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Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status.

ABSTRACT: BACKGROUND: Allium sativum leaf agglutinin (ASAL) is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL. METHODOLOGY/PRINCIPAL FINDINGS: By introduction of 5 site-specific mutations (-DNSNN-), a ? turn was incorporated between the 11(th) and 12(th) ? strands of subunits of ASAL, resulting in a stable monomeric mutant ASAL (mASAL). mASAL was cloned and subsequently purified from a pMAL-c2X system. CD spectroscopic analysis confirmed the conservation of secondary structure in mASAL. Mannose binding assay confirmed that molecular mannose binds efficiently to both mASAL and ASAL. In contrast to ASAL, the hemagglutination activity of purified mASAL against rabbit erythrocytes was lost. An artificial diet bioassay of Lipaphis erysimi with mASAL displayed an insignificant level of insecticidal activity compared to ASAL. Fascinatingly, mASAL exhibited strong antifungal activity against the pathogenic fungi Fusarium oxysporum, Rhizoctonia solani and Alternaria brassicicola in a disc diffusion assay. A propidium iodide uptake assay suggested that the inhibitory activity of mASAL might be associated with the alteration of the membrane permeability of the fungus. Furthermore, a ligand blot assay of the membrane subproteome of R. solani with mASAL detected a glycoprotein receptor having interaction with mASAL. CONCLUSIONS/SIGNIFICANCE: Conversion of ASAL into a stable monomer resulted in antifungal activity. From an evolutionary aspect, these data implied that variable quaternary organization of lectins might be the outcome of defense-related adaptations to diverse situations in plants. Incorporation of mASAL into agronomically-important crops could be an alternative method to protect them from dramatic yield losses from pathogenic fungi in an effective manner.

SUBMITTER: Banerjee N

PROVIDER: S-EPMC3072408 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status.

Banerjee Nilanjana N Sengupta Subhadipa S Roy Amit A Ghosh Prithwi P Das Kalipada K Das Sampa S

PloS one 20110407 4

<h4>Background</h4>Allium sativum leaf agglutinin (ASAL) is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL.<h4>Methodology/principal find ...[more]

PMID: 21490929

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Project description:Background: In a recent intervention study, the daily supplementation with 200mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. Methodology/Principal Findings: Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 869 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNFα-stimulated endothelial cells. Nuclear factor-kappa B reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. However, in individuals, significant changes in DNA methylation of genes involved in leukocyte rolling, adhesion and cytoskeleton remodelling were seen. At the group level, the variation in DNA methylation related to life style and clinical parameters appeared to overrule the magnitude of effects of supplementation with MOF. Conclusion: Our study revealed that regular consumption of MOF modulates the expression of genes in immune cells which, however, cannot be correlated to alterations in the DNA methylome. Remarkably, at the individual level, genes related to leukocyte adhesion pathways present complex variation in DNA methylation. As such, the individual transcriptional and epigenetic modulation of genes involved in early manifestation of cardiovascular diseases constitutes important subcellular mechanisms by which MOF promote vascular health in humans.

Dataset Information

Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status.

Publications

Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status.

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