Genome sequence analyses predict many proteins that are structurally related to proteases but lack catalytic residues, thus making functional assignment difficult. We show that one of these proteins (ACN-1), a unique multi-domain angiotensin-converting enzyme (ACE)-like protein from Caenorhabditis elegans, is essential for larval development and adult morphogenesis. Green fluorescent protein-tagged ACN-1 is expressed in hypodermal cells, the developing vulva, and the ray papillae of the male tail. The hypodermal expression of acn-1 appears to be controlled by nhr-23 and nhr-25, two nuclear hormone receptors known to regulate molting in C. elegans. acn-1(RNAi) causes arrest of larval development because of a molting defect, a protruding vulva in adult hermaphrodites, severely disrupted alae, and an incomplete seam syncytium. Adult males also have multiple tail defects. The failure of the larval seam cells to undergo normal cell fusion is the likely reason for the severe disruption of the adult alae. We propose that alteration of the ancestral ACE during evolution, by loss of the metallopeptidase active site and the addition of new protein modules, has provided opportunities for novel molecular interactions important for post-embryonic development in nematodes. Genome sequence analyses predict many proteins that are structurally related to proteases but lack catalytic residues, thus making functional assignment difficult. We show that one of these proteins (ACN-1), a unique multi-domain angiotensin-converting enzyme (ACE)-like protein from Caenorhabditis elegans, is essential for larval development and adult morphogenesis. Green fluorescent protein-tagged ACN-1 is expressed in hypodermal cells, the developing vulva, and the ray papillae of the male tail. The hypodermal expression of acn-1 appears to be controlled by nhr-23 and nhr-25, two nuclear hormone receptors known to regulate molting in C. elegans. acn-1(RNAi) causes arrest of larval development because of a molting defect, a protruding vulva in adult hermaphrodites, severely disrupted alae, and an incomplete seam syncytium. Adult males also have multiple tail defects. The failure of the larval seam cells to undergo normal cell fusion is the likely reason for the severe disruption of the adult alae. We propose that alteration of the ancestral ACE during evolution, by loss of the metallopeptidase active site and the addition of new protein modules, has provided opportunities for novel molecular interactions important for post-embryonic development in nematodes. The large number of protease genes in animal genomes reflects the widespread importance of proteolysis to animal physiology and development. The rich functional diversity of proteases can be attributed to the variety of catalytic mechanisms and protein structures, some of which are of a modular design, offering different levels of structural and functional complexity (1Puente X.S. Sanchez L.M. Overall C.M. Lopez-Otin C. Nat. Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (725) Google Scholar). The MEROPS data base (merops.sanger.ac.uk; release 6.3) (2Rawlings N.D. O'Brien E. Barrett A.J. Nucleic Acids Res. 2002; 30: 343-346Crossref PubMed Scopus (174) Google Scholar) catalogs 551, 553, 563, 366, and 367 proteases for human, mouse, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans, respectively. The majority of these enzymes have not been studied at the physiological and biochemical level, but it has been possible to classify most of them into families based on statistically significant similarities in primary protein structure (2Rawlings N.D. O'Brien E. Barrett A.J. Nucleic Acids Res. 2002; 30: 343-346Crossref PubMed Scopus (174) Google Scholar). Surprisingly, a significant proportion (12-25%) of the total number of the predicted protease-like genes in the human, mouse, D. melanogaster, A. gambiae, and C. elegans genomes code for proteins that lack one or more catalytic residues and are therefore classified as “non-peptidase” family members. Some of these genes may be nonfunctional pseudogenes, but many have probably lost the catalytic activity of an ancestral protein while acquiring new functions. These non-peptidase proteins are structurally related to proteases distributed across all classes, but homologues of metallopeptidases are particularly well represented. One such protein is UNC-71, a C. elegans member of the ADAMs family, which lacks a zinc binding site and yet has acquired important roles in development (3Huang X. Huang P. Robinson M.K. Stern M.J. Jin Y. Development. 2003; 130: 3147-3161Crossref PubMed Scopus (58) Google Scholar). Our attention has recently been drawn to the fact that several invertebrate members of the angiotensin-converting enzyme (ACE) 1The abbreviations used are: ACEangiotensin-converting enzymeGPIglycosylphosphatidylinositoldsRNAdouble-stranded RNARNAiRNA interferenceESTexpressed sequence tagGFPgreen fluorescent protein.1The abbreviations used are: ACEangiotensin-converting enzymeGPIglycosylphosphatidylinositoldsRNAdouble-stranded RNARNAiRNA interferenceESTexpressed sequence tagGFPgreen fluorescent protein. (EC 3.4.15.1, peptidyl dipeptidase A) family from D. melanogaster and C. elegans lack crucial active site residues and are, therefore, predicted not to function as peptidases (4Isaac R.E. Siviter R.J. Stancombe P. Coates D. Shirras A.D. Biochem. Soc. Trans. 