The use of hydrophobins to functionalize surfaces - IOS Press

Bio-Medical Materials and Engineering 14 (2004) 447–454 IOS Press

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The use of hydrophobins to functionalize surfaces K. Scholtmeijer a,∗ , M.I. Janssen b , M.B.M van Leeuwen c , T.G. van Kooten c , H. Hektor a and H.A.B. Wösten d a

BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG, Groningen, The Netherlands b Groningen Biotechnology and Biomolecular Sciences Institute, Kerklaan 30, 9751 NN Haren, The Netherlands c Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands d Microbiology, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands Abstract. The physiochemical nature of surfaces can be changed by small proteins which are secreted by filamentous fungi. These proteins, called hydrophobins, are characterized by the presence of eight conserved cysteine residues and a typical hydropathy pattern. Upon contact with a hydrophilic–hydrophobic interface they self-assemble into highly insoluble amphipathic membranes. As a result, hydrophobic surfaces become hydrophilic and vice versa. Genetic engineering of hydrophobins was used to study structure–function relationships. In addition, engineered hydrophobins were constructed to increase the biocompatibility of surfaces. The glycosylated N-terminal region of the mature SC3 hydrophobin was deleted and the cell-binding domain of human fibronectin was introduced at the N-terminus. The gross properties of the hydrophobins were not affected. However, the physiochemical properties of the hydrophilic side of the assembled protein did change. Growth of fibroblasts on Teflon could be improved by coating the solid with the engineered hydrophobins. Thus, by changing the N-terminal part of hydrophobins, the physiochemical nature of the hydrophilic side of the assembled form can be altered and a variety of new functionalities introduced. The fact that hydrophobins self-assemble at any hydrophilic–hydrophobic interface, irrespective of the chemical nature of the surface, therefore provides a generic approach to modify surfaces and make them interesting candidates for the use in various technical and medical applications. Keywords:, Biocompatibility, genetic engineering, hydrophobin, surface modification

1. Introduction Hydrophobins are small (±100 amino acids), moderately hydrophobic proteins produced by filamentous fungi [1,2]. They fulfill a broad spectrum of functions in fungal growth and development. For instance, these proteins are involved in formation of hydrophobic aerial structures (e.g., aerial hyphae and fruiting bodies) and mediate attachment of hyphae to hydrophobic surfaces [2–4]. Hydrophobins are characterized by eight conserved cysteine residues and a typical hydropathy pattern. However, their amino acid sequences are quite diverse [2,5]. The length of the N-terminal sequence preceding the first cysteine residue is also variable. They range from 17 to 158 amino acids when signal sequences for secretion are taken into account [4]. Although a few hydrophobins contain post-translational * Corresponding author: K. Scholtmeijer, BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. Tel.: +31 50 3634554; Fax: +31 50 3632249; E-mail: [email protected].

0959-2989/04/$17.00  2004 – IOS Press and the authors. All rights reserved

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K. Scholtmeijer et al. / The use of hydrophobins to functionalize surfaces

modifications, their generic properties can be attributed solely by their amino acid sequences [6]. Based on differences in hydropathy pattern and biophysical properties, class I and class II hydrophobins can be distinguished [5]. Both types self-assemble at a hydrophilic–hydrophobic interface into an amphipathic film [2,7]. The membranes formed by class I hydrophobins are highly insoluble. They resist boiling in detergent and can only be dissociated by agents like trifluoroacetic acid (TFA) and formic acid [1,8]. In contrast, assemblages formed by class II hydrophobins are less stable. Those of CU and CRP can be dissociated in 60% ethanol and 2% SDS [6,9,10] and assembled CU also dissociates by applying pressure or by cooling [9]. Because of the instability of the assemblages of class II hydrophobins these proteins seem to be less interesting for changing surface properties as class I hydrophobins and will therefore not be discussed further. The hydrophobin SC3 of Schizophyllum commune is the most extensively studied class I hydrophobin and, as far as tested, other members of this class have similar properties. Contact with hydrophilic– hydrophobic interfaces results in the self-assembly of SC3 monomers into a 10 nm thick amphipathic film [11–13]. The hydrophilic and hydrophobic sides of the SC3 membrane have water contact angles (θ) of 36 and 110◦ , respectively [11,12]. Interfacial self-assembly of SC3 involves several conformational changes [6,14,15]. The β-sheet rich water soluble form of SC3, composed of dimers and tetramers [16] initially adopt a conformation with increased α-helix (α-helix state). SC3 is arrested in this intermediate state at the water–Teflon interface but at the water–air interface the protein proceeds to a form with increased β-sheet (β-sheet state). Initially, the protein has no clear ultra-structure (β-sheet I state) but as time proceeds molecules rearrange such that a film is formed with an ultrastructure composed of a mosaic of parallel 10 nm wide rodlets (β-sheet II state) [15]. The transition from the α-helix state to the β-sheet state can also occur at a water–solid interface but has to be induced by increasing the temperature and by adding detergent [6,15]. Upon self-assembly the properties of SC3 also change. SC3 in the β-sheet state is highly surface active, while the water soluble forms have no detectable surface activity [17,18]. In addition, the α-helix form appears to be less stable than the β-sheet state. Although both forms strongly adhere to hydrophobic surfaces, e.g., resisting washes with water or buffer, the α-helix form can be dissociated into the water soluble forms by treatment with cold diluted detergents. In contrast, the β-sheet state is not affected by this treatment [6,15]. The characteristic property of hydrophobins to form an amphipathic membrane upon contact with a hydrophilic–hydrophobic interface allows them to change the nature of a surface (Fig. 1). Hydrophobic surfaces of liquids (e.g., oil droplets) or solids (e.g., Teflon, polyethylene) can be made hydrophilic by suspending or submerging them into a solution of hydrophobin [12,13,19]. Conversely, by allowing such a solution to evaporate on glass or filter paper, these materials become hydrophobic [11,19].

