mouse intronless genes have orthologs in eukaryotic genomes only, and thus are specific to eukaryotic organisms. 608 of these genes have intronless human orthologs and 302 of these orthologs have a match in OMIM database. Investigation into these mouse genes will be important in generating mouse models for understanding human diseases.     

A panel of monoclonal antibodies recognizing GPI-anchored ADP-ribosyltransferase ART4, the carrier of the Dombrock blood group antigens

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.     

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework.     

Bacterial ADP-ribosylating toxins: molecular structures and signal transducing functions

Mono-ADP-ribosylation is a posttranslational modification of proteins employed by a variety of bacterial ADP-ribosylating toxins to modify the metabolism of target cells. The ADP-ribosyltransferases of bacterial toxins, in general, use NAD as a substrate for covalent modification by ADP-ribose to certain GTP-binding proteins (G proteins) as signal transducers resulting in altered enzymatic activity of the membrane enzymes as effectors. Such a mechanism has the potential of being of importance in the physiological regulation of cellular metabolism, particularly if the process is reversible. These ADP-ribosylating toxins are characterized in Table 1.     

Comparative study of apoptosis-related gene loci in human, mouse and rat genomes

Many genes are involved in mammalian cell apoptosis pathway. These apoptosis genes often contain characteristic functional domains, and can be classified into at least 15 functional groups, according to previous reports. Using an integrated bioinformatics platform for motif or domain search from three public mammalian proteomes (International Protein Index database for human, mouse, and rat), we systematically cataloged all of the proteins involved in mammalian apoptosis pathway. By localizing those proteins onto the genomes, we obtained a gene locus centric apoptosis gene catalog for human, mouse and rat.Further phylogenetic analysis showed that most of the apoptosis related gene loci are conserved among these three mammals. Interestingly, about one-third of apoptosis gene loci form gene clusters on mammal chromosomes, and exist in the three species, which indicated that mammalian apoptosis gene orders are also conserved. In addition, some tandem duplicated gene loci were revealed by comparing gene loci clusters in the three species. All data produced in this work were stored in a relational database and may be viewed at http://pcas.cbi.pku.edu.cn/database/apd.php.     

Three conserved consensus sequences identify the NAD-binding site of ADP-ribosylating enzymes, expressed by eukaryotes, bacteria and T-even bacteriophages

It has been previously reported that the three-dimensional structures of the NAD-binding and catalytic site of bacterial toxins with ADP-ribosylating activity are superimposable, and that the key amino acids for the enzymatic activity are conserved. The model includes an NAD-binding and catalytic site formed by an α-helix bent over a β-strand, surrounded by two β-strands bearing a Glu and a His, or Arg, that are required for catalysis. We show here that the model can be extended to comprise all proteins with ADP-ribosylating activity known to date, including all eukaryotic mono- and poly-ADP-ribosyltransferases, the bacterial ADP-ribosylating enzymes which do not have toxic activity, and the analogous enzymes encoded by T-even bacteriophages. We show that, in addition to the common Glu and Arg/His amino acids previously identified, the conserved motifs can be extended as follows: (i) the Arg/His motif is usually arom-His/Arg (where 'arom' is an aromatic residue); (ii) in the sequences of the CT group the β-strand forming part of the 'scaffold' of the catalytic cavity has an arom-ph-Ser-Thr-Ser-ph consensus (where 'ph' represents a hydrophobic residue); and (iii) the motif centred in the key glutamic residue is Glu/Gln-X-Glu; while (iv) in the sequences of the DT group the NAD-binding motif is Tyr-X₁₀-Tyr. We believe that the model proposed not only accounts for all ADP-ribosylating proteins known to date, but it is likely to fit other enzymes (currently being analysed) which possess such an activity.     

The vacuolar protein sorting genes in insects: A comparative genome view

In eukaryotic cells, regulated vesicular trafficking is critical for directing protein transport and for recycling and degradation of membrane lipids and proteins. Through carefully regulated transport vesicles, the endomembrane system performs a large and important array of dynamic cellular functions while maintaining the integrity of the cellular membrane system. Genetic studies in yeast Saccharomyces cerevisiae have identified approximately 50 vacuolar protein sorting (VPS) genes involved in vesicle trafficking, and most of these genes are also characterized in mammals. The VPS proteins form distinct functional complexes, which include complexes known as ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III. Little is known about the orthologs of VPS proteins in insects. Here, with the newly annotated Manduca sexta genome, we carried out genomic comparative analysis of VPS proteins in yeast, humans, and 13 sequenced insect genomes representing the Orders Hymenoptera, Diptera, Hemiptera, Phthiraptera, Lepidoptera, and Coleoptera. Amino acid sequence alignments and domain/motif structure analyses reveal that most of the components of ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III are evolutionarily conserved across yeast, insects, and humans. However, in contrast to the VPS gene expansions observed in the human genome, only four VPS genes (VPS13, VPS16, VPS33, and VPS37) were expanded in the six insect Orders. Additionally, VPS2 was expanded only in species from Phthiraptera, Lepidoptera, and Coleoptera. These studies provide a baseline for understanding the evolution of vesicular trafficking across yeast, insect, and human genomes, and also provide a basis for further addressing specific functional roles of VPS proteins in insects.     