2000; 28: 460-464Crossref PubMed Google Scholar). Mammalian ACE is a cell surface zinc metallopeptidase that cleaves a C-terminal dipeptide from angiotensin I to generate the vasoconstrictor, angiotensin II (for reviews see Refs. 5Corvol P. Williams T.A. Soubrier F. Barrett, A.J. Methods in Enzymology. Peptidyl dipeptidase A: Angiotensin I-converting Enzyme. Vol. 248. Academic Press, London1995: 283-305Google Scholar and 6Turner A.J. Hooper N.M. Trends Pharmacol. Sci. 2002; 23: 177-183Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Bradykinin, a vasodilator, and the hemoregulatory peptide N-acetyl-SDKP are also substrates for mammalian ACE. Somatic ACE comprises two catalytic units in tandem, which are anchored to the cell surface by a transmembrane region and a short C-terminal cytoplasmic domain. ACE knockout mice have, as expected, lower blood pressure but also have severe renal abnormalities and suffer from anemia (7Krege J.H. John S.W.M. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. Obrien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (602) Google Scholar, 8Esther C.R. Howard T.E. Marino E.M. Goddard J.M. Capecchi M.R. Bernstein K.E. Lab. Investig. 1996; 74: 953-965PubMed Google Scholar). In mammalian testes, a single-domain ACE (germinal ACE, 100 kDa) is expressed in developing spermatids, and the lack of this isoform results in male infertility (7Krege J.H. John S.W.M. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. Obrien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (602) Google Scholar). Sperm from ACE (-/-) mice appear normal but fail to migrate beyond the extramural uterotubal junction of the oviduct of mated wild-type females, and in vitro experiments demonstrate that they have reduced binding to zonae pellucidae. The peptide substrate for the germinal enzyme has not been identified, and it has been suggested that the role of the gACE in reproduction might depend on direct molecular interactions at the cell surface rather than the hydrolysis of a peptide (7Krege J.H. John S.W.M. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. Obrien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (602) Google Scholar). A new member of the ACE family, ACE2, has recently been characterized (9Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1572) Google Scholar, 10Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar). Although structurally similar to ACE, it is not a dipeptidyl carboxypeptidase, but a carboxypeptidase; it is expressed in cardiac cells, kidney, and the testes (6Turner A.J. Hooper N.M. Trends Pharmacol. Sci. 2002; 23: 177-183Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Mice lacking ACE2 activity have defects in both the speed and overall percentage of heart contraction on aging (11Crackower M.A. Sarao R. Oudit G.Y. Yagil C. Kozieradzki I. Scanga S.E. Oliveira-dos-Santos A.J. da Costa J. Zhang L. Pei Y. Scholey J. Ferrario C.M. Manoukian A.S. Chappell M.C. Backx P.H. Yagil Y. Penninger J.M. Nature. 2002; 417: 822-828Crossref PubMed Scopus (1338) Google Scholar). angiotensin-converting enzyme glycosylphosphatidylinositol double-stranded RNA RNA interference expressed sequence tag green fluorescent protein. angiotensin-converting enzyme glycosylphosphatidylinositol double-stranded RNA RNA interference expressed sequence tag green fluorescent protein. A D. melanogaster ACE homologue, known as Ance, has very similar enzymatic properties to those of mammalian ACE and is required for completion of embryogenesis and for spermatogenesis in adult flies (12Williams T.A. Michaud A. Houard X. Chauvet M.T. Soubrier F. Corvol P. Biochem. J. 1996; 318: 125-131Crossref PubMed Scopus (61) Google Scholar, 13Hurst D. Rylett C.M. Isaac R.E. Shirras A.D. Dev. Biol. 2003; 254: 238-247Crossref PubMed Scopus (54) Google Scholar). Acer, a second D. melanogaster ACE homologue, is also catalytically active toward peptides but does not convert angiotensin I to angiotensin II (14Houard X. Williams T.A. Michaud A. Dani P. Isaac R.E. Shirras A.D. Coates D. Corvol P. Eur. J. Biochem. 1998; 257: 599-606Crossref PubMed Scopus (76) Google Scholar, 15Siviter R.J. Nachman R.J. Dani M.P. Keen J.N. Shirras A.D. Isaac R.E. Peptides. 2002; 23: 2025-2034Crossref PubMed Scopus (32) Google Scholar). Four other ACE-like genes (Ance-2, -3, -4, -5) are present in the D. melanogaster genome, but all four conceptual proteins are missing one or more of the active site residues required for catalysis by human ACE (16Coates D. Isaac R.E. Cotton J. Siviter R. Williams T.A. Shirras A. Corvol P. Dive V. Biochemistry. 