Worm Breeder's Gazette 8(3): 58
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We had previously observed that thiol-dependent, leupeptin-sensitive proteases make a significant contribution to proteolysis by crude extracts of C. elegans at pH 4.5-5.0 (approximating the intralysosomal pH). We have now resolved two distinct thiol proteases, which we designate cathepsin L1 and cathepsin L2, by chromatography on DEAE-Sephadex. The properties of cathepsin L2 from C. elegans correspond quite closely to those of vertebrate cathepsin L with respect to substrate specificity, pH optimum and molecular weight. Both proteases are optimally active toward Z-phe-arg-7-amino-4-methyl coumarin amide (MCA) at pH 5. Both are strictly thiol-dependent and leupeptin-sensitive, and neither is inhibited by pepstatin, bestatin or PMSF. Neither shows any activity toward Z-arg-arg-MCA, a substrate for vertebrate lysosomal cathepsin B, nor towards succinyl-(ala)(ala)( ala)-MCA, a substrate for the elastase-like protease of C. elegans. Cathepsins L1 and L2 are clearly distinct enzymes, since L1 is inhibited by soybean trypsin inhibitor (STI), whereas L2 is not. L2 digests denatured FITC-casein, whereas L1 does not. Cathepsin L2 preparations show a single major band on SDS gels with M=25,000, as detected after labeling the enzyme with [125I]. L2 acitivity emerges from a gel filtration column at a position corresponding to 24 kDa, so it appears likely that cathepsin L2 is active as a monomer. We are presently developing screening procedures for isolating mutants deficient in cathepsin L2. to contain several additional MSP genes. Thus in a 50Kb region there are at least five MSP genes. Two of them correspond to one of our cDNA sequences and two others correspond to one of Michael Klass's cDNA sequences so at least four of these genes must be expressed. If we can learn where these genes are in the genome it might be possible to eliminate them with a small deletion and so discover what happens to sperm with insufficient MSP. We are currently sequencing the genomic MSP genes to see if there are conserved regions outside the coding sequence. These might be either regulatory sites or the remnants of residual translocatable elements which could have participated in dispersing these genes throughout the genome.