Novel Galectins Purified from the Sponge Chondrilla australiensis: Unique Structural Features and Cytotoxic Effects on Colorectal Cancer Cells Mediated by TF-Antigen Binding
Abstract
:1. Introduction
2. Results
2.1. Purification of Chondrilla australiensis Lectin (hRTL)
2.2. Characterization of the Primary Sequence of hRTL
2.3. hRTLs Target the Secretory Pathway by Signal Peptides
2.4. Glycan-Binding Properties of hRTL
2.5. The Cytotoxic Activity of hRTL
2.6. Prediction of the 3D Structure of hRTLs
3. Discussion
4. Materials and Methods
4.1. Purification of β-D-Galactoside-Binding Lectin from C. australiensis
4.2. Hemagglutinating Activity and Analysis of Sugar-Binding Property
4.3. Glycan-Binding Profiling of the Lectin
4.4. Molecular Mass Analysis
4.5. N-Terminal Amino Acid Sequence
4.6. RNA Sequencing
4.7. Cell Viability and Cytotoxicity Assays
4.8. 3D Structure Prediction
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Taylor, M.E.; Drickamer, K.; Imberty, A.; van Kooyk, Y.; Schnaar, R.L.; Etzler, M.E.; Varki, A. Chapter 28, Discovery and Classification of Glycan-Binding Proteins. In Essentials of Glycobiology, 4th ed.; Varki, A., Cummings, R.D., Esko, J.D., Stanley, P., Hart, G.W., Aebi, M., Mohnen, D., Kinoshita, T., Packer, N.H., Prestegard, J.H., et al., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 2022. [Google Scholar]
- Islam, M.K.; Khan, M.; Gidwani, K.; Witwer, K.W.; Lamminmäki, U.; Leivo, J. Lectins as potential tools for cancer biomarker discovery from extracellular vesicles. Biomark. Res. 2023, 11, 85. [Google Scholar] [CrossRef] [PubMed]
- Ahmmed, M.K.; Bhowmik, S.; Giteru, S.G.; Zilani, M.N.H.; Adadi, P.; Islam, S.S.; Kanwugu, O.N.; Haq, M.; Ahmmed, F.; Ng, C.C.W.; et al. An update of lectins from marine organisms: Characterization, extraction methodology, and potential biofunctional applications. Mar. Drugs 2022, 20, 430. [Google Scholar] [CrossRef] [PubMed]
- Marothia, D.; Kaur, N.; Jhamat, C.; Sharma, I.; Pati, P.K. Plant lectins: Classical molecules with emerging roles in stress tolerance. Int. J. Biol. Macromol. 2023, 244, 125272. [Google Scholar] [CrossRef]
- Bonnardel, F.; Perez, S.; Lisacek, F.; Imberty, A. Structural database for lectins and the UniLectin web platform. Methods Mol. Biol. 2020, 2132, 1–14. [Google Scholar] [CrossRef] [PubMed]
- Wood, R.A.; Grotzinger, J.P.; Dickson, J.A. Proterozoic modular biomineralized metazoan from the Nama Group, Namibia. Science 2002, 296, 2383–2386. [Google Scholar] [CrossRef]
- Peterson, K.J.; Butterfield, N.J. Origin of the Eumetazoa: Testing ecological predictions of molecular clocks against the Proterozoic fossil record. Proc. Natl. Acad. Sci. USA 2005, 102, 9547–9552. [Google Scholar] [CrossRef]
- Gardères, J.; Bourguet-Kondracki, M.L.; Hamer, B.; Batel, R.; Schröder, H.C.; Müller, W.E. Porifera lectins: Diversity, physiological roles and biotechnological potential. Mar. Drugs 2015, 13, 5059–5101. [Google Scholar] [CrossRef]
- Duarte, J.A.; Oliveira Neto, J.E.; Torres, R.C.F.; Sousa, A.R.O.; Andrade, A.L.; Chaves, R.P.; Carneiro, R.; Vasconcelos, M.A.; Teixeira, C.S.; Teixeira, E.H.; et al. Structural characterization of a galectin from the marine sponge Aplysina lactuca (ALL) with synergistic effects when associated with antibiotics against bacteria. Biochimie 2023, 214, 165–175. [Google Scholar] [CrossRef]
