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Modification of (bio)material surfaces using hydrophobinsJanssen, Meike Irene

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Chapter 5

Interaction of proteins with hydrophobin-coated Teflon

M. I. Janssen, L. Dijkhuizen and H. A. B. Wösten

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Abstract Proteins readily adsorb from an aqueous solution when they contact a solid surface. This conditioning film changes the physico-chemical properties of the surface of the solid and thus may affect the performance of e.g. a biomaterial. We here show that the non-specific adsorption of proteins to Teflon is not reduced by pre-coating the surface with the SC3 and SC4 hydrophobins of Schizophyllum commune. In fact, the extracellular matrix protein fibronectin adsorbed more strongly to Teflon coated with SC4 than to bare and SC3-coated Teflon. SC3 and SC4 coatings were stable during exposure to protein mixtures but the SC4 coating on Teflon was degraded when human fibroblast cells were grown on the coated surface.

Introduction Cell adhesion to solid surfaces is influenced by physico-chemical properties like surface hydrophobicity, surface topography and surface free energy. These properties are changed by a conditioning film of adsorbed proteins that rapidly forms when a solid contacts a protein solution [Wahlgren and Arnebrant, 1991; Bruinsma et al., 2001]. Although the composition of the conditioning film varies, all materials quickly acquire a layer of a variety of proteins in many states of orientation and denaturation [Horbett, 1993]. This layer may have a beneficial or detrimental impact on the performance of a biomaterial [Wahlgren and Arnebrant, 1991]. Non-specific protein adsorption can be effectively prevented by pre-coating the material with phospholipid polymers [Lewis, 2000], polyvinyl alcohol [Barrett et al, 2001; Amanda and Mallapragada, 2001], polyacrylamide [Park et al., 2000], hyaluronic acid [Matsuda et al., 1992], dextran [Holland et al, 1998], and especially poly(ethylene glycol) (PEG) [Scott and Murad, 1998; Zalipsky and Lee, 1992].

In the present study proteins called hydrophobins were used to coat hydrophobic Teflon surfaces. Hydrophobins such as SC3 and SC4 of Schizophyllum commune are small fungal proteins of about 100 amino acids that are characterized by eight conserved cysteine residues and a typical spacing of hydrophilic and hydrophobic regions [Wessels 1997]. They self -assemble at any hydrophilic-hydrophobic interface (e.g. between water and air, water and oil, or water and a hydrophobic solid) into an amphipathic monolayer of about 10 nm thick [Wö sten et al., 1993, 1994a, 1994b, 1995]. The monolayer strongly attaches to a hydrophobic solid like Teflon and makes it completely wettable [Wö sten et al., 1994a, 1995; Lugones et al., 1996, 1998, 1999]. During assembly at Teflon, hydrophobins are arrested in an intermediate conformation called the α-helical state. The stable β-sheet state can be induced by treatment with hot detergent [de Vocht et al., 1998; 2002]. The properties of hydrophobins make these proteins promising candidates for the use in technical and medical applications such as biomaterial devices [Wessels, 1997; Scholtmeijer et al., 2001]. Coating with natural or genetic engineered hydrophobins improved growth and morphology of fibroblasts on Teflon surfaces [Scholtmeijer et al., 2002; Janssen et al., 2002; Chapter 2; 3] and cellular activity was not

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affected when hydrophobins in the β-sheet conformation were used [Janssen et al., 2004; Chapter 4].

We here report on adsorption of proteins to Teflon coated with the SC3 and SC4 hydrophobins in the α-helical and β-sheet conformation and determined the stability of the hydrophobin coating during exposure to protein mixtures and cell cultures.

