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J. Biol. Chem., Vol. 280, Issue 34, 30367-30375, August 26, 2005

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Identification and Characterization of Human Archaemetzincin-1 and -2, Two Novel Members of a Family of Metalloproteases Widely Distributed in Archaea^*

Araceli Díaz-Perales, Víctor Quesada, Juan R. Peinado, Alejandro P. Ugalde, Jesús Álvarez, María F. Suárez, F. Xavier Gomis-Rüth¶, and Carlos López-Otín||

From the Departamentos de Bioquímica y Biología Molecular y Biología Celular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo, Spain and the ^¶Institut de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Cientiíficas, 08034 Barcelona, Spain

Systematic analysis of degradomes, the complete protease repertoires of organisms, has demonstrated the large and growing complexity of proteolytic systems operating in all cells and tissues. We report here the identification of two new human metalloproteases that have been called archaemetzincin-1 (AMZ1) and archaemetzincin-2 (AMZ2) to emphasize their close relationship to putative proteases predicted by bioinformatic analysis of archaeal genomes. Both human proteins contain a catalytic domain with a core motif (HEXXHXXGX₃CX₄CXMX₁₇CXXC) that includes an archetypal zinc-binding site, the methionine residue characteristic of metzincins, and four conserved cysteine residues that are not present at the equivalent positions of other human metalloproteases. Analysis of genome sequence databases revealed that AMZs are widely distributed in Archaea and vertebrates and contribute to the defining of a new metalloprotease family that has been called archaemetzincin. However, AMZ-like sequences are absent in a number of model organisms from bacteria to nematodes. Phylogenetic analysis showed that these enzymes have undergone a complex evolutionary process involving a series of lateral gene transfer, gene loss, and genetic duplication events that have shaped this novel family of metalloproteases. Northern blot analysis showed that AMZ1 and AMZ2 exhibit distinct expression patterns in human tissues. AMZ1 is mainly detected in liver and heart whereas AMZ2 is predominantly expressed in testis and heart, although both are also detectable at lower levels in other tissues. Both human enzymes were produced in Escherichia coli, and the purified recombinant proteins hydrolyzed synthetic substrates and bioactive peptides, demonstrating that they are functional proteases. Finally, these activities were abolished by inhibitors of metalloproteases, providing further evidence that AMZs belong to this catalytic class of proteolytic enzymes.

Received for publication, April 26, 2005 , and in revised form, June 13, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ635357, AJ635358, AJ879912, AJ879913, AJ879914, and AJ879915.

^* This work was supported by grants from Ministerio de Ciencia y Tecnología-Spain, Fundación La Caixa, the European Union (FP5 and FP6-Cancer Degradome), and the Daiichi Fine Chemical Company, Limited. (Toyama, Japan). The Instituto Universitario de Oncología is supported by Obra Social Cajastur-Asturias and Red de Centros de Cáncer-Instituto Carlos III of Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 34-985-104201; Fax: 34-985-103564; E-mail: clo{at}uniovi.es.

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