Abstract A001: Mapping the interaction between C-type lectin domain group 14A and Multimerin 2

Abstract

Abstract Introduction: Understanding the molecular basis of protein-protein interactions involved in the regulation of angiogenesis enhances our understanding of cancer pathogenesis and enables the development of novel anti-cancer therapies. In this study, we focused on the transmembrane spanning glycoprotein C-type lectin domain group 14A (CLEC14A), which is exclusively expressed on the tumor endothelium and is important in regulating tumor growth. CLEC14A promotes endothelial tube formation and migration in vitro, in addition tumor growth is retarded in CLEC14A knockout mice. CLEC14A, through its N-terminal C-type lectin domain (CTLD) interacts with the extracellular matrix protein Multimerin 2 (MMRN2) and heparan sulfate proteoglycans. Administration of antibodies which block the CLEC14A-MMRN2 interaction decreases tumor growth in vivo indicating the importance of this interaction, however the biological significance of CLEC14A’s binding to heparan sulfate proteoglycans is not known. Aims: The purpose of this study was to identify the area and key residues on CLEC14A which interact directly with MMRN2. Mapping the CLEC14A-MMRN2 interface would enable the design of small molecule inhibitors of this interaction, a potential anti-cancer strategy. Methods: We employed the Alpha fold predictive modelling algorithm to analyze the predicted structure of CLEC14A. Solvent-facing residues within a previously identified critical loop region were mutated to alanine and binding to MMRN2 was determined. Flow cytometry was used to assess the cell surface expression of these CLEC14A mutants, and far western blotting was employed to determine their capacity to bind MMRN2. Furthermore, we studied whether these mutants interacted with heparin-bound agarose beads in pulldown assays. Results: The alanine-scanning approach resulted in the identification of specific residues of CLEC14A which directly interacted with MMRN2. Furthermore, we identified the epitope on CLEC14A for a biologically active antibody previously reported to block the CLEC14A-MMRN2 interaction. Additionally, we identified critical residues that are essential for cell surface trafficking of CLEC14A. Importantly, the CLEC14A mutants that do not bind to MMRN2 retain their capacity to bind heparin. Conclusion: By leveraging predictive modelling software, we have characterized the molecular interaction between CLEC14A and MMRN2. Our findings may inform anti-angiogenesis drug design in the future. Citation Format: Aleen Baber, Victoria L Birmingham, Roy Bicknell, Philip R Morrison. Mapping the interaction between C-type lectin domain group 14A and Multimerin 2 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A001.