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Cited by 42Supramolecular Self-Assembly in MaterialsProtein Structure and DynamicsRNA Research and Splicing

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Phase separation and ageing of glycine-rich protein from tick adhesive

Open access
Ganar, Ketan A., Nandy, Manali, Turbina, Polina, Chen, Chang, Suylen, Dennis, Nihoul, Elisa, Pascoe, Emily Louise, van der Beelen, Stan, Plaum, Maarten, van den Bos, Leendert, Koenraadt, Constantianus J. M., Dijkgraaf, Ingrid, Deshpande, Siddharth·2025·Nature Chemistry
DOI 10.1038/s41557-024-01686-8PMC PMC11794139

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Abstract

Hard ticks feed on their host for multiple days. To ensure firm attachment, they secrete a protein-rich saliva that eventually forms a solid cement cone. The underlying mechanism of this liquid-to-solid transition is currently not understood. This study focuses on the phase transitions of a disordered glycine-rich protein (GRP) found in tick saliva. We show that GRP undergoes liquid–liquid phase separation via simple coacervation to form biomolecular condensates in salty environments. Cation– π and π – π interactions mediated by periodically placed arginine and aromatic amino-acid residues are the primary driving forces that promote phase separation. Interestingly, GRP condensates exhibit ageing by undergoing liquid-to-gel transition over time and exhibit adhesive properties, similar to the naturally occurring cement cone. Finally, we provide evidence for protein-rich condensates in natural tick saliva. Our findings provide a starting point to gain further insights into the bioadhesion of ticks, to develop novel tick control strategies, and towards achieving biomedical applications such as tissue sealants.

