Summary
1. A cDNA that encodes a Kex2-like prohormone convertase (PC) containing an active site similar to that of mammalian PC2 has been isolated fromC. elegans. Total RNA was isolated from a mixed population of strain BA713 worms. After poly-(A)-selection and reverse transcription, degenerate/nested polymerase chain reactions (PCR) were performed using primers based on conserved regions within the active sites of the known vertebrate and invertebrate endoproteases.
2. Two distinct 300-bp PCR products that shared homologies with the active sites of known Kex2-like endoproteases were isolated. These two PCR products were used to screen aC. elegans cDNA library.
3. The complete cDNA for a Kex2-like endoprotease, designated CELPC2, was isolated and determined to be 2527 bp in length. This size was confirmed by northern analysis. The deduced amino acid sequence for the CELPC2 cDNA is very similar to the known Kex2-like endoproteases, especially at conserved regions within the active sites, but not identical to any one of them. The strongest structural homology was to vertebrate and invertebrate PC2 sequences.
4.In situ hybridization suggests that CELPC2 is synthesized primarily in cells associated with the circumpharyngeal nerve ring and the dorsorectal ganglion.
Similar content being viewed by others
References
Bloomquist, B. T., Eipper, B. A., and Mains, R. E. (1991). Prohormone-converting enzymes: Regulation and evaluation of function using antisense RNA.Mol. Endocrinol. 5:2014–2024.
Braks, J. A. M., Guldemond, K. C. W., van Riel, M. C. H. M., Coenen, A. J. M., and Martens, G. J. M. (1992). Structure and expression ofXenopus prohormone convertase PC2.FEBS Lett. 305:45–50.
Bresnahan, P. A., Leduc, R., Thomas, L., Thorner, J., Gibson, H. L., Brake, A. J., Barr, P. J., and Thomas, G. (1990). Humanfur gene encodes a yeast KEX2-like endoprotease that cleaves pro-β-NGF in vivo.J. Cell Biol. 111:2851–2859.
Burglin, T. R., and Li, C. (1990). Update on the FMRFamide-like peptides inC. elegans.Worm Breeder's Gazette 11:43.
Chan, S. J., Oliva, A. A., LaMendola, J., Grens, A., Bode, H., and Steiner, D. F. (1992). Conservation of the prohormone convertase gene family in metazoa: Analysis of cDNAs encoding a PC3-like protein from hydra.Proc. Natl. Acad. Sci. USA 89:6678–6682.
del Castillo, J., de Mello, W. C., and Morales, T. (1964). Inhibitory action of γ-aminobutyric acid (GABA) onAscaris muscle.Experientia 20:141–143.
Dickerson, I. M., and Mains, R. E. (1990). Cell-type specific postranslational processing of peptides by different pituitary cell lines.Endocrinology 127:133–140.
Dickerson, I. M., and Noël, G. (1991). Tissue-specific peptide processing. InPeptide Biosynthesis and Processing (L. D. Fricker, Ed.), CRC Press, Boca Raton, FL, pp. 71–109.
Gómez-Saladín, E., Wilson, D., and Dickerson, I. M. (1993). Isolation of CELPC2, a cDNA that encodes a putative Kex2-like prohormone convertase inC. elegans.Worm Breeder's Gazette 13:51.
Hatsuzawa, K., Hosaka, M., Nakagawa, T., Nagase, M., Shoda, A., Murakami, K., and Nakayama, K. (1990). Structure and expression of mouse furin, a yeast Kex2-related protease.J. Biol. Chem. 265:22075–22078.
Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada K., Murakami, K., and Nakayama, K. (1992). Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells.J. Biol. Chem. 267:16094–16099.
Hodgkin, J. (1983). Male phenotypes and mating efficiency inCaenorhabditis elegans.Genetics 103:43–64.
Horvitz, H. R., Chalfie, M., Trent, C., Sulston, J., and Evans, P. D. (1983). Serotonin and octopamine in the nematodeCaenorhabditis elegans.Science 216:1012–1014.
Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984). Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-α-factor.Cell 37:1075–1089.
Keifer, M. C., Tucker, J. E., Joh, R., Landsberg, K. E., Saltman, D., and Barr, P. J. (1991). Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome 15.DNA Cell Biol. 10:757–769.
Korner, J., Chun, J., O'Bryan, L., and Axel, R. (1991). Prohormone processing inXenopus oocytes: Characterization of cleavage signals and cleavage enzymes.Proc. Natl. Acad. Sci. 88:11393–11397.
Krause, M., and Hirsh, D. (1987). Atrans-spliced leader sequence on Actin mRNA inC. elegans.Cell 49:753–761.
Lewis, J. A., Wu, C. H., Levine, J. H., and Berg, H. (1980). Levamisole-resistant mutants of the nematodeCaenorhabditis elegans appear to lack pharmacological acetylcholine receptors.Neuroscience 5: 967–989.
Li, C., and Chalfie, M. (1987). FRMFamide-like immunoreactivity inC. elegans.Worm Breeder's Gazette 9:110.
Lusson, J., Vieau, D., Hamelin, J., Day, R., Chrétien, M., and Seidah, N. G. (1993). cDNA structure of mouse and rat subtilisin/kexin-like PC5: A candidate proprotein convertase expressed in endocrine and nonendocrine cells.Proc. Natl. Acad. Sci. USA 90:6691–6695.
Mains, R. E., Dickerson, I. M., May, V., Stoffers, D. A., Perkins, S. N., Ouafik, L., Husten, E. J., and Eipper, B. A. (1990). Cellular and molecular aspects of peptide hormone biosynthesis.Front. Neuroendocr. 11:52–89.
Misumi, Y., Sohoda, M., and Ikehara, Y. (1990). Sequence of the cDNA encoding rat furin, a possible propeptide-processing protease.Nucleic Acids Res. 18:6719–6719.
Nakagawa, T., Hosaka, M., Torii, S., Watanabe, T., Murakami, K., and Nakayama, K. (1993). Identification and functional expression of a new member of the mammalian Kex2-like processing endoprotease family: its striking structural similarity to PACE4.J. Biochem. 113:132–135.
Nakayama, K., Kim, W. S., Torii, S., Hosaka, M., Nakagawa, T., Ikemizu, J., Baba, T., and Murakami, K. (1992). Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease.J. Biol. Chem. 267:5897–5900.
Orci, L., Ravazzola, M., Storch, M. J., Anderson, R. G. W., Vasalli, J. D., and Perrelet, A. (1987). Proteolytic maturation of insulin is a post golgi event which occurs in acidifying clathrin-coated secretory vesicles.Cell 49:865–868.
Ouimet, T., Mammarbachi, A., Cloutier, T., Seidah, N. G., and Castellucci, V. F. (1993). cDNA structure andin situ localization of theAplysia californica pro-hormone convertase PC2.FEBS Lett. 330:343–346.
Peters, K., and Rose, A. M. (1991).bli-4 encodes a protein structurally related to the yeast serine endoprotease Kex2.Worm Breeder's Gazette 11:28.
Peters, K., McDowell, J., and Rose, A. M. (1991). Mutations in thebli-4 (I) locus ofCaenorhabditis elegans disrupt both adult cuticle and early larval development.Genetics 129:95–102.
Ray, C., and McKerrow, J. H. (1992). Gut-specific and developmental expression of aCaenorhabditis elegans cysteine protease gene.Mol. Biochem. Parasitol. 51:239–250.
Roebroek, A. J. M., Schalken, J. A., Leunissen, J. A. M., Onnekink, C., Bloemers, H. P. J., and Van de Ven, W. J. M. (1986). Evolutionary conserved close linkage of the c-fed/fps protooncogene and genetic sequences encoding a receptor-like protein.EMBO J. 5: 2197–2202.
