CATHEPSIN L IS AN INTRACELLULAR AND EXTRACELLULAR PROTEASE IN PARAMECIUM-TETRAURELIA - PURIFICATION, CLONING, SEQUENCING AND SPECIFIC-INHIBITION BY ITS EXPRESSED PROPEPTIDE

The ciliate Paramecium tetraurelia secretes large amounts of a cystein e protease into the growth medium, presumably for extracellular food d igestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respec tively), both present in cell homogenates and in spent growth medium w ere purified to homogeneity. Peptide sequence analysis revealed that t hese isozymes share identities at the amino acid level but are probabl y differently processed. Enzymatic characterization of the isolated pr oteases and sequencing of the cloned cDNA demonstrated that the enzyme s belong to the cathepsin-L protease subfamily, Although the identity with mammalian and other protozoan L cathepsins was only around 30%, a ll important signature sequences for cathepsin L in the preproregion a s 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 characte ristic conserved ERFNIN motif, was fused with a His(6) tag, expressed in Escherichia call, and purified. Both cathepsin L isozymes of Parame cium tetraurelia were inhibited by their cognate propeptide in the nan omolar concentration range. All other cysteine proteases tested (papai n and mammalian cathepsin B, G and H) were unaffected by the propeptid e up to 10 mu M.