Elsevier

Gene

Volume 206, Issue 2, 12 January 1998, Pages 273-282
Gene

ADAM 20 and 21; two novel human testis-specific membrane metalloproteases with similarity to fertilin-α

https://doi.org/10.1016/S0378-1119(97)00597-0Get rights and content

Abstract

Two novel membrane disintegrin–metalloproteases, ADAM 20 and ADAM 21 were cloned from a human testis cDNA library. Their predicted translation products share 50% sequence identity with each other. Among previously characterized ADAMs, the best similarity was to sperm cell-specific fertilins-α and -β, and meltrin-γ (ADAM 9) which is ubiquitously expressed. Both ADAM 20 and 21 mRNAs are exclusively expressed in testis, presumably, in analogy to all other testis-specific ADAMs, on mature spermatocytes. Both cDNAs were mapped on the genome, and found to be tightly linked to the same marker (SHGC-36001) on chromosome 14q24.1. This region is not syntenic with the loci of mouse sperm-specific ADAMs 1–5. ADAM 20, but not 21, encodes a consensus Zn2+ binding site of active adamalysin metzincin metalloproteases, and both 20 and 21 encode putative cell-fusion peptides, required for sperm–egg fusion. Based on these characteristics it is possible that ADAM 20 and/or 21 is the functional equivalent of sperm fertilin-α, as it was recently reported that this gene is non-functional in humans.

Introduction

Recently a new family of proteins, called ADAMs, was identified whose members are type I integral membrane proteins characterized by a disintegrin and metalloprotease domain (see Blobel, 1997; Wolfsberg et al., 1995a, Wolfsberg et al., 1995b; Huovila et al., 1996for recent reviews). The ADAMs are related to the crotalid snake venom disintegrin metalloproteases (SVMPs) and matrix metalloprotease (MMP) families, whose members lack transmembrane domains. Full-length ADAM cDNAs all encode a signal peptide followed by proprotein, Zn2+-metalloprotease, disintegrin, transmembrane region and cytoplasmic tail. Members of this family have been found in many animal species including mammals, Xenopus (Alfandari et al., 1997), Drosophila (Fambrough et al., 1996; Rooke et al., 1996) and nematodes (Podbilewicz, 1996; GenBank H89394), but none has, as yet, been found in unicellular eukaryotes or plants. Nor are ADAM genes found in the sequenced genomes of bacteria or S. cerevisiae.

In spite of the ADAM's conservation of structural domains, they appear to play rather diverse roles in development.

ADAM 12 (meltrin-α) was shown to be involved in myoblast, and perhaps also osteoclast fusion (Yagami-Hiromasa et al., 1995).

Mammalian ADAM 10 (MADAM) can degrade myelin basic protein (Howard et al., 1996). Its insect homologue Kuzbanian is involved in axonal extension in Drosophila (Fambrough et al., 1996; Rooke et al., 1996). The proteolytic substrate for Kuzbanian, and perhaps also for mammalian ADAM 10 is the Notch receptor, which regulates neuron proliferation (see Blobel, 1997for a recent discussion and references).

The ADAM 11 encoding gene is rearranged in primary breast tumours (Emi et al., 1993).

Another ADAM, TACE (TNF-α convertase), is required for cleavage of TNF-α from its membrane-bound precursor (Black et al., 1997; Moss et al., 1997a, Moss et al., 1997b).

ADAM 1 and -2, also named fertilin-α/β or PH30-α/β are expressed as heterodimers on the posterior head of mammalian spermatocytes and play a role in oocyte adhesion and fusion (Wolfsberg et al., 1993; Myles et al., 1994; Carroll et al., 1995; Wolfsberg and White, 1996; Myles and Primakoff, 1997). In mouse, it has been shown that fertilin β binds the α6/β1 integrin on oocytes, and that this interaction is essential for sperm–egg binding (Almeida et al., 1995). This subunit encodes a non-functional metalloprotease domain, which is absent from the mature protein (Blobel et al., 1990). The α subunit encodes an active protease domain and encodes a putative fusion peptide postulated to be involved in cell–cell fusion (Blobel et al., 1992; White, 1992; Muga et al., 1994). Like fertilin β, fertilin α may also be directly involved in sperm–egg adhesion (Evans et al., 1997). Fertilins α and β have been found in rat, rabbit, mouse, macaque and two species of guinea pig. In humans only the β subunit has been cloned (Gupta et al., 1996; Vidaeus et al., 1997). Surprisingly, the only human fertilin α gene is non-functional (Jury et al., 1997). This leads to the question as to what gene(s), then, are involved in human sperm–egg fusion, since fertilin β lacks potential fusion peptides. The fertilins are of considerable interest because of their essential role in fertilization and application as contraceptive vaccine (Herr, 1996) or drug target. It has been shown that vaccination with fertilin results in complete infertility in a male animal model (Ramarao et al., 1996).

Here we report the cDNA cloning of two novel, closely related human ADAM members which are expressed exclusively in testis. Their encoded products show good sequence similarity and share other characteristics with fertilin-α from other species; it is possible that they functionally replace fertilin-α.

Section snippets

RT–PCR

OligodT-primed, ds cDNA was prepared (Marathon kit, ClonTech, Palo Alto, CA) from polyA-RNA isolated from tonsillar B-cells which had been purified by rosetting on sheep red blood cells. Ten nanograms of DNA was used as template in a PCR containing 30 mM Tricine (pH 8.4), 2 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mg/ml gelatin, 0.1% Thesit (Ponce and Micol, 1992), 0.2 mM of each of the four dNTPs (freshly prepared from buffered 100 mM solution; Pharmacia, Uppsala, Sweden), 0.3 pmol/μl of each

cDNA cloning of ADAM 20 and -21

The original aim of this study was to identify novel ADAMs that might be involved in shedding of adhesion molecules and cytokines, such as L-selectin, ICAM-3 and TNF-α. As there is evidence, and one concrete example (TACE; Black et al., 1997; Moss et al., 1997a) that disintegrin–metalloproteases, or ADAMs are involved in these processes, degenerate oligonucleotides were designed that encode the Zn2+-binding pocket (HEXGHNFG) consensus and conserved disintegrin domain (GEECDXG). An RT–PCR on

Discussion

We describe here the cDNA cloning of two novel, closely related members of the ADAM family of membrane metalloproteases, ADAM 20 and ADAM 21. Comparison of their predicted translation products with known ADAMs showed they are related to fertilin α, fertilin β and meltrin-γ (ADAM 1, 2 and 9). Meltrin-γ mRNA is ubiquitously expressed (Yagami-Hiromasa et al., 1995) and not particularly up-regulated in testis (data not shown). ADAM 20 and ADAM 21's exclusive expression in human testis and sequence

Acknowledgements

I thank Dr J.-F. Gauchat for B-cell cDNA and Dr J.-Y. Bonneyfoy for initiating this work and stimulating discussions. The ADAM 20 and ADAM 21 sequences have been desposited with GenBank under accession numbers AF029899 and AF029900.

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