Elsevier

Journal of Neuroimmunology

Volume 72, Issue 2, February 1997, Pages 127-129
Journal of Neuroimmunology

Structural features and biochemical properties of TNF-α converting enzyme (TACE)

https://doi.org/10.1016/S0165-5728(96)00180-4Get rights and content

Abstract

Tumor necrosis factor-α is a potent cytokine, secreted primarily by activated monocytes and macrophages, that possesses a broad range of immunomodulating properties. Involvement of this cytokine has been validated in disease states such as arthritis and Crohn's disease and implicated in diverse neuroimmunological pathologies such as multiple sclerosis, Alzheimers and stroke. TNF-α is initially synthesized as a 26 kDa precursor molecule that is subsequently processed to the mature form by cleavage of the Ala76–Val77 bond. The 17 kDa carboxy-terminal protein is then secreted to function in a paracrine manner. The enzyme that processes precursor TNF-α has previously been identified as a microsomal metalloprotease called TNF-α converting enzyme (TACE). We have now purified and partially cloned the enzyme. TACE represents a novel target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases.

Introduction

Two years ago, we discovered that a hydroxamic acid, GW 9471 (Fig. 1), inhibited TNF secretion from a monocytic cell line, THP-1(McGeehan et al., 1994). The inhibitor is a British Biotech compound that was previously demonstrated to be a broad spectrum inhibitor of the matrix metalloproteases (MMPs) (Campion et al., 1990). The MMPs are a family of enzymes that can be divided into three domains: pro, catalytic, and hemopexin (Fig. 2). Membrane bound MMPs (MT-MMPs), have a transmembrane region in addition to the above domains. The pro region contains a cysteine switch, a feature that maintains the enzyme in an inactive state. The pro domain can be autocatalytically removed to generate an active MMP. The catalytic domain contains a standard zinc ligand binding motif and the hemopexin domain is important for recognition of natural substrates of the enzymes such as collagen and aggrecan.

The MMPs are involved in normal processes such as tissue remodeling, wound repair and endometrial cycling (Talhouk et al., 1992; Jeffrey, 1991; Salamonsen, 1994). However, the MMPs are associated with disease states as well, such as cancer metastasis, multiple sclerosis, arthritis and restenosis (Stetler-Stevenson et al., 1993; Gijbels et al., 1994; Matrisian, 1990; Galis et al., 1995).

TNF-α has also been implicated in diverse pathologies such as arthritis and multiple sclerosis. For example, Centecor has demonstrated that in clinical trials, antibody treatment of patients with rheumatoid arthritis results in improvement in several clinical scores such as joint pain and tenderness and grip strength (Elliot et al., 1994).

In addition, neutralizing antibodies to TNF-α inhibit the development of experimental auto-immune encephalomyelitis (EAE) in rodent models (Selmaj et al., 1991). Other neuroimmunological conditions where the presence of TNF-α is well documented and may play a role in the etiology of the diseases are Alzheimers and stroke (Cochran and Vitek, 1996; Reiss et al., 1993).

Section snippets

Identification of TNF-α converting enzyme as a metalloprotease

Unlike the substrates for the MMPs, TNF-α exists as a membrane bound precursor form that is processed to yield a 17 kDa secreted form (Fig. 3). Processing of precursor TNF-α is required for TNF secretion (Perez et al., 1990; Utsumi et al., 1993). Therefore, we thought that the target of GW 9471, was the processing metalloprotease for precursor TNF-α (TNF-α converting enzyme or TACE). Attempts at identifying a convertase activity were initiated using a macrophage like cell line called Mono-Mac

Purification of TACE

Purification of TACE was accomplished from Mono-Mac 6 cell membrane fractions but there was not enough protein for sequence analysis. Therefore, we switched to a tissue source for purification of the enzyme. Pig spleen was chosen because the cleavage sequence of porcine is similar to the human cleavage sequence and spleen contains a variety of immune competent cell types. Purification was accomplished by the scheme described in Fig. 4. Briefly, a particulate fraction was washed with 0.05% NP-40

Complementary cDNA of TACE

Sequence analysis was performed on the Lys-C digested enzyme. A 41 amino acid residue was identified. Data base searches indicated that the sequence was unique. Therefore, the porcine enzyme was partially cloned from a cDNA library made from spleen mRNA. Results from the cloning indicated the presence of a zinc ligand binding motif. Therefore we had cloned a metalloprotease. Unlike the matrix metalloproteases, there was a cytoplasmic tail that followed the transmembrane region and a cysteine

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1

Present address: Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305, USA.

2

Present address: RPR, 600 Arcola Rd., Collegville, PA 19426, USA.

3

Present address: Dow Corning Corporation, Midland, MI 48686-0994, USA.

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