Patrick Nusbaum

The Cleavage of Neutrophil Leukosialin (CD43) by Cathepsin G Releases Its Extracellular Domain and Triggers Its Intramembrane Proteolysis by Presenilin/γ-Secretase

The highly negatively charged membrane sialoglycoprotein leukosialin, CD43, is shed during neutrophil activation. This is generally thought to enhance cell adhesion. We here describe two novel consequences of this shedding, during neutrophil activation by phorbol esters or by chemoattractants after TNF-α priming. CD43 proteolysis was investigated by Western blotting, using a polyclonal antibody to CD43 intracellular domain. Our data emphasize the importance of a juxtamembranous cleavage of about 50% of membrane CD43 molecules by cathepsin G. Indeed, it is inhibited by α1-antichymotrypsin and cathepsin G inhibitor I and is reproduced by exogenous purified cathepsin G. The resulting membrane-anchored C-terminal fragment, CD43-CTF, becomes susceptible to presenilin/γ-secretase, which releases CD43 intracytoplasmic domain: preincubation with three different γ-secretase inhibitors, before PMN treatment by agonists or by purified cathepsin G, results in the accumulation of CD43-CTF. Because CD43 binds E-selectin, we also investigated the effect of the soluble extracellular domain CD43s, released by cathepsin G juxtamembranous cleavage, on neutrophil adhesion to endothelial cells. A recombinant CD43s-Fc fusion protein inhibited neutrophil E selectindependent adhesion to endothelial cells under flow conditions, while it had no effect on neutrophil static adhesion. We thus propose that, in addition to its potential pro-adhesive role, CD43 proteolysis results in: (i) the release, by cathepsin G, of CD43 extracellular domain, able to inhibit the adhesion of flowing neutrophils on endothelial cells and thus to participate to the natural control of inflammation; (ii) the release and/or the clearance, by presenilin/γ-secretase, of CD43 intracellular domain, thereby regulating CD43-mediated signaling.

Characterization of a recombinant proteinase 3, the autoantigen in Wegener's granulomatosis and its reactivity with anti-neutrophil cytoplasmic autoantibodies

Using the baculovirus/insect cells system, we have expressed a recombinant proteinase 3 (PR3) — the neutrophil‐derived serine protease autoantigen in Wegeners granulomatosis — as a glycosylated intracellular and membrane‐associated protein. Ollgosaccharides accounted for the difference in molecular weights between recombinant (34 kDa) and neutrophil‐PR3 (29 kDa). Whereas rabbit‐anti‐PR3 IgG recognized both recombinant and neutrophil‐derived PR3, autoantibodies from Wegener patient sera recognized only neutrophil‐derived PR3. Although oligosaccharides were not involved in PR3 epitope recognition, autoantibodies did not recognize the amino acid primary structure of recombinant PR3. Improper disulfide bond formation and/or lack of post‐translational events in insect cells, may affect the conformation of PR3, precluding its reactivity with sera from WG patients.