Materials and Methods Growth conditions for Schizophyllum commune, and isolation and purification of hyd rophobins S. commune was grown in 1 L shaken cultures (225 rpm) for 5 – 7 days at 24 °C in production medium [Scholtmeijer et al., 2002; Chapter 2]. The SC3 hydrophobin was produced using a strain with a deleted SC15 gene [Lugones 1999], while a dikaryon resulting from a cross between S. commune strains M4.58 [van Wetter et al, 2000] and 72-3 [Wö sten et al, 1994a; van Wetter et al, 1996] was used to produce SC4. In strains M4.58 and 72-3 the SC3 gene has been deleted, while strain M4.58 expresses a SC4 gene under control of the SC3 regulatory sequences. SC3 and SC4 were purified from the culture medium as described [Wö sten et al., 1993; Wessels 1997]. Coating of Teflon with hydrophobins Teflon sheets (FEP; 0,25 mm thick; Norton Fluorplast, Raamsdonkveer, The Netherlands) cleaned with 70 % ethanol were incubated overnight at 20 °C in aqueous solutions of SC3 or SC4 (50 µg ml-1). In this way, the hydrophobins adsorbed to the Teflon in the intermediate α-helical conformation. After washing 3 times 10 min with distilled water using a test tube rotator at 10 rpm, the sheets were allowed to dry. To induce the transition to the stable β-sheet conformation, coated Teflon sheets were heated for 10 min in 2 % SDS at 100 ºC. After the SDS treatment sheets were washed as described above. Bare Teflon was treated in the same way serving as a control. Protein adsorption from protein solutions Bare and hydrophobin-coated Teflon sheets (2.5 cm x 0.8 cm) were incubated concurrently in protein solutions contained in 2 ml eppendorf tubes for 16 h at 37 ºC. After incubation, the Teflon sheets were transferred to clean tubes. They were dried at 20 ºC using a concentrator (Eppendorf 5301). This was either or not preceded by 3 washes with distilled water of 10 min each. BSA was adsorbed from PBS (50 µg ml-1; Sigma) or from RPMI 1640 medium supplemented with 1 % L-glutamine and 10 % calf serum, in which BSA is present (Life Technologies). Human fibronectin (25 - 50 µg ml-1; Sigma) was dissolved in PBS or supplemented RPMI 1640 medium (see above). All experiments were done at least in duplicate in three independent experiments.

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Protein adsorption from cell cultures Human fibroblast strain Hs 44.fs (ATCC CRL-7024) was cultured in 25 cm2 T- flasks in Dulbecco’s modified Eagle’s medium (ATCC 30-2002) supplemented with penicillin and streptomycin (1 % each) and 10 % fetal calf serum. Cells were cultured at 37 °C in a humidified atmosphere containing 5 % CO2 and were routinely passaged by trypsinization. Bare and hydrophobin-coated Teflon was cut into discs (1.75 cm2) to fit into wells of tissue culture polystyrene (TCPS) 24 well plates. Cells were seeded in the test wells either or not containing Teflon sheets to a density of 3000 cells cm-2. After 24, 48 and 72 h cells were removed from the surfaces by a 5 min treatment with a solution of 0.25 % trypsin and 0.3 % EDTA in PBS. Samples were either or not washed with water (3 x 10 min) and air dried. Protein analysis Proteins adsorbed to bare and hydrophobin-coated Teflon were extracted with trifluoroacetic acid (TFA). After evaporation of the solvent by a stream of air, extracts were taken up in 1 x SDS sample buffer with 5 % β-mercaptoethanol and loaded on 12.5 % SDS PAA gels. After separation, proteins were stained with Fast Silver (Geno Technologies Inc.) or blotted onto PVDF membrane for immuno-detection. Immuno-detection was performed using IgG fractions of rabbit antisera against BSA (Abcam) and human fibronectin (Sigma). Goat-anti-rabbit conjugated with alkaline phosphatase (Sigma) was used as a second antibody with NBT/BCIP as substrates for detection [Harlow and Lane, 1988]. Gels and blots were digitized in 256 gray scale using Gene Genius Bioimaging System (Syngene, Synoptics Ltd).

Results Stability of hydrophobin coatings in protein solutions and cell culture Teflon coated with SC3 or SC4 in the intermediate α-helical conformation or the β-sheet end form were incubated for 16 h in PBS containing BSA (50 µg ml-1). Samples were washed with water followed by an extraction with TFA. After removing the solvent, proteins were analyzed by SDS PAGE. SC3 and SC4 in either conformation were neither replaced by the BSA during the overnight incubation nor removed by the washing procedure (Figure 1). Similar results were obtained when SC3- and SC4-coated Teflon were incubated in RPMI medium or artificial tear fluid (data not shown). Previously, it was shown that 1.5 mg m-2 of SC3 [Wö sten et al., 1994a] and 2.7 mg m-2 of SC4 [Janssen et al., 2004; Chapter 4] adsorb to Teflon. Our results show that this was only about 0.1 mg m-2 for BSA and fibronectin (data not shown). From this it is concluded that BSA and fibronectin have a much lower affinity to bare Teflon than the hydrophobins.

