Assay System Based on Metabolic Glycoengineering-Enabled Click Chemistry for the Efficient Evaluation of Complement-Regulatory Peptide Coatings on Endothelial Cells

Protective cell-surface coatings with complement-regulatory activity emerge as promising therapeutic strategies, particularly for preventing thromboinflammatory complications during transplantation. However, their development and in vitro evaluation require specialized assay systems. This study establishes an efficient platform to coat endothelial cells with peptides that recruit the physiological complement regulator factor H (FH) and test their activity. Azide-groups are introduced into cell-surface glycans of human and porcine endothelial cells via metabolic glycoengineering to enable covalent attachment of alkyne-labeled FH-binding peptides via click chemistry. By optimizing the type and concentration of azido-sugars, effective incorporation of accessible click handles can be achieved with minimal impact on cell viability. This approach results in uniform, controllable peptide coatings that efficiently recruit FH from purified sources and human serum to endothelial cell surfaces and enhance their resistance to complement-mediated opsonization. This model not only served as validation for the efficacy of FH-recruiting peptides but also provides a versatile platform to evaluate and optimize various parameters that define coating efficacy and gain insight into mechanisms of complement activation. As such, it may facilitate the screening of modulators and the development of protective coatings for future applications in biomedical or preclinical research.