Abstract
BackgroundWe have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity.ResultsFollowing our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the α-chain of fibrinogen as well as pro-urokinase-type plasminogen activator (pro-uPA). Northern blot analysis showed that polyserase-3 exhibits a unique expression pattern among human polyserases, being predominantly detected in testis, liver, heart and ovary, as well as in several tumor cell lines.ConclusionThese findings contribute to define the growing group of human polyserine proteases composed at present by three different proteins. All of them share a complex structural design with several catalytic units in a single polypeptide but also show specific features in terms of enzymatic properties, expression patterns and post-translational maturation mechanisms.
Highlights
We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain
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 [9]
Identification and molecular cloning of a new human polyserine protease We used the polyserase-2 cDNA as a query and the BLAST algorithm to search regions in the human genome that could encode new proteases with several serine protease domains within the same polypeptide chain. This search allowed us to identify a region in chromosome 16p11.2 containing two serine protease domains closely linked. Conceptual translation of these domains showed that they were different to the serine protease domains of polyserase-2 and prostasin, which are serine protease genes located at the same chromosomal region [3,22]
Summary
We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. As part of our studies on mammalian degradomes – the entire protease complement of these organisms [14,15,16,17] –, we have identified and characterized a cDNA encoding an unusual mosaic protease called polyserase-1 [9] (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. 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 [3] 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 [3]
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