- Email: [email protected]
Insect Carboxypeptidases
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[82]
[83]
[84]
[85]
[86]
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(CGRP), and neuropeptide Y by vascular smooth muscle cells. Peptides 17(4), 709 720. Palmieri, F.E., Bausback, H.H., Churchill, L., Ward, P.E. (1986). Kinin and enkephalin conversion by an endothelial, plasma membrane carboxypeptidase. Biochem. Pharmacol. 35(16), 2749 2756. Zhang, Y., Brovkovych, V., Brovkovych, S., Tan, F., Lee, B.S., Sharma, T., Skidgel, R.A. (2007). Dynamic receptor-dependent activation of inducible nitric-oxide synthase by ERK-mediated phosphorylation of Ser745. J. Biol. Chem. 282(44), 32453 32461. Brovkovych, V., Zhang, Y., Brovkovych, S., Minshall, R.D., Skidgel, R.A. (2011). A novel pathway for receptor-mediated posttranslational activation of inducible nitric oxide synthase. J. Cell. Mol. Med. 15(2), 258 269. Skidgel, R.A., Stanisavljevic, S., Erdo¨s, E.G. (2006). Kinin- and angiotensin-converting enzyme (ACE) inhibitor-mediated nitric oxide production in endothelial cells. Biol. Chem. 387(2), 159 165. Zhang, X., Tan, F., Brovkovych, V., Zhang, Y., Skidgel, R.A. (2011). Crosstalk between carboxypeptidase M and the kinin B1 receptor mediates a new mode of G protein-coupled receptor signaling. J. Biol. Chem. .
M14A | 305. Insect Carboxypeptidases
[87] Lendeckel, U., Arndt, M., Wrenger, S., Nepple, K., Huth, C., Ansorge, S., Klein, H.U., Goette, A. (2001). Expression and activity of ectopeptidases in fibrillating human atria. J. Mol. Cell. Cardiol. 33(6), 1273 1281. [88] Hall, J.L., Grindle, S., Han, X., Fermin, D., Park, S., Chen, Y., Bache, R.J., Mariash, A., Guan, Z., Ormaza, S., Thompson, J., Graziano, J., de Sam Lazaro, S.E., Pan, S., Simari, R.D., Miller, L.W. (2004). Genomic profiling of the human heart before and after mechanical support with a ventricular assist device reveals alterations in vascular signaling networks. Physiol. Genomics 17(3), 283 291. [89] Milligan, G., Smith, N.J. (2007). Allosteric modulation of heterodimeric G-protein-coupled receptors. Trends Pharmacol. Sci. 28(12), 615 620. [90] Song, L., Fricker, L.D. (1995). Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J. Biol. Chem. 270(42), 25007 25013. [91] Reznik, S.E., Fricker, L.D. (2001). Carboxypeptidases from A to Z: implications in embryonic development and Wnt binding. Cell. Mol. Life Sci. 58(12 13), 1790 1804.
Xianming Zhang Department of Pharmacology, University of Illinois College of Medicine at Chicago, 835 S. Wolcott Ave. (m/c 868), Chicago, IL 60612, USA. Email: [email protected]
Randal A. Skidgel Department of Pharmacology, University of Illinois College of Medicine at Chicago, 835 S. Wolcott Ave. (m/c 868), Chicago, IL 60612, USA. Email: [email protected] © 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00305-7
Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
Chapter 305
Insect Carboxypeptidases DATABANKS MEROPS name: insect gut carboxypeptidase-1 MEROPS classification: clan MC, family M14, subfamily M14A, peptidase M14.024 Tertiary structure: Available Species distribution: order Lepidoptera Reference sequence from: Helicoverpa armigera MEROPS name: insect gut carboxypeptidase-2 MEROPS classification: clan MC, family M14, subfamily M14A, peptidase M14.031
Species distribution: family Noctuidae Reference sequence from: Helicoverpa armigera
Name and History The presence of carboxypeptidases in insects was unambiguously demonstrated in 1976 by the purification of a protein with carboxypeptidase activity from clothes moth (Lepidoptera; Tineolla bisselliella) [1], and partial purification of enzymes with carboxypeptidase A and B activities from Tsetse fly (Diptera; Glossinia morsitans) [2].