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Figures

Fig. 1 Glycine-rich protein present in tick saliva is intrinsically disordered and shows a high propensity for LLPS. a, Schematic overview showing the consequence of a tick bite. The tick inserts its hypostome into the host epidermis and secretes a protein-rich saliva, abundant in GRPs. The saliva undergoes a liquid-to-solid transition, forming a hard cement cone, allowing the tick to feed on the host over several days and facilitating pathogen transmission (the shown ‘bull’s-eye’ rash is typical in the case of Borrelia infection, causing Lyme borreliosis). b, Amino-acid composition of tick-GRP77 shows a high proportion of non-polar amino acids (44%, of which 26% are glycine). c, AlphaFold correctly predicts the N-terminal signal peptide of GRP as an α-helix, while the rest of the sequence (tick-GRP77) remains unstructured, indicating a disordered region. d, The IUPred2A long disorder mode scores of the entire tick-GRP77 sequence are above 0.5, while the short disorder score shows prominent disorder near the termini, overall indicating tick-GRP77 to be a highly disordered protein.
Fig. 1 Glycine-rich protein present in tick saliva is intrinsically disordered and shows a high propensity for LLPS. a, Schematic overview showing the consequence of a tick bite. The tick inserts its hypostome into the host epidermis and secretes a protein-rich saliva, abundant in GRPs. The saliva undergoes a liquid-to-solid transition, forming a hard cement cone, allowing the tick to feed on the host over several days and facilitating pathogen transmission (the shown ‘bull’s-eye’ rash is typical in the case of Borrelia infection, causing Lyme borreliosis). b, Amino-acid composition of tick-GRP77 shows a high proportion of non-polar amino acids (44%, of which 26% are glycine). c, AlphaFold correctly predicts the N-terminal signal peptide of GRP as an α-helix, while the rest of the sequence (tick-GRP77) remains unstructured, indicating a disordered region. d, The IUPred2A long disorder mode scores of the entire tick-GRP77 sequence are above 0.5, while the short disorder score shows prominent disorder near the termini, overall indicating tick-GRP77 to be a highly disordered protein.CC BY · PubMed Central
Fig. 2 Tick-GRP77 undergoes LLPS via simple coacervation to form liquid-like condensates. a, Schematic of the droplet evaporation assay, where a droplet of a buffered tick-GRP77 solution is allowed to evaporate at room temperature, continuously increasing the protein concentration at the droplet boundary. b,c, Evaporation of a 2-µl tick-GRP77 droplet leads to the formation of spherical condensates near the phase boundary (ti ≈ 15 min). Subsequently, condensate formation rapidly spreads inward. Bright-field images are shown in b, with corresponding fluorescence images shown in c. d, Evaporation of tick-GRP77 on a surface-passivated glass slide showing numerous spherical tick-GRP77 condensates. e, Two similar-sized condensates coalescing and relaxing into a bigger condensate over a time span of a few seconds. f, For a typical example, change in aspect ratio as a function of time during a fusion event. The line shows an exponential decay fit (R2 = 0.99), giving a fusion timescale of τ ≈ 3 s. g, A linear fit for the decay time (τ) against the characteristic length scale (l) for several fusion events (n = 16) gives an inverse capillary velocity (η/γ) of 0.92 s µm−1 (R2 = 0.47). h, The phase-separated solution eventually dries out, forming salt crystals. However, rehydration of the dried sample resolubilizes the condensates. Evaporation of the resolubilized sample again leads to LLPS, demonstrating the reversibility of the phase separation process. The starting concentration of tick-GRP77 was 32 µM in PBS for all the experiments. In the case of fluorescence imaging, the samples were doped with 5 mol% OG488-GRP77. Source data
Fig. 2 Tick-GRP77 undergoes LLPS via simple coacervation to form liquid-like condensates. a, Schematic of the droplet evaporation assay, where a droplet of a buffered tick-GRP77 solution is allowed to evaporate at room temperature, continuously increasing the protein concentration at the droplet boundary. b,c, Evaporation of a 2-µl tick-GRP77 droplet leads to the formation of spherical condensates near the phase boundary (ti ≈ 15 min). Subsequently, condensate formation rapidly spreads inward. Bright-field images are shown in b, with corresponding fluorescence images shown in c. d, Evaporation of tick-GRP77 on a surface-passivated glass slide showing numerous spherical tick-GRP77 condensates. e, Two similar-sized condensates coalescing and relaxing into a bigger condensate over a time span of a few seconds. f, For a typical example, change in aspect ratio as a function of time during a fusion event. The line shows an exponential decay fit (R2 = 0.99), giving a fusion timescale of τ ≈ 3 s. g, A linear fit for the decay time (τ) against the characteristic length scale (l) for several fusion events (n = 16) gives an inverse capillary velocity (η/γ) of 0.92 s µm−1 (R2 = 0.47). h, The phase-separated solution eventually dries out, forming salt crystals. However, rehydration of the dried sample resolubilizes the condensates. Evaporation of the resolubilized sample again leads to LLPS, demonstrating the reversibility of the phase separation process. The starting concentration of tick-GRP77 was 32 µM in PBS for all the experiments. In the case of fluorescence imaging, the samples were doped with 5 mol% OG488-GRP77. Source dataCC BY · PubMed Central
Fig. 3 Arginine and aromatic residues are the key drivers of phase separation of tick-GRP77. a, The four fractions of tick-GRP77: N terminus (1–32), C terminus (33–77), C terminus mutant without aromatic amino acids (ΔFY mutant) and C terminus mutant without arginine residues (ΔR mutant). They all have a similar glycine content but varying basic (green), acidic (red) and aromatic (blue) amino-acid residues. b, Net charge per residue as a function of amino-acid position for the two termini, obtained using CIDER42. The N terminus is strongly negatively charged, and the C terminus is moderately positively charged. c, Droplet evaporation assay of the N terminus leads to phase separation and the formation of micrometre-sized condensates on a similar timescale as that of tick-GRP77 (ti ≈ 13 min). d, Droplet evaporation assay of the C terminus leads to quicker phase separation (ti ≈ 9 min), shown in the inset. The formed condensates readily wet the glass surface. e, Performing the evaporation assay on a PVA-passivated glass slide prevented surface wetting, leading to the formation of spherical C terminus condensates. OG488-GRP77 (5 mol%) readily partitioned in the condensates. f, Droplet evaporation assay of the ΔFY mutant led to weak phase separation, with ti ≈ 7 min. g, Droplet evaporation assay of the ΔR mutant led to negligible phase separation, with ti ≈ 7 min. The starting peptide concentrations were 50 µM for all panels. All experiments were performed in PBS.
Fig. 3 Arginine and aromatic residues are the key drivers of phase separation of tick-GRP77. a, The four fractions of tick-GRP77: N terminus (1–32), C terminus (33–77), C terminus mutant without aromatic amino acids (ΔFY mutant) and C terminus mutant without arginine residues (ΔR mutant). They all have a similar glycine content but varying basic (green), acidic (red) and aromatic (blue) amino-acid residues. b, Net charge per residue as a function of amino-acid position for the two termini, obtained using CIDER42. The N terminus is strongly negatively charged, and the C terminus is moderately positively charged. c, Droplet evaporation assay of the N terminus leads to phase separation and the formation of micrometre-sized condensates on a similar timescale as that of tick-GRP77 (ti ≈ 13 min). d, Droplet evaporation assay of the C terminus leads to quicker phase separation (ti ≈ 9 min), shown in the inset. The formed condensates readily wet the glass surface. e, Performing the evaporation assay on a PVA-passivated glass slide prevented surface wetting, leading to the formation of spherical C terminus condensates. OG488-GRP77 (5 mol%) readily partitioned in the condensates. f, Droplet evaporation assay of the ΔFY mutant led to weak phase separation, with ti ≈ 7 min. g, Droplet evaporation assay of the ΔR mutant led to negligible phase separation, with ti ≈ 7 min. The starting peptide concentrations were 50 µM for all panels. All experiments were performed in PBS.CC BY · PubMed Central
