The presence of several catalytic domains embedded in the same polypeptide chain is an unusual molecular feature, whose evolutionary or functional advantages are still unclear . However, recent works have provided evidence that a number of proteins from different organisms exhibit these complex architectures. Among these multidomain enzymes there are luciferases, xylanases, chitinases, endoglucanases, kinases, and diverse hydrolases including proteases [2-9]. The occurrence of human proteases with different catalytic domains in the same translation product was first reported for two metalloproteinases: angiotensin-converting enzyme (ACE) and carboxypeptidase D [10,11]. ACE is a type-I membrane-bound metalloproteinase with two enzymatically active domains which shows specific catalytic constants and interact differently with several competitive inhibitors . Carboxypeptidase D is also a type-I membrane metalloproteinase that contains three catalytic domains, two of them being catalytically active and showing optimal activities at different pHs . More recently, and as part of our studies on mammalian degradomes – the entire protease complement of these organisms [14-17] –, we have identified and characterized a cDNA encoding an unusual mosaic protease called polyserase-1  (polyserine protease-1). This protein shows a complex domain organization including a type-II transmembrane motif, a low-density lipoprotein receptor A module, and three tandem serine protease domains. Interestingly, analysis of post-translational processing mechanisms of polyserase-1 revealed that it is synthesized as a membrane-bound protein which undergoes a series of proteolytic processing events to generate three independent serine protease domains . Further studies of the human degradome have revealed the occurrence of another gene coding for a protein with three tandem serine protease domains in a single polypeptide chain . This protein – called polyserase-2 – is an extracellular glycosylated enzyme, whose three serine protease domains are not proteolytically cleaved and remain as an integral part of the same polypeptide chain. Enzymatic analysis of polyserase-2 has demonstrated that only its first protease domain is catalytically active, whereas the second and third domains are inactive due to the lack of critical residues present in the catalytic triad of serine proteases .
The finding of two polyserine proteases in the human genome, together with the presence of similar mosaic structures in serine proteases from other organisms such as Xenopus laevis, Bufo japonicus, Drosophila melanogaster and Caenorhabditis elegans [18-21], prompted us to explore the possibility that additional yet uncharacterized polyserases could be produced by human tissues. In this work, we report the finding of a novel human protein, tentatively called polyserase-3, which contains two serine protease domains in its amino acid sequence. We describe the molecular cloning of a full-length cDNA for this protein, and perform a structural and enzymatic analysis of this new protease. We also examine the expression pattern of polyserase-3 in human tissues and tumor cell lines, and perform a comparative analysis between this enzyme and the previously described polyserases-1 and -2 in terms of structural design, phylogenetic relationships, cellular location and post-translational maturation mechanisms. On the basis of the obtained results, we conclude that polyserase-3 is more closely related to polyserase-2 than to polyserase-1, but also exhibits a series of characteristic features unique for this novel polyprotein among all other polyserine proteases described to date.