Role of Glutamic Acid 988 of Human Poly-ADP-ribose Polymerase in Polymer Formation: EVIDENCE FOR ACTIVE SITE SIMILARITIES TO THE ADP-RIBOSYLATING TOXINS

Gerald T Marsischky,Brenda A Wilson,R.John Collier

doi:10.1074/jbc.270.7.3247

Abstract

Sequence similarities between the enzymatic region of poly-ADP-ribose polymerase and the corresponding region of mono-ADP-ribosylating bacterial toxins suggest similarities in active site structure and catalytic mechanism. Glu988 of the human polymerase aligns with the catalytic glutamic acid of the toxins, and replacement of this residue with Gln, Asp, or Ala caused major reductions in synthesis of enzyme-linked poly-ADP-ribose. Replacement of any of 3 other nearby Glu residues had little effect. The Glu988 mutations produced similar changes in activity in the carboxyl-terminal 40-kDa catalytic fragment fused to maltose-binding protein: E988Q and E988A reduced polymer elongation > 2000-fold, and E988D approximately 20-fold. Smaller changes were seen in chain initiation. The mutations had little effect on the Km of NAD, indicating a predominantly catalytic function for Glu988. The results support the concept of similar active sites of the polymerase and the ADP-ribosylating toxins. Glu988 may function in polymer elongation similarly to the toxins' active site glutamate, as a general base to activate the attacking nucleophile (in the case of the polymerase, the 2'-OH of the terminal adenosine group of a nascent poly-ADP-ribose chain).

Highlights

Systemic sclerosis (SSc), a rheumatic disease characterised by autoimmunity, tissue fibrosis and vasculopathy, has a high mortality rate compared with other rheumatic diseases.[1 2]
What is already known about this subject? ►► Capturing the complexity and heterogeneity of systemic sclerosis in clinical trials is difficult. ►► The widely used modified Rodnan skin score failed in recent clinical trials as a surrogate parameter for universal disease progression
A patient was considered to have organ worsening if he or she fulfilled any of the following criteria within 12±3 months of the baseline visit: new-onset renal crisis; decrease in forced vital capacity (FVC)≥10%; new left ventricular ejection fraction (LVEF)10% for patients with baseline LVEF

Summary

Introduction

Systemic sclerosis (SSc), a rheumatic disease characterised by autoimmunity, tissue fibrosis and vasculopathy, has a high mortality rate compared with other rheumatic diseases.[1 2] Mortality in SSc is the result of organ involvement, with lung disease (either interstitial lung disease or pulmonary arterial hypertension (PAH)) being the most prominent risk factor for death.[3] Skin fibrosis is a hallmark of SSc and is measurable in a standardised manner using the modified Rodnan skin score (mRSS), which has good inter-rater and intra rater variability.[4,5,6,7] The mRSS correlates with Key messages. ►► The widely used modified Rodnan skin score failed in recent clinical trials as a surrogate parameter for universal disease progression. What does this study add? ►► We have identified factors that are associated with disease progression and lead to organ failure

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