Fig. 4 Tick-GRP77 forms condensates in the presence of phosphate salts. a, Phase diagram showing the LLPS behaviour of tick-GRP77 in the presence of 1 M Na2HPO4 as a function of protein concentration and incubation time. b, Phase diagram demonstrating that the addition of crowding agents (PEG, 8 kDa) drastically lowers the critical salt concentration required for the onset of LLPS of 125 µM tick-GRP77. The red-outlined regions in a and b indicate the condensation regime. c, Top left: bright-field image showing a flow-focusing junction of the microfluidic device. The inner stream containing 63 µM tick-GRP77 in Milli-Q water is focused by two streams of 2 M Na2HPO4. As the three streams flow side by side, GRP condensates are observed to form exclusively at the GRP–salt interface. The condensates wet the channel wall and increase in size over time. d, Fluorescence intensity plot, corresponding to the dotted line in c (bottom left), shows the intensity profile across the interface of GRP–salt streams. e, GRP condensates stuck to the channel walls downstream of the junction are deformed into tear-shaped droplets because of the fluid flow, indicating their liquid-like nature.
Fig. 4 Tick-GRP77 forms condensates in the presence of phosphate salts. a, Phase diagram showing the LLPS behaviour of tick-GRP77 in the presence of 1 M Na2HPO4 as a function of protein concentration and incubation time. b, Phase diagram demonstrating that the addition of crowding agents (PEG, 8 kDa) drastically lowers the critical salt concentration required for the onset of LLPS of 125 µM tick-GRP77. The red-outlined regions in a and b indicate the condensation regime. c, Top left: bright-field image showing a flow-focusing junction of the microfluidic device. The inner stream containing 63 µM tick-GRP77 in Milli-Q water is focused by two streams of 2 M Na2HPO4. As the three streams flow side by side, GRP condensates are observed to form exclusively at the GRP–salt interface. The condensates wet the channel wall and increase in size over time. d, Fluorescence intensity plot, corresponding to the dotted line in c (bottom left), shows the intensity profile across the interface of GRP–salt streams. e, GRP condensates stuck to the channel walls downstream of the junction are deformed into tear-shaped droplets because of the fluid flow, indicating their liquid-like nature.CC BY · PubMed Central
Fig. 5 GRP condensates form viscoelastic networks and solid-like aggregates and exhibit adhesive properties. a, Droplet evaporation assay of a concentrated tick-GRP77 solution (500 µM in PBS) resulted in the formation of condensate fibres, leading to interconnected gel-like networks. b, Time-lapse showing the assembly of the condensates into fibre-like structures. Two such events are indicated by black arrows. c, Fluorescence image of an interconnected stretched network formed during an evaporation assay of tick-GRP77 (32 µM in five-times-concentrated PBS) on a PVA-passivated glass slide. d, Incubating tick-GRP77 solution (125 µM) in 1 M Na2HPO4 for 5.5 h at room temperature formed stable condensate clusters. The zoom-in shows physically connected condensates with complete arrest of coalescence. e, Time-lapse showing the fluorescence recovery of GRP condensates (125 µM) for fresh (0.5 h) and older (18 h) samples. The condensates were formed in the presence of 1 M Na2HPO4 salt. f, Fluorescence recovery curve showing that the freshly formed condensates (n = 6) have a higher fraction of the mobile phase compared with matured GRP condensates (n = 4). The solid line shows an exponential fit to the dataset (R2 = 0.99). Samples in c–f were doped with 5 mol% OG488-GRP77. g, Schematic of adhesion measurements showing the force spectroscopy set-up. h, Approach (red) and retract (blue) force–distance curves for the surface coated with tick-GRP77 condensates (125 µM) formed in the presence of 1 M Na2HPO4 (n = 3 different samples). The work of adhesion is four magnitudes higher than that of the negative control (n = 3 different samples). The inset shows approach and retract force–distance curves for a non-adhesive tick-GRP77 solution (125 µM) in Milli-Q water acting as a negative control. The plots in f and h are represented as mean ± s.d., with the dotted curves showing individual datasets. Source data
Fig. 5 GRP condensates form viscoelastic networks and solid-like aggregates and exhibit adhesive properties. a, Droplet evaporation assay of a concentrated tick-GRP77 solution (500 µM in PBS) resulted in the formation of condensate fibres, leading to interconnected gel-like networks. b, Time-lapse showing the assembly of the condensates into fibre-like structures. Two such events are indicated by black arrows. c, Fluorescence image of an interconnected stretched network formed during an evaporation assay of tick-GRP77 (32 µM in five-times-concentrated PBS) on a PVA-passivated glass slide. d, Incubating tick-GRP77 solution (125 µM) in 1 M Na2HPO4 for 5.5 h at room temperature formed stable condensate clusters. The zoom-in shows physically connected condensates with complete arrest of coalescence. e, Time-lapse showing the fluorescence recovery of GRP condensates (125 µM) for fresh (0.5 h) and older (18 h) samples. The condensates were formed in the presence of 1 M Na2HPO4 salt. f, Fluorescence recovery curve showing that the freshly formed condensates (n = 6) have a higher fraction of the mobile phase compared with matured GRP condensates (n = 4). The solid line shows an exponential fit to the dataset (R2 = 0.99). Samples in c–f were doped with 5 mol% OG488-GRP77. g, Schematic of adhesion measurements showing the force spectroscopy set-up. h, Approach (red) and retract (blue) force–distance curves for the surface coated with tick-GRP77 condensates (125 µM) formed in the presence of 1 M Na2HPO4 (n = 3 different samples). The work of adhesion is four magnitudes higher than that of the negative control (n = 3 different samples). The inset shows approach and retract force–distance curves for a non-adhesive tick-GRP77 solution (125 µM) in Milli-Q water acting as a negative control. The plots in f and h are represented as mean ± s.d., with the dotted curves showing individual datasets. Source dataCC BY · PubMed Central
Fig. 6 Salivary gland extracts from ticks hint at the presence of protein-rich phase-separated droplets. a, Schematic overview showing the extraction of the salivary gland from a non-blood-fed tick (I. ricinus). The collected supernatant was used in the following experiments. b, Bright-field visualization of the supernatant showing numerous micrometre-sized spherical droplets. c, Fusion of two droplets, indicating their liquid nature. d, Spiking the supernatant with fluorescently labelled GRP77 (OG488-GRP77) readily led to its partitioning inside the phase-separated droplets (marked with arrows in the bright-field image) indicating their protein-rich nature. e, Fluorescence image showing fibre-like structures observed in the supernatant in the presence of 0.75 M Na2HPO4 with OG488-GRP77 (6 µM), showing strong partitioning in these structures. Insets in d and e are zoom-ins from the respective images.
Fig. 6 Salivary gland extracts from ticks hint at the presence of protein-rich phase-separated droplets. a, Schematic overview showing the extraction of the salivary gland from a non-blood-fed tick (I. ricinus). The collected supernatant was used in the following experiments. b, Bright-field visualization of the supernatant showing numerous micrometre-sized spherical droplets. c, Fusion of two droplets, indicating their liquid nature. d, Spiking the supernatant with fluorescently labelled GRP77 (OG488-GRP77) readily led to its partitioning inside the phase-separated droplets (marked with arrows in the bright-field image) indicating their protein-rich nature. e, Fluorescence image showing fibre-like structures observed in the supernatant in the presence of 0.75 M Na2HPO4 with OG488-GRP77 (6 µM), showing strong partitioning in these structures. Insets in d and e are zoom-ins from the respective images.CC BY · PubMed Central

