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Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil
L E Donnelly1, N B Rendell, S Murray
1Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, U.K.
Abstract:
An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino-(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1-10 mM) did not yield ADP-ribosyl-DEA-BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD+ by an NAD+ glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The Km of the enzyme for NAD+ was 100.1 30.4 microM and the Vmax 1.4 0.2 pmol of ADP-ribosylagmatine/h per 10(6) cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98% decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [32P]NAD+. Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5'-AMP.
Insights
Human neutrophils possess surface mono(ADP-ribosyl)transferase activity, confirmed using a novel acceptor (DEA-BAG). This enzyme, likely GPI-anchored, modifies cell surface proteins, offering insights into neutrophil signaling pathways.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Human polymorphonuclear neutrophil leucocytes (PMNs) possess enzymatic activities on their surface.
- Mono(ADP-ribosyl)transferase enzymes play roles in cellular signaling and DNA repair.
- Understanding PMN surface enzymes is crucial for immune response research.
Purpose of the Study:
- To confirm and characterize Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human PMNs.
- To identify the substrate specificity and kinetic properties of the identified enzyme.
- To investigate the enzyme's localization and potential anchoring mechanism on the cell surface.
Main Methods:
- Utilized diethylamino-(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor to detect enzyme activity.
- Employed High-Performance Liquid Chromatography (HPLC) and electrospray mass spectrometry for product identification.
- Assessed enzyme kinetics using agmatine as a substrate and investigated cell surface anchoring with phosphoinositol-specific phospholipase C (PI-PLC).
Main Results:
- Confirmed the presence of Arg-specific mono(ADP-ribosyl)transferase activity on human PMN surfaces.
- ADP-ribosyl-DEA-BAG was synthesized in the presence of PMNs, distinct from non-enzymatic reactions.
- Enzyme kinetics revealed a Km for NAD+ of 100.1 ± 30.4 μM and Vmax of 1.4 ± 0.2 pmol/h/10^6 cells.
- PI-PLC treatment reduced enzyme activity by 98%, indicating a glycosylphosphatidylinositol (GPI) anchor.
- Identified cell surface proteins (79, 67, 46, 36, 26 kDa) labeled with [32P]NAD+ as mono(ADP-ribosyl)ated.
Conclusions:
- Human PMNs exhibit surface mono(ADP-ribosyl)transferase activity, likely mediated by a GPI-anchored enzyme.
- This activity modifies specific cell surface proteins, suggesting a role in neutrophil function.
- The findings provide a basis for further investigation into the biological significance of PMN surface ADP-ribosylation.