Isolation of Two Novel Metalloproteinase-Disintegrin (ADAM) cDNAs That Show Testis-Specific Gene Expression Douglas Pat Cerretti, 1 Robert F. DuBose, Roy A. Black, and Nicole Nelson Immunex Corporation, 51 University Street, Seattle, Washington 98101 Received August 10, 1999 Metalloproteinase-disintegrins (ADAMs) are type 1 transmembrane proteins that contain a unique do- main structure including a zinc-binding metallopro- teinase domain. We have isolated cDNAs encoding two novel members of this family, ADAM29 and ADAM30 which show testis-specific expression. Three forms of ADAM29 were found that encode proteins of 820, 786 and 767 amino acids. All of the amino acid differences are located in the cytoplasmic domain. Two forms of ADAM30 were isolated that encode proteins of 790 and 781 amino acids, with the difference in the coding region occurring in the cytoplasmic domain. ADAM29 and ADAM30 map to human chromosome 4q34 and 1p11-13, respectively. An ancestral analysis of all known mammalian ADAMs indicates that the zinc- binding motif in the catalytic domain arose once in a common ancestor and was subsequently lost by those members lacking this motif. © 1999 Academic Press Metalloproteinase-disintegrins are a family of zinc- binding metalloproteinases that were originally iso- lated from the venom of snakes (1, 2). They are closely related to the matrix metalloproteinases (3). The metalloproteinase-disintegrins or ADAMs (a disinte- grin and metalloproteinase) have a unique domain structure composed of a signal sequence, pro-domain with a Cys switch, catalytic domain with a zinc-binding motif, disintegrin domain, cysteine-rich domain, a transmembrane domain, and a cytoplasmic domain (4, 5). Thus, ADAMs are type 1 transmembrane proteins expressed on the cell surface. ADAMs have been iso- lated from mammalian species, Caenorhabditis, Xeno- pus, and Drosophila. Approximately half of the ADAMs do not contain the zinc-binding motif HEXXHX- XGXXHD which is required for enzymatic activity. However, all ADAMs contain the disintegrin domain, which is approximately 80 amino acids in length with 15 highly conserved Cys residues. In some members this region has been found to bind integrins (6 – 8), although the role of this domain for the majority of the family members is unknown. Over two dozen ADAMs have been identified but only a few have had their biological roles elucidated. Tumor necrosis factor- converting enzyme (TACE/ ADAM17) was isolated as the proteinase required for the shedding of TNF- from the plasma membrane (9, 10). More recently TACE/ADAM17 has been found to be required for the ectodomain shedding of other cell surface proteins including L-selectin and TGF- (11). Fertilin-/ADAM1 and fertilin-/ADAM2 are required for sperm-egg fusion (12) while meltrin-/ADAM12 has a role in muscle cell fusion (13). In addition MDC/ ADAM11 is a candidate tumor suppressor gene (14) and Kuz/ADAM10 plays an important role in neuro- genesis (15, 16). Some ADAMs are ubiquitously expressed such as ADAM9, ADAM10, ADAM15, and ADAM17 and may have pleiotropic effects, as has been found for ADAM15 and ADAM17. Many of the other ADAMs, however, show tissue-specific expression: ADAM12 and ADAM19 in muscle (13), ADAM22 in brain, and ADAM23 in brain and heart (17). The largest group of ADAMs (1, 2, 3, 4, 5, 6, 7, 18, 20, 21, 24, 25, 26, and 27) is predomi- nately expressed in testis and is thought to be involved in spermatogenesis and fertilization (18 –20). Indeed, the first mammalian ADAMs discovered, ADAM1 and ADAM2, were found to be required for sperm-egg fu- sion (21). In this report we describe the identification of two novel human ADAMs, called ADAM29 and ADAM30, that show testis-specific expression. Both proteins have all of the domains common to the ADAM family. However, ADAM29 has a variant amino acid in the zinc-binding domain and thus may be proteolytically inactive. Because of alternative RNA splicing, three forms of ADAM29 and two forms of ADAM30 were 1 To whom correspondence should be addressed. Fax: 1-206-233- 9733. E-mail: cerretti@immunex.com. Biochemical and Biophysical Research Communications 263, 810 – 815 (1999) Article ID bbrc.1999.1322, available online at http://www.idealibrary.com on 810 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.