Worm Breeder's Gazette 7(2): 70

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The Cathepsin D of C. elegans

L. Jacobson, L. Jen-Jacobson, M. Bolanowski, M. Shah

We are interested in the biochemical pathways of intracellular 
protein turnover and in the possible roles of proteolytic processes in 
development.  We have been concentrating on the enzyme Cathepsin D 
because preliminary experiments with crude extracts of C.  elegans 
showed that proteolysis at acid pH was >95% inhibited by pepstatin, a 
known inhibitor of carboxyl proteases.
We have purified the wild-type enzyme in a single step by affinity 
chromatography on pepstatin-agarose.  We can obtain about 5mg of 
'pure' enzyme from 15gm of N2 in one afternoon.  The purified enzyme 
shows 6-7 bands (MW = 32-42 Kdal) on SDS gels.  We believe, on the 
basis of digestion studies with exo- and endo-glycosidases, that 
Cathepsin D is a complex (sialylated) glycoprotein and that this 
multiplicity of bands represents heterogeneity of the oligosaccharide 
sidechains.  It is not yet clear whether this heterogeneity exists in 
vivo or represents partial degradation during purification.  Gel 
filtration of crude extracts indicates that the enzyme is a monomer.  
The proteolytic specificity of the enzyme is similar, but not 
identical, to that of Cathepsin D from bovine spleen.
We have isolated one mutant deficient in Cathepsin D, and have (
unofficially) named the gene cad-1.  Mutant homozygotes have 10-20% of 
wildtype enzyme levels.  The cad-1 gene has been assigned to the right 
extremity of LG II, 33% from sqt-2, 22% from dpy-10 and 23% from unc-4.
Other markers on the right extremity of LG II have been useless for 
reasons of shared phenotypes, so more precise mapping remains to be 
The cad-1 gene appears to be a structural gene on two grounds: 1) 
The enzyme level in cad-1/+ heterozygotes is precisely the mean of the 
levels in mutant and wild-type homozygotes.  2) The pure enzyme from 
the cad-1 mutant has one-third the activity per molecule of the wild-
type enzyme.  A search for electrophoretic differences is in progress.
Our cad-1 mutant has an 'odd' appearance of the intestine, looking 
less dark than wild-type under the dissecting microscope, and having 
highly vacuolated intestinal cells in the EM.  It has a reasonably 
high tendency to egg retention and internal hatching (frequency 
similar to a lin-7; nzyme level) and lays 
only unfertiIized eggs at 25 C.  Reproduction is not rescued by N2 
maIes at 25 C.  Conversely, cad-1 males preincubated for 24 hr.  at 25 
C remain fertile.  Growth of the mutant at 25 C is quite poor, even 
though the in vitro activity of its Cathepsin D shows a temperature 
coefficient (Q10 = 1.9 in the range 16 C to 37 C) no different from 
that of wild-type.  It is not yet totally clear that the ts properties 
are attributable directly to the cad-1 mutation.