- Sousa, A.R.O.; Andrade, F.R.N.; Chaves, R.P.; Sousa, B.L.; Lima, D.B.; Souza, R.O.D.S.; da Silva, C.G.L.; Teixeira, C.S.; Sampaio, A.H.; Nagano, C.S.; et al. Structural characterization of a galectin isolated from the marine sponge Chondrilla caribensis with leishmanicidal potential. Biochim. Biophys. Acta Gen. Subj. 2021, 1865, 129992. [Google Scholar] [CrossRef]
- Marques, D.N.; Almeida, A.S.; Sousa, A.R.O.; Pereira, R.; Andrade, A.L.; Chaves, R.P.; Carneiro, R.F.; Vasconcelos, M.A.; Nascimento-Neto, L.G.D.; Pinheiro, U.; et al. Antibacterial activity of a new lectin isolated from the marine sponge Chondrilla caribensis. Int. J. Biol. Macromol. 2018, 109, 1292–1301. [Google Scholar] [CrossRef]
- Ueda, T.; Nakamura, Y.; Smith, C.M.; Copits, B.A.; Inoue, A.; Ojima, T.; Matsunaga, S.; Swanson, G.T.; Sakai, R. Isolation of novel prototype galectins from the marine ball sponge Cinachyrella sp. guided by their modulatory activity on mammalian glutamate-gated ion channels. Glycobiology 2013, 23, 412–425. [Google Scholar] [CrossRef]
- Freymann, D.M.; Nakamura, Y.; Focia, P.J.; Sakai, R.; Swanson, G.T. Structure of a tetrameric galectin from Cinachyrella sp. (ball sponge). Acta Crystallogr. D Biol. Crystallogr. 2012, 68, 1163–1174. [Google Scholar] [CrossRef] [PubMed]
- Kawsar, S.M.; Fujii, Y.; Matsumoto, R.; Ichikawa, T.; Tateno, H.; Hirabayashi, J.; Yasumitsu, H.; Dogasaki, C.; Hosono, M.; Nitta, K.; et al. Isolation, purification, characterization and glycan-binding profile of a D-galactoside specific lectin from the marine sponge, Halichondria okadai. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2008, 150, 349–357. [Google Scholar] [CrossRef] [PubMed]
- Schröder, H.C.; Boreiko, A.; Korzhev, M.; Tahir, M.N.; Tremel, W.; Eckert, C.; Ushijima, H.; Müller, I.M.; Müller, W.E. Co-expression and functional interaction of silicatein with galectin: Matrix-guided formation of siliceous spicules in the marine demosponge Suberites domuncula. J. Biol. Chem. 2006, 281, 12001–12009. [Google Scholar] [CrossRef] [PubMed]
- Pfeifer, K.; Haasemann, M.; Gamulin, V.; Bretting, H.; Fahrenholz, F.; Müller, W.E. S-type lectins occur also in invertebrates: High conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium. Glycobiology 1993, 3, 179–184. [Google Scholar] [CrossRef] [PubMed]
- Wagner-Hülsmann, C.; Bachinski, N.; Diehl-Seifert, B.; Blumbach, B.; Steffen, R.; Pancer, Z.; Müller, W.E. A galectin links the aggregation factor to cells in the sponge (Geodia cydonium) system. Glycobiology 1996, 6, 785–793. [Google Scholar] [CrossRef]
- Gundacker, D.; Leys, S.P.; Schröder, H.C.; Müller, I.M.; Müller, W.E. Isolation and cloning of a C-type lectin from the hexactinellid sponge Aphrocallistes vastus: A putative aggregation factor. Glycobiology 2001, 11, 21–29. [Google Scholar] [CrossRef] [PubMed]
- Watari, H.; Kageyama, H.; Masubuchi, N.; Nakajima, H.; Onodera, K.; Focia, P.J.; Oshiro, T.; Matsui, T.; Kodera, Y.; Ogawa, T.; et al. A marine sponge-derived lectin reveals hidden pathway for thrombopoietin receptor activation. Nat. Commun. 2022, 13, 7262. [Google Scholar] [CrossRef]
- Schröder, H.C.; Ushijima, H.; Krasko, A.; Gamulin, V.; Thakur, N.L.; Diehl-Seifert, B.; Müller, I.M.; Müller, W.E. Emergence and disappearance of an immune molecule, an antimicrobial lectin, in basal metazoa. A tachylectin-related protein in the sponge Suberites domuncula. J. Biol. Chem. 2003, 278, 32810–32817. [Google Scholar] [CrossRef]
