Dr Alan Shirras
Senior Lecturer
Office C12
Biological Sciences Building
Lancaster University
Lancaster
LA1 4YQ
UK
Tel: +44 1524 592597
Fax: +44 1524 593192
E-mail: a.shirras@lancaster.ac.uk
Research Interests
My main area of interest is insect molecular biology, particularly of the fruit fly, Drosophila melanogaster.
Angiotensin-converting enzyme (ACE) homologues in Drosophila and other insects
with Elwyn Isaac, University of Leeds
Mammalian angiotensin I-converting enzyme (ACE, peptidyl dipeptidase A, EC 3.4.15.1) is a zinc metallopeptidase that cleaves dipeptides from the C-terminus of oligopeptides and is best known for its pivotal role in blood homeostasis, as part of the Renin-Angiotensin and Kinin-Kininogen systems. Endothelial ACE is responsible for the synthesis of the vasoconstrictor, angiotensin II, by the hydrolysis of the penultimate peptide bond of angiotensin I. It is also involved in the inactivation of the vasodilatory peptide, bradykinin, and the degradation of the haemoregulatory peptide, N-acetyl-Ser-Asp-Lys-Pro. The somatic form of mammalian ACE (180-210 kDa) comprises two catalytic ectodomains (N- and C-domains) in tandem, anchored to the cell-surface by a trans-membrane region and a short C-terminal cytoplasmic domain. An alternative promoter is used in spermatocytes to generate a single-domain ACE (gACE, 100 kDa), found exclusively in male germ cells. The primary structure of gACE is identical to the C-domain of sACE, apart from a 67 residue N-terminal peptide which is rich in serine and threonine and is an important region for O-glycosylation.
ACE activity is found in invertebrates from several phyla, including all insect species studied so far. Since invertebrates do not possess peptide hormones structurally related to angiotensin, bradykinin and N-acetylSDKP, understanding the physiological roles of these invertebrate ACEs is likely to reveal novel functions of ACE-like peptidases in biological processes. Two Drosophila homologues of human ACE (ANCE and ACER) have been the subject of genetic and biochemical studies in our labs. They are both single domain proteins which, unlike mammalian ACE, are secreted and do not possess a protein membrane anchor. ANCE can remove dipeptides from a wide range of peptide substrates and, therefore, might have a general role in peptide metabolism. However, it is quite clear that selected peptides are cleaved with very high efficiency (e.g. locust tachykinin I and mammalian bradykinin) and, therefore, the enzyme can also be a specialist, regulating the activity of key signalling peptides. Ance null mutants die at the end of embryogenesis, apparently as a result of abnormal hatching behaviour, which prevents the developed embryo from emerging from the vitelline membrane. Weak Ance alleles permit survival to the adult stage but males are infertile as a result of a failure in spermatid differentiation. As well as the testes, Ance is expressed in male accessory glands where it may play a role in processing peptides that are passed to the female during mating.
ACER is a more enigmatic enzyme. Despite having a full complement of active site residues, it has only weak peptidase activity against most substrates. Strongest expression of the Acer gene is in adult flies - large amounts of protein are present in the adult head and in haemolymph. It is also expressed in the testes and ovarian follicle cells. Despite this strong expression, Acer null mutants are viable and fertile. An Acer gene is present in all 12 Drosophila genomes sequenced so far where it is well-conserved, suggesting an important function, but it is absent from other insect genomes. The lack of obvious mutant phenotype may caused by redundancy with ANCE or a subtle requirement that is not obvious under stress-free laboratory conditions.
Sequencing of the Drosophila melanogaster genome identified a further four ACE-like genes, called Ance-2, Ance-3, Ance-4, and Ance-5. Any enzymatic function for the protein products of these genes is doubtful, since the conceptual proteins lack one or more key active site residues.
ACE activity has been found in the gonads and accessory glands of several insect species in addition to D. melanogaster, suggesting a conserved role for this enzyme in insect reproduction. ACE activity in the adult female Anopheles stephensi mosquitoes increases by 260% following a blood meal, perhaps in order to regulate peptide signalling in response to feeding. Feeding two different ACE inhibitors to male A. stephensi in their glucose diet, results in an approx. 80% reduction in the number of eggs laid. In addition, ACE inhibitors introduced in the blood meal, results in a dose-dependent effect on brood-size, but has no effect on oocyte development, nor the rate of digestion of the blood. The only observable difference between inhibitor-fed and control insects is that the inhibitor-fed females do not lay eggs, even 8 days after the blood meal, suggesting that the blood-induced ACE is involved in the control of egg-laying.
