Protein identification from the parotoid macrogland secretion of Duttaphrynus melanostictus

Abstract

Background: Bufonid parotoid macrogland secretion contains several low molecular mass molecules, such as alkaloids and steroids. Nevertheless, its protein content is poorly understood. Herein, we applied a sample preparation methodology that allows the analysis of viscous matrices in order to examine its proteins. Methods: Duttaphrynus melanostictus parotoid macrogland secretion was submitted to ion-exchange batch sample preparation, yielding two fractions: salt-displaced fraction and acid-displaced fraction. Each sample was then fractionated by anionic-exchange chromatography, followed by in-solution proteomic analysis. Results: Forty-two proteins could be identified, such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin and histone. Moreover, de novo analyses yielded 153 peptides, whereas BLAST analyses corroborated some of the proteomic-identified proteins. Furthermore, the de novo peptide analyses indicate the presence of proteins related to apoptosis, cellular structure, catalysis and transport processes. Conclusions: Proper sample preparation allowed the proteomic and de novo identification of different proteins in the D. melanostictus parotoid macrogland secretion. These results may increase the knowledge about the universe of molecules that compose amphibian skin secretion, as well as to understand their biological/physiological role in the granular gland.

Comprehensive Analysis of Bufadienolide and Protein Profiles of Gland Secretions from Medicinal Bufo Species

“…The protein component, which is another typical active substance of TV, has been receiving growing attention. Most investigations have focused on the qualitative identification of proteins, and few publications displayed the total protein content of TVs [ 4 , 6 ]. Therefore, the total protein content of 28 TV samples from the four Bufo samples was determined by the Bradford method, and the results are shown in Figure 3 A and Table 2 .…”

“…Only BgC and BrS samples were found to contain probable E3 ubiquitin-protein ligase makorin-2 with antitumor activity. It is worth noting that certain homologous proteins were identified as distinct entities due to the utilization of different databases [ 6 , 21 ]. For instance, we identified serpin B5 and serpin B8, while serpin B6 was reported previously [ 5 ].…”

“…However, there is still a gap in distinguishing different species by protein markers. Researchers from Brazil reported 42 identified proteins such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin, and histone from the soluble protein fractions of the parotoid macro-gland secretion of Duttaphrynus melanostictus , namely BmS [ 6 ]. An irreversible serine protease inhibitor called baserpin, with an apparent molecular weight of about 60 kDa, and a BA-lysozyme with molecular weight of about 15 kDa, were purified from the skin secretions of B. andrewsi [ 25 , 26 ].…”

“…Over 140 bufadienolides and indole alkaloids have been identified from Bufo species, along with sterols and amino acids [ 2 , 3 ]. Additionally, increasing attention has started being paid to the functional proteins and peptides in TV, and a series of active macromolecular components have been identified, including proteins with anti-inflammatory, analgesic, antioxidant, catalytic, and transport activities [ 4 , 5 , 6 ] as well as polypeptides associated with antitumor and antimicrobial properties [ 7 , 8 ].…”

Monitoring the Diversity of Frogs and Toads (Amphibia: Anura) along Code River Province of Daerah Istimewa Yogyakarta

“…Ingerophrynus biporcatus prefer primary forest habitat, but much less in secondary and degraded habitats (Iskandar, 1998;Kurniati, 2013) such as the upstream part of the Code River and some areas downstream, which have more dense riparian vegetation but also more dense household waste. Both toads have parotoid glands on their skin that could protect them from potential predators (e.g., x, y, z) and also microbial infections (Neerati & Yanamala, 2013;Mariano et al, 2019;Wulandari et al, 2013), which are likely to be found on disturbed areas or areas with many household wastes. Phrynoidis asper is commonly found in the upstream area (Subeno, 2018).…”