Abstract
In Leishmania mexicana, the protease gp63 has been documented as the protein responsible for cyclooxygenase (COX) activity. The present work aimed to obtain a monoclonal antibody capable of recognizing this protein without blocking the COX-like enzymatic activity. The antibody produced by the selected hybridoma was named D12 mAb. The antigen recognized by the D12 mAb was characterized by the determination of COX activity associated with immune complexes in the presence of exogenous arachidonic acid (AA) using the commercial Activity Assay Abcam kit. LSM-SMS analysis validated the identity of the antigen associated with the D12 mAb as the L. mexicana protease gp63. Confocal microscopy assays with the D12 mAb detected, by cross-recognition, similar proteins in other protozoan parasites. COX-like molecules are located in vesicular structures, homogeneously distributed throughout the cytoplasm in amastigotes (intracellular infectious phase) and promastigotes of L. mexicana, and trophozoites of Entamoeba histolytica, Acanthamoeba castellanii, and Naegleria fowleri. However, in Giardia duodenalis trophozoites, the distribution of the COX-like molecule was also in perinuclear areas. In comparison, in Trypanosoma cruzi trypomastigotes, the distribution was mainly observed in the plasma membrane. Structural analyses of COX-2-like antigens revealed continuous and discontinuous epitopes for B cells, which could be relevant in the cross-reaction of D12 mAb with the analyzed parasites. These results indicate that the D12 mAb against the L. mexicana gp63 also recognizes a COX-like molecule in several protozoan parasites, suggesting that this D12 mAb could potentially be used in combined therapies against infectious diseases.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
Not applicable.
References
Alves-Ferreira EVC, Toledo JS, de Oliveira AHC et al (2015) Differential gene expression and infection profiles of cutaneous and mucosal Leishmania braziliensis isolates from the same patient. PLoS Negl Trop Dis 9:e0004018. https://doi.org/10.1371/journal.pntd.0004018
Alves-Ferreira EVC, Ferreira TR, Walrad P et al (2020) Leishmania braziliensis prostaglandin F2α synthase impacts host infection. Parasit Vectors 13:9. https://doi.org/10.1186/s13071-020-3883-z
Araújo-Santos T, Rodríguez NE, Moura-Pontes S, Dixt UG, Abánades DR, Bozza PT, Wilson ME, Borges VM (2014) Role of prostaglandin F2α production in lipid bodies from Leishmania infantum chagasi: insights on virulence. J Infect Dis 210:1951–1961. https://doi.org/10.1093/infdis/jiu299
Bhattacharjee S, Bhattacharjee A, Majumder S et al (2012) Glycyrrhizic acid suppresses Cox-2-mediated anti-inflammatory responses during Leishmania donovani infection. J Antimicrob Chemother 67:1905–1914. https://doi.org/10.1093/jac/dks159
Barbour SE, Marciano-Cabral F (2001) Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2). Biochim Biophys Acta 1530:123–133. https://doi.org/10.1016/s1388-1981(00)00069-x
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K Madden TL (2009) BLAST+ architecture and applications. BMC Bioinformatics 10:421. https://doi.org/10.1186/1471-2105-10-421
Cuevas IC, Cazzulo JJ, Sánchez DO (2003) gp63 homologs in Trypanosoma cruzi: surface antigens with metalloprotease activity and a possible role in host cell infection. Infect Immun 71:5739–5749. https://doi.org/10.1128/IAI.71.10.5739-5749.2003
de Sousa Andrade YMF, de Castro MV, de Souza Tavares V, et al (2022) Polyunsaturated fatty acids alter the formation of lipid droplets and eicosanoid production in <em>Leishmania</em> promatigotes. bioRxiv 2022.07.01.498279. https://doi.org/10.1101/2022.07.01.498279
Dey I, Keller K, Belley A, Chadee K (2003) Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica. Proc Natl Acad Sci 100:13561–13566. https://doi.org/10.1073/pnas.1835863100
Diamond LS, Harlow DR, Cunnick CC (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72:431–432. https://doi.org/10.1016/0035-9203(78)90144-X
Díaz-Viraqué F, Chiribao ML, Trochine A et al (2018) Old Yellow enzyme from Trypanosoma cruzi exhibits in vivo prostaglandin F2α synthase activity and has a key role in parasite infection and drug susceptibility. Front Immunol 9. https://doi.org/10.3389/fimmu.2018.00456
