Karin Scholtmeijer

Structural Characterization of the Hydrophobin SC3, as a Monomer and after Self-Assembly at Hydrophobic/Hydrophilic Interfaces

Hydrophobins are small fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes that, in the case of Class I hydrophobins, can be disassembled only by treatment with agents like pure trifluoroacetic acid. Here we characterize, by spectroscopic techniques, the structural changes that occur upon assembly at an air/water interface and upon assembly on a hydrophobic solid surface, and the influence of deglycosylation on these events. We determined that the hydrophobin SC3 from Schizophyllum commune contains 16-22 O-linked mannose residues, probably attached to the N-terminal part of the peptide chain. Scanning force microscopy revealed that SC3 adsorbs specifically to a hydrophobic surface and cannot be removed by heating at 100 degrees C in 2% sodium dodecyl sulfate. Attenuated total reflection Fourier transform infrared spectroscopy and circular dichroism spectroscopy revealed that the monomeric, water-soluble form of the protein is rich in beta-sheet structure and that the amount of beta-sheet is increased after self-assembly on a water-air interface. Alpha-helix is induced specifically upon assembly of the protein on a hydrophobic solid. We propose a model for the formation of rodlets, which may be induced by dehydration and a conformational change of the glycosylated part of the protein, resulting in the formation of an amphipathic alpha-helix that forms an anchor for binding to a substrate. The assembly in the beta-sheet form seems to be involved in lowering of the surface tension, a potential function of hydrophobins.

Fungal hydrophobins in medical and technical applications

Abstract. Class I and class II hydrophobins are small secreted fungal proteins that self-assemble at hydrophilic–hydrophobic interfaces into amphipathic films. Apart from eight conserved cysteine residues, the amino acid sequences between and within both classes have diverged considerably, and this is reflected in the biophysical properties of these proteins. For instance, assemblages of class I hydrophobins are highly insoluble, while those of class II hydrophobins readily dissolve in a variety of solvents. The properties of hydrophobins make them interesting candidates for use in a wide range of medical and technical applications. Each application has its own requirements, which may be met by using specific natural variants of hydrophobins or by modifying hydrophobins chemically or genetically. Applications also require high production systems for hydrophobins. In this respect, filamentous fungi that naturally secrete hydrophobins into the medium seem to be the hosts of choice.

Introns are necessary for mRNA accumulation in Schizophyllum commune

The cDNA coding sequence of the Agaricus bisporus hydrophobin gene ABH1 under the regulation sequences of the Schizophyllum commune SC3 hydrophobin gene gave no expression in S. commune. In contrast, the genomic coding sequence (containing three introns) produced high levels of ABH1 mRNA when transformed to S. commune in the same configuration. Apparently, introns were needed for the accumulation of mRNAs from the ABH1 gene. When the effect of intron deletion on expression of the homologous genes SC3 and SC6 was examined, it was observed that only the genomic coding sequences were expressed in S. commune. Run‐on analysis with nuclei harbouring intron‐containing and intronless SC6 showed that this effect did not occur at the level of transcription initiation: genomic and cDNA sequences were equally active in this respect. When a 50 bp artificial intron containing the consensus splice and branch sites of S. commune introns, in addition to random‐generated sequences, was introduced in the right orientation into the intronless SC3 transcriptional unit, accumulation of SC3 mRNA was restored. By polymerase chain reaction amplification, no unspliced SC3 mRNA species could be detected. Furthermore, the addition of an intron into the transcriptional unit of the gene for green fluorescent protein (GFP) effected clear fluorescence of the transgenic hyphae. Apparently, splicing is required for the normal processing of primary transcripts in S. commune.

Surface Modifications Created by Using Engineered Hydrophobins

ABSTRACT Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.

Highly Efficient Production of Laccase by the Basidiomycete Pycnoporus cinnabarinus

ABSTRACT An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml−1 in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter−1 was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml−1. These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter−1 when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml−1 (i.e., 360 mg liter−1) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter−1. In this case, maximal activities were 3,900 and 4,660 nkat ml−1, respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.