2. Genetic engineering of hydrophobins In order to examine structure-function relationships we have started genetic engineering of hydrophobins [20]. The class I hydrophobins SC4 of S. commune and ABH1 and ABH3 of Agaricus bisporus have similar properties as SC3 [19,21,22]. However, the mature forms of these hydrophobins have only 8 to 9 amino acids preceding the first cysteine residue and are not glycosylated [19,21,22] whereas this region in SC3 has a length of 31 amino acids and contains 16–22 mannose residues [14]. This indicates that this part does not determine the gross properties of hydrophobins. However, it may determine the surface properties of the hydrophilic side of the assemblage since the N-terminus of SC3 is exposed at this side after assembly [23]. To study the effect of modification of the N-terminal region


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Fig. 1. Hydrophobins can change the wettability of surfaces by their property to self-assemble at hydrophilic–hydrophobic interfaces. (A) Air bubbles or oil droplets in an aqueous solution of hydrophobin become coated with an amphipathic film that stabilizes them in water. (B) Similarly, when a sheet of hydrophobic plastic such as Teflon (θ 110◦ ) is immersed in such a solution it is coated with a strongly adhering protein film that makes the surface wettable (θ 22–63◦ ). (C) In contrast, hydrophobin monomers dried down on a hydrophilic surface make the surface hydrophobic (θ 110◦ ). Adapted from [7].

on hydrophobin properties, derivatives of SC3 were constructed (Fig. 2) [20]. To construct a SC3 variant more resembling hydrophobins such as SC4, ABH1 and ABH3 with respect to the length of the N-terminal region and glycosylation, 25 out of the 31 amino acids preceding the first cysteine residue of mature SC3 were deleted (TrSC3). Moreover, a tripeptide (RGD) was introduced at the N-terminal end of SC3 and TrSC3 (RGD-SC3 and RGD-TrSC3, respectively) in such a way that RGD is preceded by G and followed by SP, creating the GRGDSP of fibronectin. These derivatives were produced in a strain of S. commune with a disrupted SC3-gene. The SC3 derivatives were purified from the culture medium in mg quantities to allow characterization [20]. SDS-PAGE revealed a protein band at 24 kDa in the RGDSC3 strain which was similar to the position of SC3 in the wild-type strain. Both proteins reacted with the SC3-antiserum. In contrast, TrSC3 and RGD-TrSC3 had apparent molecular weights of 6.4 kDa and did not react with the SC3 antiserum. The higher mobility in SDS-PAGE and the fact that the anti-serum did not react with these proteins can be explained by the absence of glycosylation in these derivatives of SC3. Indeed, in contrast to SC3 and RGD-SC3, TrSC3 and RGD-TrSC3 did not react with PAS-staining. N-terminal sequencing analysis confirmed the identity of the proteins. MALDI-TOF mass spectroscopy of SC3 and its derivatives were in agreement with the expected masses based on N-terminal sequencing and assuming the absence of mannose residues in TrSC3 and RGD-TrSC3 and the presence of 16–22 mannose residues in SC3 and RGD-SC3.

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Fig. 2. N-terminal sequences preceding the first cysteine residues of SC3 and its derivatives. The N-terminal signal sequences that are cleaved off upon entry in the endoplasmic reticulum are not shown. In RGD-SC3 the RGD sequence is introduced behind Gly26 and His27 is substituted for serine, creating the GRGDSP peptide sequence of fibronectin. In TrSC3 the amino acids Gly29-Gly53 of SC3 are removed. RGD-TrSC3 is a derivative of TrSC3 in which the RGD sequence is introduced behind Gly26 and His27 is substituted for serine. –M indicates putative O-glycosylation sites. The introduced amino acids in RGD-SC3 and RGD-TrSC3 and the amino acids of SC3 that were deleted in TrSC3 are indicated in bold.