Human fibroblasts were grown on bare Teflon, Teflon coated with SC3 or SC4 in α-helical conformation and on TCPS. After 24, 48 and 72 h the cells were removed by a brief trypsin treatment. Proteins adsorbed at the surfaces were extracted with TFA (before and after washing with water) and analyzed by SDS PAGE (Figure 2). The amount of SC3 extracted from

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the Teflon surfaces did not decrease during the growth of the cells. In contrast, while SC4 was still present after 24 h of culturing, no SC4 could be detected after 72 h. SC4 and SC3 in α-helical and β-sheet conformation were not degraded when incubated for 3 h in a trypsin solution used to release the cells from the Teflon surfaces (see Materials and Methods; data not shown). This suggests that the disappearance of SC4 during growth of fibroblasts was due to proteases released by the cells. Figure 1. Hydrophobin extracted from Teflon before (1 - 4) and after (5 - 8) incubating the coated solid for 16 h in PBS containing 50 µg ml –1 BSA. Teflon was coated with SC3 in α-helical (1, 5) and β-sheet conformation (2, 6) or with SC4 in α-helical (3, 7) and β-sheet conformation (4, 8). The band in (9) represents 1 µg of BSA. Proteins were separated by SDS PAGE and stained with silver. The low molecular weight bands are expected to be degradation products of the hydrophobins. Adsorption of BSA and fibronectin to hydrophobin-coated surfaces Bare and hydrophobin-coated Teflon were incubated in PBS containing BSA or fibronectin (50 µg ml-1). Proteins adsorbed to the surfaces were extracted with TFA before and after washing with water and separated with SDS PAGE. Silver staining (not shown) and immuno-detection showed that similar amounts of BSA adsorbed to the SC3- and SC4-coated Teflon surfaces, irrespective of their conformation (Figure 3). Part of the adsorbed BSA was removed by the washing procedure. Also in the case of fibronectin, no differences could be observed between adsorption to the different coatings of SC3 and SC4. However, washing removed less fibronectin from SC4-coated surfaces. Adsorption of BSA and fibronectin to bare Teflon before washing with water was similar to that on hydrophobin-coated Teflon (Figure 3). Removal of these proteins by washing was similar to that from SC3-coated surfaces.

Bare Teflon and Teflon coated with SC3 or SC4 in α-helical or β-sheet conformation were incubated for 16 h in RPMI medium supplemented with calf serum (10 %), L-glutamine (1

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%) and fibronectin (25 µg ml-1). Adsorption of proteins was analyzed by SDS PAGE after extraction of the proteins from the Teflon sheets with TFA (with or without prior washing with Figure 2. SDS PAGE analysis of proteins adsorbed to bare Teflon (1, 5), Teflon coated with SC3 (2, 6) and SC4 (3, 7) in α-helical conformation and on TCPS (4, 8) during growth of human fibroblast cultures for 24 h (A) and 72 h (B) before (1 - 4) or after (5-8) washing with water. 1 µl of fresh medium supplemented with 10 % calf serum and 1 µg fibronectin was used as a standard (9). Bands which could be assigned to specific proteins are indicated. water) (Figure 4). Compared to adsorption from PBS, relatively high amounts of BSA were detected at bare and hydrophobin-coated Teflon sheets when adsorbed from RPMI medium (compare Figures 3 and 4). However, the protein was efficiently removed by washing with water. Levels of fibronectin adsorbing to bare and hydrophobin-coated Teflon were similar when adsorbed from RPMI medium. However, no immuno-signal for fibronectin was detected after washing the sheets with water. Similar results were obtained when bare and hydrophobin-

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coated Teflon sheets were incubated in supplemented Dulbecco’s modified Eagle’s medium instead of RPMI medium. Figure 3. Immuno-detection of BSA (A) and fibronectin (B) adsorbed from PBS at bare or hydrophobin-coated Teflon sheets before (1 - 4) and after (5 - 8) washing with water. Teflon was coated with SC3 in α-helical (1, 5) and β-sheet conformation (2, 6) or with SC4 in α-helical (3, 7) and β-sheet conformation (4, 8). Bare Teflon sheets were treated identical to those in the corresponding lanes of the hydrophobin-coated sheets. 1 µg of BSA or fibronectin served as standards. Figure 4. Immuno-detection of BSA and fibronectin adsorbed at hydrophobin-coated (A) or bare (B) Teflon sheets from supplemented RPMI medium with (5 - 8) or without (1 - 4) washing with water before extraction of the proteins with TFA. Teflon was coated with SC3 in α-helical (1, 5) and β-sheet conformation (2, 6) or with SC4 in α-helical (3, 7) and β-sheet conformation (4, 8). Bare Teflon sheets were treated identical to those in the corresponding lanes of the

