Worm Breeder's Gazette 9(3): 119

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.

Cathepsins Ce1, Ce2 and Ce3 in C. elegans

G.J. Sarkis, J.D. Ashcom and L.A. Jacobson

Two acid thiol proteases previously designated as cathepsins L 1 and 
L2 [Kurpiewski et al., Gazette Vol. 8, No. 3, 1984] have now been 
renamed cathepsins Ce1 and Ce2, since further studies have shown that 
they are not precisely analogous to the mammalian cathepsin L. Both 
proteases prefer Z-phe-arg-aminomethylcoumarin (AMC) to Z-arg-arg-AMC, 
and in this respect resemble mammalian cathepsin L rather than 
cathepsins B or H. On the other hand, both Ce1 and Ce2 show 
carboxydipeptidase activity toward the model substrate leu-trp-met-arg-
phe-ala, and in this respect resemble cathepsin B rather than 
cathepsin L. Like the mammalian thiol proteases, both Ce1 and Ce2 are 
inhibited by EP-64, leupeptin and antipain. Both are insensitive to 
the trypsin inhibitors from soybean and pancreas, to alpha- 1 -
antitrypsin and to pepstatin. Three lines of evidence indicate that 
Ce1 and Ce2 are distinct gene products. First, Ce1 has much more 
endoprotease activity (using FITC-casein as substrate) than Ce2, 
relative to the activity of each against Z-phe-arg-AMC. Second, 
isoelectric focusing in nondenaturing slab gels shows that Ce1 has a 
pI of 6.8, whereas Ce2 has a pI of 4.7. This seems too large a 
difference to attribute to variant post-translational processing. 
Finally, the age-dependences of Ce1 and Ce2 are different (Sarkis et 
al., this issue). We are still working on the development of 
differential screens for mutants deficient in each of these enzymes. A 
third protease has also been separated during purification of Ce1 and 
Ce2 on DEAE-cellulose. We call this enzyme cathepsin Ce3. It has 
strong endoproteolytic activity toward FITC-casein at pH 5.5, but no 
activity toward Z-phe-arg-AMC, Z-arg-arg-AMC, arg-AMC, N-succinyl-(ala)
3-AMC, glutaryl-phe-AMC, or any other model substrate we have tried. 
It does not hydrolyze the model hexapeptide leu-trp-met-arg-phe-ala. 
Unlike Ce1 and Ce2, it does not require a thiol for activity, but is 
inhibited by millimolar concentrations of DTT. It is insensitive to 
leupeptin, EP-64, pepstatin, antipain, STI and PTI. Its pI is about 7.
4. The major protein band in our Ce3 preparations reacts covalently 
with alpha-2-macroglobulin, a general protease-trapping inhibitor. 
This suggests that the major band is cathepsin Ce3. We have looked at 
crude extracts -- which have been depleted of cathepsin D by affinity 
chromatography -- by IEF and solid-phase casein zymography. The 
predominant activities are in the positions of cathepsins Ce 1 and Ce3,
with a minor band at the position of cathepsin Ce2. Thus, most of the 
endoprotease activity at acid pH is attributable to cathepsins D, Ce1 
and Ce3. For those of you whose interest in proteases is limited to 
protecting your favorite proteins or enzymes during isolation or assay,
we recommend a mixture of pepstatin (an inhibitor of cathepsin D) and 
EP-64. The former is a queer pentapeptide; the latter is an active-
site directed inhibitor based upon an active epoxide group. Neither is 
likely to interfere with your pet enzyme or protein. Both are 
available from Sigma. We suggest concentrations of about 10  M.