CD-spectrometry revealed that the modifications did not affect the conformation of the water soluble forms of the derivatives and the conformational changes that accompany self-assembly. Moreover, all derivatives lowered the water surface tension from 72 to 30–32 mJ m−2 at 100 µg ml−1 , indicating that the surface activity was also not affected by the modifications [20]. Surface shadowing revealed that the hydrophobic side of the assembled derivatives was characterized by rodlets similar to those formed by SC3. However, the diameter of the TrSC3 and RGD-TrSC3 rodlets ranged between 8–10 nm instead of the 10 nm observed for SC3 and RGD-SC3. Yet, wettability of the hydrophobic side of the assembled hydrophobins was not affected. In contrast, hydrophilicity of the hydrophilic side of the assembled hydrophobins did change. Assembled TrSC3 and RGD-TrSC3 had water contact angles of 73◦ and 68◦ at their hydrophilic sides, respectively, which is considerably higher than those of SC3 (44◦ ) and RGD-SC3 (44◦ ). The reduced wettability of the truncated SC3 forms is probably due to the absence of the mannose residues because chemically deglycosylated SC3 had a similar water contact angle (66◦ ) as TrSC3 and RGD-TrSC3. 3. SC3 and TrSC3 are not cytotoxic In medical applications, a molecule should not be toxic. To examine whether (genetically engineered) hydrophobins are potentially cytotoxic, water soluble and assembled SC3 or TrSC3 (up to 125 µg ml−1 ) were added to the medium of 24-h-old fibroblast cultures growing in 2 ml RPMI-1640 medium complemented with penicillin and streptomycin (1% each), 1% glutamine and 10% fetal calf serum [24]. Since 1.5 mg of SC3 is sufficient to coat 1 m2 of Teflon [12], the amount added can coat a surface of 1700 cm2 . At all concentrations used, SC3 and TrSC3 did not affect cell morphology, confluence or cell numbers. Furthermore, cell lysis was not increased. Only at the highest concentration of water soluble TrSC3 (125 µg ml−1 ) an effect on cell numbers was observed after 24 h. No effects were seen when a ten-fold lower concentration of water soluble TrSC3 was used. From this it was concluded that at least water soluble and assembled SC3 as well as assembled TrSC3 are not cytotoxic. Since the assembled form of hydrophobins is of interest for medical applications, the low cytotoxicity of water soluble TrSC3 should not pose a problem, especially since the assemblage hardly, if at all, dissociates in aqueous solutions. The effect of water soluble and assembled SC3 and TrSC3 on mitochondrial activity (MTT) of the fibroblasts was also tested [24]. After 24 h of growth, the mitochondrial activity showed a decrease of 50% compared to the control at the highest concentrations of both water soluble and assembled SC3. This reduction was less with water soluble and assembled TrSC3 (30%). After 72 h similar reductions


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(30–40%) of mitochondrial activities were observed with water soluble and assembled forms of SC3 and TrSC3. Taken together, these results supported the potential to use hydrophobins in medical applications.

4. Increased biocompatibility of surfaces by using hydrophobins For replacement or support of body parts (e.g., bone, spinal, cardiac and dental tissues) implants made of artificial materials can be used. However, these materials often poorly integrate into human tissue. This makes it necessary to develop implant materials that have improved biocompatibility. By modifying the surface properties of the material its interaction with cells can be improved [25]. Another approach to improve biocompatibility is to modify the material surface with molecules involved in receptor-mediated cellular functions [26], such as fibronectin. The Arg-Gly-Asp (RGD) sequence of this adhesin is sufficient to bind integrins in the cell plasma membrane [27–29]. Indeed, by immobilizing this tripeptide at an implant surface, cell attachment is promoted in a way similar to that of fibronectin [30– 36]. Both approaches to increase biocompatibility of surfaces may be addressed by using the derivatives of SC3. Coating of a hydrophobic surface with SC3 or TrSC3 results in a hydrophilic and a moderately hydrophilic surface, respectively, and the introduction of the RGD-tripeptide at the N-terminus of these hydrophobins provides a surface exposing this integrin binding peptide. To examine biocompatibility of hydrophobin coatings, fibroblasts (mouse strain L929, ATTC CCL1) were grown for 96 h in cell cultures on Teflon coated with SC3 or its derivatives [20,24]. Cells grew poorly on bare Teflon or Teflon coated with SC3 and showed a rounded morphology compared to the spreaded, flat morphology observed in control wells without Teflon (Table 1). Fusion of the RGD peptide to SC3 improved the biocompatibility of the hydrophobin (Table 1). Whether this is due to the slightly decreased wettability or to the interaction of fibroblasts with the RGD peptide remains to be established. Increase in cell numbers due to coating with TrSC3 was similar to that observed with RGD-SC3. However, the morphology of cells exposed to TrSC3 was improved (Table 1). Biocompatibility of TrSC3 was thus improved compared to that of RGD-SC3. No further improvement of biocompatibility was observed for RGD-TrSC3 as far as cell numbers and cell morphology are concerned. The increased biocompatibility of TrSC3 when compared to SC3 may be due to decreased wettability of the former hydrophobin, being closer to the optimal water contact angle range with respect to cell adhesion and spreading [37]. In agreement, the SC4 hydrophobin of S. commune similarly promoted cell growth as TrSC3 [24]. As mentioned, the wettability of the hydrophilic side of these hydrophobins is similar although they share only 45% amino-acid identity. Mitochondrial activity (MTT) of cells Table 1 Growth of fibroblasts on Teflon coated with SC3 or its derivatives. Cells were grown for 96 h. Bare Teflon and a well (tissue culture polystyrene) without Teflon served as controls Coating None SC3 RGD-SC3 TrSC3 RGD-TrSC3 SC4 Control well