Clan MC
M14A | 305. Insect Carboxypeptidases
Subsequent work identified carboxypeptidases in other insect orders, including Hymenoptera, Coleoptera, and Hemiptera, and evidence suggests they are found in all insects (reviewed in Terra & Ferreira [3]). Similar activities are present in other arthropods, and had been identified before carboxypeptidases in insects. Carboxypeptidase activity is widely present in insect digestive systems, since most insects rely on protein digestion to provide nitrogen necessary for biosynthesis. Digestive carboxypeptidases in insects have been classified as similar in activity to mammalian carboxypeptidase A (Chapter 289) and B (Chapter 296), as determined by assays with synthetic substrates (see below). However, proteolytic and other hydrolytic enzymes can vary greatly between different orders of insects, and even between different species within an order, reflecting specialization in feeding strategies. Consequently, some insects contain digestive carboxypeptidases with sequence similarity to the carboxypeptidase A family, but with activity specific to C-terminal glutamate [4]; these enzymes are referred to as ‘glutamate carboxypeptidases’ in this chapter. Carboxypeptidases are also involved in the molting process in insects, where proteolytic activity is necessary to separate the old exoskeleton from the underlying epidermal cells. Carboxypeptidase activity was shown to be present in molting fluid in tobacco hornworm (Lepidoptera; Manduca sexta) [5], and a molting enzyme with carboxypeptidase A-like activity is present in silk worm (Lepidoptera; Bombyx mori) [6]. Carboxypeptidases in higher animals are also involved in processing of preproteins and peptide hormones, and insects contain genes encoding enzymes predicted to show these activities. The fruit fly (Diptera; Drosophila melanogaster) svr gene encodes a protein with similarities to carboxypeptidases E, M and N [7] containing multiple carboxypeptidase domains like duck carboxypeptidase D (Chapter 303) [8]. Similar proteins are present in other insects. Carboxypeptidase activity may also be involved in signaling pathways and pattern recognition [9]. The availability of complete genome sequences for a number of insects has allowed carboxypeptidase gene families to be analyzed, demonstrating considerable diversity in the numbers and activities of carboxypeptidases present. For example, the mosquito (Diptera; Anopheles gambiae) genome predicts 23 sequences coding for zinc-carboxypeptidases [10]. The Drosophila genome contains 20 genes of the carboxypeptidase A subfamily, six predicted to show carboxypeptidase A activity, five carboxypeptidase B activity, four glutamate carboxypeptidase activity, and five predicted to be inactive [4]; in addition, two genes encoding proteins of the multidomain carboxypeptidase D type are present. In contrast, the genome in pea aphid (Hemiptera; Acyrthosiphon pisum) contains only two carboxypeptidase A-type genes, one annotated as a molting carboxypeptidase A enzyme,
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the other as a carboxypeptidase B enzyme. The absence of a multigene family encoding digestive carboxypeptidases reflects the minimal role protein digestion plays in this insect, which derives nitrogen from free amino acids in its diet, plant sap.
Activity and Specificity Carboxypeptidase activity in extracts from insect tissues has been detected with synthetic substrates used for assaying mammalian enzymes, such as N-(3-[2-furyl] acryloyl)-Phe-Phe (FAPP) and N-(3-[2-furyl]acryloyl)Ala-Lys (FAAK). However, these assays are not entirely satisfactory, due to lack of sensitivity and interference by extract components, and reliable data for specificity and kinetic constants for enzymes can only be obtained from purified proteins. The small size of many insects makes purification of carboxypeptidases technically challenging, and recombinant expression systems are necessary to produce pure proteins for study. The data available suggest that specificities of insect carboxypeptidases A and B are broadly similar to their mammalian counterparts, although a systematic study has yet to be undertaken. Some carboxypeptidase A enzymes may differ slightly in specificity from mammalian enzymes due to differing active site residues (see below). Similarly, a carboxypeptidase B has been identified in bollworm (Lepidoptera; Helicoverpa armigera) that is highly specific for C-terminal Lys residues [11]. The insect glutamate carboxypeptidase has a high degree of specificity towards C-terminal Glu residues. It cleaves the synthetic substrate N-(3-[2-furyl]acryloyl)-Glu-Glu (FAEE), but not FAPP or FAAK, and will remove Cterminal glutamate residues from peptides [4]. Very low levels of activity towards C-terminal Asp in peptides were also observed. Gut conditions in insects are highly variable, with lepidoptera having high gut pH (up to 10.5), whereas some dipteran species have regions in the gut where local pH is as low as 3.0. However, where pH optima for insect carboxypeptidases have been determined, there is generally a broad pH optimum in the alkaline range; for example, the digestive carboxypeptidase A enzyme from bollworm (Lepidoptera; Helicoverpa armigera) has an optimal range of pH 7.5 10 [12]. Insect metallocarboxypeptidases are inhibited by zinc chelators such as 1,10-phenanthroline, like their mammalian counterparts; the digestive enzymes are generally strongly inhibited by specific protein proteinase inhibitors, such as potato carboxypeptidase inhibitor, with the resulting complexes only dissociable under denaturing conditions [4]. An exception is the Lys-specific carboxypeptidase B in bollworm, which is insensitive to this inhibitor [11]. The carboxypeptidase D-type enzymes are not inhibited by these protein proteinase inhibitors [13].
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Structural Chemistry Sequences for many insect metallocarboxypeptidases have been deduced from cDNA sequences, or genome sequences. They have predicted signal peptides, as expected for secreted enzymes, and are of similar size to their mammalian counterparts, with a pro-region of approximately 90 amino acids, and an active enzyme domain of approximately 300 amino acids. The sequences of digestive and molting carboxypeptidases can be mapped onto the ‘model’ sequence for human carboxypeptidase A, using sequence similarity, which shows that the features necessary for zinc binding and catalysis are present; His69, Glu72 and His196, residues which bind the catalytic zinc ion; Arg71, Arg145, Tyr248 and Glu270, residues responsible for substrate binding and cleavage (using mammalian carboxypeptidase A numbering). The presence of genes predicting proteins with strong sequence similarity to carboxypeptidases in the Drosophila genome, which would be inactive as enzymes due to the absence of some of these key residues has been noted above. X255, the residue which determines substrate specificity, is neutral in carboxypeptidase A enzymes, to interact with neutral hydrophobic side chains on the substrate, but besides the hydrophobic residues found in mammalian carboxypeptidases, can be Ser, potentially extending the substrate range of the enzymes [14]. X255 is Asp in most carboxypeptidase B enzymes, and is Lys or Arg in glutamate carboxypeptidases, to interact with basic and acidic side chains on the substrate, respectively [4]. The lysine-specific carboxypeptidase B from bollworm has a Glu residue at position 255, which is partly responsible for its substrate specificity [11]. The carboxypeptidase D-type enzymes have a complex multidomain structure with large numbers of variants generated by alternate splicing. Crystal structures are available for carboxypeptidase A- and B-type proteases from bollworm (Lepidoptera; Helicoverpa armigera) [11,14], and for one domain of the carboxypeptidase D-type enzyme from Drosophila [13]. The insect carboxypeptidase A contains a pro-region of 91 residues, and an active enzyme of 318 residues, with both regions showing strong structural similarities to mammalian digestive carboxypeptidases. The pro-region remains bound to the enzyme after activation, which is probably carried out by a trypsin-like proteinase [14]; subsequent digestion of the pro-region in the gut or molting fluid would result in full activation of the carboxypeptidase A family enzymes. The structure of the inhibitor-insensitive carboxypeptidase B enzyme is very similar, with only minor changes to surface loops accounting for its insensitivity to inhibition by potato carboxypeptidase inhibitor [11]. In contrast, the structure of a splice variant of the carboxypeptidase D contains no pro-region,
Clan MC
M14A | 305. Insect Carboxypeptidases
having an N-terminal catalytic domain of 311 residues, and a C-terminal regulatory domain of 84 residues [13]. The activity of the enzyme is proposed to be regulated by localization at cell membranes, using exposed cysteine side chains as sites for farnesylation to provide one or more membrane anchors, and by proteolysis in exposed loops. The structure of this enzyme can again be fitted to its mammalian orthologs.
