Identification and Characterization of a Pro-tumor Necrosis Factor-α-processing Enzyme from the ADAM Family of Zinc Metalloproteases

Abstract

Tumor necrosis factor-␣ (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 Ala 76 and Val 77 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. Tumor necrosis factor-␣ (TNF)1 is a cytokine that is produced primarily by activated monocytes and macrophages in response to a variety of physiological stresses such as infection or injury (1). Clinical and experimental evidence has also identified TNF as a mediator of chronic autoimmune diseases such as rheumatoid arthritis (2) and Crohn's disease (3), as well as being involved in the pathology associated with sepsis (1). Accordingly, TNF has become a primary target for therapeutic intervention of several inflammatory diseases.TNF is initially synthesized as a 26-kDa membrane-bound protein (pro-TNF) that is subsequently cleaved to release soluble 17-kDa TNF with an NH 2 terminus of Val 77 (4). The identity of the protease(s) responsible for TNF processing remains controversial. Robache-Gallea et al. (5) detected a serine protease activity (PR3) in monocyte membrane preparations which was able to generate a 17-kDa active TNF with an NH 2 terminus of Arg 78 . In 1994, the partial isolation and characterization of a membrane-bound activity capable of generating the 17-kDa form of TNF were reported (6). The TNF-processing enzyme was thought to be a non-matrix metalloprotease since it was not inhibited by TIMP-1,2 or phosphoramidon, and no calcium requirement was detected. More recently, two members of the ADAM family (TNF-␣ converting enzyme (TACE) and bovine ADAM 10 (BoAD10)) have been shown to possess pro-TNF processing activity (7-9).In this report we describe the isolation, cloning, and characterization of a TNF-processing enzyme from the human monocytic cell line THP-1. The purified recombinant enzyme, rHuAD10, specifically recognizes the authentic cleavage site in pro-TNF and is sensitive to metalloprotease inhibitors that block soluble TNF production (6). MATERIALS AND METHODSReagents-Dinitrophenol-labeled polypeptides were synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl-based solid phase peptide chemistry method using an Applied Biosystems, Inc. 431A peptide synthesizer (10). All peptides were purified by reversed phase HPLC, and molecular weights were verified by mass spectrometry.HPLC Peptide Assay-TNF processing activity was measured as the ability to cleave a 12-residue peptide spanning the Ala 76 -Val 77 site in pro-TNF. The chromatopho...

Expression of a disintegrin and metalloprotease (ADAM and ADAMTS) enzymes in human non-small-cell lung carcinomas (NSCLC)

“…Indeed, ADAMs and ADAMTS belong to the adamalysin family, related to snake venom proteases, among which many members are implicated in different loops of reciprocal interactions with some mediators of inflammation such as TNF-a (Rosendahl et al, 1997;Moss et al, 2001) and growth factors including TGF-a and -b (Hinkle et al, 2003;Le Pabic et al, 2003).…”

Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

“…Of the 17 ADAMs that have a catalytic site consensus sequence, to date four have been shown to have catalytic activity [7][8][9][10][11][12][13][14]. Little is known about the activity or functions of the other 13 ADAMs with a catalytic site consensus sequence, except that seven of them appear to be expressed predominantly in the testis, suggesting a role in sperm function [4,[15][16][17][18][19][20][21][22][23].…”

“…Little is known about the activity or functions of the other 13 ADAMs with a catalytic site consensus sequence, except that seven of them appear to be expressed predominantly in the testis, suggesting a role in sperm function [4,[15][16][17][18][19][20][21][22][23]. ADAMs harbouring a catalytic site that are known to be expressed in somatic cells include MDC9 [24], ADAM10\KUZ [8,9,25], ADAM8\ MS2 [26], ADAM12\meltrin α [27], MDC15 [28][29][30], ADAM17\ TACE (tumour necrosis factor converting enzyme) [10,11] and ADAM19\meltrin β [31,32]. Here we report the cDNA cloning and initial biochemical characterization of the mouse protein ADAM28, a catalytic-site-containing ADAM that is expressed in at least two somatic tissues, mouse epididymis and lung.…”

The Increase in TNF-α Levels Is Implicated in NF-κB Activation and Inducible Nitric Oxide Synthase Expression in Brain Cortex after Immobilization Stress

“…This cytokine is released in its soluble form from its membrane-bound precursor by a membraneanchored zinc metalloproteinase, identified as a disintegrin and metalloproteinase (ADAM) called TNF-␣ convertase (TACE/ADAM17) (Gearing et al 1994;Black et al 1997;Moss et al 1997;Rosendahl et al 1997). TNF-␣ exerts some of its effects through the activation of a proinflammatory transcription factor, nuclear factor B (NF-B) (rev.…”