Roebroek, A. J. M., Creemers, J. W. M., Pauli, I. G. L., Kurzik-Dumke, U., Rentrop, M., Gateff, E. A. F., Leunissen, J. A. M., and Van de Ven, W. J. M. (1992). Cloning and functional expression of Dfurin2, a subtilisin-like proprotein processing enzyme ofDrosophila melanogaster with multiple repeats of a cysteine motif.J. Biol. Chem. 267:17208–17215.
Sakanari, J. A., Staunton, C. E., Eakin, A. E., and Craik, C. S. (1989). Serine proteases from nematode and protozoan parasites: Isolation of sequence homologues using generic molecular probes.Proc. Natl. Acad. Sci. USA 86:4863–4867.
Seidah, N. G., and Chrétien, M. (1992). Proprotein and prohormone convertases of the subtilisin family.TEM 3: 133–140.
Seidah, N. G., Gaspar, L., Mion, P., Marcinkiewicz, M., Mbikay, M., and Chrétien, M. (1990). cDNA sequence of two distinct pituitary proteins homologous to Kex2 and Furin gene products: Tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases.DNA Cell Biol. 9:415–424.
Seidah, N. G., Marcinkiewicz, M., Benjannet, S., Gaspar, L., Beaubien, G., Mattei, M. G., Lazure, C., Mbikay, M., and Chrétien, M. (1991). Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin and Kex2: Distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2.Mol. Endcrinol. 5:111–122.
Seidah, N. G., Hamelin, J., Gaspar, A. M., Day, R., and Chrétien, M. (1992a). The cDNA sequence of the human pro-hormone and pro-protein convertase PC1.DNA Cell Biol. 11:283–289.
Seidah, N. G., Fournier, H., Boileau, G., Benjannet, S., Rondeau, N., and Chrétien, M. (1992b). The cDNA structure of the porcine pro-hormone convertase PC2 and the comparative processing by PC1 and PC2 of the N-terminal glycopeptide segment of porcine POMC.FEBS Lett. 310:235–239.
Smeekens, S. P., and Steiner, D. F. (1990). Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2.J. Biol. Chem. 265: 2997–3000.
Smeekens, S. P., Avruch, A. S., LaMendola, J., Chan, S. J., and Steiner, D. F. (1991). Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans.Proc. Natl. Acad. Sci. USA 88:340–344.
Smit, A. B., Spijker, S., and Geraerts, P. M. (1992). Molluscan putative prohormone convertases: Structural diversity in the central nervous system ofLymnaea stagnalis.FEBS Lett. 312:213–218.
Steiner, D. F., Smeekens, S. P., Ohagi, S., and Chan, S. J. (1992). The new enzymology of precursor processing endoproteases.J. Biol. Chem. 267:23435–23438.
van den Ouweland, A. M. W., van Groningen, J. J. M., Roebroek, A. J. M., Onnekink, C., and Van de Ven, W. J. M. (1989). Nucleotide sequence analysis of the humanfur gene.Nucleic Acids Res. 17:7101–7102.
van den Ouweland, A. M. W., van Duijnhoven, H. L. P., Keizer, G. D., Dorssers, C. J., and Van de Ven, W. J. M. (1990). Structural homology between the humanfur gene product and the subtilisin-like protease encoded by yeast KEX2.Nucleic Acids Res. 18:664.
Von Heijne, G. (1983). Patterns of amino acids near signal-sequence cleavage sites.Eur. J. Biochem. 133:17–21.
Wood, B. W. (1988).The nematode Caenorhabditis elegans, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Gómez-Saladín, E., Wilson, D.L. & Dickerson, I.M. Isolation andin Situ localization of a cDNA encoding a Kex2-like prohormone convertase in the nematodeCaenorhabditis elegans . Cell Mol Neurobiol 14, 9–25 (1994). https://doi.org/10.1007/BF02088586
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02088586