Preparation The purification of an insect carboxypeptidase by a multistep process, culminating in preparative electrophoresis, described by Ward [1] was a considerable achievement, but is not practical for routine use. Affinity chromatography on immobilized potato carboxypeptidase inhibitor has been used to purify mammalian carboxypeptidases, and can be used for insect enzymes, although the denaturing conditions necessary for elution normally result in loss of activity [4]. The inhibitor-insensitive carboxypeptidase B from bollworm can be successfully purified by this method as it does not require denaturing conditions for elution [15]. Recombinant protein expression systems have superseded earlier methods as the technique of choice for preparation of functionally active insect carboxypeptidases; the ease with which cDNAs encoding specific proteins can be isolated from insects has made this technology feasible. Full-length cDNAs are used as templates for preparing expression constructs, which can be used to express native or modified protein sequences. Digestive carboxypeptidase A from bollworm has been expressed in insect cells as a native protein, using baculovirus vectors containing the entire preproprotein coding sequence, although amounts were not sufficient for subsequent purification [12]. The molting carboxypeptidase A from silkworm was also expressed using this system, again only yielding small amounts of product, which was purified by gel filtrationHPLC [6]. Similar methods have been used to produce small amounts of Drosophila carboxypeptidase D [8]. The yeast Pichia pastoris can be used to produce quantities of insect carboxypeptidases sufficient for full functional and structural analysis. Carboxypeptidases A [14] and B [11], glutamate carboxypeptidase [4] from bollworm, and carboxypeptidase D from Drosophila [13] have been produced using this system. The proproteins are expressed as fusions with an N-terminal yeast peptide (α-mating factor) which directs secretion of the processed recombinant proprotein out of the yeast cells [4,11,13,14]. The recombinant protein can be purified from the culture supernatant by hydrophobic interaction chromatography [11,14], or by affinity chromatography using a fused ‘tag’ sequence at the C-terminus of the protein [4,13]. Yields of recombinant protein are up to 5 mg l21 of culture supernatant [4].
Clan MC
M14A | 305. Insect Carboxypeptidases
Biological Aspects The roles played by carboxypeptidases in insects have been outlined above. Insect digestive carboxypeptidases conform to the general principle of digestive adaptation; the presence of large gene families of digestive proteinases allows adaptation to a range of different foodstuffs. The presence of digestive glutamate carboxypeptidases in some insects exemplifies this adaptation process. Carboxypeptidase gene expression responds to changes in the dietary status in many insects; for example, 11 of the mosquito (Diptera: Aedes aegypti) carboxypeptidase genes were induced in the midgut in response to blood meal feeding, with increases in mRNA level up to 40-fold [16]. The endogenous carboxypeptidase A promoter has been used to drive RNAi expression in transgenic mosquitoes to produce endogenous resistance to dengue virus [17]. Carboxypeptidases are also involved in responses to protein proteinase inhibitors present in the diet; carboxypeptidase A gene expression is upregulated when bollworm larvae are fed protein proteinase inhibitors active against serine proteinases [12]. Feeding potato carboxypeptidase inhibitor downregulates expression of the gene encoding inhibitor-sensitive carboxypeptidase A in bollworm, but has no effect on inhibitor-insensitive carboxypeptidase B gene expression [15]. Protein carboxypeptidase inhibitors have no negative effects on insect mortality or growth, whether added to diet or expressed in transgenic plants [15,18]. Carboxypeptidases have been shown to be present in the epidermis of bollworm larvae [19], but the molting carboxypeptidase A in silkworm is specifically upregulated during the molt, and is present in the molting fluid between the old cuticle and the epidermis during molting. The enzyme is activated in the fluid after secretion [6]. Besides degrading proteins in the old cuticle to allow separation from the epidermis, the enzyme may be involved in recycling amino acids for synthesis of new proteins. The biological functions of carboxypeptidase D-type enzymes in insects are diverse. The svr gene encoding carboxypeptidase D is required for viability in Drosophila, but many non-lethal mutants are known with varying phenotypes (the gene symbol refers to the phenotype silver, where cuticle shows a lack of blackening due to melanization). Overexpression of carboxypeptidase D domains in transgenic flies decreased levels of partially processed hormone intermediates, confirming the role of this enzyme in proprotein processing [20]; the varying phenotypes observed in mutants of this gene reflect the multiple protein and peptide substrates the enzyme is active against. Besides metallocarboxypeptidases, insects also contain serine carboxypeptidases (carboxypeptidase C-type), which have been detected in salivary glands and fat
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bodies of wheat blossom midge (Diptera; Sitodiplosis mosellana) [21]. These proteins may play roles in digestion, both in the gut, and in turnover of vitellogenin, a precursor protein for egg yolk used as a storage protein in insects. They are also present in venoms in bees, wasps and other insects and arthropods. Detailed characterization of these proteins has yet to be carried out.