A new monoclonal antibody detects a developmentally regulated mouse ecto-ADP-ribosyltransferase on T cells: subset distribution, inbred strain variation, and modulation upon T cell activation

ADP-ribosylation of membrane proteins on mouse T cells by ecto-ADP-ribosyltransferase(s) (ARTs) can down-regulate proliferation and function. The lack of mAbs against mouse ARTs has heretofore prevented analysis of ART expression on T cell subsets. Using gene gun technology, we immunized a Wistar rat with an Art2b expression vector and produced a novel mAb, Nika102, specific for ART2.2, the Art2b gene product. We show that ART2.2 is expressed as a GPI-anchored protein on the surface of mature T cells. Inbred strain-dependent differences in ART2.2 expression levels were observed. C57BL/6J and C57BLKS/J express the Ag at high level, with up to 70% of CD4+ and up to 95% of CD8+ peripheral T cells expressing ART2.2. CBA/J and DBA/2J represent strains with lowest expression levels. T cell-deficient mice and NZW/LacJ mice with a defective structural gene for this enzyme were ART2.2 negative. In the thymus, ART2.2 expression is restricted to subpopulations of mature cells. During postnatal ontogeny, increasing percentages of T cells express ART2.2, reaching a peak at 6-8 wk of age. Interestingly, ART2.2 and CD25 are reciprocally expressed: activation-induced up-regulation of CD25 is accompanied by loss of ART2.2 from the cell surface. Nika102 thus defines a new differentiation/activation marker of thymic and postthymic T cells in the mouse and should be useful for further elucidating the function of the ART2.2 cell surface enzyme.     

Laboratory mouse models for the human genome-wide associations

The agnostic screening performed by genome-wide association studies (GWAS) has uncovered associations for previously unsuspected genes. Knowledge about the functional role of these genes is crucial and laboratory mouse models can provide such information. Here, we describe a systematic juxtaposition of human GWAS-discovered loci versus mouse models in order to appreciate the availability of mouse models data, to gain biological insights for the role of these genes and to explore the extent of concordance between these two lines of evidence. We perused publicly available data (NHGRI database for human associations and Mouse Genome Informatics database for mouse models) and employed two alternative approaches for cross-species comparisons, phenotype- and gene-centric. A total of 293 single gene-phenotype human associations (262 unique genes and 69 unique phenotypes) were evaluated. In the phenotype-centric approach, we identified all mouse models and related ortholog genes for the 51 human phenotypes with a comparable phenotype in mice. A total of 27 ortholog genes were found to be associated with the same phenotype in humans and mice, a concordance that was significantly larger than expected by chance (p<0.001). In the gene-centric approach, we were able to locate at least 1 knockout model for 60% of the 262 genes. The knockouts for 35% of these orthologs displayed pre- or post-natal lethality. For the remaining non-lethal orthologs, the same organ system was involved in mice and humans in 71% of the cases (p<0.001). Our project highlights the wealth of available information from mouse models for human GWAS, catalogues extensive information on plausible physiologic implications for many genes, provides hypothesis-generating findings for additional GWAS analyses and documents that the concordance between human and mouse genetic association is larger than expected by chance and can be informative.     

