Rink, Rick

Robillard, George T.

Scholtmeijer, Karin

Vocht, Marcel L. de

Wösten, Han A.B.

English

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 µg ml-1) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (β(1–3),β(1–6)-glucan) and β(1–3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment

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   University of GroningenAssembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on ItsConcentration and Is Promoted by Cell Wall PolysaccharidesScholtmeijer, Karin; Vocht, Marcel L. de; Rink, Rick; Robillard, George T.; Wösten, Han A.B.Published in:The Journal of Biological ChemistryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2009Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Scholtmeijer, K., Vocht, M. L. D., Rink, R., Robillard, G. T., & Wösten, H. A. B. (2009). Assembly of theFungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted byCell Wall Polysaccharides. The Journal of Biological Chemistry, 284(39), 26309-26314.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.LEGENDS SUPPLEMENTARY DATA  Fig. 1. Circular dichroism (CD) shows that SC3 in the α-helical state can be removed from a hydrophobic solid by addition of 0.1 % Tween 20 pH 7, while the β-sheet state remains adsorbed. (A) Addition of PTFE to an aqueous SC3 solution results in a change in the CD spectrum from that of the water-soluble form (black line) to that of the α-helical state (dotted line). Subsequent addition of Tween-20 at pH 7 reverses the spectrum back to that of the soluble state (grey line). (B) Treatment of SC3 adsorbed to PTFE with 0.1% Tween-20 at pH 2 results in the β-sheet spectrum (black line), which is not affected by increasing the pH to pH 7 (grey line).  Fig. 2. Schizophyllan promotes the transition of SC3 to the β-sheet state at the water-PTFE interface. WCA measurements of PTFE-sheets coated with SC3 at concentrations of 1, 2.5 and 5 µg ml-1 (pH 2) after washing with water (5W-0W) or 0.1 % Tween-20 pH 7 (5T-0T). Coating was performed for 16 h in the absence (white bars) or presence of 0.3 mg ml-1 schizophyllan (black bars). Incubation of Teflon in the absence of SC3 served as a control (samples 0 W and 0 T).  Fig. 3. Schizophyllan promotes the transition of SC3 to the β-sheet state at the water-PTFE interface at concentrations ≥ 0.3 mg ml-1. WCA measurements of PTFE-sheets coated with 5 µg ml-1 SC3 (pH 2) washed with water (5WW-5WT) or treated with 1 % SDS at 95°C (5 SDSW-5 SDST) followed by washing with either water (5WW-5 SDSW) or 0.1 % Tween-20 pH 7 (5WT-5 SDST). Coating was performed for 16 h in the absence (white bars) or presence of 0.04 mg ml-1 (light grey bars), 0.08 mg ml-1 (hatched bars), 0.17 mg ml-1 (dark grey bars) and 0.30 mg ml-1 (black bars) schizophyllan.   Fig. 4.  Schizophyllan accelerates the formation of SC3 into the β-sheet II amyloid state. ThT fluorescence of colloidal PTFE coated with an aqueous solution of SC3 in the absence (W) or presence of 0.3 mg ml-1 schizophyllan (SCH). Fluorescence was measured immediately after addition of ThT (white bars), or after 2 h (light grey bars) or 16 h (dark grey bars) of incubation. The black bars represent the total fluorescence obtained after addition of Tween 20 to a final concentration of 0.1% (pH2), which induces formation of amyloid fibrils at the PTFE surface.   Fig. 5. Scleroglucan promotes the transition of SC3 to the β-sheet state at the water-PTFE interface. WCA measurements of PTFE-sheets coated with 5 µg ml-1 SC3 washed with water (W) or treated with 0.1 % Tween-20 pH 7 (T). Coating was performed for 16 h in the absence (5W-5T) or presence of 1 mg ml-1 scleroglucan (5W Sc-5T Sc). Coating was performed at pH 2 (white bars), pH 5 (grey bars) or pH 7 (black bars).  Suppl. Figure 1                       Suppl. Figure 2     Suppl. Figure 3                             Suppl. Figure 4     Suppl. Figure 5     

Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

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Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

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