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 β turn was incorporated between the 11(th) and 12(th) β 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.

Figure 1. Alignment analysis of the deduced amino acid sequence of ASAL with closely related mannose binding homologues.

Sequence alignment among ASAL, Amorphophallus paeonifolius lectin (ACL), Arum maculatum lectin (AML), Allium cepa lectin (ACAL), Galanthus nivalis agglutinin (GNA) and Gastrodianin (GAFP). All identities and similarities are indicated by (*) and (:). Blue blocks and the grey box represent all mannose binding domains and the stretch of amino acids that differ in the monomer, respectively. The two residues at positions 98 and 99 (GNA numbering) are shown with two down triangles, where a trans-peptide bond is present in monomers instead of the cis-peptide bond found in all oligomers.

Figure 2. Comparison between the homology models of ASAL and mASAL.

(A) The homology model of ASAL to mASAL, showing that after incorporation of the loop between the 11^th and 12^th β-strand, the flanking C-terminal peptide folds back towards the central axis and thereby maintains the overall β-prism II fold. (B) Superimposition of mASAL (red) and the counterpart of ASAL (blue). The inflection point for the radical shift appears at position 98 (GNA numbering), from which point the C-terminal peptide moves in a completely different direction. In ASAL, the C-terminal peptide protrudes from the central axis of the molecule, whereas in mASAL, the 12^th β-strand is folded to form a homogeneous β-sheet.

Figure 3. Elution profile and expression analysis of mASAL.

(A) Expression and purification of mASAL in 15% SDS-PAGE analysis; lane 1 represents MBP fusion protein; lane 2 represents fusion protein digested with Factor Xa; lane 3 represents hydroxyapatite column purified mASAL; lane 4 represents Western blotting of purified mASAL against an anti-ASAL antibody showing a band at 12.5 kDa; lane M represents a standard protein molecular weight marker. (B) The corresponding elution profiles from size exclusion chromatography results of the mutant protein from a Biosep-SEC-S-2000 column of Phenomenex at the flow rate 2 ml/min. The figure shows fused mASAL with MBP (pick at 3.5 min), MBP and mASAL after factor Xa digestion (picks at 4.8 min and 6.15 min, respectively) and pure mASAL (pick at 6.1 min).

Figure 4. Size determination, Molecular characterization and secondary structure determination of mASAL and ASAL.

(A) Dimeric ASAL and monomeric mASAL were resolved in 15% native gel; Lane 1 represents purified ASAL showing a band in the 25-kDa region, lane 2 represents purified monomeric mASAL with band of size 12.5 kDa. (B) Proteins were resolved in 15% SDS-PAGE, lane 1 and lane 2 represent dimeric ASAL and mASAL, respectively. Both show bands in the 12.5-kDa region. Lane M represents the Standard protein molecular weight marker. Conservation of the secondary structure of (C) ASAL was determined by comparing the circular dichroism spectra with (D) mASAL. CD spectra were recorded over a wave length range of 200 to 260 nm. Spectra were obtained as an average of 10 scans and measured in PBS (pH 7.4) at a temperature of 25°C. The protein concentrations were approximately 0.2 mg/ml in PBS (pH 7.4). (E) Fluorescence spectra of Native ASAL and mASAL, where protein concentrations were 0.15 mg/ml. Excitation was performed at 295 nm and emission was scanned in the wavelength range of 300 to 400 nm. The slight red shift in the fluorescence spectra of mASAL observed was due to a change in the sub-domain organization after C-terminal self assembly in the monomeric form.

Figure 5. Determination of dissociation constant (K_d) for the interaction of mannose with mASAL and ASAL.

The protein concentration used was 0.15 mg/ml. The dissociation constant was calculated from the linear plot of ΔF/C against ΔF, where ΔF represents the increase or decrease in fluorescence intensity at a given concentration of mannose. (A) and (B) represent the binding of mannose with mASAL and ASAL where the dissociation constants were 0.12 µM and 0.06 µM, respectively.

Figure 6. Hemagglutination assay of rabbit erythrocyte with different doses of ASAL and mASAL.

Microtiter wells represent an agglutination pattern of 100 µl of 1% rabbit erythrocytes with various doses of ASAL and mASAL. Well 1 in vertical rows 1 and 2 represent negative controls containing PBS buffer (pH 7.4). Wells 2–5 in vertical rows one and rows two represent ASAL and mASAL with different doses (50 µg/ml to 6.12 µg/ml), respectively.

Figure 7. In vitro disc diffusion assay of mASAL.

In vitro antifungal activity of mASAL against (A) Fusarium oxysporum f.sp. cicero, (B) Fusarium lycopersici, and (C) Alternaria brassicicola. mASAL (5, 10, 15 µg) was applied to the filter discs numbered 3–5 of panel A and 3–6 of panel B and panel C (with 5,10,15,20 µg of mASAL). Discs 1 and 2 of panels A, B and C are 10-mM sodium phosphate buffer and purified ASAL (20 µg), respectively.

Figure 8. Fluorescent microscopic analysis of a propidium iodide uptake assay.

(A, B, C) and (D, E, F) are respective light microscope images and fluorescent images of mASAL treated R. solani, F. oxysporum and A. brassicicola. (G, H, I) and (J, K, L) are light microscope images and fluorescent images of mASAL pre-saturated with excess α-D mannose treated R solani, F. oxysporum and A. brassicicola, respectively. (M, N, O) and (P, Q, R) are light microscope images and fluorescent images of ASAL-treated R solani, F. oxysporum and A. brassicicola, respectively. (S, T, U) and (V, W, X) are respective light microscope images and fluorescent images of untreated R solani, F. oxysporum and A. brassicicola. Fungi were grown for 40 hours in the presence of mASAL and/or ASAL at peptide concentrations of 4 µg. Untreated fungi were taken as control. Afterwards, fungal hyphae were stained with Propidium iodide for 10 min, washed with 1× PBS, and subjected to fluorescent microscopic analysis. Bar = 15 µm. Images were captured with the AxioCam ICc3 digital camera and AxioVision imaging software system (Carl Zeiss Micro Imaging, GmbH, Germany).

Figure 9. Immunolocalization of mASAL in R. solani hyphae.

(A) Strong signals appeared in the hyphae of mASAL treated R. solani. In contrast, in (B), no signals were generated in the hyphae treated with ASAL. (C) Negative control (PBS buffer, pH 7.4). White arrows indicated presence of signals. Bars represent 10 µm.

Figure 10. Identification and characterization of fungal receptor.

(A) Staining of the subproteome of R. solani using a Sypro ruby stain. (B) An approximately 37 kDa putative receptor was identified in a ligand blot assay of the membrane subproteome of R. solani with mASAL when probed with an anti-ASAL antibody. (C) Ligand blot analysis of the subproteome with mASAL pre-saturated with excess α- D mannose and probed with an anti-ASAL antibody showed an absence of signal (D) Glycospecific-staining of subproteome indicated the glycoprotein nature of the putative receptor (arrowhead denoting an approximately 37-kDa receptor) (E) Staining of deglycosidase-treated subproteome by Sypro ruby stain (F) Ligand blot analysis of de-glycosylated subproteome probed with an anti-ASAL antibody showed no signal (G) A ligand blot assay of a subproteome without mASAL as a negative control (M) represents a standard Protein molecular weight marker.