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 cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, 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. Enzymatic 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 His6 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 microM.
Völkel
H
H
Fakultät für Chemie und Pharmazie, Universität Tübingen, Germany.
Kurz
U
U
Linder
J
J
Klumpp
S
S
Gnau
V
V
Jung
G
G
Schultz
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000201:enzymology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016805:Paramecium tetraurelia / Q000737:chemistry / Q000201:enzymology*
D003470:Culture Media; D004792:Enzyme Precursors; D007527:Isoenzymes; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L