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  • In this report we characterized MRJP containing

    2021-11-23

    In this report, we characterized MRJP-containing mct inhibitor and other (glyco)proteins from honey. Honey glycoproteins demonstrated potent anti-C. elegans activity. It has been reported that molecules that kill C. elegans are likely to be toxic to parasitic nematodes (Burns et al., 2015). To examine molecular mechanisms underlying nematicidal activity of honey and MRJP-containing aggregates, expressional analyses of gene transcripts of housekeeping that are critical for the growth, development, reproduction, and movement of C. elegans were carried out. These findings provided novel information regarding the function of MRJP1 as a major nematicidal protein of honey.
    Material and methods
    Results and discussion Previously, we have reported nematicidal activity of natural honey (LD90 = 1.5%) using nematode C. elegans which is widely considered as a useful model for anthelmintic discovery (Azim and Sajid, 2009). Anti-C. elegans molecules are likely to kill parasitic nematodes particularly those from the same phylogenetic clade as C. elegans (e.g. C. onchophora and H. contortus) (Burns et al., 2015). The nematicidal activity of honey was found to be comparable with WHO recommended anthelmintic drugs (e.g. Levamisole) (Hagel and Giusti, 2010). We also showed that the nematicidal effect of honey was due to the reproductive anomaly as manifested by defects in egg-laying and -hatching by C. elegans (Sajid and Azim, 2012). A glycoconjugate with a molecular mass of 5.5 kDa was identified as among the nematicidal molecules mct inhibitor present in honey (Sajid and Azim, 2012). However, detail characterization of nematicidal components of honey and underlying biochemical pathways remained elusive. The natural honey predominantly comprised of simple sugars i.e. D-fructose, D-glucose, maltose, and sucrose. These sugars were not found to be involved in the nematicidal activity (Sajid and Azim, 2012). During the present study, characterization of nematicidal effects of honey and honey (glyco)proteins were carried out using microscopy, gene expression analysis, and protein chemistry. C. elegans based nematicidal assays were carried out using ammonium sulphate precipitated proteins of honey. For this purpose, 200 adult hermaphrodite C. elegans were incubated for 24 h at 20 °C with 50, 100, 150, 200 and 300 ng/μL of ammonium sulphate precipitated honey protein. Effects of honey protein mixture was checked by inverted as well as fluorescence microscope (using acridine orange (AO) dye). Acridine orange is used to detect apoptosis in vitro (Ribble et al., 2005). In vitro immersion tests showed that honey proteins exhibited potent nematicidal effects on adult C. elegans with LD50 = 100 ng proteins/μL (Fig. 1g). Honey glycoproteins can enter into C. elegans body via ingestion through mouth or uptake by skin (Kaletta and Hengartner, 2006). Green fluorescence in AO-stained adult worms [treated with extracted honey proteins (50, 100, 150, 200, 300 ng proteins/μL)] indicated protein-acridine orange complex formation. Several studies reported that AO has tendency to bind with proteins (Chatterjee and Kumar, 2016) (Fig. 1 a–f). This is in agreement with previously reported honey-induced toxic effect in intestine and gonads of C. elegans (Sajid and Azim, 2012). These observations clearly indicated the involvement of honey proteins in anti-C. elegans activity.
    Conclusion Overall, results of the present study led to a number of important conclusions. First, (glyco)proteins from natural honey exhibited potent anti-C. elegans activity with LD50 of 100 ng proteins/μL. Chromatographically separated honey (glyco)proteins also showed potent nematicidal activity. Second, honey glycoproteins complexes with nematicidal activity (with molecular masses of 260 kD and 160 kD) comprised of ‘major royal jelly protein-1’-containing aggregates. MRJP1 exists in different glycosylation states in these complexes. Third, exposure of honey and honey (glyco)proteins caused regulation of gene transcripts involved in the citric acid cycle, cytoskeleton, body morphogenesis, embryo development, meiotic chromosome segregation etc. The quantitative gene expression results presented are at the transcript level, and therefore preliminary pending confirmation at the whole transcriptome as well as protein level. Our studies suggest that regulation of individual genes that contribute key molecular functions can be confidently elucidated using quantitative PCR approach.