The Low Affinity IgE Receptor (CD23) Is Cleaved by the Metalloproteinase ADAM10

George A Lemieux,Fernando Blumenkron,Nolan Yeung,Pei Zhou,Jason Williams,Amrie C Grammer,Robert Petrovich,Peter E Lipsky,Marcia L Moss,Zena Werb

doi:10.1074/jbc.m608414200

Abstract

The low affinity IgE receptor, FcepsilonRII (CD23), is both a positive and negative regulator of IgE synthesis. The proteinase activity that converts the membrane-bound form of CD23 into a soluble species (sCD23) is an important regulator of the function of CD23 and may be an important therapeutic target for the control of allergy and inflammation. We have characterized the catalytic activity of ADAM (a disintegrin and metalloproteinase) 10 toward human CD23. We found that ADAM10 efficiently catalyzes the cleavage of peptides derived from two distinct cleavage sites in the CD23 backbone. Tissue inhibitors of metalloproteinases and a specific prodomain-based inhibitor of ADAM10 perturb the release of endogenously produced CD23 from human leukemia cell lines as well as primary cultures of human B-cells. Expression of a mutant metalloproteinase-deficient construct of ADAM10 partially inhibited the production of sCD23. Similarly, small inhibitory RNA knockdown of ADAM10 partially inhibited CD23 release and resulted in the accumulation of the membrane-bound form of CD23 on the cells. ADAM10 contributes to CD23 shedding and thus could be considered a potential therapeutic target for the treatment of allergic disease.

Highlights

The low affinity IgE receptor Fc⑀RII (CD23) is a 46-kDa type II membrane protein that is expressed on B-cells and cells of the myeloid lineage [1]
The specificity constant of 250 MϪ1 sϪ1 indicated that the efficiency of the cleavage reaction was comparable to that of DNP-EVHHGKLVTFFAE (350 MϪ1 sϪ1), a peptide derived from the ␣-secretase cleavage site of amyloid precursor protein (APP), which is a known ADAM10 substrate [25]
The peptide corresponding to the 33-kDa cleavage site sequence of CD23, DNPRAEQQRLKSQDL, was cleaved in two places by rhADAM10 having specificity constants of 190 and 90 MϪ1 sϪ1

Summary

EXPERIMENTAL PROCEDURES

Reagents—Commercial reagents were obtained from the following sources: Recombinant human IL-4 and recombinant human catalytic/disintegrin domains of ADAMs 8, 10, and 17 were from R&D Systems (Minneapolis, MN). The resulting metalloproteinase domain-deleted (amino acids 228 –397) ADAM8 (ADAM8⌬MP) construct was directionally recombined into a modified pQCXIP (Clontech, Mountain View, CA) that contained a Gateway acceptor cassette (Invitrogen) followed by a 3Ј FLAG epitope tag. Cells were washed with cold PBS and lysed in radioimmune precipitation assay buffer (140 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% glycerol, 2 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, and Complete proteinase inhibitor mixture). Radioimmune precipitation assay buffer lysates and concentrated conditioned media were separated by SDS-PAGE (12% acrylamide gels) and transferred onto PVDF membrane for Western blotting using a monoclonal anti-HA antibody (HA., Covance); this was followed by secondary detection using a goat anti-mouse IgG-horseradish peroxidase conjugate and chemiluminescence detection using the ECL substrate (Amersham Biosciences). DNP-LPQLENVKGTED DNP-SPLAQAVRSSSR a Nonphysiological cleavage. b TNF receptor I

90 Ϯ 10a 250 Ϯ 100

RESULTS

DISCUSSION