BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3619

Peptides:

Sequences
CRGAMWMYKRC(5/6) CEGLAIQVKQC(1/6)

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (competitive panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 36070465

Target Name:

Hyaluronan-binding protein 2

Template Name:

Not determined.

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

CX9C phage display library

Affinity Measurement Method:

Affinity Measurement Description:

Brief Description:

Tubes and streptavidin-coated Dynabeads (Thermo Fischer Scientific, Oslo, Norway) were blocked with PBS with 4% (w/v) skim milk powder. Approximately 1 × 10^13 virions of library and 1−10 μg (R1, 10 μg; R2, 3:1 μg) of biotinylated pro-FSAP were incubated with the appropriate amount of Dynabeads for 60 min at room temperature (RT). The beads/ protein/phage complexes were separated from the supernatant using a magnetic rack and washed with PBS-T20 (0.1% (v/v) Tween-20 in phosphate-buffered saline (PBS)). The complexes were then incubated with an excess amount of unbiotinylated pro-FSAP for 60 min at RT to remove phages bound with low-affinity and retain only high-affinity binding clones. The beads/protein/phage complexes were then separated from the supernatant containing the soluble FSAP using a magnet rack and washed with PBS-T20 (0.1% (v/v) Tween-20 in PBS) and with PBS pH 7.4. This was applied to both libraries and in all rounds. The remaining phages were then eluted from the beads with 500 μL of 100 mM triethylamine (pH 11) and neutralized in Tris−HCl pH 7.4.

BiopanningDataSet Comments:

Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide (CRGAMWMYKRC), NNKC9/41, that activates pro-factor VII Activating protease (FSAP) in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30−35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.

Curator:

Bowen Li

Curation Date:

2023-10-13

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