The Metalloprotease Disintegrin ADAM8

Abstract

ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu 330 to Gln exchange (EQ-A8) in the Zn 2؉ binding motif (HE 330 LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substratebound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC 50 ϳ50 nM) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion. ADAM1 (a disintegrin and metalloprotease domain) proteins constitute a family of transmembrane glycoproteins and serve essential physiological roles in fertilization, myogenesis, and neurogenesis. These functions are due to distinct protein domains involved in cell-cell fusion, cell-cell interaction, or proteolysis of membrane proteins, a process termed ectodomain shedding (1). To date, the family of ADAM proteinases comprises more than 30 members in different species (2, 3), and 24 ADAM genes were found in the mouse genome. Fourteen of the murine ADAMs contain the catalytic consensus sequence HEXXHXXGXXHD in their metalloprotease domains and are therefore predicted to be proteolytically active (4). The cleavage of myelin basic protein (MBP) by ADAM10/MADM was the first demonstration of proteolysis by ADAMs (5). The tumor necrosis factor-␣ convertase (ADAM17) was purified on the basis of its ability to cleave tumor necrosis factor-␣ (6, 7) and a number of other peptide and protein substrates in vitro (1, 8). Proteolysis of membrane-bound surface molecules was also demonstrated for heparin-binding epidermal growth factor (9) and amyloid precursor protein (10,11), which are cleaved by ADAMs 9 and 10, respectively.Catalytically active ADAMs are usually activated by furincatalyzed removal of the prodomain or by other proprotein convertases. For cleavage by furin-like...

Catalytic Activity of Human ADAM33

“…For example, ADAM10 was inhibited by TIMP-1 and TIMP-3 but not TIMP-2 (35); ADAM12 (27) was sensitive to TIMP-3 but not TIMP-1 and -2. In contrast, ADAM9 was not inhibited by TIMP-1, -2, and -3 (7), whereas ADAM8 was not inhibited by any of the four TIMPs (6). Mouse ADAM17 catalytic domain was reported to be inhibited by human TIMP-3 but not by human TIMP-1 and -2 and mouse TIMP-4 (34).…”

ADAM10, the Rate-limiting Protease of Regulated Intramembrane Proteolysis of Notch and Other Proteins, Is Processed by ADAMS-9, ADAMS-15, and the γ-Secretase

“…A VP16-Gal4 sequence (50) was subcloned into mADAM10 cDNA after introduction of an HpaI restriction site in the ADAM10 C terminus via site-directed mutagenesis (Stratagene) at positions G745V,H746N. A mADAM10 construct lacking the ectodomain (containing a signal peptide sequence (amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] joined to amino acids 669 -749) was FLAG-tagged (CTTGTCATCGTCGTC-CTTGTAGTC) before the stop codon at the C terminus. The PCR product was ligated into a pcDNA3.1 vector (ADAM10⌬E-flag).…”

“…A second group (ADAMs 2,7,11,18,22,23,and 29) is characterized by an inactive protease domain, and they seem to be mainly important for cell adhesion and fusion. A large third group of ADAMs displays a broad expression profile and has demonstrated (ADAMs 8,9,10,12,17,19, and 28) or predicted (ADAMs 15,20,21,30, and 33) proteolytic activity. These proteases play a major role in the ectodomain shedding of proteins involved in paracrine signaling, cell adhesion, and intracellular signaling (reviewed in Refs.…”

Structure of human ADAM-8 catalytic domain complexed with batimastat

“…The activation mechanism is autolytic and was recently determined in vitro using recombinant proteins to yield a final product consisting of only the catalytic domain . Inhibitors such as batimastat can bind the pro-form of the enzyme to inhibit all steps of this process (Schlomann et al, 2002). Thus, a selective inhibitor of ADAM-8 could have potentially far-reaching effects beyond those related to proteolysis.…”

“…The specific ions used in refinement were chosen after thorough evaluation of biochemical evidence, anomalous scattering, coordination geometry, bond distances and electron density (Primakoff & Myles, 2000;Schlomann et al, 2002;Becherer & Blobel, 2003).…”