Conserved roles for peptidases in the processing of invertebrate neuropeptides.

Invertebrates use a wide range of peptides as transmitters and hormones to regulate complex behaviour, physiology and development. These animals, especially those that are amenable to genetic study and are the subject of genome-sequencing projects, provide powerful model systems for understanding the functions of peptidases in controlling the bioactivity of peptides. Neprilysin, a zinc metallopeptidase and a key enzyme in the metabolism of mammalian peptides, is also implicated in the inactivation of peptides at synapses and of circulating peptide hormones in insects and nematodes. A family of neprilysin-like genes are present in the genomes of both Drosophila melanogaster and Caenorhabditis elegans; in C. elegans it seems that individual family members have evolved to take on different physiological functions, because they are expressed in a tissue-specific manner. Angiotensin I-converting enzymes (peptidyl dipeptidase A, angiotensin-converting enzyme) are another group of zinc metallopeptidases found in some invertebrates that lack angiotensin peptides. In D. melanogaster there are two functional angiotensin-converting enzymes that are essential for normal development. One of these (Acer) is expressed in the embryonic heart, whereas the second enzyme (Ance) is expressed in several tissues at different stages of the life cycle. The accumulation of Ance within secretory vesicles of some peptide-synthesizing cells suggests a role for the enzyme in the intracellular processing of insect peptides. Ance is very efficient at cleaving pairs of basic residues from the C-terminus of partly processed peptides, suggesting a novel role for the enzyme in prohormone processing. Invertebrates will continue to provide insights into the evolutionarily conserved functions of known peptidases and of those additional family members that are expected to be identified in the future from genome-sequencing projects.

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.

Insect angiotensin-converting enzyme. A processing enzyme with broad substrate specificity and a role in reproduction.

Insect angiotensin-converting enzyme (ACE) is a peptidyl dipeptidase that removes dipeptides and dipeptideamides from the C-terminus of a broad range of in vitro oligopeptide substrates. In mammals, ACE has important roles in blood homeostasis and a recently recognized, but as yet undefined, role in the fertility of male mice. High levels of ACE are found in the male reproductive tissues of several insect species, and emerging data indicates an important role for the enzyme in insect reproduction. In this paper we review some of the recent findings about insect ACE, and we speculate as to the physiological role of this enzyme in insect reproduction.

The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance--distinct enzymic characteristics and alternative expression during pupal development.

Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of primary structure similarity. We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance. Purified Acer and Ance expressed in yeast were used to raise anti-Acer Ig and anti-Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves the C-terminal dipeptides from benzoylglycyl-histidyl-leucine and [Leu5]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from [Leu5]enkephalinamide. However, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C-terminus with a kcat 15-fold higher than [Leu5]enkephalinamide. In addition, Acer did not cleave angiotensin I. In contrast, Ance hydrolysed 25% of this substrate at an 8-fold lower enzyme concentration. Furthermore, Acer did not hydrolyse the synthetic substrates Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis. Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent Ki values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages P1 (white puparium)-P15 (eclosion) of pupal development; Ance is expressed predominantly during stages P4-P7, whereas the ACE activity expressed during stages P9-P12 is mainly due to Acer suggesting different roles for the two enzymes during pupal development.

The Orct gene of Drosophila melanogaster codes for a putative organic cation transporter with six or 12 transmembrane domains.

Mutations at the lemming (lmg) locus of Drosophila melanogaster cause apoptotic cell death in dividing imaginal cells. Genomic DNA flanking the P element insertion corresponding to the lmg allele lmg03424 has been cloned and found to give rise to multiple transcripts. Several cDNA clones corresponding to this genomic region were isolated and shown to differ due to alternative splicing. The complete nucleotide sequences of two of the longest cDNAs were determined and found to encode proteins with similarity to mammalian organic cation transporter (OCT) proteins. One cDNA potentially encodes a protein with six transmembrane (TM) domains, corresponding to the N-terminal half of a mammalian OCT protein, whereas the other cDNA potentially encodes a protein with 12 TM domains, corresponding to the complete mammalian OCT protein. The gene giving rise to these alternative transcripts has been named Organic cation transporter-like (Orct). The previously identified Acer gene (Taylor, C.A.M., Coates, D., Shirras, A.D., 1996. The Acer gene of Drosophila codes for an angiotensin-converting enzyme homologue. Gene 181, 191 197) appears to lie within an intron of the Orct gene.

The Acer gene of Drosophila codes for an angiotensin-converting enzyme homologue.

