The essential role of aggregation for the emulsifying ability of a fungal CYS-rich protein (2024)

Fungal growth

Fungal strains were maintained through periodic transfer on agar plates at 25° C, using malt extract agar medium. Mycelium discs (10mm diameter) were taken from the margin of the actively growing colonies and inoculated in 250ml flasks containing the medium (0.3g/L KH₂PO₄, 0.3g/L MgSO₄, 3g/L NaNO₃, 2g/L yeast extract) enriched with two different amount of olive oil, precisely 5% and 1% (v/v). The flasks were inoculated in triplicates and incubated in the dark at 25 ° C for 10 days.

BS extraction

At the end of incubation time, mycelia were separated from the cultural broths with a Whatman paper 3mm filters. The culture broth was centrifuged (4250 xg for 15min) and filtrated using a Stericup GP vacuum filtration system (Merck KGaA, Darmstadt, Germany). BS were extracted from the culture broth by adding MetOH: chloroform at a 1:1:0.5 v/v ratio, respectively. The sample was then centrifuged (1420 xg for 1h) and BS were recovered in the MetOH -water phase. The extract was then concentrated to the desired volume using a Rotavapor (Hei-Vap Core, Heidolph KG, Schwabach, Germany).

Protein concentration was evaluated using the BioRad Protein Assay kit (Hercules, California, USA) using bovine serum albumin as standard.

SDS-PAGE

Samples were first treated with Sample Buffer 4 × (2% sodium dodecyl sulfate (SDS), 80 mM Tris-HCl pH 6.8, 10% glycerol, 0.002% bromphenol blue, 0.05M dithiothreitol (DTT), and then they were further denatured boiling them at 100°C for 10 min. Electrophoretic runs were performed loading the samples on 12.5% acrylamide gels (12.5% acrylamide, 0.1% w/v bis-acrylamide, 0.4M Tris-HCl pH 9.2, 0.1% w/v SDS, 0.1% w/v ammonium persulfate (APS), 0.001% v/v tetramethyl ethylenediamine (TEMED)) and soaking it in 0.1M Tris-glycine pH 8.3. Silver stain coloration involves multiple steps and solutions reagent: first, in pH 6.5 solution A (0.5% glutaraldehyde, 0.1g/L sodium tiosulphate, 30% ethanol, 0.4M NaCl) was incubated for 1 hour followed by 3 water washings of 10 min each. Then the gel in a solution B (AgNO₃ 1g/L and formaldehyde 125 µl) was incubated for 30 min. During the last step the gel was stained by the solution C (3% Na₂CO₃ and formaldehyde 0.015%). When not specified, gels were dyed with Colloidal Coomassie G-250, BioRad. In the case of harsher denaturation treatment, samples were diluted in 4M urea, 0.1M DTT and then incubated at 60°C for 1h. At the end of the incubation time, 0.5M iodoacetamine (IAM) was added to the solution and incubated in the dark for 45’. To stop the reaction, 10% formic acid was added. Protein was then recovered using methanol-chloroform-water extraction (3:1:1 v/v).

Thin-layer chromatography (TLC)

Silica gel on TLC aluminum foil was used as a stationary phase. A mixture of toluene-chloroform-acetone (7:2:1) was used as the mobile phase, phosphom*olybdic acid (10% w/v in pure ethanol) and sulfuric acid (3% v/v in 10% ethanol) were used as a color developer for the detection of lipids and sugars respectively, through dipping and heating. 20µl of the extracted broths (MetOH 50%) were loaded on TLC with multiple deposition of 2µl; 10% olive oil in MetOH 50% was used as a control.

Surface activity

Drop collapse consists of depositing a sample drop onto a hydrophobic surface (parafilm M®, Amcor, Melbourne, Australia), and then evaluating the collapsing area. BS solutions should spread on the surface more extensively than water; pictures were acquired after 30min.

The surface tension, γ, of the extracts was measured with a Sigma 70 tensiometer (KSV, Stockholm, Sweden) using the Du Noüy ring method (Russo et al. 2017). γ was related to the force required to lift the ring from the surface of the air/liquid interface. 7ml of sample were added to the vessel and allowed to balance 5min prior to measuring surface tension. Water has been used to calibrate the tensiometer (72 mN/m).

Emulsification test

The emulsification capability of each sample was investigated. 2ml of Dectol (a mix of decane-toluene 65:35, v/v) were added as emulsifying agent to 1ml of each surfactant protein suspended in 10 mM phosphate buffer (pH 7.0) in 5ml glass vials (Blesic et al. 2018). This mixture was hom*ogenized using the IKA T-10 Basic Ultra Turrax hom*ogenizer (IKA-Werke GmbH, Staufen, Germany) for 3min, then the stability of the emulsions during the time was evaluated. Stability is reported as emulsification index after 24h, E₂₄.