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Cites

Research works printed but not linked: 1

  1. Sonenshine, D. E. & Roe, R. M. Biology of Ticks Vol. 2 (Oxford Univ. Press, 2014).

Metadata

Authors
Ganar, K. A., Nandy, M., Turbina, P., Chen, C., Suylen, D., Nihoul, E., Pascoe, E. L., van der Beelen, S., Plaum, M., van den Bos, L., Koenraadt, C. J. M., Dijkgraaf, I., & Deshpande, S.
Affiliations
  • Wageningen University & Research (NL)
  • Maastricht University (NL)
  • Fondazione Edmund Mach (IT)
Corresponding author
  • Dijkgraaf, Ingrid
  • Deshpande, Siddharth
ORCID
Venue
Nature Chemistry
Publisher
Springer Science and Business Media LLC
Published
2024-11-29
Volume
17
Issue
2
Pages
186-197
ISSN
  • 1755-4330 (ISSN-L)
  • 1755-4349
DOI
10.1038/s41557-024-01686-8
PMC
PMC11794139
OpenAlex
W4404860401
Cited by
42
References
97
Citations by year
2026: 26 · 2025: 14 · 2024: 1
FWCI
3.86
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Chemistry (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Socio-Environmental Systems Modeling (submittedVersion, cc-by)
  • Research Publications (Maastricht University) (submittedVersion, cc-by)
Funders
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (OCENW.KLEIN.465)
Topics
  • Physical Sciences › Materials Science › Biomaterials › Supramolecular Self-Assembly in Materials
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
glycine-rich proteins, tick saliva, liquid-liquid phase separation, coacervation, biomolecular condensates, cation-π interactions, π-π stacking, bioadhesion, tick control, tissue sealants
MeSH terms
Phase Separation, Adhesives, Animals, Glycine, Saliva, Salivary Proteins and Peptides, Ticks, Phase Transition

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