Mammalian angiotensin-converting enzyme (ACE) exists as two forms, somatic (sACE), controlling blood pressure via angiotensin II, and testicular (tACE), whose function is unknown. The former has two highly homologous N- and C-terminal Zn2+ metallopeptidase active sites, whereas the latter only has one, which is identical to the C-terminal domain of sACE. We have sequenced 2452 bases of a 3.1-kb mRNA whose predicted translation product shows 40% identity with mammalian testicular ACE, and 48% identity with an already identified Drosophila homologue of ACE (Ance). We have termed this gene Acer (Angiotensin converting enzyme-related). Acer mRNA is found in the developing dorsal vessel (heart) during embryogenesis. Phylogenetic analysis indicates that duplication of an ancestral ACE gene occurred in the lineage leading to the arthropods, independently of the duplication which gave rise to the two domain somatic ACE of mammals.

Cell fates in the adult abdomen of Drosophila are determined by wingless during pupal development.

wingless activity is required for the patterning of tergites and sternites in the adult abdomen of Drosophila melanogaster. In the absence of wg, tergite and sternite cuticular differentiation is replaced by that of pleura. Temperature shift analysis of a wg temperature-sensitive allele has shown that sensory bristles and tergite/sternite histotype are determined independently and that wg is required between 15 and 20 hr after pupariation for bristle formation. The determination of sensory mother cells at this stage of development was confirmed by expression of the neuralized gene in a subset of the proliferating histoblasts. Ectopic expression of wg leads to the appearance of ectopic bristles and expanded tergite and sternite, indicating that wg expression is sufficient to promote both bristle formation and tergite/sternite differentiation. wg is expressed in the dividing and spreading histoblast population in a restricted pattern which may determine the spatial arrangement of cuticular elements.

Control of imaginal cell development by the escargot gene of Drosophila.

Mutations in the escargot (esg) locus, which codes for a zinc-finger-containing protein with similarity to the product of the snail gene, cause a variety of defects in adult structures such as loss of abdominal cuticle and malformation of the wings and legs. esg RNA is expressed in wing, haltere, leg and genital imaginal discs and in abdominal histoblast nests in the embryo. Expression in imaginal tissues is also found in third instar larvae. In esg mutant larvae, normally diploid abdominal histoblasts replicate their DNA without cell division and become similar in appearance to the polytene larval epidermal cells. A similar phenotype was also found in imaginal discs of larvae mutant for both esg and the Drosophila raf gene. These results suggest that one of the normal functions of esg may be the maintenance of diploidy in imaginal cells.

cricklet: A locus regulating a number of adult functions of Drosophila melanogaster.

During a screen for mutations in trans-acting genes regulating yolk protein synthesis in Drosophila melanogaster, we have isolated a mutant (cricklet, clt) that is defective in yolk protein synthesis, histolysis of the larval fat body, vitellogenesis, and synthesis of larval serum protein 2 in the adult, larval synthesis occurring normally. Larval serum protein 2 was previously thought to be synthesized only in the third-instar larva. We suggest that the clt locus may encode a protein essential for mediating the response of adult tissues to juvenile hormone.

Separate DNA sequences are required for normal female and ecdysone-induced male expression of Drosophila melanogaster yolk protein 1.

Drosophila melanogaster flies were transformed with a yp1-Adh fusion gene with 890 bp of yp1 5' flanking sequence. In an Adh- background these flies show a stage, tissue and sex-specific pattern of alcohol dehydrogenase (ADH) activity characteristic of yolk protein genes. ADH activity is not present in dsxD/dsx pseudomales indicating that this fragment contains sites where the dsx gene product exerts its effect. Transformed male flies do not exhibit ADH activity when injected with 20-hydroxyecdysone while synthesis of native yolk proteins is induced. Thus the hormone inducibility and sex regulation have been separated in this construct.

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3.

The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies.

Molecular cloning and characterisation of cDNAs complementary to mRNAs from wounded potato (Solanum tuberosum) tuber tissue.

Five cDNA clones complementary to mRNAs representing different abundances and responses to wounding have been isolated from a library of Sau 3A fragments in the bacteriophage M13 mp8. These were characterised by hybrid-release translation and hybridisation to RNA blots. The levels of RNA complementary to two of the clones show a marked increase during the 24h after wounding, one shows a small increase and two show no appreciable changes except that caused by a general increase in the total amount of polyadenylated RNA per microgram of total RNA which increases 2.5-fold during the same period. The would-induced RNAs are not induced in diluted suspension-culture cells, but RNA complementary to each clone is present in varying levels in stems, leaves and roots of intact potato plants.