Proteins samples, 0.05mg/mL, were incubated with Proteinase K (from Tritirachium album, Sigma-Aldrich, St. Louis, MO, USA) 1mg/mL at 37°C for 30min. Then, an emulsification test was performed as described above.

Protein identification by Mass Spectrometry (MS)

Protein identification by mass spectrometry was performed on Coomassie Blue stained bands excised from mono-dimensional gels. Protein bands corresponding to proteins of interest (molecular mass about 10kDa), were excised from gel, destained by washes with 0.1M NH₄HCO₃ pH 7.5 and acetonitrile, reduced for 45min in 100 µL of 10 mM dithiothreitol, 0.1M NH₄HCO₃, pH 7.5, and carboxyamidomethylated for 30min in the dark by the addition of 100 µL of 55 mM iodoacetamide (IAM) dissolved in the same buffer. Enzymatic digestion was performed and was then analyzed with 6520 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Palo Alto, CA, USA) equipped with a 1200 HPLC (high-performance liquid chromatography) system and a chip cube (Agilent Technologies). The acquired MS/MS spectra were transformed in Mascot Generic format (mgf) and used to query the NCBInr database, with taxonomy restriction to Fungi (http://www.ncbi.nlm.nih.gov).

UV-Vis analysis

Fluorescence spectra were recorded at 25°C with a HORIBA Scientific Fluoromax-4 spectrofluorometer (Horiba, Rome, Italy). Slits were set to 3 and 6nm spectral band-passes in excitation and emission monochromators, respectively. Tryptophan fluorescence emission was observed using λ_exc of 280nm (emission range 300–500nm); fluorescence emission of the putative fluorophore compounds was observed using λ_exc of 325nm (emission range 350–550nm).

Protein purification

5mg of total proteins were loaded on a Superdex 75pg HiLoad column (Merck KGaA, Darmstadt, Germany), with a flow of 0.3ml/min and monitoring the wavelength at 280nm. 10 mM sodium phosphate with 150 mM NaCl was used as elution buffer, fractions of 1ml were collected and then analysed. Successive chromatography purification was performed using anionic exchange column. 0.3mg of sample were first dialyzed with 10 mM sodium phosphate buffer 10 mM (RC dialysis tube, 3.5kDa cut-off, Merck KGaA, Darmstadt, Germany). Emulsification ability of the gel filtration peaks was verified after this process, and then the sample was loaded on HiTrap Q XL, 1ml column, and eluted using a linear gradient with 0.5M NaCl as final salt concentration, 0.5M using a linear gradient at 1ml/min flux and monitoring the absorbance at 280nm. Fractions were collected and further analysed.

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis

Samples were diluted 1:10 in MetOH, filtered and centrifuged (10,000rpm for 10min). The supernatant was then directly transferred into HPLC auto sampler and 1µl of supernatant was analysed by using an AB-sciex 5500 QTRAP® system with a HPLC chromatography system Exion LC™ (Agilent Technologies, Palo Alto, CA, USA). The mobile phase was generated by mixing eluent A (0.1% formic acid in water) and eluent B (0.1% formic acid in acetonitrile) and the flow rate was 0.2 mL/min. Chromatographic gradient was from 20 to 90% in 4min, hold for 2min, then return to 20% in 1min. Tandem mass spectrometry was performed using Turbo VTM ion source operated in positive ion mode, and the multiple reaction monitoring (MRM) mode was used for the selected analytes. Qualitative analysis and identification of the phenolic compounds contained in samples were carried out by using LC-MS/MS in negative (ESI-) ionization modes. A set of targeted molecules were explored by mass spectrometry in multiple reaction monitoring as reported in the supplementary section (Supplementary Table S1) taking advantage of high performances of triple quadrupole mass spec. For quantification of analytes, standard calibration curves for the selected set of molecules, were constructed by plotting peak areas against concentration (µg/L), and linear functions were applied to the calibration curves. The coefficients of determination (R2) were greater than 0.99 for all analytes. The extracted mass chromatogram peaks of metabolites were integrated using Skyline software for data processing. For each molecule specific transitions precursor ion/fragment ion were selected (supplementary Table S1). The LCK331/LCK332/LCK334 (Hach Lange GMBH, Linate, Italy) tests were used for the determination of anionic, cationic, and non -ionic surfactants, through a titration with bromine blue phenol. The concentration is determined by spectrophotometric analysis.