Further Reading For structure of insect digestive carboxypeptidase A, see Estebanez-Perpina et al. [14]. For insect digestive glutamate carboxypeptidase, see Bown & Gatehouse [4]. For inhibitor-insensitive insect digestive carboxypeptidase B, see Bayes et al. [11], and for molting fluid carboxypeptidase, see Ote et al. [6].
References [1] Ward, C.W. (1976). Properties of the major carboxypeptidase in the larvae of the webbing clothes moth Tineolla bisselliella. Biochim. Biophys. Acta 429, 564 572. [2] Gooding, R.H., Rolset, B.M. (1976). Digestive processes of hematophagous insects. 11. Partial purification and some properties of 6 proteolytic enzymes from tsetse fly Glossinia morsitans morsitans Westwood. Canadian J. Zoology 54, 1950 1959. [3] Terra, W.R., Ferreira, C. (1994). Insect digestive enzymes: properties, compartmentalization and function. Comp. Biochem. Physiol. B 109, 1 62. [4] Bown, D.P., Gatehouse, J.A. (2004). Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues. Eur. J. Biochem. 271, 2000 2011. [5] Brookhart, G.L., Kramer, K.J. (1990). Proteinases in molting fluid of the tobacco hornworm, Manduca sexta. Insect Biochem. 20, 467 477. [6] Ote, M., Mita, K., Kawasaki, H., Daimon, T., Kobayashi, M., Shimada, T. (2005). Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori. Comp. Biochem. Physiol. B 141, 314 322. [7] Settle, S.H., Green, M.M., Burtis, K.C. (1995). The silver gene of Drosophila melanogaster encodes multiple carboxypeptidases similar to mammalian prohormone-processing enzymes. Proc. Natl. Acad. Sci. USA 92, 9470 9474. [8] Sidyelyeva, G., Fricker, L.D. (2002). Characterization of Drosophila carboxypeptidase D. J. Biol. Chem. 277, 49613 49620. [9] Chang, C.I., Pili-Floury, S., Herve, M., Parquet, C., Chelliah, Y., Lemaitre, B., Mengin-Lecreulx, D., Deisenhofer, J. (2004). A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D-carboxypeptidase activity. PLOS Biology 2, 1293 1302. [10] Lavazec, C., Bonnet, S., Thiery, I., Boisson, B., Bourgouin, C. (2005). cpbAg1 encodes an active carboxypeptidase B expressed in the midgut of Anopheles gambiae. Insect Mol. Biol. 14, 163 174. [11] Bayes, A., Comellas-Bigler, M., de la Vega, M.R., Maskos, K., Bode, W., Aviles, F.X., Jongsma, M.A., Beekwilder, J.,
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[12]
[13]
[14]
[15]
[16]
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Vendrell, J. (2005). Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors. Proc. Natl. Acad. Sci. USA 102, 16602 16607. Bown, D.P., Wilkinson, H.S., Gatehouse, J.A. (1998). Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression. Insect Biochem. Mol. Biol. 28, 739 749. Tanco, S., Arolas, J.L., Guevara, T., Lorenzo, J., Aviles, F.X., Gomis-Ruth, F.X. (2010). Structure-function analysis of the short splicing variant carboxypeptidase encoded by Drosophila melanogaster silver. J. Mol. Biol. 401, 465 477. Estebanez-Perpina, E., Bayes, A., Vendrell, J., Jongsma, M.A., Bown, D.P., Gatehouse, J.A., Huber, R., Bode, W., Aviles, F.X., Reverter, D. (2001). Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera. J. Mol. Biol. 313, 629 638. Bayes, A., de la Vega, M.R., Vendrell, J., Aviles, F.X., Jongsma, M.A., Beekwilder, J. (2006). Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B. Insect Biochem. Mol. Biol. 36, 654 664. Isoe, J., Zamora, J., Miesfeld, R.L. (2009). Molecular analysis of the Aedes aegypti carboxypeptidase gene family. Insect Biochem. Mol. Biol. 39, 68 73.
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[17] Franz, A.W.E., Sanchez-Vargas, I., Adelman, Z.N., Blair, C.D., Beaty, B.J., James, A.A., Olson, K.E. (2006). Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. Proc. Natl. Acad. Sci. USA 103, 4198 4203. [18] Quilis, J., Meynard, D., Vila, L., Aviles, F.X., Guiderdoni, E., San Segundo, B. (2007). A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice. Plant Biotech. J. 5, 537 553. [19] Dong, D.-J., He, H.-J., Chai, L.-Q., Jiang, X.-J., Wang, J.-X., Zhao, X.-F. (2007). Identification of genes differentially expressed during larval molting and metamorphosis of Helicoverpa armigera. BMC Dev. Biol. 7, 73. [20] Sidyelyeva, G., Wegener, C., Schoenfeld, B.P., Bell, A.J., Baker, N.E., McBride, S.M.J., Fricker, L.D. (2010). Individual carboxypeptidase D domains have both redundant and unique functions in Drosophila development and behavior. Cell. Mol. Life Sci. 67, 2991 3004. [21] Mittapalli, O., Wise, I.L., Shukle, R.H. (2006). Characterization of a serine carboxypeptidase in the salivary glands and fat body of the orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae). Insect Biochem. Mol. Biol. 36, 154 160.
