Computer-aided engineering of staphylokinase toward enhanced affinity and selectivity for plasmin

Nikitin, Dmitri; Mičan, Jan; Toul, Martin; Bednář, David; Pešková, Michaela; Kittová, Patrícia; Thalerová, Sandra; Víteček, Jan; Damborský, Jiřı́; Mikulík, Robert; Fleishman, Sarel J.; Prokop, Zbyněk; Marek, Martin

doi:10.1016/j.csbj.2022.03.004

Cited by 4 publications

(2 citation statements)

References 49 publications

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“…Channels of a freshly prepared microfluidic chip were modified with plasma for 1 min (for details, see the previous chapter). Channels were subsequently coated with rhodamine b-labeled BSA solution (0.87 mg mL –1 ), prepared as described in Nikitin et al, 33 for 1 h at 37 °C. After coating, channels were thoroughly washed with PBS and were imaged under a microscope (AxioObserver Z1, Zeiss, Germany) in 1 mm sections.…”

Section: Materials and Methodsmentioning

confidence: 99%

Luminal Surface Plasma Treatment of Closed Cylindrical Microchannels: A Tool toward the Creation of On-Chip Vascular Endothelium

Černík

Poláková

Kubala

et al. 2023

ACS Biomater. Sci. Eng.

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On-chip vascular microfluidic models provide a great tool to study aspects of cardiovascular diseases in vitro. To produce such models, polydimethylsiloxane (PDMS) has been the most widely used material. For biological applications, its hydrophobic surface has to be modified. The major approach has been plasma-based surface oxidation, which has been very challenging in the case of channels enclosed within a microfluidic chip. The preparation of the chip combined a 3D-printed mold with soft lithography and commonly available materials. We have introduced the high-frequency low-pressure air-plasma surface modification of seamless channels enclosed within a PDMS microfluidic chip. The plasma treatment modified the luminal surface more uniformly than in previous works. Such a setup enabled a higher degree of design freedom and a possibility of rapid prototyping. Further, plasma treatment in combination with collagen IV coating created a biomimetic surface for efficient adhesion of vascular endothelial cells as well as promoted long-term cell culture stability under flow. The cells within the channels were highly viable and showed physiological behavior, confirming the benefit of the presented surface modification.

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Section: Materials and Methodsmentioning

confidence: 99%

Luminal Surface Plasma Treatment of Closed Cylindrical Microchannels: A Tool toward the Creation of On-Chip Vascular Endothelium

Černík

Poláková

Kubala

et al. 2023

ACS Biomater. Sci. Eng.

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“…The second aspect we considered was the role that SAK could play in modulating the proteolytic susceptibility of S. aureus adhesins of the A region. Recent computational modeling efforts allowed engineering of SAK for better thrombolytic behavior; however, its native role as a virulence factor in S. aureus infections has been poorly investigated. We evaluated the impact of SAK binding to plasmin on the digestion patterns observed within the adhesins.…”

Section: Introductionmentioning

confidence: 99%

Adhesive Virulence Factors of Staphylococcus aureus Resist Digestion by Coagulation Proteases Thrombin and Plasmin

Risser

Lopez-Morales

Nash

2022

ACS Bio Med Chem Au

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Staphylococcus aureus (S. aureus) is an invasive and life-threatening pathogen that has undergone extensive coevolution with its mammalian hosts. Its molecular adaptations include elaborate mechanisms for immune escape and hijacking of the coagulation and fibrinolytic pathways. These capabilities are enacted by virulence factors including microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and the plasminogen-activating enzyme staphylokinase (SAK). Despite the ability of S. aureus to modulate coagulation, until now the sensitivity of S. aureus virulence factors to digestion by proteases of the coagulation system was unknown. Here, we used protein engineering, biophysical assays, and mass spectrometry to study the susceptibility of S. aureus MSCRAMMs to proteolytic digestion by human thrombin, plasmin, and plasmin/SAK complexes. We found that MSCRAMMs were highly resistant to proteolysis, and that SAK binding to plasmin enhanced this resistance. We mapped thrombin, plasmin, and plasmin/SAK cleavage sites of nine MSCRAMMs and performed biophysical, bioinformatic, and stability analysis to understand structural and sequence features common to protease-susceptible sites. Overall, our study offers comprehensive digestion patterns of S. aureus MSCRAMMs by thrombin, plasmin, and plasmin/SAK complexes and paves the way for new studies into this resistance and virulence mechanism.

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C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin

Kaur

Sethi

Hade

et al. 2023

Archives of Biochemistry and Biophysics

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Computer-aided engineering of staphylokinase toward enhanced affinity and selectivity for plasmin

Cited by 4 publications

References 49 publications

Luminal Surface Plasma Treatment of Closed Cylindrical Microchannels: A Tool toward the Creation of On-Chip Vascular Endothelium

Luminal Surface Plasma Treatment of Closed Cylindrical Microchannels: A Tool toward the Creation of On-Chip Vascular Endothelium

Adhesive Virulence Factors of Staphylococcus aureus Resist Digestion by Coagulation Proteases Thrombin and Plasmin

C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin

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