BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.

Center for Discovery and Innovation in Parasitic Diseases Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego 9500 Gilman Dr La Jolla CA 92093 USA

Department of Parasitology Faculty of Science Charles University Viničná 7 12844 Prague 2 Czech Republic

Department of Zoology and Fisheries Center of Infectious Animal Diseases Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Kamýcká 129 16521 Prague 6 Czech Republic

Faculty of Environmental Sciences Czech University of Life Sciences Kamýcká 129 16521 Prague 6 Czech Republic

Institute of Organic Chemistry and Biochemistry The Czech Academy of Sciences Flemingovo N 2 16610 Prague 6 Czech Republic

Laboratory of Structural Biology Institute of Biotechnology Czech Academy of Sciences BIOCEV Průmyslová 595 252 42 Vestec Czech Republic

Zobrazit více v PubMed

Lockyer AE, Olson PD, Ostergaard P, Rollinson D, Johnston DA, Attwood SW, et al. The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858. Parasitology. 2003;126:203–224. doi: 10.1017/S0031182002002792. PubMed DOI

Caffrey CR. Schistosomiasis and its treatment. Future Med Chem. 2015;7:675–676. doi: 10.4155/fmc.15.27. PubMed DOI

Wilson RA. Schistosomiasis then and now: what has changed in the last 100 years? Parasitology. 2020;147:507–515. doi: 10.1017/S0031182020000049. PubMed DOI PMC

Pearce EJ, MacDonald AS. The immunobiology of schistosomiasis. Nat Rev Immunol. 2002;2:499–511. doi: 10.1038/nri843. PubMed DOI

Sombetzki M, Rabes A, Bischofsberger M, Winkelmann F, Koslowski N, Schulz C, et al. Preventive CTLA-4-Ig treatment reduces hepatic egg load and hepatic fibrosis in Schistosoma mansoni-infected mice. Biomed Res Int. 2019;2019:1704238. doi: 10.1155/2019/1704238. PubMed DOI PMC

Da'dara A, Skelly PJ. Manipulation of vascular function by blood flukes? Blood Rev. 2011;25:175–179. doi: 10.1016/j.blre.2011.04.002. PubMed DOI PMC

Mas-Coma S, Bargues MD, Valero MA. Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol. 2005;35:1255–1278. doi: 10.1016/j.ijpara.2005.07.010. PubMed DOI

Piedrafita D, Spithill TW, Smith RE, Raadsma HW. Improving animal and human health through understanding liver fluke immunology. Parasite Immunol. 2010;32:572–581. PubMed

Fairweather I. Triclabendazole: new skills to unravel an old(ish) enigma. J Helminthol. 2005;79:227–234. doi: 10.1079/JOH2005298. PubMed DOI

Mas-Coma S, Valero MA, Bargues MD. Climate change effects on trematodiases, with emphasis on zoonotic fascioliasis and schistosomiasis. Vet Parasitol. 2009;163:264–280. doi: 10.1016/j.vetpar.2009.03.024. PubMed DOI

Howe KL, Bolt BJ, Shafie M, Kersey P, Berriman M. WormBase ParaSite—a comprehensive resource for helminth genomics. Mol Biochem Parasitol. 2017;215:2–10. doi: 10.1016/j.molbiopara.2016.11.005. PubMed DOI PMC

Wendt G, Zhao L, Chen R, Liu C, O'Donoghue AJ, Caffrey CR, et al. A single-cell RNA-seq atlas of Schistosoma mansoni identifies a key regulator of blood feeding. Science. 2020;369:1644–1649. doi: 10.1126/science.abb7709. PubMed DOI PMC

Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 2018;46:D624–D632. doi: 10.1093/nar/gkx1134. PubMed DOI PMC

Lambert LA, Mitchell SL. Molecular evolution of the transferrin receptor/glutamate carboxypeptidase II family. J Mol Evol. 2007;64:113–128. doi: 10.1007/s00239-006-0137-4. PubMed DOI

Tykvart J, Barinka C, Svoboda M, Navratil V, Soucek R, Hubalek M, et al. Structural and biochemical characterization of a novel aminopeptidase from human intestine. J Biol Chem. 2015;290:11321–11336. doi: 10.1074/jbc.M114.628149. PubMed DOI PMC

Tykvart J, Schimer J, Barinkova J, Pachl P, Postova-Slavetinska L, Majer P, et al. Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery. Bioorg Med Chem. 2014;22:4099–4108. doi: 10.1016/j.bmc.2014.05.061. PubMed DOI