John A. Gatehouse School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00306-9
[82]
[83]
[84]
[85]
[86]
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(CGRP), and neuropeptide Y by vascular smooth muscle cells. Peptides 17(4), 709 720. Palmieri, F.E., Bausback, H.H., Churchill, L., Ward, P.E. (1986). Kinin and enkephalin conversion by an endothelial, plasma membrane carboxypeptidase. Biochem. Pharmacol. 35(16), 2749 2756. Zhang, Y., Brovkovych, V., Brovkovych, S., Tan, F., Lee, B.S., Sharma, T., Skidgel, R.A. (2007). Dynamic receptor-dependent activation of inducible nitric-oxide synthase by ERK-mediated phosphorylation of Ser745. J. Biol. Chem. 282(44), 32453 32461. Brovkovych, V., Zhang, Y., Brovkovych, S., Minshall, R.D., Skidgel, R.A. (2011). A novel pathway for receptor-mediated posttranslational activation of inducible nitric oxide synthase. J. Cell. Mol. Med. 15(2), 258 269. Skidgel, R.A., Stanisavljevic, S., Erdo¨s, E.G. (2006). Kinin- and angiotensin-converting enzyme (ACE) inhibitor-mediated nitric oxide production in endothelial cells. Biol. Chem. 387(2), 159 165. Zhang, X., Tan, F., Brovkovych, V., Zhang, Y., Skidgel, R.A. (2011). Crosstalk between carboxypeptidase M and the kinin B1 receptor mediates a new mode of G protein-coupled receptor signaling. J. Biol. Chem. .
M14A | 305. Insect Carboxypeptidases
[87] Lendeckel, U., Arndt, M., Wrenger, S., Nepple, K., Huth, C., Ansorge, S., Klein, H.U., Goette, A. (2001). Expression and activity of ectopeptidases in fibrillating human atria. J. Mol. Cell. Cardiol. 33(6), 1273 1281. [88] Hall, J.L., Grindle, S., Han, X., Fermin, D., Park, S., Chen, Y., Bache, R.J., Mariash, A., Guan, Z., Ormaza, S., Thompson, J., Graziano, J., de Sam Lazaro, S.E., Pan, S., Simari, R.D., Miller, L.W. (2004). Genomic profiling of the human heart before and after mechanical support with a ventricular assist device reveals alterations in vascular signaling networks. Physiol. Genomics 17(3), 283 291. [89] Milligan, G., Smith, N.J. (2007). Allosteric modulation of heterodimeric G-protein-coupled receptors. Trends Pharmacol. Sci. 28(12), 615 620. [90] Song, L., Fricker, L.D. (1995). Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J. Biol. Chem. 270(42), 25007 25013. [91] Reznik, S.E., Fricker, L.D. (2001). Carboxypeptidases from A to Z: implications in embryonic development and Wnt binding. Cell. Mol. Life Sci. 58(12 13), 1790 1804.
Xianming Zhang Department of Pharmacology, University of Illinois College of Medicine at Chicago, 835 S. Wolcott Ave. (m/c 868), Chicago, IL 60612, USA. Email: [email protected]
Randal A. Skidgel Department of Pharmacology, University of Illinois College of Medicine at Chicago, 835 S. Wolcott Ave. (m/c 868), Chicago, IL 60612, USA. Email: [email protected] © 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00305-7
Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
Chapter 305
Insect Carboxypeptidases DATABANKS MEROPS name: insect gut carboxypeptidase-1 MEROPS classification: clan MC, family M14, subfamily M14A, peptidase M14.024 Tertiary structure: Available Species distribution: order Lepidoptera Reference sequence from: Helicoverpa armigera MEROPS name: insect gut carboxypeptidase-2 MEROPS classification: clan MC, family M14, subfamily M14A, peptidase M14.031
Species distribution: family Noctuidae Reference sequence from: Helicoverpa armigera
Name and History The presence of carboxypeptidases in insects was unambiguously demonstrated in 1976 by the purification of a protein with carboxypeptidase activity from clothes moth (Lepidoptera; Tineolla bisselliella) [1], and partial purification of enzymes with carboxypeptidase A and B activities from Tsetse fly (Diptera; Glossinia morsitans) [2].
