Caeruloplasmin as a Specific Inhibitor of Myeloperoxidase

Abstract

Myeloperoxidase (MPO) released from activated neutrophils has been demonstrated to localise to the glomerular basement membrane, probably by a charge mediated process, and may play a major role in the development of vasculitic injury by catalysis of the production of hypochlorous acid and hence reactive oxygen species, known to be potently cytotoxic. The pathways for the metabolism and degradation of MPO are therefore of central importance in understanding the mechanisms of cellular injury. The passage of sera from both patients with anti-MPO antibodies and normal controls over columns bearing cyanogen bromide activated sepharose beads results in co-purification of both anti-MPO antibodies and caeruloplasmin (CP), identified by N terminal sequencing of the eluted protein. CP has known antioxidant activity and binds specifically to native and recombinant MPO in both ELISA and Westem blotting, and complexation has been demonstrated by gel filtration. The binding of CP to MPO in an ELISA is inhibited by preincubation with MPO but not by preincubation with other cationic neutrophil enzymes. The presence of CP partially inhibits the enzymatic activity of MPO in a dose dependent manner as measured by oxidation of tetramethylbenzidine, with maximal inhibition of 49.2 -+ 3.2 % seen following incubation of MPO at a concentration of IOug/ml with CP at 5Oug/ml. Further inhibition of activity may be limited by the susceptibility of CP itself to oxidative damage by hydrogen peroxide. The affinity of the MPO-CP interaction has been characterised using a biosensor (Pharmacia BIAlite ") with the association rate constant (ka) measured as 2.78 X lo3 ?r. 587 and dissociation rate constant (kd) as 2.15 X l o 3 2 1.2 X 10".These values suggest a low affinity interaction compared to that between MPO and several mouse monoclonal antibodies (MoAbs) and human affinity purified antibodies (ka l.lOX lo5 -1.85 X lo6 ; kd 1.5 X 10" -9.2 X 10" ). Affinity ranking has been confirmed by ammonium thiocyanate (ATC) disruption ELISA with 50% inhibition of binding between MPO and CP by 0.66 2 0.02 M ATC,' whereas 50% inhibition of binding between MPO and a mouse MoAb required 0.96 2 0.2M ATC (~0.03). The specific nature of the interaction between CP and MPO may indicate a novel function in the regulation of MPO activity which may be deranged and allow the development of endothelial cell damage in systemic vasculitis. Epidemiological data suggests that Crohn's disease (CD) and ulcerative colitis (UC) share some susceptibility loci, but diffcr at others. Recent nome-wide searches have focussed interest on the location of such susce t%li loci.(l 2) Hugot et al reported linka e of a 40 cM region of Chrlc?to C# we ha& now studied tlus area in a & dataset DNA frdm 186 affected sibling pairs (CD=81; UC=M; mixed=4l) was am lified by PCR, using fluoresence labelled primers for microsatellites witiin the 40 CM reeion of Chr 16. The uroducts were woled. __..__._. ...-._ -.~ electrophoresed on 6%%z-~limid; elsi and g e h o t F d using th'e semi: automated GEN...