To understand the mechanism of ACE induction in blood-fed mosquitoes and the role of the enzyme in mosquito reproductive physiology, we have characterised the ACE gene family in Anopheles gambiae, the mosquito responsible for the transmission of the human malaria parasite. TBLASTN and sequence analysis of cDNAs revealed that the A. gambiae genome contains nine genes (AnoACE genes) which code for proteins with similarity to mammalian ACE. Eight of these genes code for putative single domain enzymes similar to other insect ACEs described so far. AnoACE9, however, has several features in common with mammalian somatic ACE such as a two domain structure and a hydrophobic C terminus. Four of the AnoACE genes (2, 3, 7 and 9) were shown to be expressed at a variety of developmental stages. Expression of AnoACE3, AnoACE7 and AnoACE9 is induced by a blood meal, with AnoACE7 showing the largest (approximately 10-fold) induction. Click here to see the AnoACE annotations on Ensembl.
Phylogenetic analysis of insect ACE-like proteins (ACELPs) reveals five groups of proteins. Group 1 contains single-domain, secreted proteins with no apparent membrane anchor, similar to D. melanogaster ANCE and ACER. Genes coding for group 2 proteins are found in all insect genomes which have been completely sequenced. These proteins are characterized by an extended N-terminus and an alanine at position 4 of the HExxH active site motif. The D. melanogaster member of this family, ANCE-3, has a potential glycosylphosphatidylinositol (GPI) membrane anchor but it is unclear whether this is a general feature of this group. Group 3 proteins are currently represented only in the mosquitoes A. gambiae and Aedes aegypti, and in the beetle Tribolium castaneum. The mosquito proteins have duplicated active site domains and a C-terminal hydrophobic potential membrane anchor region reminiscent of mammalian somatic ACE. Group 4 has representatives from Tribolium, Aedes, Anopheles and Apis mellifera, but D. melanogaster lacks a group 4 ACELP. Group 4 ACELPs appear to be single domain secreted proteins, like group 1, but they greater sequence similarity to group 2 proteins. While proteins belonging to group 5 form a separate sequence family, they are very divergent and have been evolving rapidly compared to other insect ACEs, indicating that there are fewer constraints on their sequence. Their function is, however, unknown.
Click
to see full size phylogram
Downloads: ACELP phylogram: .dnd; .pdf ACELP sequences: fasta. These are "core" sequences corresponding to amino acids 145–553 of D. melanogaster ANCE. Species codes are: Dm, Drosophila melanogaster; Dg, Drosophila grimshawi; Ll: Lutzomyia longipalpis; Aa, Aedes aegypti; Ag, Anopheles gambiae; Hi, Haematobia irritans; Tc, Tribolium castaneum; Am, Apis mellifera; Bm, Bombyx mori; Lm, Locusta migratoria.
Publications
Ahmet Carhan, Ke Tang, K, Christine A. Shirras, Alan D. Shirras, and R. Elwyn Isaac (2011) Loss of Angiotensin-converting enzyme-related (ACER) peptidase disrupts night-time sleep in adult Drosophila melanogaster. J. Exp. Biol. 214, 680-686.
R. Elwyn Isaac, Chenxi X. Li, Amy E. Leedale and Alan D. Shirras (2010) Drosophila male sex peptide inhibits siesta sleep and promotes locomotor activity in the post-mated female. Proc. Roy. Soc. B 277, 65-70
Richard E Isaac, Erik C Johnson, Neil Audlsey, and Alan D Shirras (2007) Metabolic inactivation of the circadian transmitter, PDF, by neprilysin-like peptidases in Drosophila. J. Exp. Biol. 210, 4465-4470.
Caroline M. Rylett, Michael J. Walker, Gareth J. Howell, Alan D. Shirras, and R. Elwyn Isaac (2007) Male accessory glands of Drosophila melanogaster make a secreted angiotensin I-converting enzyme (ANCE), suggesting a role for the peptide-processing enzyme in seminal fluid. J. Exp. Biol. 210, 3601-3606.
Lyndsay Davies, Ian P. Anderson, Philip C. Turner, Alan D. Shirras, Huw H. Rees, Daniel J. Rigden (2007). An unsuspected ecdysteroid/steroid phosphatase activity in the key T-cell regulator, Sts-1: Surprising relationship to insect ecdysteroid phosphate phosphatase. Proteins: Structure, Function, and Bioinformatics 67, 720-731.