- Araújo, T.A.T.; de Souza, A.; Santana, A.F.; Braga, A.R.C.; Custódio, M.R.; Simões, F.R.; Araújo, G.M.; Miranda, A.; Alves, F.; Granito, R.N.; et al. Comparison of different methods for spongin-like collagen extraction from marine sponges (Chondrilla caribensis and Aplysina fulva): Physicochemical properties and in vitro biological analysis. Membranes 2021, 11, 522. [Google Scholar] [CrossRef]
- Santarsia, S.; Grosso, A.S.; Trovão, F.; Jiménez-Barbero, J.; Carvalho, A.L.; Nativi, C.; Marcelo, F. Molecular recognition of a Thomsen-Friedenreich antigen mimetic targeting human Galectin-3. ChemMedChem 2018, 13, 2030–2036. [Google Scholar] [CrossRef] [PubMed]
- Karsten, U.; Goletz, S. What controls the expression of the core-1 (Thomsen-Friedenreich) glycotope on tumor cells? Biochemistry 2015, 80, 801–807. [Google Scholar] [CrossRef] [PubMed]
- Kurtenkov, O. Profiling of naturally occurring antibodies to the Thomsen-Friedenreich antigen in health and cancer: The diversity and clinical potential. Biomed. Res. Int. 2020, 2020, 9747040. [Google Scholar] [CrossRef]
- Inamdar, S.R.; Savanur, M.A.; Eligar, S.M.; Chachadi, V.B.; Nagre, N.N.; Chen, C.; Barclays, M.; Ingle, A.; Mahajan, P.; Borges, A.; et al. The TF-antigen binding lectin from Sclerotium rolfsii inhibits growth of human colon cancer cells by inducing apoptosis in vitro and suppresses tumor growth in vivo. Glycobiology 2012, 22, 1227–1235. [Google Scholar] [CrossRef] [PubMed]
- Steet, R.A.; Melancon, P.; Kuchta, R.D. 3′-Azidothymidine potently inhibits the biosynthesis of highly branched N-linked oligosaccharides and poly-N-acetyllactosamine chains in cells. J. Biol. Chem. 2000, 275, 26812–26820. [Google Scholar] [CrossRef]
- Swift, F.V.; Bhat, K.; Younghusband, H.B.; Hamada, H. Characterization of a cell type-specific enhancer found in the human papilloma virus type 18 genome. EMBO J. 1987, 6, 1339–1344. [Google Scholar] [CrossRef]
- Hirabayashi, J.; Kasai, K. Complete amino acid sequence of a beta-galactoside-binding lectin from human placenta. J. Biochem. 1988, 104, 1–4. [Google Scholar] [CrossRef] [PubMed]
- Goddard, T.D.; Huang, C.C.; Meng, E.C.; Pettersen, E.F.; Couch, G.S.; Morris, J.H.; Ferrin, T.E. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 2018, 27, 14–25. [Google Scholar] [CrossRef] [PubMed]
- Popa, S.J.; Stewart, S.E.; Moreau, K. Unconventional secretion of annexins and galectins. Semin. Cell Dev. Biol. 2018, 83, 42–50. [Google Scholar] [CrossRef]
- Nemoto-Sasaki, Y.; Hayama, K.; Ohya, H.; Arata, Y.; Kaneko, M.K.; Saitou, N.; Hirabayashi, J.; Kasai, K. Caenorhabditis elegans galectins LEC-1-LEC-11: Structural features and sugar-binding properties. Biochim. Biophys. Acta. 2008, 1780, 1131–1142. [Google Scholar] [CrossRef]
- Ditgen, D.; Anandarajah, E.M.; Reinhardt, A.; Younis, A.E.; Witt, S.; Hansmann, J.; Lorenz, E.; García-Hernández, M.; Paclik, D.; Soblik, H.; et al. Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides. Mol. Biochem. Parasitol. 2018, 225, 73–83. [Google Scholar] [CrossRef]
- Gerdol, M.; Nerelli, D.E.; Martelossi, N.; Ogawa, Y.; Fujii, Y.; Pallavicini, A.; Ozeki, Y. Taxonomic distribution and molecular evolution of mytilectins. Mar. Drugs. 2023, 21, 614. [Google Scholar] [CrossRef] [PubMed]