Hlouchova K, Navratil V, Tykvart J, Sacha P, Konvalinka J. GCPII variants, paralogs and orthologs. Curr Med Chem. 2012;19:1316–1322. doi: 10.2174/092986712799462676. PubMed DOI

Rovenska M, Hlouchova K, Sacha P, Mlcochova P, Horak V, Zamecnik J, et al. Tissue expression and enzymologic characterization of human prostate specific membrane antigen and its rat and pig orthologs. Prostate. 2008;68:171–182. doi: 10.1002/pros.20676. PubMed DOI

Silver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 1997;3:81–85. PubMed

Navratil M, Ptacek J, Sacha P, Starkova J, Lubkowski J, Barinka C, et al. Structural and biochemical characterization of the folyl-poly-gamma-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II. FEBS J. 2014;281:3228–3242. doi: 10.1111/febs.12857. PubMed DOI PMC

Slusher BS, Vornov JJ, Thomas AG, Hurn PD, Harukuni I, Bhardwaj A, et al. Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury. Nat Med. 1999;5:1396–1402. doi: 10.1038/70971. PubMed DOI

Rajasekaran AK, Anilkumar G, Christiansen JJ. Is prostate-specific membrane antigen a multifunctional protein? Am J Physiol Cell Physiol. 2005;288:C975–C981. doi: 10.1152/ajpcell.00506.2004. PubMed DOI

Barinka C, Rojas C, Slusher B, Pomper M. Glutamate carboxypeptidase II in diagnosis and treatment of neurologic disorders and prostate cancer. Curr Med Chem. 2012;19:856–870. doi: 10.2174/092986712799034888. PubMed DOI PMC

Bacich DJ, Ramadan E, O'Keefe DS, Bukhari N, Wegorzewska I, Ojeifo O, et al. Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate. J Neurochem. 2002;83:20–29. doi: 10.1046/j.1471-4159.2002.01117.x. PubMed DOI

Hlouchova K, Barinka C, Konvalinka J, Lubkowski J. Structural insight into the evolutionary and pharmacologic homology of glutamate carboxypeptidases II and III. FEBS J. 2009;276:4448–4462. doi: 10.1111/j.1742-4658.2009.07152.x. PubMed DOI

Jeitner TM, Babich JW, Kelly JM. Advances in PSMA theranostics. Transl Oncol. 2022;22:101450. doi: 10.1016/j.tranon.2022.101450. PubMed DOI PMC

Tsai G, Stauch-Slusher B, Sim L, Hedreen JC, Rothstein JD, Kuncl R, Coyle JT. Reductions in acidic amino acids and N-acetylaspartylglutamate in amyotrophic lateral sclerosis CNS. Brain Res. 1991;556:151–156. doi: 10.1016/0006-8993(91)90560-I. PubMed DOI

Plaitakis A. Glutamate dysfunction and selective motor neuron degeneration in amyotrophc lateral sclerosis A hypothesis. Ann Neurol. 1990;28:3–8. doi: 10.1002/ana.410280103. PubMed DOI

Sacha P, Zamecnik J, Barinka C, Hlouchova K, Vicha A, Mlcochova P, et al. Expression of glutamate carboxypeptidase II in human brain. Neuroscience. 2007;144:1361–1372. doi: 10.1016/j.neuroscience.2006.10.022. PubMed DOI

Fricker AC, Mok MH, de la Flor R, Shah AJ, Woolley M, Dawson LA, et al. Effects of N-acetylaspartylglutamate (NAAG) at group II mGluRs and NMDAR. Neuropharmacology. 2009;56:1060–1067. doi: 10.1016/j.neuropharm.2009.03.002. PubMed DOI

Leontovyc A, Ulrychova L, O'Donoghue AJ, Vondrasek J, Maresova L, Hubalek M, et al. SmSP2: a serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties. PLoS Negl Trop Dis. 2018;12:e0006446. doi: 10.1371/journal.pntd.0006446. PubMed DOI PMC

Dvorak J, Fajtova P, Ulrychova L, Leontovyc A, Rojo-Arreola L, Suzuki BM, et al. Excretion/secretion products from Schistosoma mansoni adults, eggs and schistosomula have unique peptidase specificity profiles. Biochimie. 2016;122:99–109. doi: 10.1016/j.biochi.2015.09.025. PubMed DOI PMC