Duong-Ly KC, Gabelli SB (2014) Salting out of proteins using ammonium sulfate precipitation. pp 85–94
Eida A (2015) Prostanoids and parasitic diseases. Parasitologists United Journal 8:38. 10.4103/1687-7942.163408
Eliopoulos AG (2002) Induction of COX-2 by LPS in macrophages is regulated by Tpl2-dependent CREB activation signals. EMBO J 21:4831–4840. https://doi.org/10.1093/emboj/cdf478
Ellis JE, Jarrollb L, Kaneshiro ES (1996) Changes in lipid composition during in vitro encystation and fatty acid desaturase activity of Giardia lamblia. 81:13–25
Estrada-Figueroa LA, Díaz-Gandarilla JA, Hernández-Ramírez VI et al (2018) Leishmania mexicana gp63 is the enzyme responsible for cyclooxygenase (COX) activity in this parasitic protozoa. Biochimie 151:73–84. https://doi.org/10.1016/j.biochi.2018.05.016
Faleiro RJ, Kumar R, Bunn PT et al (2016) Combined immune therapy for the treatment of visceral leishmaniasis. PLoS Negl Trop Dis 10:1–21. https://doi.org/10.1371/journal.pntd.0004415
Fam SS, Murphey LJ, Terry ES et al (2002) Formation of highly reactive A-ring and J-ring isoprostane-like compounds (A4/J4-neuroprostanes) in vivo from docosahexaenoic acid. J Biol Chem 277:36076–36084. https://doi.org/10.1074/jbc.M205638200
Garavaglia PA, Cannata JJB, Ruiz AM et al (2010) Identification, cloning and characterization of an aldo-keto reductase from Trypanosoma cruzi with quinone oxido-reductase activity. Mol Biochem Parasitol 173:132–141. https://doi.org/10.1016/j.molbiopara.2010.05.019
Hadas E, Mazur T (1997) Biosynthesis of prostaglandins in pathogenic and nonpathogenic strains of Acanthamoeba spp. Parasitol Res 83:296–299. https://doi.org/10.1007/s004360050250
Hashemi Goradel N, Najafi M, Salehi E et al (2019) Cyclooxygenase-2 in cancer: a review. J Cell Physiol 234:5683–5699. https://doi.org/10.1002/jcp.27411
Jahangeer M, Mahmood Z, Munir N et al (2020) Naegleria fowleri : sources of infection, pathophysiology, diagnosis, and management; a review. Clin Exp Pharmacol Physiol 47:199–212. https://doi.org/10.1111/1440-1681.13192
Kubata BK, Duszenko M, Kabututu Z et al (2000) Identification of a novel prostaglandin f (2alpha) synthase in Trypanosoma brucei. J Exp Med 192:1327–1338. https://doi.org/10.1084/jem.192.9.1327
Kubata BK, Duszenko M, Martin KS, Urade Y (2007) Molecular basis for prostaglandin production in hosts and parasites. Trends Parasitol 23:325–331. https://doi.org/10.1016/j.pt.2007.05.005
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evolution 35(6):1547–1549. https://doi.org/10.1093/molbev/msy096
Mabbott NA (2018) The influence of parasite infections on host immunity to co-infection with other pathogens. Front Immunol 9:2579. https://doi.org/10.3389/fimmu.2018.02579
Noverr MC, Phare SM, Toews GB, Coffey MJ, Huffnagle GB (2001) Pathogenic yeasts Cryptococcus neoformans and Candida albicans produce immunomodulatory prostaglandins. Infect Immun 69:2957–2963. https://doi.org/10.1128/IAI.69.5.2957-2963.2001
Paloque L, Perez-Berezo T, Abot A, Dalloux-Chioccioli J, Bourgeade-Delmas S, Le Faouder P, Pujo J, Marie-Ange T, Jean-Marie F, Nils Helge S, Mainka M, Rolland C, Blanpied C, Dietrich G, Bertrand-Michel J, Deraison C, Valentin A, Cenac N (2019) Polyunsaturated fatty acid metabolites: biosynthesis in Leishmania and role in parasite/host interaction. J Lipid Res 60:636–647. https://doi.org/10.1194/jlr.M091736
Roberts AJ, Dunne J, Scullion P et al (2018) A role for trypanosomatid aldo-keto reductases in methylglyoxal, prostaglandin and isoprostane metabolism. Biochem J 475:2593–2610. https://doi.org/10.1042/BCJ20180232
Roberts JL, Milne GL (2009) Isoprostanes. J Lipid Res 50 Suppl(Suppl):S219–23. https://doi.org/10.1194/jlr.R800037-JLR200
Rogerio AP, Anibal FF (2012) Role of leukotrienes on protozoan and helminth infections. Mediat Inflamm 2012:. https://doi.org/10.1155/2012/595694
Segovia-Gamboa NC, Chávez-Munguía B, Medina-Flores Y et al (2010) Entamoeba invadens, encystation process and enolase. Exp Parasitol 125:63–69. https://doi.org/10.1016/j.exppara.2009.12.019
Sengupta K, Das P, Johnson TM, Chaudhuri PP, Das DNGB (1993) Production and characterization of monoclonal antibodies against a highly immunogenic fraction of Entamoeba histolytica (NIH:200) and their application in the detection of current amoebic infection. J Eukaryot Microbiol 40:722–726. https://doi.org/10.1111/j.1550-7408.1993.tb04465.x