- Żukowska, D.; Chorążewska, A.; Ciura, K.; Gędaj, A.; Kalka, M.; Poźniak, M.; Porębska, N.; Opaliński, Ł. The diverse dependence of galectin-1 and -8 on multivalency for the modulation of FGFR1 endocytosis. Cell Commun. Signal 2024, 22, 270. [Google Scholar] [CrossRef]
- Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. [Google Scholar] [CrossRef] [PubMed]
- Yasumitsu, H.; Ozeki, Y.; Kawsar, S.M.; Toda, T.; Kanaly, R. CGP stain: An inexpensive, odorless, rapid, sensitive, and in principle in vitro methylation-free Coomassie Brilliant Blue stain. Anal. Biochem. 2010, 406, 86–88. [Google Scholar] [CrossRef]
- Wiechelman, K.J.; Braun, R.D.; Fitzpatrick, J.D. Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal. Biochem. 1988, 175, 231–237. [Google Scholar] [CrossRef] [PubMed]
- Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76–85. [Google Scholar] [CrossRef]
- Gourdine, J.P.; Cioci, G.; Miguet, L.; Unverzagt, C.; Silva, D.V.; Varrot, A.; Gautier, C.; Smith-Ravin, E.J.; Imberty, A. High affinity interaction between a bivalve C-type lectin and a biantennary complex-type N-glycan revealed by crystallography and microcalorimetry. J. Biol. Chem. 2008, 283, 30112–30120. [Google Scholar] [CrossRef]
- Kuno, A.; Uchiyama, N.; Koseki-Kuno, S.; Ebe, Y.; Takashima, S.; Yamada, M.; Hirabayashi, J. Evanescent-field fluorescence-assisted lectin microarray: A new strategy for glycan profiling. Nat. Methods 2005, 2, 851–856. [Google Scholar] [CrossRef]
- Hirabayashi, J.; Yamada, M.; Kuno, A.; Tateno, H. Lectin microarrays: Concept, principle and applications. Chem. Soc. Rev. 2013, 42, 443–458. [Google Scholar] [CrossRef]
- Miyanishi, N.; Nishi, N.; Abe, H.; Kashio, Y.; Shinonaga, R.; Nakakita, S.; Sumiyoshi, W.; Yamauchi, A.; Nakamura, T.; Hirashima, M.; et al. Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family. Glycobiology 2007, 17, 423–432. [Google Scholar] [CrossRef]
- Hewick, R.M.; Hunkapiller, M.W.; Hood, L.E.; Dreyer, W.J. A gas-liquid solid phase peptide and protein sequenator. J. Biol. Chem. 1981, 256, 7990–7997. [Google Scholar] [CrossRef] [PubMed]
- Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [PubMed]
- Grabherr, M.G.; Haas, B.J.; Yassour, M.; Levin, J.Z.; Thompson, D.A.; Amit, I.; Adiconis, X.; Fan, L.; Raychowdhury, R.; Zeng, Q.; et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 2011, 29, 644–652. [Google Scholar] [CrossRef] [PubMed]
- Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R.D.; Bairoch, A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003, 31, 3784–3788. [Google Scholar] [CrossRef] [PubMed]
- Teufel, F.; Almagro Armenteros, J.J.; Johansen, A.R.; Gíslason, M.H.; Pihl, S.I.; Tsirigos, K.D.; Winther, O.; Brunak, S.; von Heijne, G.; Nielsen, H. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat. Biotechnol. 2022, 40, 1023–1025. [Google Scholar] [CrossRef]
- Manni, M.; Berkeley, M.R.; Seppey, M.; Simão, F.A.; Zdobnov, E.M. BUSCO Update: Novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol. Biol. Evol. 2021, 38, 4647–4654. [Google Scholar] [CrossRef]
- Kuznetsov, D.; Tegenfeldt, F.; Manni, M.; Seppey, M.; Berkeley, M.; Kriventseva, E.V.; Zdobnov, E.M. OrthoDB v11: Annotation of orthologs in the widest sampling of organismal diversity. Nucleic. Acids Res. 2023, 51D, 445–451. [Google Scholar] [CrossRef]
