Worm Breeder's Gazette 13(1): 51 (October 1, 1993)

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.

Isolation of CELPC2 ,a cDNA that encodes a putative K ex2 -likeprohormone convertase in C. elegans.

Eduardo G--mez-Salad'n, David L. Wilson, Ian M. Dickerson

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Department of Physiology & Biophysics
University of Miami School of Medicine, and Department of Biology, Univ. of Miami, Miami, Fl 33136.

A novel family of subtilisin-like serine endoproteases has been determined to be responsible for propeptide cleavage. The first such endoprotease discovered was K ex2 ,found in yeast. Several other members of this family have been isolated from both vertebrate and invertebrate organisms. The gene bli-4 has been reported to encode a protein related to K ex2 in C elegans (Peters and Rose, WBG 11:28, 1991). We initiated studies on the K ex2 -likeserine endoproteases of C. elegans by isolating total RNA from a mixed culture of N2 wild type worms. After poly-A selection, we performed degenerate/nested PCR, using primers based on conserved regions within the active sites of known vertebrate and invertebrate endoproteases, and isolated two distinct 300 bp PCR products that shared homologies with the active sites of known K ex2 -likeendoproteases. These two PCR products were used to screen a C. elegans cDNA library. Several cDNA clones were isolated and a 2 kb cDNA was selected for sequencing. This clone, designated CELPC2 ,contained most of the coding sequence for a K ex2 -likeendoprotease, but lacked approximately 500 bp of 5' coding sequence. The missing 5' region was obtained by rapid amplification of cDNA ends (RACE). The full length cDNA is approximately 2.5 kb in length. The deduced amino acid sequence for the CELPC2 cDNA was found to be very similar to the known K ex2 -likeendoproteases, especially at conserved regions within the active sites, but not identical to any one of them. For example, the amino acid sequence contains the HGTR CAGEI/V, VGV/IAY, and GI/VRML motifs characteristic of subtilisin endoproteases. Moreover, CELPC2 has several similarities to PC2 ,such as the Met (M) at the gap before position 245, the Thr (T) within the SWGPT motif at position 275, and Arg (R) within the VDGPR motif at position 285. Most notable is the sequence similarity between CELPC2 and mouse PC2 in positions 186-205 and 266-280. A search of protein databases revealed significant homologies of CELPC2 to known invertebrate and vertebrate PC2 . CELPC2 is 89% similar to Aplysia californica PC2 ,88% similar to Lymnaea stagnalis PC2 ,86% similar to Xenopus laevis PC2 and human PC2 ,and 85% similar to Sus scrofa PC2 .The similarity of CELPC2 to Drosophila melanogaster furin is only 50%. No perfect match was found for the CELPC2 amino acid sequence during this search. We are currently using the two 300 bp probes for northern blots and in situ hybridization. This work was supported in part by training grants HD07129 and NS07044 from the NIH and by grant 91II/6 from the AHA.

Figure 1