An automated comparative analysis of 17 complete microbial genomes

MOTIVATION: As sequenced genomes become larger and sequencing becomes faster, there is a need to develop accurate automated genome comparison techniques and databases to facilitate derivation of genome functionality; identification of enzymes, putative operons and metabolic pathways; and to derive phylogenetic classification of microbes. RESULTS: This paper extends an automated pair-wise genome comparison technique (Bansal et al., Math. Model. Sci. Comput., 9, 1-23, 1998, Bansal and Bork, in First International Workshop of Declarative Languages, Springer, pp. 275-289, 1999) used to identify orthologs and gene groups to derive orthologous genes in a group of genomes and to identify genes with conserved functionality. Seventeen microbial genomes archived at ftp://ncbi.nlm.nih.gov/genbank/genomes have been compared using the automated technique. Data related to orthologs, gene groups, gene duplication, gene fusion, orthologs with conserved functionality, and genes specifically orthologous to Escherichia coli and pathogens has been presented and analyzed. AVAILABILITY: A prototype database is available at ftp://www.mcs.kent.edu/arvind/intellibio / orthos.html. The software is free for academic research under an academic license. The detailed database for every microbial genome in NCBI is commercially available through intellibio software and consultancy corporation (Web site: http://www.mcs.kent.edu/?rvind/intellibio . html). CONTACT: arvind@mcs.kent.edu.     

Two Novel Human Members of an Emerging Mammalian Gene Family Related to Mono-ADP-Ribosylating Bacterial Toxins

Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes. Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts. Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2). We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designatedART3andART4). The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32–41% identity vs the muscle and T cell enzymes. They contain signal peptide sequences characteristic of GPI anchorage. Southern Zoo blot analyses suggest the presence of related genes in other mammalian species. By PCR screening of somatic cell hybrids and byin situhybridization, we have mapped the two genes to human chromosomes 4p14–p15.1 and 12q13.2–q13.3. Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively. Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins. Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases. It is possible that the four human gene family members identified so far represent the “tip of an iceberg,” i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation.     

Gene expression in placentation of farm animals: an overview of gene function during development

Eutherian mammals share a common ancestor that evolved into two main placental types, i.e., hemotrophic (e.g., human and mouse) and histiotrophic (e.g., farm animals), which differ in invasiveness. Pregnancies initiated with assisted reproductive techniques (ART) in farm animals are at increased risk of failure; these losses were associated with placental defects, perhaps due to altered gene expression. Developmentally regulated genes in the placenta seem highly phylogenetically conserved, whereas those expressed later in pregnancy are more species-specific. To elucidate differences between hemotrophic and epitheliochorial placentae, gene expression data were compiled from microarray studies of bovine placental tissues at various stages of pregnancy. Moreover, an in silico subtractive library was constructed based on homology of bovine genes to the database of zebrafish — a nonplacental vertebrate. In addition, the list of placental preferentially expressed genes for the human and mouse were collected using bioinformatics tools (Tissue-specific Gene Expression and Regulation [TiGER] — for humans, and tissue-specific genes database (TiSGeD) — for mice and humans). Humans, mice, and cattle shared 93 genes expressed in their placentae. Most of these were related to immune function (based on analysis of gene ontology). Cattle and women shared expression of 23 genes, mostly related to hormonal activity, whereas mice and women shared 16 genes (primarily sexual differentiation and glycoprotein biology). Because the number of genes expressed by the placentae of both cattle and mice were similar (based on cluster analysis), we concluded that both cattle and mice were suitable models to study the biology of the human placenta.     

Rho-specific Bacillus cereus ADP-ribosyltransferase C3cer cloning and characterization

C3-like ADP-ribosyltransferases represent an expanding family of related exoenzymes, which are produced by Clostridia and various Staphylococcus aureus strains. Here we report on the cloning and biochemical characterization of an ADP-ribosyltransferase from Bacillus cereus strain 2339. The transferase encompasses 219 amino acids; it has a predicted mass of 25.2 kDa and a theoretical isoelectric point of 9.3. To indicate the relationship to the family of C3-like ADP-ribosyltransferases, we termed the enzyme C3cer. The amino acid sequence of C3cer is 30 to 40% identical to other C3-like exoenzymes. By site-directed mutagenesis, Arg(59), Arg(97), Tyr(151), Arg(155), Thr(178), Tyr(180), Gln(183), and Glu(185) of recombinant C3cer were identified as pivotal residues of enzyme activity and/or protein substrate recognition. Precipitation experiments with immobilized RhoA revealed that C3cerTyr(180), which is located in the so-called "ADP-ribosylating toxin turn-turn" (ARTT) motif, plays a major role in the recognition of RhoA. Like other C3-like exoenzymes, C3cer ADP-ribosylates preferentially RhoA and RhoB and to a much lesser extent RhoC. Because the cellular accessibility of recombinant C3cer is low, a fusion toxin (C2IN-C3cer), consisting of the N-terminal 225 amino acid residues of the enzyme component of C2 toxin from Clostridium botulinum and C3cer was used to study the cytotoxic effects of the transferase. This fusion toxin caused rounding up of Vero cells comparable to the effects of Rho-inactivating toxins.