Clan MC
M14A | 305. Insect Carboxypeptidases
Subsequent work identified carboxypeptidases in other insect orders, including Hymenoptera, Coleoptera, and Hemiptera, and evidence suggests they are found in all insects (reviewed in Terra & Ferreira [3]). Similar activities are present in other arthropods, and had been identified before carboxypeptidases in insects. Carboxypeptidase activity is widely present in insect digestive systems, since most insects rely on protein digestion to provide nitrogen necessary for biosynthesis. Digestive carboxypeptidases in insects have been classified as similar in activity to mammalian carboxypeptidase A (Chapter 289) and B (Chapter 296), as determined by assays with synthetic substrates (see below). However, proteolytic and other hydrolytic enzymes can vary greatly between different orders of insects, and even between different species within an order, reflecting specialization in feeding strategies. Consequently, some insects contain digestive carboxypeptidases with sequence similarity to the carboxypeptidase A family, but with activity specific to C-terminal glutamate [4]; these enzymes are referred to as ‘glutamate carboxypeptidases’ in this chapter. Carboxypeptidases are also involved in the molting process in insects, where proteolytic activity is necessary to separate the old exoskeleton from the underlying epidermal cells. Carboxypeptidase activity was shown to be present in molting fluid in tobacco hornworm (Lepidoptera; Manduca sexta) [5], and a molting enzyme with carboxypeptidase A-like activity is present in silk worm (Lepidoptera; Bombyx mori) [6]. Carboxypeptidases in higher animals are also involved in processing of preproteins and peptide hormones, and insects contain genes encoding enzymes predicted to show these activities. The fruit fly (Diptera; Drosophila melanogaster) svr gene encodes a protein with similarities to carboxypeptidases E, M and N [7] containing multiple carboxypeptidase domains like duck carboxypeptidase D (Chapter 303) [8]. Similar proteins are present in other insects. Carboxypeptidase activity may also be involved in signaling pathways and pattern recognition [9]. The availability of complete genome sequences for a number of insects has allowed carboxypeptidase gene families to be analyzed, demonstrating considerable diversity in the numbers and activities of carboxypeptidases present. For example, the mosquito (Diptera; Anopheles gambiae) genome predicts 23 sequences coding for zinc-carboxypeptidases [10]. The Drosophila genome contains 20 genes of the carboxypeptidase A subfamily, six predicted to show carboxypeptidase A activity, five carboxypeptidase B activity, four glutamate carboxypeptidase activity, and five predicted to be inactive [4]; in addition, two genes encoding proteins of the multidomain carboxypeptidase D type are present. In contrast, the genome in pea aphid (Hemiptera; Acyrthosiphon pisum) contains only two carboxypeptidase A-type genes, one annotated as a molting carboxypeptidase A enzyme,
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the other as a carboxypeptidase B enzyme. The absence of a multigene family encoding digestive carboxypeptidases reflects the minimal role protein digestion plays in this insect, which derives nitrogen from free amino acids in its diet, plant sap.
Activity and Specificity Carboxypeptidase activity in extracts from insect tissues has been detected with synthetic substrates used for assaying mammalian enzymes, such as N-(3-[2-furyl] acryloyl)-Phe-Phe (FAPP) and N-(3-[2-furyl]acryloyl)Ala-Lys (FAAK). However, these assays are not entirely satisfactory, due to lack of sensitivity and interference by extract components, and reliable data for specificity and kinetic constants for enzymes can only be obtained from purified proteins. The small size of many insects makes purification of carboxypeptidases technically challenging, and recombinant expression systems are necessary to produce pure proteins for study. The data available suggest that specificities of insect carboxypeptidases A and B are broadly similar to their mammalian counterparts, although a systematic study has yet to be undertaken. Some carboxypeptidase A enzymes may differ slightly in specificity from mammalian enzymes due to differing active site residues (see below). Similarly, a carboxypeptidase B has been identified in bollworm (Lepidoptera; Helicoverpa armigera) that is highly specific for C-terminal Lys residues [11]. The insect glutamate carboxypeptidase has a high degree of specificity towards C-terminal Glu residues. It cleaves the synthetic substrate N-(3-[2-furyl]acryloyl)-Glu-Glu (FAEE), but not FAPP or FAAK, and will remove Cterminal glutamate residues from peptides [4]. Very low levels of activity towards C-terminal Asp in peptides were also observed. Gut conditions in insects are highly variable, with lepidoptera having high gut pH (up to 10.5), whereas some dipteran species have regions in the gut where local pH is as low as 3.0. However, where pH optima for insect carboxypeptidases have been determined, there is generally a broad pH optimum in the alkaline range; for example, the digestive carboxypeptidase A enzyme from bollworm (Lepidoptera; Helicoverpa armigera) has an optimal range of pH 7.5 10 [12]. Insect metallocarboxypeptidases are inhibited by zinc chelators such as 1,10-phenanthroline, like their mammalian counterparts; the digestive enzymes are generally strongly inhibited by specific protein proteinase inhibitors, such as potato carboxypeptidase inhibitor, with the resulting complexes only dissociable under denaturing conditions [4]. An exception is the Lys-specific carboxypeptidase B in bollworm, which is insensitive to this inhibitor [11]. The carboxypeptidase D-type enzymes are not inhibited by these protein proteinase inhibitors [13].
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Structural Chemistry Sequences for many insect metallocarboxypeptidases have been deduced from cDNA sequences, or genome sequences. They have predicted signal peptides, as expected for secreted enzymes, and are of similar size to their mammalian counterparts, with a pro-region of approximately 90 amino acids, and an active enzyme domain of approximately 300 amino acids. The sequences of digestive and molting carboxypeptidases can be mapped onto the ‘model’ sequence for human carboxypeptidase A, using sequence similarity, which shows that the features necessary for zinc binding and catalysis are present; His69, Glu72 and His196, residues which bind the catalytic zinc ion; Arg71, Arg145, Tyr248 and Glu270, residues responsible for substrate binding and cleavage (using mammalian carboxypeptidase A numbering). The presence of genes predicting proteins with strong sequence similarity to carboxypeptidases in the Drosophila genome, which would be inactive as enzymes due to the absence of some of these key residues has been noted above. X255, the residue which determines substrate specificity, is neutral in carboxypeptidase A enzymes, to interact with neutral hydrophobic side chains on the substrate, but besides the hydrophobic residues found in mammalian carboxypeptidases, can be Ser, potentially extending the substrate range of the enzymes [14]. X255 is Asp in most carboxypeptidase B enzymes, and is Lys or Arg in glutamate carboxypeptidases, to interact with basic and acidic side chains on the substrate, respectively [4]. The lysine-specific carboxypeptidase B from bollworm has a Glu residue at position 255, which is partly responsible for its substrate specificity [11]. The carboxypeptidase D-type enzymes have a complex multidomain structure with large numbers of variants generated by alternate splicing. Crystal structures are available for carboxypeptidase A- and B-type proteases from bollworm (Lepidoptera; Helicoverpa armigera) [11,14], and for one domain of the carboxypeptidase D-type enzyme from Drosophila [13]. The insect carboxypeptidase A contains a pro-region of 91 residues, and an active enzyme of 318 residues, with both regions showing strong structural similarities to mammalian digestive carboxypeptidases. The pro-region remains bound to the enzyme after activation, which is probably carried out by a trypsin-like proteinase [14]; subsequent digestion of the pro-region in the gut or molting fluid would result in full activation of the carboxypeptidase A family enzymes. The structure of the inhibitor-insensitive carboxypeptidase B enzyme is very similar, with only minor changes to surface loops accounting for its insensitivity to inhibition by potato carboxypeptidase inhibitor [11]. In contrast, the structure of a splice variant of the carboxypeptidase D contains no pro-region,
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having an N-terminal catalytic domain of 311 residues, and a C-terminal regulatory domain of 84 residues [13]. The activity of the enzyme is proposed to be regulated by localization at cell membranes, using exposed cysteine side chains as sites for farnesylation to provide one or more membrane anchors, and by proteolysis in exposed loops. The structure of this enzyme can again be fitted to its mammalian orthologs.
