Abstract
The ADAM family of disintegrin metalloproteases plays important roles in "ectodomain shedding," the process by which biologically active, soluble forms of cytokines, growth factors, and their receptors are released from membrane-bound precursors. Whereas ADAM8, ADAM15, and MDC-L (ADAM28) are expressed in specific cell types and tissues, their in vivo functions and substrates are not known. By screening a library of synthetic peptides as potential substrates, we show that soluble recombinant forms of these enzymes have similar proteolytic substrate specificity, clearly distinct from that of ADAM17 (TNFalpha-converting enzyme). A number of tumor necrosis factor (TNF) family proteins and CD23 were screened as potential substrates for ectodomain cleavage. We found that ADAM8, ADAM15, and MDC-L, but not ADAM17, catalyzed ectodomain shedding of CD23, the low affinity IgE receptor. ADAM8-dependent, soluble CD23 release required proteolytically active ADAM8, and a physical association of ADAM8 was observed with the membrane-bound form of CD23. The ADAM8-dependent release of sCD23 and the endogenous release from B cell lines could be similarly inhibited by a hydroxamic acid, metalloprotease inhibitor compound. We conclude that ADAM8 could contribute to ectodomain shedding of CD23 and may thus be a potential target for therapeutic intervention in allergy and inflammation.
Highlights
EXPERIMENTAL PROCEDURESPCR and cDNA Cloning—The pro-domain and protease domain of human ADAM 8 were amplified by PCR with Pfu polymerase and the primers 5A8, 5Ј-GGAATCCGCCATGCGCGGCCTCGGGCTC-3Ј, and 3FLAGA8, 5Ј-CGGGATCCTCTAGACTACCCCTTGTCATCGTCGTCCTTGTAGTCCCCGGCGAGGCACACCGACTGCGGCCGCTCCAAAAAGCTCTC-3Ј
The disintegrin metalloprotease1 family of cell surface and secreted proteolytic enzymes is known to play roles in sperm-egg binding and fusion, muscle cell fusion, neurogenesis, modulation of Notch receptor and ligand processing, and processing of the pro-inflammatory cytokine, tumor necrosis factor (TNF)␣
By screening a library of synthetic peptides as potential substrates, we show that soluble recombinant forms of these enzymes have similar proteolytic substrate specificity, clearly distinct from that of ADAM17 (TNF␣-converting enzyme)
Summary
PCR and cDNA Cloning—The pro-domain and protease domain of human ADAM 8 were amplified by PCR with Pfu polymerase and the primers 5A8, 5Ј-GGAATCCGCCATGCGCGGCCTCGGGCTC-3Ј, and 3FLAGA8, 5Ј-CGGGATCCTCTAGACTACCCCTTGTCATCGTCGTCCTTGTAGTCCCCGGCGAGGCACACCGACTGCGGCCGCTCCAAAAAGCTCTC-3Ј. Expression and Purification of Recombinant Soluble Human ADAM Proteins from the Medium of Sf9 Cells—Recombinant baculovirus for expression of the prodomain and protease domain of ADAM8 was generated from the pFastBac construct using the Bac-to-Bac system (Invitrogen). The peptides comprised the following: (i) a collection of substrates for other proteases and (ii) a number of sequences corresponding to membrane proximal cleavage sites of various proteins postulated to be released by metalloproteases (including those published by Roghani et al [21] for ADAM9/MDC9). The media, cell lysates, or immunoprecipitates were subjected to SDS-PAGE (5–15% acrylamide) and transferred to nitrocellulose, followed by immunoblotting with the antiADAM8 polyclonal antiserum or anti-V5 antibody (for proteins expressed from Genestorms clones), secondary antibodies conjugated to horseradish peroxidase, and fluorography using ECL substrate (Amersham Biosciences). RPMI8866 cells, JY cells, or HEK293 cells transfected with ADAM8 and CD23 were incubated overnight at 37 °C, in the absence or presence of 0.4 –12.5 M MMP Inhibitor II (Calbiochem), after which the supernatant was analyzed for the presence of soluble CD23 by enzyme-linked immunosorbent assay (Pharmingen)
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