Worm Breeder's Gazette 13(2): 81 (February 1, 1994)
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.
Matrix-degrading proteases participate in normal development and differentiation of most, if not all, multicellular organisms. We have been looking for such proteases in C elegans, using gelatin and casein zymograms (1993 worm meeting, page 145). We are interested in identifying both proteases involved in restructuring of basement membranes and those involved in modification or molting of larval cuticles. The cuticle of C. elegans could provide a model system for investigating the restructuring/modification of an extracellular matrix by proteolysis. In addition, understanding of the molting process in C. elegans could be applicable to attempts to understand similar processes in parasitic nematodes, where a therapeutic application is desired. We have identified some activities on gelatin and casein zymograms that are potential extracellular matrix proteases We have analyzed samples from mixed populations, isolated eggs and from time courses on synchronized L1 's,using salt extraction of french pressed worms/eggs. Two types of reproducible activity have been observed one in late stage embryos, ~50 kDa, and one whose appearance correlates to molting worms, with two major species, ~90 and ~70 kDa. Both types of activity appear to have some affinity for zinc-chelated iminodiacetic acid columns, suggesting they may be metalloproteinases. The 50 kDa activity is reproducibly obtained from egg preps. It is active on both casein and gelatin zymograms. The amount of activity appears to be correlated to the number of eggs in later stages. Analysis of this activity is complicated by the fact that it seems to be proteolytically activated in the course of sample preparation. If protease inhibitors are added during sample preparation (E-64 PMSF, and Pepstatin A for sulfhydryl, serine, and acid proteases, respectively) to a duplicate sample no activity is obtained. However addition of these inhibitors to an already active sample does not inhibit its activity. Also, when samples of the activity are concentrated on zinc-chelate resin, a "ladder" of fainter higher molecular weight bands can sometimes be seen, supporting the notion that the 50 kDa band results from proteolysis of a higher molecular weight precursor. When time courses of synchronized L1 'sare analyzed on casein zymograms, a number of bands of activity are observed that correlate to the L1 /L2molt, peaking 12-13 hours after food addition to the starved L1 's.It is not yet clear which bands are distinct species, some appear to be proteolytic fragments of higher molecular weight precursors. At least two appear to be distinct in that one (~70 kDa) appears to be temporally offset, by about one hour later, from the other (~90 kDa) band. All of the bands are similar in that they show little, if any, activity on gelatin zymograms. All the bands appear to be metalloproteases, as they are inhibited by 1, 10 phenanthroline, EDTA and DTT, but not by PMSF, E-64 or Pepstatin A. They may be analogous to molting enzymes identified in parasitic nematodes (S. Lustigman, 1993, Parasitology Today, vol. 9, no. 8, 294-297). We are currently determining the feasibility of using a biochemical approach to obtain peptide sequences for the activities