Coating with genetic engineered hydrophobin promotes growth of fibroblasts on a hydrophobic solid

Class I Hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into a highly insoluble amphipathic film. Upon self-assembly of these fungal proteins hydrophobic solids turn hydrophilic, while hydrophilic materials can be made hydrophobic. Hydrophobins thus change the nature of a surface. This property makes them interesting candidates to improve physio- and physico-chemical properties of implant surfaces. We here show that growth of fibroblasts on Teflon can be improved by coating the solid with genetically engineered SC3 hydrophobin. Either deleting a stretch of 25 amino acids at the N-terminus of the mature hydrophobin (TrSC3) or fusing the RGD peptide to this end (RGD-SC3) improved growth of fibroblasts on the solid surface. In addition, we have shown that assembled SC3 and TrSC3 are not toxic when added to the medium of a cell culture of fibroblasts in amounts up to 125 microg ml(-1).

Effect of Introns and AT-Rich Sequences on Expression of the Bacterial Hygromycin B Resistance Gene in the Basidiomycete Schizophyllum commune

ABSTRACT Previously, it was shown that introns are required for efficient mRNA accumulation in Schizophyllum commune and that the presence of AT-rich sequences in the coding region of genes can result in truncation of transcripts in this homobasidiomycete. Here we show that intron-dependent mRNA accumulation and truncation of transcripts are two independent events that both affect expression of the bacterial hygromycin B resistance gene in S. commune.

Use of hydrophobins in formulation of water insoluble drugs for oral administration

The poor water solubility of many drugs requires a specific formulation to achieve a sufficient bioavailability after oral administration. Suspensions of small drug particles can be used to improve the bioavailability. We here show that the fungal hydrophobin SC3 can be used to make suspensions of water insoluble drugs. Bioavailability of two of these drugs, nifedipine and cyclosporine A (CyA), was tested when administered as a SC3-based suspension. SC3 (in a 1:2 (w/w) drug:SC3 ratio) or 100% PEG400 increased the bioavailability of nifedipine to a similar degree (6+/-2- and 4+/-3-fold, respectively) compared to nifedipine powder without additives. Moreover, SC3 (in a 7:1 (w/w) drug:hydrophobin ratio) was as effective as a 20-fold diluted Neoral formulation by increasing bioavailability of CyA 2.3+/-0.3-fold compared to CyA in water. Interestingly, using SC3 in the CyA formulation resulted in a slower uptake (p<0.001 in T(max)) of the drug, with a lower peak concentration (C(max) 1.8 mg ml(-1)) at a later time point (T(max) 9+/-2 h) compared to Neoral (C(max) 2.2 mg ml(-1); T(max) 3.2+/-0.2). Consequently, SC3 will result in a more constant, longer lasting drug level in the body. Taken together, hydrophobins are attractive candidates to formulate hydrophobic drugs.

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

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.

Creating Surface Properties Using a Palette of Hydrophobins

Small secreted proteins called hydrophobins play diverse roles in the life cycle of filamentous fungi. For example, the hydrophobin SC3 of Schizophyllum commune is involved in aerial hyphae formation, cell-wall assembly and attachment to hydrophobic surfaces. Hydrophobins are capable of self-assembly at a hydrophilic-hydrophobic interface, resulting in the formation of an amphipathic film. This amphipathic film can make hydrophobic surfaces of a liquid or a solid material wettable, while a hydrophilic surface can be turned into a hydrophobic one. These properties, among others, make hydrophobins of interest for medical and technical applications. For instance, hydrophobins can be used to purify proteins from complex mixtures; to reduce the friction of materials; to increase the biocompatibility of medical implants; to increase the solubility of water insoluble drugs; and to immobilize enzymes, for example, biosensor surfaces.