Masoud Yadollahi-Farsani

Inactivation of platelet‐derived growth factor‐BB following modification by ADP‐ribosyltransferase

Arginine‐specific ADP‐ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP‐ribose from NAD+ to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline‐inducible expression of human ART. From a panel of growth factors, platelet‐derived growth factor‐BB (PDGF‐BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF‐AA was not a substrate. Under conditions of maximum labelling 5 mol ADP‐ribose was incorporated per mol of PDGF‐BB. Purified (ADP‐ribosyl)‐PDGF‐BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF‐BB. PDGF‐dependent [3H‐methyl]‐thymidine incorporation was measured in the ART1‐transfected fibroblast cell line at physiological concentrations of PDGF‐BB, and without addition of extracellular NAD+. Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF‐BB, but not to PDGF‐AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate). In conclusion, we propose that PDGF‐BB‐dependent signalling may be regulated by post‐translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1‐transfected fibroblasts.

Enzymic and Interindividual Differences in the Human Metabolism of Heterocyclic Amines

Heterocyclic amines (HAs) present in cooked meat (PhIP and MeIQx) are activated only by CYP1A2 in the liver of most species, including man. This enzyme exhibits marked interindividual differences in its expression, due to induction and possibly also genetically. The absence of CYP1A2 appears to protect from HA-(PhIP and MeIQx) induced cancer, as exemplified by results in the cynomolgus monkey. Differences in the potency of these HAs are not due to differences in the kinetics of their activation. The catalytic efficiency of CYP1A2 towards HAs and their oxidative fate varies amongst species, in both cases increasing the susceptibility of humans compared to that of the rat. Interindividual and inter-organ differences in the further metabolism of N-hydroxy-HAs appear to be important determinants of cancer susceptibility, as does the glutathione S-transferase catalysed detoxication of esters of N-hydroxy-PhIP. There is a need for an effective means of quantifying the in vivo activation of HAs in man to enable the possible risk posed by these compounds to be assessed effectively.

ARGININE-SPECIFIC ADP-RIBOSYLTRANSFERASES IN LEUKOCYTES

The posttranslational modification of proteins by the addition of an ADP‐ribose group is mediated by ADP‐ribosyltransferases, which are expressed widely in many eukaryotic tissues, including leukocytes. DNA encoding arginine‐specific ADP‐ribosyltransferases has been cloned from both polymorphonuclear neutrophil leukocytes and lymphocytes, and their primary structures are widely conserved, particularly in those domains of the enzyme implicated in NAD+ binding and catalysis. In most cases the enzymes are tethered to the outer aspect of the cell surface or are released directly from the cell surface. The protein substrates of some of the ADP‐ribosyltransferases have been identified and the catalytic activity of these enzymes has been implicated in several immune responses as well as white cell chemotaxis. This review describes recent significant advances in this field, and it seems likely that additional leukocyte functions, most particularly those linked to the activity of surface integrins, will be assigned to this class of enzymes. J. Leukoc. Biol. 63: 15–21; 1998.

Arginine-Specific Mono(ADP-Ribosyl)Transferase Activity in Human Neutrophil Polymorphs

Mono(ADP-ribosyl)transferase activity has been detected on the external surface of human polymorphonuclear neutrophil leucocytes (PMNs). The corresponding cDNA has been cloned and shown to be identical to that derived from human skeletal muscle. Our results suggest that mono(ADP-ribosyl)transferase is involved in the transduction pathway mediating (i) receptor-dependent re-alignment of cytoskeletal actin and (ii) chemotaxis of PMNs.

Inhibition of chemotaxis in A7r5 rat smooth muscle cells by a novel panel of inhibitors.

1 Arginine‐specific ADP‐ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudo‐substrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF‐BB. 2 The IC50 values for nicotinamide (12 mM) and novobiocin (165 μM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 μM) and K1 (IC50=95 μM) were less potent than previously described in PMNs. The pseudo‐substrates for the enzyme (DEA‐BAG, agmatine and arginine‐methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3 A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4 Evidence for ART activity on the surface of A7r5 cells was investigated using 32P‐NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P‐AMP, and hydrolysis with NaOH yielded 32P‐NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP‐ribosylation of agmatine. 5 We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.