Preparation The purification of an insect carboxypeptidase by a multistep process, culminating in preparative electrophoresis, described by Ward [1] was a considerable achievement, but is not practical for routine use. Affinity chromatography on immobilized potato carboxypeptidase inhibitor has been used to purify mammalian carboxypeptidases, and can be used for insect enzymes, although the denaturing conditions necessary for elution normally result in loss of activity [4]. The inhibitor-insensitive carboxypeptidase B from bollworm can be successfully purified by this method as it does not require denaturing conditions for elution [15]. Recombinant protein expression systems have superseded earlier methods as the technique of choice for preparation of functionally active insect carboxypeptidases; the ease with which cDNAs encoding specific proteins can be isolated from insects has made this technology feasible. Full-length cDNAs are used as templates for preparing expression constructs, which can be used to express native or modified protein sequences. Digestive carboxypeptidase A from bollworm has been expressed in insect cells as a native protein, using baculovirus vectors containing the entire preproprotein coding sequence, although amounts were not sufficient for subsequent purification [12]. The molting carboxypeptidase A from silkworm was also expressed using this system, again only yielding small amounts of product, which was purified by gel filtrationHPLC [6]. Similar methods have been used to produce small amounts of Drosophila carboxypeptidase D [8]. The yeast Pichia pastoris can be used to produce quantities of insect carboxypeptidases sufficient for full functional and structural analysis. Carboxypeptidases A [14] and B [11], glutamate carboxypeptidase [4] from bollworm, and carboxypeptidase D from Drosophila [13] have been produced using this system. The proproteins are expressed as fusions with an N-terminal yeast peptide (α-mating factor) which directs secretion of the processed recombinant proprotein out of the yeast cells [4,11,13,14]. The recombinant protein can be purified from the culture supernatant by hydrophobic interaction chromatography [11,14], or by affinity chromatography using a fused ‘tag’ sequence at the C-terminus of the protein [4,13]. Yields of recombinant protein are up to 5 mg l21 of culture supernatant [4].
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Biological Aspects The roles played by carboxypeptidases in insects have been outlined above. Insect digestive carboxypeptidases conform to the general principle of digestive adaptation; the presence of large gene families of digestive proteinases allows adaptation to a range of different foodstuffs. The presence of digestive glutamate carboxypeptidases in some insects exemplifies this adaptation process. Carboxypeptidase gene expression responds to changes in the dietary status in many insects; for example, 11 of the mosquito (Diptera: Aedes aegypti) carboxypeptidase genes were induced in the midgut in response to blood meal feeding, with increases in mRNA level up to 40-fold [16]. The endogenous carboxypeptidase A promoter has been used to drive RNAi expression in transgenic mosquitoes to produce endogenous resistance to dengue virus [17]. Carboxypeptidases are also involved in responses to protein proteinase inhibitors present in the diet; carboxypeptidase A gene expression is upregulated when bollworm larvae are fed protein proteinase inhibitors active against serine proteinases [12]. Feeding potato carboxypeptidase inhibitor downregulates expression of the gene encoding inhibitor-sensitive carboxypeptidase A in bollworm, but has no effect on inhibitor-insensitive carboxypeptidase B gene expression [15]. Protein carboxypeptidase inhibitors have no negative effects on insect mortality or growth, whether added to diet or expressed in transgenic plants [15,18]. Carboxypeptidases have been shown to be present in the epidermis of bollworm larvae [19], but the molting carboxypeptidase A in silkworm is specifically upregulated during the molt, and is present in the molting fluid between the old cuticle and the epidermis during molting. The enzyme is activated in the fluid after secretion [6]. Besides degrading proteins in the old cuticle to allow separation from the epidermis, the enzyme may be involved in recycling amino acids for synthesis of new proteins. The biological functions of carboxypeptidase D-type enzymes in insects are diverse. The svr gene encoding carboxypeptidase D is required for viability in Drosophila, but many non-lethal mutants are known with varying phenotypes (the gene symbol refers to the phenotype silver, where cuticle shows a lack of blackening due to melanization). Overexpression of carboxypeptidase D domains in transgenic flies decreased levels of partially processed hormone intermediates, confirming the role of this enzyme in proprotein processing [20]; the varying phenotypes observed in mutants of this gene reflect the multiple protein and peptide substrates the enzyme is active against. Besides metallocarboxypeptidases, insects also contain serine carboxypeptidases (carboxypeptidase C-type), which have been detected in salivary glands and fat
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bodies of wheat blossom midge (Diptera; Sitodiplosis mosellana) [21]. These proteins may play roles in digestion, both in the gut, and in turnover of vitellogenin, a precursor protein for egg yolk used as a storage protein in insects. They are also present in venoms in bees, wasps and other insects and arthropods. Detailed characterization of these proteins has yet to be carried out.