Nicholas D. Bland, Josie E. Thomas, Neil Audsley, Alan.D. Shirras, Anthony J. Turner and R. Elwyn Isaac (2007) Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster Peptides 28, 127-135.
R. Elwyn Isaac, Nazarius S. Lamango, Uma Ekbote, Christine A. Taylor, Debra Hurst, Robert J. Weaver, Ahmet Carhan, Susan Burnham and Alan D. Shirras (2007) Angiotensin-converting enzyme as a target for the development of novel insect growth regulators Peptides 28, 153-162
Michael J Walker, Caroline M Rylett, Jeff N Keen, Neil Audsley, Mohammed Sajid, Alan D Shirras and Richard E Isaac (2006) Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble gamma-glutamyl transpeptidase. Proteome Science 4:9
Richard J. Bingham, Vincent Dive, Simon E. V. Phillips, Alan D. Shirras and R. Elwyn Isaac (2006) Structural diversity of angiotensin-converting enzyme: Insights from structure-activity comparisons of two Drosophila enzymes. FEBS J. 273, 362–373.
Susan Burnham, Judith
A Smith, Alison
J Lee, R. Elwyn Isaac, Alan D Shirras (2005) The
angiotensin-converting enzyme (ACE) gene family of Anopheles
gambiae
BMC Genomics 6, 172
Thomas,J. E., Rylett, C. M., Carhan, A., Bland, N. D., Bingham, R. J., Shirras, A. D., Turner, A. J. and Isaac, R. E. (2005). Drosophila melanogaster NEP2 is a new soluble member of the neprilysin family of endopeptidases with implications for reproduction and renal function. Biochem. J. 386, 357-366.
Isaac, R. E., Taylor, C. A. M., Hamasaka, Y., Nässel, D. R. and Shirras, A. D. (2004) Proctolin in the post-genomic era: new insights and challenges. Invertebrate Neuroscience. 5, 51-64.
Taylor, C. A. M., Winther, Å. M. E., Siviter, R. J., Shirras, A. D., Isaac, R. E. and Nässel, R. R. (2004) Identification of a Proctolin Preprohormone Gene (Proct) of Drosophila melanogaster: Expression and Predicted Prohormone Processing. J. Neurobiol. 58, 379-391.
Hurst, D., Rylett, C. M., Isaac, R. E. and Shirras, A. D. (2003) The Drosophila angiotensin-converting enzyme homologue Ance is required for spermiogenesis. Developmental Biology 254, 238-247.
Siviter, R. J.,
Nachman, R. J., Dani, P. M., Keen, J. N., Shirras,
A. D. and Isaac, R. E. (2002) Peptidyl
dipeptidases (Ance and Acer) of Drosophila melanogaster:
major
differences in the substrate specificity of two homologs of human
angiotensin
I-converting enzyme, Peptides 23,
Wilson, C. L., Shirras,
A. D. and Isaac, R. E. (2002) Extracellular
peptidases of imaginal discs of Drosophila melanogaster
Peptides 23,
Siviter, R. J.,
Taylor, C. A. M., Cottam, D. M., Denton, A.,
Dani, P. M., Milner, M. J., Shirras, A. D. and
Isaac, R. E. (2002) Ance,
a Drosophila angiotensin-converting enzyme homologue, is expressed in
imaginal
cells during metamorphosis and is regulated by the steroid,
20-hydroxyecdysone. The
Biochemical Journal, 367,
Isaac, R.
E., Parkin, E. T., Jeffrey N. Keen, J.
K., Nässel, R. R., Siviter, R. J. and Shirras, A. D.
(2002) Inactivation
of a tachykinin-related peptide: identification of four
neuropeptide-degrading
enzymes in neuronal membranes of insects from four different orders.
Peptides 23,
Siviter, R. J., Coast, G. M. , Winther, A. M., Nachman, R. J., Taylor, C. A., Shirras, A. D., Coates, D., Isaac, R. E. and Nässel, R. R. (2000) Expression and functional characterisation of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A. J. Biol. Chem. 275, 23273-23280.
Coates, D., Isaac, R. E., Cotton, J., Siviter, R., Williams, T. A., Shirras, A. D., Corvol, P. and Dive, V. (2000) Functional conservation of the active sites of human and Drosophila angiotensin I-converting enzyme. Biochemistry. 39, 8963-8969.
Isaac, R. E., Siviter,
R. J., Stancombe, P., Coates, D. and Shirras,
A. D. (2000) Conserved
roles for peptidases in the processing of invertebrate neuropeptides. Biochemical
Society Transactions 28,