Identification and Characterization of a Pro-tumor Necrosis Factor-α-processing Enzyme from the ADAM Family of Zinc Metalloproteases*
- Mary S. Rosendahl‡,
- S. Christine Ko,
- Dana L. Long,
- Michael T. Brewer,
- Bradley Rosenzweig,
- Erik Hedl,
- Lana Anderson,
- Suzanne M. Pyle,
- Julie Moreland,
- Mark A. Meyers,
- Tadahiko Kohno,
- David Lyons and
- Henri S. Lichenstein
Abstract
Tumor necrosis factor-α (TNF) is initially expressed as a 26-kDa membrane-bound precusor protein (pro-TNF) that is shed proteolytically from the cell surface, releasing soluble 17-kDa TNF. We have identified human ADAM 10 (HuAD10) from THP-1 membrane extracts as a metalloprotease that specifically clips a peptide substrate spanning the authentic cleavage site between Ala76 and Val77 in pro-TNF. To confirm that HuAD10 has TNF processing activity, we cloned, expressed, and purified an active, truncated form of HuAD10. Characterization of recombinant HuAD10 (rHuAD10) suggests that this enzyme has many of the properties (i.e. substrate specificity, metalloprotease activity, cellular location) expected for a physiologically relevant TNF-processing enzyme.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF009615.
The first two authors contributed equally to this work.
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↵‡ To whom correspondence should be addressed: Amgen Inc., 3200 Walnut St., Boulder, CO 80301. Tel.: 303-401-1855; Fax: 303-938-6241; E-mail: maryr{at}amgen.com.
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↵1 The abbreviations used are: TNF, tumor necrosis factor α; ADAM, a disintegrin andmetalloprotease; BoAD10, bovine ADAM 10; bp, base pair(s); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DNP, dinitrophenol; HA, hydroxyapatite; HPLC, high performance liquid chromatography; HuAD10, human ADAM 10; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; rHuAD10, recombinant human ADAM 10; TACE, TNF-α convertingenzyme; TIMP, tissue inhibitor of metalloprotease; TNFR, tumor necrosis factor receptor.
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↵2 Amgen cDNA data base, unpublished results.
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- Received March 17, 1997.
- Revision received June 4, 1997.
