Figure 6

CLEC14A and CD93 bind MMRN2 in long-loop region of the CTLD. (a) CLEC14A-CTLD iTASSER generated molecular model, displaying long-loop region in red and numbered α-helices and β-sheets. (b) CLEC14A-CTLD model displaying predicted arrangement of cysteine residues. Cysteines C31, C37, C54, C143, C162 and C171 are canonical CTLD fold cysteines and are in close proximity to form disulphide bonds. C103 and C138 are non-canonical cysteines present in the long-loop region. (c) Solved crystal structure of tetranectin CTLD (1TN3), displaying long-loop region in red and numbered α-helices and β-sheets. (d) CLEC14A-CTLD model with predicted antibody epitopes 1–42 (showing 21–42 in the model, blue), 122–142 (orange) and region 97–108 (grey). (e) Flow cytometry analysis of HEK293T transfected with CLEC14A^{THBD(97–108)}, CLEC14A^C103S or CLEC14A^C138S. Only C2 and C3 bind to CLEC14A^{THBD(97–108)}, all C1–5 bind CLEC14A^C103S and CLEC14A^C138S all CLEC14A mutant proteins fail to bind MMRN2^495–674. (f) Flow cytometry analysis of HEK239T transfected with CD93 wt, CD93^C104S or CD93^C136S. All proteins bind R139 antibody showing correct conformational folding and presence at the cell surface, although both CD93^C104S and CD93^C136S fail to bind MMRN2^495–674.