Further Reading For structure of insect digestive carboxypeptidase A, see Estebanez-Perpina et al. [14]. For insect digestive glutamate carboxypeptidase, see Bown & Gatehouse [4]. For inhibitor-insensitive insect digestive carboxypeptidase B, see Bayes et al. [11], and for molting fluid carboxypeptidase, see Ote et al. [6].
References [1] Ward, C.W. (1976). Properties of the major carboxypeptidase in the larvae of the webbing clothes moth Tineolla bisselliella. Biochim. Biophys. Acta 429, 564 572. [2] Gooding, R.H., Rolset, B.M. (1976). Digestive processes of hematophagous insects. 11. Partial purification and some properties of 6 proteolytic enzymes from tsetse fly Glossinia morsitans morsitans Westwood. Canadian J. Zoology 54, 1950 1959. [3] Terra, W.R., Ferreira, C. (1994). Insect digestive enzymes: properties, compartmentalization and function. Comp. Biochem. Physiol. B 109, 1 62. [4] Bown, D.P., Gatehouse, J.A. (2004). Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues. Eur. J. Biochem. 271, 2000 2011. [5] Brookhart, G.L., Kramer, K.J. (1990). Proteinases in molting fluid of the tobacco hornworm, Manduca sexta. Insect Biochem. 20, 467 477. [6] Ote, M., Mita, K., Kawasaki, H., Daimon, T., Kobayashi, M., Shimada, T. (2005). Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori. Comp. Biochem. Physiol. B 141, 314 322. [7] Settle, S.H., Green, M.M., Burtis, K.C. (1995). The silver gene of Drosophila melanogaster encodes multiple carboxypeptidases similar to mammalian prohormone-processing enzymes. Proc. Natl. Acad. Sci. USA 92, 9470 9474. [8] Sidyelyeva, G., Fricker, L.D. (2002). Characterization of Drosophila carboxypeptidase D. J. Biol. Chem. 277, 49613 49620. [9] Chang, C.I., Pili-Floury, S., Herve, M., Parquet, C., Chelliah, Y., Lemaitre, B., Mengin-Lecreulx, D., Deisenhofer, J. (2004). A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D-carboxypeptidase activity. PLOS Biology 2, 1293 1302. [10] Lavazec, C., Bonnet, S., Thiery, I., Boisson, B., Bourgouin, C. (2005). cpbAg1 encodes an active carboxypeptidase B expressed in the midgut of Anopheles gambiae. Insect Mol. Biol. 14, 163 174. [11] Bayes, A., Comellas-Bigler, M., de la Vega, M.R., Maskos, K., Bode, W., Aviles, F.X., Jongsma, M.A., Beekwilder, J.,
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[12]
[13]
[14]
[15]
[16]
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Vendrell, J. (2005). Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors. Proc. Natl. Acad. Sci. USA 102, 16602 16607. Bown, D.P., Wilkinson, H.S., Gatehouse, J.A. (1998). Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression. Insect Biochem. Mol. Biol. 28, 739 749. Tanco, S., Arolas, J.L., Guevara, T., Lorenzo, J., Aviles, F.X., Gomis-Ruth, F.X. (2010). Structure-function analysis of the short splicing variant carboxypeptidase encoded by Drosophila melanogaster silver. J. Mol. Biol. 401, 465 477. Estebanez-Perpina, E., Bayes, A., Vendrell, J., Jongsma, M.A., Bown, D.P., Gatehouse, J.A., Huber, R., Bode, W., Aviles, F.X., Reverter, D. (2001). Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera. J. Mol. Biol. 313, 629 638. Bayes, A., de la Vega, M.R., Vendrell, J., Aviles, F.X., Jongsma, M.A., Beekwilder, J. (2006). Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B. Insect Biochem. Mol. Biol. 36, 654 664. Isoe, J., Zamora, J., Miesfeld, R.L. (2009). Molecular analysis of the Aedes aegypti carboxypeptidase gene family. Insect Biochem. Mol. Biol. 39, 68 73.
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[17] Franz, A.W.E., Sanchez-Vargas, I., Adelman, Z.N., Blair, C.D., Beaty, B.J., James, A.A., Olson, K.E. (2006). Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. Proc. Natl. Acad. Sci. USA 103, 4198 4203. [18] Quilis, J., Meynard, D., Vila, L., Aviles, F.X., Guiderdoni, E., San Segundo, B. (2007). A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice. Plant Biotech. J. 5, 537 553. [19] Dong, D.-J., He, H.-J., Chai, L.-Q., Jiang, X.-J., Wang, J.-X., Zhao, X.-F. (2007). Identification of genes differentially expressed during larval molting and metamorphosis of Helicoverpa armigera. BMC Dev. Biol. 7, 73. [20] Sidyelyeva, G., Wegener, C., Schoenfeld, B.P., Bell, A.J., Baker, N.E., McBride, S.M.J., Fricker, L.D. (2010). Individual carboxypeptidase D domains have both redundant and unique functions in Drosophila development and behavior. Cell. Mol. Life Sci. 67, 2991 3004. [21] Mittapalli, O., Wise, I.L., Shukle, R.H. (2006). Characterization of a serine carboxypeptidase in the salivary glands and fat body of the orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae). Insect Biochem. Mol. Biol. 36, 154 160.
John A. Gatehouse School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00306-9