Fungal growth

F. solani (MUT 4426 and MUT 6181) were grown in the presence of olive oil as a carbon source, to stimulate BS production. Two different amounts of olive oil were used, 1% and 5% (v/v), to better exploit BS induction and surface tension was monitored during the fungal growth in the two conditions. Culture media, enriched with the same amounts of olive oil, used as a control, led to a minimal surface tension decrease compared to that of water (68 mN/m versus 73 mN/m). Figure1 shows that the lowest value is achieved when 5% olive oil is present in the culture media. On the other hand, no relevant amount of BS seemed to be produced when 1% of olive oil was added, as verified by the constant and high value of surface tension in this condition.

Mycelia were separated from the culture broth, freeze-dried, washed with hexane to remove adhered oil, and weighed. A much higher amount of fungal biomass (about fourfold for both strains) was obtained when culture broth was implemented with 5% olive oil. Thus, 5% olive oil and 10 days of growth were hence used as the best-growing conditions for all the successive experiments. BS production was also checked by emulsion tests in the presence of Dectol, finding neglectable results in both tested conditions.

BS characterization and protein identification

To purify BS produced by the strains, a commonly used MetOH -chloroform extraction protocol was adopted. To determine the chemical nature of the compounds present in these raw extracts, thin-layer chromatography was used, as a fast and simple technique (Supplementary Fig. S1). No lipids or sugars were detectable by the staining methods used. On the other hand, protein concentrations of both extracts were evaluated and found to be 60¸80µg/ml, hence the SDS-PAGE was carried out (Fig.2). Only one protein band was visible in each of the raw extracts loaded on the SDS PAGE. These bands were excised and submitted to a classical proteomic approach to identify proteins. The same protein could be confidently identified in the respective protein bands as C7Z2B1 from F. solani, a “common in fungal extracellular membrane (CFEM)” domain-containing protein. The identified peptides and the protein sequence are reported in Fig. S2A, B. Analysis of the protein aminoacidic sequence showed an isoelectric point of 7.45 and a significant hydrophobic character (grand average of hydropathy, GRAVY + 0.487) (Gasteiger et al. 2005) where three sequence portions showed a score > 1 (Kyle Doolittle scale, Fig. S2C). Eight Cys-residues are usually found in the CFEM domain, while the protein C7Z2B1 displays an even higher Cys content (10 Cys).

The raw extracts were distilled to remove MetOH and concentrated (tenfold the initial culture broth volume). Afterwards, the same amounts of proteins (1µg) were loaded again on the gel, and surprisingly other protein bands at higher molecular weight could be detected (Fig.2).

The appearance of several new bands at high molecular weights may suggest that formation of aggregates occurs after sample concentration. This phenomenon is not related to denaturation of these proteins since their structures, determined by circular dichroism analysis, did not change when the proteins were purified by ultrafiltration and dialysis, a protocol that led to a scarce protein yield (data not shown).

Emulsifying and surface activity of the raw extracts

At first surface activity of the concentrated raw extracts was qualitatively tested using drop collapse test. This first measurement validated the hypothesis that water-soluble surface-active compounds were present in the extracts, as the drop shape of the samples on a hydrophobic surface was flattened compared to the control. Quantitative measurements, using the tensiometer, confirmed the lower surface activity of these extracts, showing surface tension values of about 45 mN/m for both extracts. Emulsification tests were also carried out, revealing that both samples can efficiently stabilize emulsions exhibiting an E₂₄ value of about 80%. The different behavior of these raw extracts respect to that of the culture broths (no emulsifying ability) can be ascribed to the higher concentration.

A hydrolytic treatment with Proteinase K was performed to ensure that the observed low surface tension and emulsifying capability were related to the presence of proteins. Indeed, after this enzymatic treatment, no emulsion was observed, thus proving the crucial role of proteins in forming and stabilizing emulsions (Fig.3). On the other hand, reduction of the surface tension was still achieved even after the hydrolytic treatment. These results indicate that the protein components in the extracts are responsible for the emulsifying ability, while the surface tension reduction should be due to different compounds. Hence, further drop collapse tests and emulsification tests as a function of protein concentrations were performed (Fig.4). Experiments revealed that both samples can efficiently stabilize emulsions reaching a maximum value of E₂₄ of 80% at the highest concentration tested. Emulsions at higher protein concentration were not tested because of the rapid protein precipitation occurring when further concentrated. The samples from MUT6181 showed a higher emulsification index at low concentration, while the E₂₄ of the samples from MUT4426 increased slower as a function of concentration.

