The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDWPAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatik characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His, tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 pM.Keywords: cathepsin L; propeptide; expression ; Paramecium tetraurelia.Proteases serve many distinct functions in the cellular metabolism. Intracellularly, most obvious roles are in degradation of ingested food and turnover of cellular proteins. This usually occurs in lysosomes or digestive vacuoles. Other functions exist in the developmental and metabolic control of specific proteins through activation or inactivation by limited proteolysis [ 11. Extracellularly, secreted proteases are used to access solid proteinacous food or to facilitate tissue invasion via collagenolytic and elastinolytic activities as reported for some pathogenic bacteria, parasitic protozoa, and ras-transformed tumor cells [2-41. In the unicellular ciliate Paramecium tetraurelia the formation and processing of digestive vacuoles and lysosomes has been investigated predominantly by electron microscopy [5]. In these studies, 16 hydrolases of presumed lysosomal localization have been superficially identified more than a decade ago. Among those were the proteolytic cathepsins B, C, and D [ 5 ] . Nothing is known about extracellular proteases. During attempts to identify and study protein phosphatases from P. tetraurelia, we noticed the presence of a highly active protease in cell homogenates that hydrolyzed the '*P-labelled phosphorylase a which was used as a substrate for the protein phosphatases, to smaller ['*P]phosphopeptides. These labelled peptides were not acid-precipitable and thus quenched the phosphatase assay. Because hitherto no detailed enzymatic study of any protease from this ciliate had been reported, we decided to examine the nature of this protease activity. After separation from protein phosphatase two prot...

Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin Llike subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K i of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure.z 2000 Federation of European Biochemical Societies.

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His 6 -tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a-and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu 1681 and Ser 1748 in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by G␣ s /GTP or forskolin, and P site inhibitors were only slightly effective.Adenylyl and guanylyl cyclases are key proteins in intracellular signaling of essentially all eukaryotic cells. In the freshwater protozoans Paramecium and Tetrahymena cAMP and cGMP levels depend on the ion composition of the extracellular milieu. Whereas adenylyl cyclase activity in vivo is stimulated by membrane hyperpolarization (1, 2), cGMP formation is enhanced by a depolarizing Ca 2ϩ inward current (3). Recently, we reported on a group of guanylyl cyclases of 280 kDa that is present in the ciliates Paramecium and Tetrahymena but also in the genome of the malaria parasite Plasmodium (4). This novel signal transduction protein is bifunctional. It has a 155-kDa N-terminal P-type ATPase-like domain and a 115-kDa C-terminal guanylyl cyclase domain. Both are linked by a cytosolic loop of about 110 amino acids (see Fig. 1A). Surprisingly, the guanylyl cyclase domain is topologically identical to mammalian adenylyl cyclases, i.e. it is composed of two pseudosymmetric modules of six putative transmembrane spans (M1 and M2) that serve as membrane anchors for the cytosolic, catalytic segments C1 and C2 (4). The sequences of both cytosolic subdomains are similar to each other and resemble the catalytic regions of metazoan adenylyl and, to a lesser extent, guanylyl cyclases. Sequence comparisons show that the order of the protozoan C1a-and C2-positioned loops is reversed compared with the corresponding mammalian adenylyl cyclase loops (4). Data based on the crystal structure of a soluble adenylyl cyclase type VC1a/type IIC2 chimera demonstrated that Lys 938 and Asp 1018 in the catalytic pocket of the C2 domain (adenylyl cyclase type II numbering) are responsible for ATP substrate specificity (5, 6). In the Paramecium guanylyl cyclase, those two amino acids have evolved as Glu 1681 ...

SummaryThe adenylyl cyclase Rv1625c from Mycobacterium tuberculosis codes for a protein with six transmembrane spans and a catalytic domain, i.e. it corresponds to one half of the pseudoheterodimeric mammalian adenylyl cyclases (ACs). Rv1625c is active as a homodimer. We investigated the role of the Rv1625c membrane domain and demonstrate that it efficiently dimerizes the protein resulting in a 7.5-fold drop in K m for ATP. Next, we generated a duplicated Rv1625c AC dimer by a head-to-tail concatenation. This produced an AC with a domain order exactly as the mammalian pseudoheterodimers. It displayed positive cooperativity and a 60% increase of v max compared with the Rv1625c monomer. Further, we probed the compatibility of mycobacterial and mammalian membrane domains. The second membrane anchor in the Rv1625c concatamer was replaced with membrane domain I or II of rabbit type V AC. The mycobacterial and either mammalian membrane domains are compatible with each other and both recombinant proteins are active. A M. tuberculosis Rv1625c knockout strain was assayed in a mouse infection model. In vitro growth characteristics and in vivo organ infection and mortality were unaltered in the knockout strain indicating that AC Rv1625c alone is not a virulence factor.

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