- Ukeda, H.; Kawana, D.; Maeda, S.; Sawamura, M. Spectrophotometric assay for superoxide dismutase based on the reduction of highly water-soluble tetrazolium salts by xanthine-xanthine oxidase. Biosci. Biotechnol. Biochem. 1999, 63, 485–488. [Google Scholar] [CrossRef]
- Mirdita, M.; Schütze, K.; Moriwaki, Y.; Heo, L.; Ovchinnikov, S.; Steinegger, M. ColabFold: Making protein folding accessible to all. Nat. Methods 2022, 19, 679–682. [Google Scholar] [CrossRef]
- Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 596, 583–589. [Google Scholar] [CrossRef]
- Deluca, S.; Khar, K.; Meiler, J. Fully Flexible docking of medium sized ligand libraries with RosettaLigand. PLoS ONE 2015, 10, e0132508. [Google Scholar] [CrossRef] [PubMed]
- Combs, S.A.; Deluca, S.L.; DeLuca, S.H.; Lemmon, G.H.; Nannemann, D.P.; Nguyen, E.D.; Willis, J.R.; Sheehan, J.H.; Meiler, J. Small-molecule ligand docking into comparative models with Rosetta. Nat. Protoc. 2013, 8, 1277–1298. [Google Scholar] [CrossRef]
- Kothiwale, S.; Mendenhall, J.L.; Meiler, J. BCL::Conf: Small molecule conformational sampling using a knowledge based rotamer library. J. Cheminformatics 2015, 7, 47. [Google Scholar] [CrossRef] [PubMed]
- Lyskov, S.; Chou, F.C.; Conchúir, S.Ó.; Der, B.S.; Drew, K.; Kuroda, D.; Xu, J.; Weitzner, B.D.; Renfrew, P.D.; Sripakdeevong, P.; et al. Serverification of molecular modeling applications: The Rosetta Online Server that Includes Everyone (ROSIE). PLoS ONE 2013, 8, e63906. [Google Scholar] [CrossRef]
Fraction | Titer (HU) | Volume (mL) | Total Activity a | Protein Conc. (mg mL−1) | Protein Amount (mg) | Specific Activity b | Purification Ratio (Fold) c | Recovery of Activity (%) d |
---|---|---|---|---|---|---|---|---|
Crude extract | 65,536 | 500 | 32,768,000 | 9.5 | 4750 | 13.79 | 1 | 100 |
Purified lectin | 262,144 | 80 | 20,971,520 | 0.86 | 68.8 | 3810.23 | 276 | 64 |
Saccharides | Minimum Inhibitory Concentration (mM) |
---|---|
TF-antigen | 1.6 |
Lactose | 3.2 |
Melibiose | 50 |
Sucrose | N.I. b |
D-galactose | 25 |
D-GalNAc | >50 |
D-GlcNAc | N.I. |
D-mannose | N.I. |
Glycoproteins | Minimum inhibitory concentration (mg/mL) |
Fetuin | 0.125 |
Porcine stomach mucin | 0.125 |
Bovine submaxillary mucin | N.I. c |
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Hayashi, R.; Kamata, K.; Gerdol, M.; Fujii, Y.; Hayashi, T.; Onoda, Y.; Kobayashi, N.; Furushima, S.; Ishiwata, R.; Ohkawa, M.; et al. Novel Galectins Purified from the Sponge Chondrilla australiensis: Unique Structural Features and Cytotoxic Effects on Colorectal Cancer Cells Mediated by TF-Antigen Binding. Mar. Drugs 2024, 22, 400. https://doi.org/10.3390/md22090400
Hayashi R, Kamata K, Gerdol M, Fujii Y, Hayashi T, Onoda Y, Kobayashi N, Furushima S, Ishiwata R, Ohkawa M, et al. Novel Galectins Purified from the Sponge Chondrilla australiensis: Unique Structural Features and Cytotoxic Effects on Colorectal Cancer Cells Mediated by TF-Antigen Binding. Marine Drugs. 2024; 22(9):400. https://doi.org/10.3390/md22090400
Chicago/Turabian StyleHayashi, Ryuhei, Kenichi Kamata, Marco Gerdol, Yuki Fujii, Takashi Hayashi, Yuto Onoda, Nanae Kobayashi, Satoshi Furushima, Ryuya Ishiwata, Mayuka Ohkawa, and et al. 2024. "Novel Galectins Purified from the Sponge Chondrilla australiensis: Unique Structural Features and Cytotoxic Effects on Colorectal Cancer Cells Mediated by TF-Antigen Binding" Marine Drugs 22, no. 9: 400. https://doi.org/10.3390/md22090400