Protein purification

Spectrophotometric analyses were carried out on the two samples. Absorption spectra of both samples surprisingly showed another intense peak at 325nm, besides the peak corresponding to Trp (280nm). Thus, fluorescence spectra were recorded using 280nm and 325nm as λ_exc (Fig.5).

When the λ_exc was 280nm, two peaks were detected: a first one around 360nm, corresponding to the Trp emission, the second one, near 450nm suggesting the presence of a fluorophore different from Trp. When λ_exc was 325nm, indeed, an intense emission peak appeared at λ around 430nm. The spectra at 280nm λ_exc of the two samples, exhibited similar patterns, except for the relative intensity of the two peaks, indicating different abundance of the protein respect to the other unknown fluorophore.

Intending to understand if the fluorophores are free or protein-bound, and the role of each component, a gel filtration chromatography was tried out. Each raw extract was concentrated fivefold just before loading to avoid protein precipitation. The chromatograms showed three main peaks for both samples, that were collected and characterized as shown in Fig.6.

As reported in Fig.6, very similar results were obtained from the two chromatography experiments. In peak 1 (P1) of both samples, protein concentration was two-fold compared to that one of peak 2 (P2), about 0.1mg/ml and 0.05mg/ml respectively, whereas no protein at all was detected in P3. SDS-PAGE analysis, moreover, highlighted the presence of the single protein band near 10kDa in P2, such as that one previously identified in the raw extract. In P3 no protein band was detected, while multiple protein bands were visible in P1 whose molecular weights were higher than 25kDa. Analysis of fluorescence emission of these fractions showed that Trp emission was only observed for P1 and P2 and not for P3 (black spectra in Fig.6A-B), as expected. On the other hand, the emission peak near 450nm relative to the other putative fluorophore, (λ_exc 325nm, red spectra in Fig.6A-B), was only present in P3.

Moving to the analyses of surface and emulsification activity of the chromatographic fractions, results indicated that only P1 retains the ability to stabilize emulsions, while unexpectedly P2 does not (at the same protein concentrations, 0.05mg/ml). On the other hand, the decrease in surface tension was only related to P3. These observations seem to indicate that emulsification and surface activity are linked to two different components; the former can be attributed to high molecular weight proteins and the latter to unknown smaller molecules, also responsible for the fluorescence emission at 450nm.

Considering that chromatograms and relative peak characterizations revealed that the two strains exhibit very similar behavior, herein after results are only reported for MUT6181.

To further study the nature of the compounds eluted as P3, several analyses were performed. Using kits for the analysis of anionic, cationic and non-ionic surfactants only a cationic surfactant was found in this sample (9mg/L), confirming what was previously determined by measuring the surface tension.

The analysis carried out with LC-MS/MS in MRM mode, showed the presence of different polyphenols (Supplementary Table S2). It is worth noting that the most represented compound among those explored was naringenin in P3 (about 5mg/L). Anyway, examining the literature data on this compound, naringenin should be not responsible neither for the observed fluorescence emission of P3 (Uivarosi et al. 2016; Li et al. 2021) nor for the surface tension decrease.

As P1 concerns, the unique fraction showing emulsification activity, SDS-PAGE analysis revealed the presence of multiple bands. After the hydrolytic treatment with Proteinase K, no emulsion was formed (Supplementary Fig. S3). Hence, P1 sample underwent an anionic exchange chromatographic step, to try to purify the protein responsible for the emulsification activity.

Three different fractions were collected and named P1a, P1b, and P1c. Only P1b retained an emulsification ability similar to that of the loaded sample, the SDS-PAGE shows again multiple bands relating to this peak (Fig.7). Hence this sample was subjected to proteomic analysis (Supplementary Fig. S4). The presence of the C7Z2B1 protein in this sample led to the hypothesis that aggregation phenomena could occur, as a consequence of the concentration step performed before the gel filtration, moreover the aggregates should not be effectively broken during the SDS-PAGE sample preparation. According to this hypothesis, emulsification activity should be only ascribable to aggregates of that protein, and not to its monomeric form.

To validate this hypothesis P2 sample (from gel filtration) was concentrated, while P1 (from gel filtration) was subjected to a harsher denaturation treatment (with urea, DTT, and IAM), and then both samples were loaded on SDS PAGE. In Fig.8a unique band near 10kDa, relative to P1 after urea denaturation and cys carboxyamidomethylation was detected, but its emulsification activity was lost. On the other hand, the presence of several bands at higher molecular weight raised after P2 sample concentration, and the formation of a stable emulsion further confirms that hypothesis.

Supplementary Information

Below is the link to the electronic supplementary material.

Conflict of interest

The authors declare no competing interests.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

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