Expression and Enzymatic Activity of Human Disintegrin and Metalloproteinase ADAM19/Meltrin Beta Ping Wei,* Yun-Ge Zhao,† Li Zhuang,* Steve Ruben,* and Qing-Xiang Amy Sang† ,1 *Human Genome Sciences, Inc., Rockville, Maryland 20850; and †Department of Chemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306-4390 Received December 5, 2000 The adamalysins are involved in proteolysis, adhe- sion, fusion, and intracellular signaling. Human ADAM19/adamalysin-19 (A disintegrin and metallo- proteinase 19) was identified from primary dendritic cell cDNA libraries. It has a signal sequence, a pro- domain with a “cysteine-switch” residue, a metallo- proteinase domain with a zinc-binding site, a disinte- grin, a cysteine-rich domain, an epidermal-growth- factor-like domain, a transmembrane domain, and a cytoplasmic domain with putative SH3 ligand binding sites. Its mRNA was expressed in the placenta, heart, bladder, lymph nodes, and leukocytes, colorectal ade- nocarcinoma SW 480, and other organs/cells. The hADAM19 recombinant protein was expressed in hu- man cells. It formed a complex with and cleaved alpha-2 macroglobulin (2-M). Its proteolytic activity was blocked by 1,10-phenanthroline, EDTA, EGTA, and a synthetic matrix metalloproteinase (MMP) in- hibitor and not by the tissue inhibitors of metallopro- teinases TIMP-1 and TIMP-2. It did not cleave the MMP substrates tested, e.g., type I collagen and gela- tin, casein, and four peptide substrates. Thus, hADAM19 is an active metalloproteinase and may have a specific substrate profile. © 2001 Academic Press Key Words: adamalysin; metzincin; metalloproteinase/ disintegrin/cysteine-rich (MDC); active enzyme; type 1a transmembrane protein; gene expression; metallopro- teinase inhibitors; alpha-2 macroglobulin. Adisintegrin and metalloproteinase (ADAM) is a member of a large family of metalloproteinases that have all or some of the following domain structures: a signal peptide, a propeptide domain, a metalloprotein- ase domain, a disintegrin domain, a cysteine-rich do- main, an epidermal growth factor (EGF)-like domain, a transmembrane region, and a cytoplasmic tail (1– 4). Some ADAMs have a putative fusion peptide in their cysteine-rich domain (1), signaling motifs including Src-homologous 3 (SH3) ligand sequences in the cyto- plasmic domain (5–7), or a thrombospondin motif in the C-terminal domain (2, 8). More than 30 genes en- coding the proteins of the ADAM family have been identified in different species (3, 9), but the biological functions of most members are still unclear. Their do- main structures suggest that they may have many potential functions during physiological and patholog- ical processes, including proteolysis, adhesion, fusion, and intracellular signaling. The proteins of the ADAM family are widely distributed in many organs, tissues, and cells, including the testes, epididymis, placenta, ovary, breast, skeletal muscle, heart, liver, kidney, co- lon, thymus, spleen, sperm, monocytes, macrophages and leukocytes (10, 11). The proteins have been impli- cated in a variety of important processes, such as sperm-egg and muscle cell binding and fusion, degra- dation of type IV collagen, shedding of tumor necrosis factor  (TNF-), myogenesis, osteogenesis, spermato- genesis, cleavage of aggrecan, tumor cell adhesion, an- giogenesis inhibition, and signal transduction (1–5, 11–13). Mouse ADAM19 (mouse meltrin ) was cloned and the gene product was characterized by Inoue et al. (14). Supported in part by grants from National Institutes of Health CA78646, Elsa U. Pardee Foundation, and Gustavus and Louise Pfeiffer Research Foundation (to Q.-X.A.S.). The nucleotide sequence(s) reported in this paper has been sub- mitted to the GenBank/EBI Data Bank with Accession No. AF311317. Abbreviations used: ADAM, Adisintegrin and metalloproteinase; hADAM19, human adisintegrin and metalloproteinase 19; 2-M, alpha-2-macroglobulin; Brij-35, polyoxyethylene lauryl ether; Dnp, 2,4-dinitrophenyl; Dpa, N-3-(2,4-dinitrophenyl)-L-2, 3-diamino- propionyl; ECM, extracellular matrix; EDTA, ethylenediamine- tetraacetic acid; EGTA, ethyleneglycol-bis-(-amino-ethyl ether) N,N'-tetra-acetic acid; EST, expressed sequence tag; HEPES, n-2- hydroxyethylpiperazine-N'-2-ethane sulfonate; IL-2 (-6), inter- leukin-2 (-6); MDC, metalloproteinase/disintegrin/cysteine-rich; MMPs, matrix metalloproteinases; PAGE, polyacrylamide gel elec- trophoresis; SH3, Src-homologous 3; TACE, tumor necrosis factor- converting enzyme; TBS, Tris buffered saline; TIMP, tissue inhibitor of metalloproteinase; TNF-, tumor necrosis factor-. 1 To whom correspondence should be addressed. Fax: 850-644- 8281. E-mail: sang@chem.fsu.edu. Web page: http://www.chem.fsu. edu/editors/sang/sang.htm. Biochemical and Biophysical Research Communications 280, 744 –755 (2001) doi:10.1006/bbrc.2000.4200, available online at http://www.idealibrary.com on 744 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.