Cell number [% of control] 50 40 60 65 70 70 100

Estimated confluence [% of surface] 80 70 80 95 95 95 95

Morphology rounded rounded somewhat rounded flat, spreaded flat, spreaded flat, spreaded flat, spreaded

MTT activity [% of control] 30 25 40 60 30 50 100

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growing on the various substrates showed a reduction varying between 40–75% of that of the control (Table 1) [24]. The biocompatibility assays described above were all performed using hydrophobins in their α-helical state. As mentioned, hydrophobins at solid surfaces can undergo the conformational change to the β-sheet end state by treatment with hot detergent. This results in a more moderate wettability compared to the α-helical state (M.I. Janssen, in preparation). Thus, cell growth may be different on Teflon coated with hydrophobin in either conformation. However, no changes in cell growth or morphology were observed in case of SC3 or SC4, although we cannot exclude an effect in case RGD is fused to the N-terminus (M.I. Janssen, in preparation). However, the reduction of the mitochondrial activity of the β-sheet state forms of both SC3 and SC4 was 2-fold lower compared to that of the α-helical state forms. This strongly indicates that the β-sheet state forms are the preferred forms for coating medical implants. Yet, previous studies have shown that mitochondrial activity is not necessarily indicating a better biocompatibility [38–40]. Therefore studies on hydrophobins have to be extended.

5. Conclusion The reported data show that by changing the N-terminal part of a hydrophobin the physio-chemical nature of the hydrophilic side of hydrophobin assembled on a hydrophobic surface can be altered [20]. In addition, it was shown that new functionalities can be introduced such as modulation of cell growth on a hydrophobic surface [20,24]. The fact that hydrophobins self-assemble at any hydrophilic–hydrophobic interface, irrespective of the chemical nature of the surface and via non-covalent interaction, is also an advantage. They thus present a generic way to change hydrophobic surfaces. Moreover, the strong interaction of hydrophobins with hydrophobic solids offers advantages over other proteins capable of coating surfaces. Furthermore, hydrophobins do not seem to be toxic (they are ingested upon consumption of mushrooms and fungus-fermented foods) or cytotoxic. Therefore, hydrophobins are interesting candidates for use in various medical and technical applications [2,20]. Naturally occurring hydrophobin variants and/or chemically or genetically modified variants can be used to change the biophysical properties of surfaces. In this way binding of molecules or cells to surfaces can be controlled. Apart from changing the biophysical properties, hydrophobins can be used to attach molecules to surfaces that otherwise do not have a high affinity for this surface. Attachment can be achieved by chemical cross-linking after the hydrophobin has been assembled on the surface or even via non-covalent interactions [41]. In addition, in case of proteins or peptides, fusion proteins can be made and assembled on the surface of interest. An additional advantage of the self-assembly properties of hydrophobins is the fact that they belong to the most surface-active molecules identified to date [6]. Their surface activity is similar to that of traditional biosurfactants (e.g., glycolipids, lipopeptides/lipoproteins, phospholipids, neutral lipids, substituted fatty acids and lipopolysaccharides) [42] used in a wide range of industrial applications like in emulsions and dispersions. Surface activity of hydrophobins is solely caused by the amino acid sequence and is thus not dependent on a lipid molecule as in traditional surfactants. Hydrophobins are, in contrast to traditional surfactants [42], encoded by small genes of about 400 bp, simplifying genetic modification to change the properties in such a way that they meet required characteristics. Hydrophobin monomers are expected to be 3 nm in diameter assuming a globular protein. This small diameter would allow patterning of molecules on a surface with nanometer accuracy. Since different hydrophobins are capable of co-assembly (X. Wang, unpublished), a mixed membrane could be made


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with a hydrophobin exposing a functional group and a hydrophobin carrying a different functional group or contributing a certain biophysical property.

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