Siddiqui R, Lakhundi S, Iqbal J, Khan NA (2016) Effect of non-steroidal anti-inflammatory drugs on biological properties of Acanthamoeba castellanii belonging to the T4 genotype. Exp Parasitol 168:45–50. https://doi.org/10.1016/j.exppara.2016.06.011
Sievers F, Wilm A, Dineen D et al (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. https://doi.org/10.1038/msb.2011.75
Simmons DL, Botting RM, Hla T (2004) Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev 56:387–437. https://doi.org/10.1124/pr.56.3.3
van Huijsduijnen RH, Kojima S, Carter D et al (2020) Reassessing therapeutic antibodies for neglected and tropical diseases. PLoS Negl Trop Dis 14:1–20. https://doi.org/10.1371/JOURNAL.PNTD.0007860
Vargas-villarreal AJ, Escobedo-guajardo BL, David B, et al (2007) Activity of intracellular phospholipase A1 and A2 in Giardia lamblia Adriana Sampayo-Reyes and Salvador Said-Fernández Published by : Allen Press on behalf of The American Society of Parasitologists Stable URL : http://www.jstor.com/stable/40058815 REFERE. 93:979–984
Visvesvara GS, Moura H, Schuster FL (2007) Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol 50:1–26. https://doi.org/10.1111/j.1574-695X.2007.00232.x
Waterhouse A, Bertoni M, Bienert S et al (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296–W303. https://doi.org/10.1093/nar/gky427
Yao C, Donelson JE, Wilson ME (2003) The major surface protease (MSP or GP63) of Leishmania sp. Biosynthesis, regulation of expression, and function. Mol Biochem Parasitol 132:1–16. https://doi.org/10.1016/S0166-6851(03)00211-1
Zidar N, Odar K, Glavac D et al (2009) Cyclooxygenase in normal human tissues - is COX-1 really a constitutive isoform, and COX-2 an inducible isoform? J Cell Mol Med 13:3753–3763. https://doi.org/10.1111/j.1582-4934.2008.00430.x
Acknowledgements
We thank Dr. Sergio Encarnación-Guevara and Magdalena Hernández-Ortiz from Centro de Ciencias Genómicas, UNAM, for processing samples by LC/MS/MS. We also thank Angélica Leticia Serrano-Ahumada, ascribed to the Bioinformatics, Experimental Medicine Research Unit, Faculty of Medicine, for her valuable participation in the processing and presentation of immunofluorescence images; Jessica Márques-Dueñas for her secretarial assistance, and MVZ Benjamín Emmanuel Chávez-Alvarez for his advice on the mouse immunization process. Axenic amastigotes (MNYC/BZ/62/M379) of L. mexicana were donated, cultured, and maintained by Dr. Alma Escalona-Montaño. Trophozoites of Acanthamoeba castellanii, Entamoeba dispar, Entamoeba invadens, and Naegleria fowleri were cultured and maintained by Biol. Lizbeth Salazar-Villatoro. The trophozoites of Giardia duodenalis were donated and cultured by Dr. Raúl Argüello. Promastigotes of Trypanosoma cruzi were cultured and maintained by Biol. Lidia Baylon.
Funding
This work was supported by CONACyT grant nos. 104108 and A1-S-15223 to PT-R. LAE-F was the recipient of a fellowship from CONACyT (204814).
Author information
Authors and Affiliations
Contributions
VIHR was responsible for interpretation of data, and writing of the manuscript; LEF carried out the D12 mAb production and characterization, in addition, was responsible for COX activity analysis; YM collaborated with the monoclonal antibody production; MRLT was responsible of the determination of COX activity and immunofluorescence assays; ATL was responsible of interpretation of the Mass spectrometry assays; COT carried out the culture of the cells and was responsible of the determination of COX activity present in parasite extracts; PTR and VIHR were responsible of the concept and of the coordination of the experiments. DMM was responsible of the analyzes of the continuous and discontinuous antigenic determinants. PTR was responsible of funding acquirement. All authors contributed critically to the drafts and gave final approval.
Corresponding author
Ethics declarations
Ethics approval
All animal procedures were approved by CINVESTAV’s Institutional Animal Care and Use Committee (CINVESTAV-IACUC). Experiments were performed with animals provided by the Animal Production and Experimentation Unit (UPEAL-CINVESTAV) following the Mexican National Norm (NOM-062-ZOO-1999) specifications, a version of the guide for the care and use of laboratory animals 2011.