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hydrophobin-coated sheets. 1 µl of RPMI supplemented with 10 % calf serum served as a standard (9). Adsorption of proteins from fibroblast cultures to bare and coated Teflon Human fibroblast cells were grown on bare Teflon discs or Teflon discs coated with SC3 or SC4 in α-helical conformation. Tissue culture polystyrene (TCPS) served as a control. The adsorbed proteins were analyzed by SDS PAGE after removing the cells with a brief trypsin treatment. During growth of the cells the total amount of protein that had adsorbed to bare and coated Teflon and to the TCPS increased (Figure 2). Washing with water removed most of the proteins except for the hydrophobins. BSA was the main protein that adsorbed to all surfaces (Figure 2). Immuno-detection confirmed that BSA adsorbed equally well at these surfaces after 24, 48 (not shown) and 72 h (Figure 5). In contrast, fibronectin was most abundant on the hydrophobin-coated Teflon and on TCPS (Figure 5). Figure 5. Immuno-detection of fibronectin and BSA adsorbed from human fibroblast cultures after 24 h (A) and 72 h (B) of growth to bare Teflon (1), Teflon coated with SC3 (2) and SC4 (3) in α-helical conformation and on TCPS (4). Surfaces were not washed with water prior to extraction with TFA. 1 µl of fresh medium supplemented with 10 % calf serum and 1µg fibronectin was used as a standard (5).

Discussion Hydrophobin coatings can be used to improve growth, morphology and activity of cells on hydrophobic solids [Scholtmeijer et al., 2002; Janssen et al., 2002; 2004; Chapter 2; 3; 4]. So far it was not known how stable the hydrophobin film was in a protein solution or in cell culture and whether proteins efficiently adsorbed to this coating.

The hydrophobins SC3 and SC4 have a much higher affinity to bare Teflon than BSA or fibronectin. After over night incubation only 0.002 mmol m-2 of BSA or fibronectin had adsorbed to the solid. In contrast, this was 0.1 mmol m-2 and 0.3 mmol m-2 for SC3 and SC4, respectively (Wö sten et al., 1994a; Janssen et al., 2004; Chapter 4). Scanning electron microscopy revealed that hydrophobins cover Teflon with a homogeneous layer [Janssen et al., 2004; Chapter 4]. In contrast, albumin and fibronectin adsorb to polymer surfaces like Teflon in a less uniform fashion, covering only part of the surface and leaving bare areas [Murthy et al., 1987].

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BSA, fibronectin and proteins in RPMI medium could not replace the hydrophobins at the Teflon surface in a competition experiment. The often reported displacement of initially adsorbed proteins from blood plasma to surfaces by high molecular-weight-proteins is referred to as the so-called Vroman effect [Schmaier et al., 1984; Turbill et al., 1996]. We found the SC3 film also to be stable when fibroblasts were grown at this coating. In contrast, SC4 was degraded, probably due to proteases released by the fibroblasts. It is not clear why cells grow better on SC4 coated Teflon even though apparently removing the hydrophobin from the surface. Assembled hydrophobins have been reported to be very protease resistant [W ö sten and Wessels, 1997]. However, in most cases resistance of the hydrophobic side of assembled hydrophobin in β-sheet state was determined. In contrast, in this study the hydrophilic side of assembled hydrophobins in α-helical state was exposed.

Proteins bound efficiently to hydrophobin-coated Teflon. Amounts of BSA, fibronectin and proteins in RPMI medium that adsorbed to the wettable hydrophobin-coated Teflon were similar to those on the non wettable bare Teflon. Comparable results were obtained for proteins adsorbed from artificial tear fluid (data not shown). This is interesting since the adsorption of plasma proteins was shown to increase at surfaces with increasing wettabilities [Lee and Lee, 1998]. In the absence of other proteins, only fibronectin adsorbed more strongly to SC4-coated Teflon than to bare and SC3-coated Teflon. However, in case fibronectin was adsorbed from supplemented RPMI medium it was efficiently removed. Probably, other proteins in the medium affected the interaction of fibronectin with SC4.

The results presented in this study show that the composition of the conditioning films formed at bare Teflon and hydrophobin-coated Teflon is similar, both quantitatively and qualitatively. Only in the presence of fibroblast cells fibronectin was more abundant at the hydrophobin-coated surfaces and at the TCPS control than at bare Teflon. This may explain why these fibroblasts grow better on hydrophobin-coated Teflon than on bare Teflon [see Chapter 3]. Additionally, the adsorbed proteins may adopt different conformations at each surface, which could affect the activity of the protein. Also the chemical composition of the surface of the bare and hydrophobin-coated solid may play a role. We expect that the hydrophobin is still partly exposed after the conditioning film has been formed. This is concluded from the fact that the amounts of BSA and fibronectin were similar on bare and hydrophobin-coated Teflon and that in the former case the surface was not homogeneously covered (see above).

Acknowledgements The authors would like to thank M. B. M. van Leeuwen for carrying out the cell growth experiments and J. G. H. Wessels for critically reading the manuscript.