Stefanic S, Dvorak J, Horn M, Braschi S, Sojka D, Ruelas DS, et al. RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening. PLoS Negl Trop Dis. 2010;4:e850. doi: 10.1371/journal.pntd.0000850. PubMed DOI PMC

Basch PF. Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. J Parasitol. 1981;67(2):179–85. PubMed

Leontovyc A, Ulrychova L, Horn M, Dvorak J. Collection of excretory/secretory products from individual developmental stages of the blood fluke Schistosoma mansoni. Methods Mol Biol. 2020;2151:55–63. doi: 10.1007/978-1-0716-0635-3_5. PubMed DOI

Klusák V, Barinka C, Plechanovová A, Mlcochová P, Konvalinka J, Rulísek L, et al. Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods. Biochemistry. 2009;48:4126–4138. doi: 10.1021/bi900220s. PubMed DOI PMC

Webb B, Sali A. Comparative protein structure modeling using MODELLER. Curr Protoc Bioinform. 2016;54:5.6.1–5.6.37. PubMed PMC

Bim D, Navratil M, Gutten O, Konvalinka J, Kutil Z, Culka M, et al. Predicting effects of site-directed mutagenesis on enzyme kinetics by QM/MM and QM calculations: a case of glutamate carboxypeptidase II. J Phys Chem B. 2022;126:132–143. doi: 10.1021/acs.jpcb.1c09240. PubMed DOI

Ustinova K, Novakova Z, Saito M, Meleshin M, Mikesova J, Kutil Z, et al. The disordered N-terminus of HDAC6 is a microtubule-binding domain critical for efficient tubulin deacetylation. J Biol Chem. 2020;295:2614–2628. doi: 10.1074/jbc.RA119.011243. PubMed DOI PMC

Barinka C, Ptacek J, Richter A, Novakova Z, Morath V, Skerra A. Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA) Protein Eng Des Sel. 2016;29:105–115. doi: 10.1093/protein/gzv065. PubMed DOI

Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–386. PubMed

Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1:1559–1582. doi: 10.1038/nprot.2006.236. PubMed DOI

Horn M, Fajtova P, Arreola LR, Ulrychova L, Bartosova-Sojkova P, Franta Z, et al. Trypsin- and chymotrypsin-like serine proteases in Schistosoma mansoni—'The Undiscovered Country'. PLoS Negl Trop Dis. 2014;8:e2766. doi: 10.1371/journal.pntd.0002766. PubMed DOI PMC

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. PubMed DOI

Ulrychova L, Horn M, Dvorak J. Sensitive fluorescence in situ hybridization on semithin sections of adult Schistosoma mansoni using DIG-labeled RNA probes. Methods Mol Biol. 2020;2151:43–53. doi: 10.1007/978-1-0716-0635-3_4. PubMed DOI

Ulrychova L, Ostasov P, Chanova M, Mares M, Horn M, Dvorak J. Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization. Parasite Vector. 2021;14(1). PubMed PMC

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–675. doi: 10.1038/nmeth.2089. PubMed DOI PMC

Jilkova A, Horn M, Rezacova P, Maresova L, Fajtova P, Brynda J, et al. Activation route of the Schistosoma mansoni cathepsin B1 drug target: structural map with a glycosaminoglycan switch. Structure. 2014;22:1786–1798. doi: 10.1016/j.str.2014.09.015. PubMed DOI

Collins JJ, 3rd, King RS, Cogswell A, Williams DL, Newmark PA. An atlas for Schistosoma mansoni organs and life-cycle stages using cell type-specific markers and confocal microscopy. PLoS Negl Trop Dis. 2011;5:e1009. doi: 10.1371/journal.pntd.0001009. PubMed DOI PMC

Dillon GP, Illes JC, Isaacs HV, Wilson RA. Patterns of gene expression in schistosomes: localization by whole mount in situ hybridization. Parasitology. 2007;134:1589–1597. doi: 10.1017/S0031182007002995. PubMed DOI

Barinka C, Hlouchova K, Rovenska M, Majer P, Dauter M, Hin N, et al. Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs. J Mol Biol. 2008;376:1438–1450. doi: 10.1016/j.jmb.2007.12.066. PubMed DOI PMC

Mesters JR, Barinka C, Li W, Tsukamoto T, Majer P, Slusher BS, et al. Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer. EMBO J. 2006;25:1375–1384. doi: 10.1038/sj.emboj.7600969. PubMed DOI PMC

