Extended Data Figure 7: Carboxylation-sensitive antibodies identify functional motifs.
a. Multiple sequence alignment of Gla-domain containing proteins (UniProt) that bind the carboxylation-sensitive Gla-motif (ExxxExC) antibody using MUSCLE110,111. Antibody epitopes for both the carboxylation-sensitive FIX-specific antibody (ω-loop) and the carboxylation-sensitive Gla-motif antibody are shown. hF9: human coagulation factor IX (P00740); hF2: human prothrombin (coagulation factor II, P00734); hF7: human coagulation factor VII (P08709); hF10: human coagulation factor X (P00742); hPC: human protein C (P04070); hPS: human protein S (P07225); hBGP: human osteocalcin (P02818); bBGP: bovine osteocalcin (P02820); hGAS6: human growth arrest-specific protein 6 (P14393); ppVPA: Pseudechis prophyriacus venom prothrombin activator porpharin-D (P58L93); nsVPA: Notechis scutatis venom prothrombin activator notecarin-D1 (P82807); osVPA: Oxyuranus scutellatus venom prothrombin activator oscutarin-C (P58L96). b-c. Fluorescence of unrecombined negative control and WT FIX-expressing cells with and without warfarin pretreatment generated by staining cells with a carboxylation-sensitive FIX-specific (b) or carboxylation-sensitive Gla-motif antibody (c). d-f. Heatmaps showing carboxylation-sensitive FIX-specific carboxylation scores (d), carboxylation-sensitive Gla-motif carboxylation scores (e), or light chain secretion scores (f) for FIX propeptide variants. Furin cleavage site (Furin CS), ω-loop, ExxxExC motif, and aromatic stack (AS) are annotated above (d). Heatmap color indicates antibody score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased). Black dots indicate the WT amino acid. Missing data are gray. g-i. Heatmaps showing carboxylation-sensitive FIX-specific carboxylation scores (g), carboxylation-sensitive Gla-motif carboxylation scores (h), or light chain secretion scores (i) for FIX Gla domain variants. ω-loop, ExxxExC motif, and aromatic stack (AS) are annotated above (g). Heatmap color indicates antibody score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased). Black dots indicate the WT amino acid. Missing data are gray.