2000; 39: 8963-8969Crossref PubMed Scopus (62) Google Scholar). In contrast to flies and mammals, the nematode, C. elegans, has only one ACE-like gene (C42D8.5), which codes for an ACE-like protein lacking not only the HEXXH consensus metallopeptidase motif but also other active site residues found in the sequence HEA(I/V)XD of mammalian and insect ACEs (4Isaac R.E. Siviter R.J. Stancombe P. Coates D. Shirras A.D. Biochem. Soc. Trans. 2000; 28: 460-464Crossref PubMed Google Scholar). 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Kohara, National Institute of Genetics, The clones were by and the data with to sequence present in the C. elegans data was from the of The of the peptide sequence and site was the J. S. G. PubMed Scopus Google The was used to predict a to the protein B. P. F. J. Biol. PubMed Scopus Google Scholar). were predicted and O. N. Williams S. J. 1998; PubMed Scopus Google Scholar). and of the fluorescent protein gene were for ACN-1 a O. 2002; PubMed Scopus Google Scholar). The acn-1 gene and of sequence was from the from the to and the sequence is the of an at °C for 5 the 25 of °C for 50 °C for and °C for The was with a from of of to and was used as the for with to generate the acn-1 The used were to a acn-1 and a The were as with the to The of this were into the of C. elegans adult with the J.M. D. V. J. PubMed Scopus Google which the were on the of the The male expression was by hermaphrodite to with wild-type Bristol strain (N2) the were on a at an of were a and and 5 of acn-1 was of °C for 5 by 25 of °C for 50 °C for and °C for from and to and The was and used as the to and with and in vitro RNA was by of and by at °C for and at °C for acn-1 was into the of adult were to for at 20 °C in to present in to and were NGM agar The were for in acn-1 a to was from the and into the site of to generate was into E. coli and expression of was by mm to a P. M. M. J. Nature. 2000; PubMed Scopus Google Scholar). a mm was to the and the were NGM agar 1 mm The C. elegans F. M. S. A. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) was to at 20 °C on the and were were in lacking as the The of on males was by adult males and to on NGM agar and the to as the of seam cell the strain E.M. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar) was on acn-1 as used to nhr-23 and were obtained from J. of P. M. M. J. Nature. 2000; PubMed Scopus Google Scholar). of in these was as the of the genes on acn-1 and expression in seam cells, adult from and E. I. K.E. Dev. 2002; PubMed Scopus Google Scholar) were to with E. coli or at 20 were for the and seam cell of not all seam cells of an expressed the were was in only one seam were for in pH and with to in (w/v) were with and for 1 in an of were with a as the and on were with to a of 50 a and and with a The of C. elegans of the ACN-1 the predicted for this gene by the C. elegans number The protein is with an peptide that is predicted to be by and 20 In with human germinal ACE and Drosophila it is that residues and a region of ACN-1 the overall structure of the protein from other ACEs in several important active ACE proteins the HEXXH and HEA(I/V)XD in ACN-1 the these have been with residues, from which is with an The of the conceptual ACN-1 is than the human ACE and Drosophila in to a rich in and residues across a region of is also a C-terminal that a region to a sequence rich in and The region has for four in addition to other predicted distributed the ACN-1 sequence and four The that ACN-1 is probably to a at B. P. F. J. Biol. PubMed Scopus Google Scholar). acn-1 in and in the during and in the of the and expression of acn-1 was studied by independent of C. elegans a C-terminal gene fusion the of the region of gene expression was first in the hypodermal seam cells during embryogenesis and in the seam cells of all larval The hypodermal of and adult also gene activity The expression in seam cells was expression was in the cells that the developing vulva in the larval stage and and expression into but was not present in the vulva of In addition to the hypodermal expression for hermaphrodites, gene activity was also in the developing ray papillae of the male tail adult males to the to the tail expression was in the but expression been to a of E. acn-1(RNAi) in and the physiological role of used RNA interference to expression of acn-1 A. S. M.K. S.E. Nature. 1998; PubMed Scopus Google Scholar). 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These structural might be for new molecular interactions that have a direct on the molting by cell and cell fusion of hypodermal or an by genes required for those for and for to are by a which is to for and to different The of and Google Scholar). The may in and structure and stage of development. it is likely that mechanisms of and be A of has of acn-1 in other animal and R. that ACN-1 was in the as a in larval development. We for Institute of Genetics, for the clones yk63f5, and yk759d02, Institute of for the laboratory and elegans of for the and of for nhr-23 and We also for to the expression for acn-1 obtained by in and for during the of this for the for and for with
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