Fungus-derived protein particles as cell-adhesive matrices for cell-cultivated food

Abstract

Cell-adhesive factors mediate adhesion of cells to substrates via peptide motifs such as the Arg-Gly-Asp (RGD) sequence. With the onset of sustainability issues, there is a pressing need to find alternatives to animal-derived cell-adhesive factors, especially for cell-cultivated food applications. In this paper, we show how data mining can be a powerful approach toward identifying fungal-derived cell-adhesive proteins and present a method to isolate and utilize these proteins as extracellular matrices (ECM) to support cell adhesion and culture in 3D. Screening of a protein database for fungal and plant proteins uncovered that ~5.5% of the unique reported proteins contain RGD sequences. A plot of fungi species vs RGD percentage revealed that 98% of the species exhibited an RGD percentage > = 1%. We observed the formation of protein particles in crude extracts isolated from basidiomycete fungi, which could be correlated to their stability towards particle aggregation at different temperatures. These protein particles were incorporated in 3D fiber matrices encapsulating mouse myoblast cells, showing a positive effect on cell alignment. We demonstrated a cell traction stress on the protein particles (from Flammulina velutipes) that was comparable to cells on fibronectin. A snapshot of the RGD-containing proteins in the fungal extracts was obtained by combining SDS-PAGE and mass spectrometry of the peptide fragments obtained by enzymatic cleavage. Therefore, a sustainable source of cell-adhesive proteins is widely available in the fungi kingdom. A method has been developed to identify candidate species and produce cell-adhesive matrices, applicable to the cell-cultivated food and healthcare industries.

Fig. 1. RGD percentage and phylogenetic relationship of edible and parasitic fungi.

A RGD percentage in the fungi and plant kingdom; B parasitic and edible fungi are represented by the blue bars. C Phylogenetic tree showing the relationship between some of the fungi in (B).

Fig. 2. Precipitation and analysis of fungal extracts.

A Comparison of crude extracts from F. velutipes after standing at 4 °C and room temperature (25 °C) for 4 days. For the extract at 25 °C, spontaneous precipitation was observed to give a suspension of white particles. B Boiling of the protein extract accelerated the formation of the particles. C Growth of particle diameters at increasing temperature for extracts from the fungal species, reflecting their respective stability towards protein aggregation. ANOVA tests were performed to compare the particle diameter between different fungi at each particular temperature. The error bars represent one standard deviation of uncertainty. The P values are represented by asterisks (*); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns: P > 0.05.

Fig. 3. Cell-alignment analysis.

A Cells encapsulated in chitin-alginate fibers incorporated with fungal particulate extracts (FV F. velutipes; LE L. edodes; PE P. eryngii), fibers with collagen (CGN) and fibers with no ECM. Scale bars represent 100 µm. B Plot of cell alignment for the various fiber types (n ≥ 12). C The frequency of cell alignment with orientation angles of 0° (range 0–10°) and 180° (range 170–180°), and 90° (range 70–110°) (n ≥ 3). The error bars represent one standard deviation of uncertainty. Chi-square tests were performed to compare the difference in frequency of alignment between the different sample types. (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). B, C were plotted using cumulative data over all 4 time points (0, 1, 2, 4 days).

Fig. 4. Traction force microscopy.

A Representative bright-field transmission images (left column) and corresponding traction stress maps (right column) indicating traction stresses for C2C12 cells at 0.1%, 1%, 5 and 10% of FVP (right panel), and with fibronectin (FN) as a positive control (left panel). Stress field maps were obtained from digital image correlation for polyacrylamide hydrogels with Young’s modulus of 14 kPa. Arrows indicate the directions of associated traction stresses. Scale bar represents 30 μm. Color bar indicates the magnitudes of the traction stress in units of Pa. B Bar plots for cell area (μm²) (top panel) and magnitude of maximum traction stress (Pa) (bottom panel) for C2C12 cells on polyacrylamide substrates coated with 0.1%, 1%, 5 and 10% of FVP or 50 μg/mL fibronectin (FN), or no ECM (sterile water) and RGD peptide with ECM (Fibronectin or FVP) as negative controls (N ≥ 14 cells). Error bars represent standard error of the mean.

Fig. 5. SDS-PAGE analysis of fungal extracts.

A SDS-PAGE gel showing bands of the protein ladder and crude extracts derived from F. velutipes (FV), P. eryngii (PE), and L. edodes (LE), respectively. RGD proteins of known molecular weight identified by the MS fragmentation experiments were mapped onto the profile. The gels were derived from the same experiment and processed in parallel. B Plot of band density vs protein molecular weight (kDa) for crude extract from F. velutipes. The putative RGD proteins, C1 protein, and beta-glucosidase have been indicated on this plot.