Barinka C, Rovenska M, Mlcochova P, Hlouchova K, Plechanovova A, Majer P, et al. Structural insight into the pharmacophore pocket of human glutamate carboxypeptidase II. J Med Chem. 2007;50:3267–3273. doi: 10.1021/jm070133w. PubMed DOI

Barinka C, Hlouchova K, Rovenska M, Majer P, Dauter M, Hin N, et al. Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs. J Mol Biol. 2008;376:1438–1450. doi: 10.1016/j.jmb.2007.12.066. PubMed DOI PMC

Barinka C, Rinnova M, Sacha P, Rojas C, Majer P, Slusher BS, et al. Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II. J Neurochem. 2002;80:477–487. doi: 10.1046/j.0022-3042.2001.00715.x. PubMed DOI

Mlcochova P, Plechanovova A, Barinka C, Mahadevan D, Saldanha JW, Rulisek L, et al. Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis. FEBS J. 2007;274:4731–4741. doi: 10.1111/j.1742-4658.2007.06021.x. PubMed DOI

Hlouchova K, Barinka C, Konvalinka J, Lubkowski J. Structural insight into the evolutionary and pharmacologic homology of glutamate carboxypeptidases II and III. FEBS J. 2009;276:4448–4462. doi: 10.1111/j.1742-4658.2009.07152.x. PubMed DOI

Barinka C, Rojas C, Slusher B, Pomper M. Glutamate carboxypeptidase II in diagnosis and treatment of neurologic disorders and prostate cancer. Curr Med Chem. 2012;19:856–870. doi: 10.2174/092986712799034888. PubMed DOI PMC

Pavlicek J, Ptacek J, Barinka C. Glutamate carboxypeptidase II: an overview of structural studies and their importance for structure-based drug design and deciphering the reaction mechanism of the enzyme. Curr Med Chem. 2012;19:1300–1309. doi: 10.2174/092986712799462667. PubMed DOI

Barinka C, Rinnova M, Sacha P, Rojas C, Majer P, Slusher BS, et al. Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II. J Neurochem. 2002;80:477–487. doi: 10.1046/j.0022-3042.2001.00715.x. PubMed DOI

Mesters JR, Barinka C, Li WX, Tsukamoto T, Majer P, Slusher BS, et al. Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer. Embo J. 2006;25:1375–1384. doi: 10.1038/sj.emboj.7600969. PubMed DOI PMC

Wahlestedt C. Natural antisense and noncoding RNA transcripts as potential drug targets. Drug Discov Today. 2006;11:503–508. doi: 10.1016/j.drudis.2006.04.013. PubMed DOI

Vasconcelos EJR, daSilva LF, Pires DS, Lavezzo GM, Pereira ASA, Amaral MS, et al. The Schistosoma mansoni genome encodes thousands of long non-coding RNAs predicted to be functional at different parasite life-cycle stages. Sci Rep. 2017;7:10508. doi: 10.1038/s41598-017-10853-6. PubMed DOI PMC

Maciel LF, Morales-Vicente DA, Silveira GO, Ribeiro RO, Olberg GGO, Pires DS, et al. Weighted gene co-expression analyses point to long non-coding RNA hub genes at different Schistosoma mansoni life-cycle stages. Front Genet. 2019;10:823. doi: 10.3389/fgene.2019.00823. PubMed DOI PMC

Guidi A, Mansour NR, Paveley RA, Carruthers IM, Besnard J, Hopkins AL, et al. Application of RNAi to genomic drug target validation in Schistosomes. PLoS Negl Trop Dis. 2015;9:e0003801. doi: 10.1371/journal.pntd.0003801. PubMed DOI PMC

Robinson MB, Blakely RD, Couto R, Coyle JT. Hydrolysis of the brain dipeptide N-acetyl-l-aspartyl-l-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain. J Biol Chem. 1987;262(30):14498–506. PubMed

Pinto JT, Suffoletto BP, Berzin TM, Qiao CH, Lin S, Tong WP, et al. Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic carcinoma cells. Clin Cancer Res. 1996;2:1445–1451. PubMed

Barinka C, Sacha P, Sklenar J, Man P, Bezouska K, Slusher BS, et al. Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity. Protein Sci. 2004;13:1627–1635. doi: 10.1110/ps.04622104. PubMed DOI PMC