Screening of Tetraspanin CD63 Protein Interactors using Alto SPR

Description

Background: CD63 is a cell surface antigen that is a member of the tetraspanin superfamily with four transmembrane domains and variable loops. CD63 is a main component of the membranes of Weibel-Palade bodies that are associated with inflamed endothelial cells. CD63 is also a biomarker for activated platelets after α-granule release during inflammation. Weibel-Palade bodies and α-granules both secrete the multimeric glycoprotein von Willebrand factor (VWF) that tethers platelets to the site of injury during inflammation. The metalloprotease ADAMTS13 regulates VWF-induced platelet aggregation to prevent clot formation.

Aim: The objective of this study was to determine protein-protein interactions between CD63 and VWF and ADAMTS13 proteins.

Methods: The Alto system integrates digital microfluidics with localized surface plasmon resonance (SPR) biosensors to study biomolecular interactions. We applied the Alto SPR platform to utilize its protein-protein interaction screening capabilities. In this study, the recombinant human CD63 was immobilized on the biosensors and probed against VWF, ADAMTS13, and related protein variants.

Results: After screening 24 VWF, ADAMTS13, and related protein variants at a low and high concentration level against the recombinant human CD63 ligand, the direct screen test yielded a heat map overview of bound/not-bound status of each analyte sample screened. In addition to the heat map, each sample had a corresponding sensorgram that provided preliminary kinetic parameters such as association rate (k_a), dissociation rate (k_d), and dissociation constant (K_D).

Conclusions: The Alto SPR system provides the advantage of performing label-free analysis of biomolecular interactions using minimal sample volumes. After identifying protein-protein interaction hits between CD63 and VWF/ADAMTS13 proteins, we will apply the Alto SPR to perform kinetic binding studies that will yield kinetic parameters between CD63 and identified protein interactors.

Period	15 Feb 2024
Event title	Oklahoma State University Center for Health Sciences Research Week 2024
Event type	Conference
Location	Tulsa, United States, OklahomaShow on map
Degree of Recognition	Regional