Consent for participate
Not applicable.
Consent to publication
Not applicable.
Conflict of interest
The authors declare no competing interests.
Additional information
Handling Editor: Una Ryan
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Suppl. Fig. 1.
Purification of COX-like activity from total promastigote extracts using a DEAE-cellulose ion-exchange column. (A) Elution profile of the fractions obtained from the column. Fractions were incubated with exogenous AA and processed according to the commercial kit protocol for COX activity detection. The graph includes the protein quantification values for each fraction eluted from the column. (B) Silver staining analysis of the immune complex. An immunoprecipitation assay was performed, using the fraction with the highest COX activity as antigen (E8-E12) and the commercial gp63 mAb (2 μg of antibody/1 mg of protein). The proteins associated with the immune complex were analyzed in a 10% SDS-PAGE. Arrows indicate the 66 kDa protein and the heavy and light chains of the antibody (IgHC and IgLC). (C) Detection of COX-like activity present in the immune complex. The readings were obtained every 2 min and ended at 20 min. Subsequently, COX activity associated with the D12 mAb was detected (D). In these analyses, an immune complex obtained with the gp63 mAb was also included, considered a positive control. The samples obtained along the immunoprecipitation process were included: material pre-cleared bound to the beads preincubated with the antigen (Pre-clear); material not bound to the beads, that is, the antigen that remains free after the formation of the immune complex (Ub anti-gp63 mAb). In the case of the D12 mAb, it was used a 1:100 dilution. The analyzes were carried out using three biological replicas (ANOVA P< 0.0001). (PNG 157 kb)
Suppl. Fig. 2.
The D12 mAb recognizes an inducible protein in mouse macrophages. (A) The J774A.1 cell line was incubated (a) in the absence of LPS (control) or (b) and (c) in the presence of LPS plus the commercial anti-COX-2 antibody (b) and the supernatant of the D12 clone (c). Image digitalization corresponds to extended focus analysis and represents one experiment with a total of two biological replicates. (B) Analysis of pixel intensity of 100 cells per field, of 3 fields analyzed (ANOVA P< 0.004). (C) Quantification of COX activity in macrophage extracts after adding AA. The results are expressed as Relative Light Units (RLU) (ANOVA P< 0.005). The images are representative of three biological replicas carried out in triplicate. (D) Immunocomplexes were obtained with the commercial anti gp63 mAb from L. mexicana, and the precipitated material was analyzed in a 10% SDS PAGE and transferred to nitrocellulose. Proteins associated with the immune complex were revealed with the D12 mAb. The positive controls were total extracts from LPS-stimulated macrophages and solubilized material from promastigotes (E). Proteins immunoprecipitated with the D12 mAb were analyzed by western blot with the commercial COX-2 pAb. (PNG 750 kb)
Suppl. Fig. 3.
Identification of the antigen precipitated by the D12 antibody. Immune complex analysis by silver-staining of a 10% SDS PAGE. Full lanes are shown. The upper arrow indicates the molecular weight of the 66 kDa protein. Additional arrows indicate the immunoglobulin heavy and light chains (IHC, ILC). (PNG 224 kb)
Suppl. Fig. 4.
Identification of COX-like in trophozoites of Entamoeba histolytica. (a) Subcellular localization of COX-like in cells fixed with 4% p-formaldehyde / PBS and permeabilized with Triton X-100 (0.01%) using the commercial anti-COX-2 polyclonal antibody. As a negative control, a preimmune antibody and trophozoites incubated only with a secondary antibody coupled to FITC were used (data not shown). The cells were analyzed by Confocal Microscopy. (b) Western blot analyses, using the D12 mAb. Soluble fractions of E. histolytica were resolved by 10% SDS-PAGE. As positive controls, total extracts of macrophages stimulated with LPS and promastigotes of L. mexicana were included. The material was transferred to nitrocellulose. Membranes were incubated with the D12 mAb and the commercial anti-COX-2 pAb. The signal developed with an anti-mouse IgG antibody coupled to HRP and detected by chemiluminescence. (PNG 716 kb)
Suppl. Fig. 5.
(A): gp63-like sequences of the five protozoa aligned in BLAST OMEGA. The putative antigenic region is marked with a green parenthesis. (B) Candidate epitopes with identical conserved residues (*) are listed with yellow/magenta/cyan pattern, sequences ID, and respective score value. (PNG 2913 kb)
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Hernández-Ramírez, V.I., Estrada-Figueroa, L.A., Medina, Y. et al. A monoclonal antibody against a Leishmania mexicana COX-like enzymatic activity also recognizes similar proteins in different protozoa of clinical importance. Parasitol Res 122, 479–492 (2023). https://doi.org/10.1007/s00436-022-07746-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-022-07746-7