doi: 10.1110/ps.0200602.
The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse
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- PMID:12070318
- PMCID: PMC2373659
- DOI: 10.1110/ps.0200602
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The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse
Gustavo Glowacki et al. Protein Sci.2002 Jul.
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Abstract
ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.
Figures
Cross-species "Zoo"-PCR analyses ofART5. Genomic DNAs were subjected to PCR-amplification with two pairs ofART5-specific primers (a and b) ending in codons for conserved amino acids. Reaction products were size fractionated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Genomic DNAs were from: rhesus monkey: lanes1–2, macaque monkey: lanes3– 4, pig: lanes5–6, rat: lanes7–8, control (H2O): lanes9–10, mouse: lanes11–12, human: lanes13–14, tree shrew: lanes15–16, house shrew: lanes17–18, cow: lanes19–20, sheep: lanes21–22, chicken: lanes23–24. M = size of marker fragments in base pairs.
Schematic diagram ofART gene exon/intron structures. Exons and flanking introns were sequenced from respective P1 and PAC genomic DNA clones. Exons are depicted as closed boxes, introns and flanking regions as lines. Genes were aligned with respect to the major exon encoding the catalytic domain (*). The sizes of exons and introns are shown in Figure 4 ▶.
Schematic diagrams of theART cDNA exon compositions. The major splice variant is depicted for eachART gene transcript. Noncoding 5` and 3` untranslated regions are white, coding regions are color coded: leader peptide: orange, native peptide: blue, GPI-anchor signal: green.
Exon-intron structures of human and mouseART genes. Exons are depicted as boxes, introns as lines. Numbers indicate lengths of exons and introns in base pairs. Corresponding exons inART orthologs are connected by arrows. Note that a small exon in the 3` coding region of humanART3 is triplicated with respect to its mouseArt3.
Amino acid sequence alignments of orthologs ARTs. Alignments of the deduced amino acid sequences of each pair of ART-orthologs are shown with the human sequence on top and mouse sequence on bottom. Conserved residues are shown in the middle of each alignment. Predicted secondary structure units lining the six conserved β strands of the active site crevice are marked below the alignment. Different colors are used for amino acids encoded by different exons. The four cysteine residues common to all ARTs are highlighted in yellow, additional ART-specific pairs of cysteine are marked by asterisks. Potential asparagine-linked glycosylation sites are highlighted in blue. Residues corresponding to the R-S-EXE motif of arginine-specific ARTs are marked in green. Three premature stop codons of human ART2AP are marked in red.
Hydropathy profiles of human and mouse ART proteins. Hydropathy profiles were calculated with a window setting of 19 amino acid residues. Putative hydrophobic amino- and carboxy-terminal signal peptides are colored orange and green, respectively. Positions of potential asparagine-linked oligosaccharide side chains are marked by forks. The four cysteine residues conserved in all mammalian ARTs are marked by circles with numbers indicating their respective positions from the amino terminus. Additional cysteine residues found only in individual ARTs are marked by shaded circles. Residues corresponding to the R-S-EXE motif of arginine specific mARTs are marked by yellow circles.
Reverse transcription PCR analyses ofART gene expression. Panels of mouse and human cDNAs were subjected to reverse transcription PCR analyses using primers from separate exons as illustrated schematically. Products after 31 cycles were size fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining. cDNAs were from: heart: lane1, brain: lane2, spleen: lane3, lung: lane4, liver: lane5, skeletal muscle: lane6, kidney: lane7, testis: lane8, 7-day embryo: lane9, 11-day embryo: lane10, 15-day embryo: lane11, 17-day embryo: lane12, control (H2O): lane13, colon: lane14, ovary: lane15, peripheral blood lymphocytes: lane16, prostate: lane17, small intestine: lane18, spleen: lane19, testis: lane20, thymus: lane21, and control (H2O): lane22.
Northern blot analyses of human and mouseART gene expression. Northern blots with mouse and human RNAs were hybridized with radiolabeledART-specific probes, and bound activity was visualized by autoradiography. Thick and thin bars indicate the migration positions of 2.4kb and 1.35kb marker fragments, respectively. Mouse RNAs were from heart: lane1; brain:2; spleen:3; lung:4; liver:5; skeletal muscle:6; kidney:7; and testis:8. Human RNAs forART1 andART2 were from heart: lane9; brain:10; placenta:11; lung:12; liver:13; skeletal muscle:14; kidney:15; and pancreas:16; forART3 andART5, pancreas: lane9; adrenal medulla:10; thyroid:11; adrenal cortex:12; testis:13; thymus:14; small intestine:15; and stomach:16; and forART4, spleen: lane9; lymph node:10; thymus:11; appendix:12; peripheral blood leukocytes:13; bone marrow:14; and fetal liver:15.
Comparative enzyme assays of purified epitope-tagged ARTs. ARTs were immunoprecipitated from the supernatants of respective baculovirus-infected insect cells with immobilized anti-FLAG monoclonal antibody M2. Purified ARTs were incubated with 32P-NAD+ for 60 min at 37°C in the presence of 2 mM agmatine (an arginine analog). Proteins were analyzed by SDS-PAGE and Western blot analyses using the anti-FLAG antibody (A). Note that human ART1 was not produced efficiently in this system (A, lane7). Enzyme reaction products were analyzed by thin-layer chromatography followed by autoradiography (B). Co: control precipitates from mock infected cells.
Matches detected by PSI-BLAST cluster in regions corresponding to the active site crevice of VIP2. Regions shown in the multiple sequence alignment (Fig. 11 ▶) are projected onto the backbone ofBacillus cereus VIP2 (Han et al. 1999) with bound NAD (cyan). The two images generated by rasmac using pdb file 1QS2 are rotated by 90° to provide side (left) and front (right) views of the active site crevice. The arrow points to the cleaved bond in NAD. The six conserved β-strands of the lower and upper jaws of the active site crevice are depicted as cartoons and are colored yellow (β4, β5, β2) and orange (β1, β3, β6), respectively. The three residues of the R-S-E motif are in red, the upstream glutamic acid of arginine-specific mARTs is in green.
Multiple sequence alignment of the catalytic cores of vertebrate and bacterial mARTs. Multiple amino sequence alignment of the region corresponding to the six conserved β-strands of the active site crevice (see Fig. 10 ▶). Nonconserved residues in connecting loops are not shown and are indicated by numbers. Known secondary structure units in the crystallized mARTs are underlined (C3, VIP2, CT, LT, PT, DT, ETA, and PARP). Color coding as in (A). Note that the last β strand in VIP2, C3, and related ARTs (marked by asterisks) has been displaced by a strand in opposite orientation relative to that in LT, PARP, and others (i.e., β5 and β6 are parallel in VIP2, whereas they are antiparallel in CT).
References
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